IMMUNOLOGY SYLLABUS 2013 University of Texas Medical School at Houston

“The Immune System is a Vital Organ System, Necessary for Life.”

IMMUNOLOGY SYLLABUS 2013 - TABLE OF CONTENTS Course Schedule

iii

Course Description

iv-vi

Essay Assignment + Exam/Essay Review Policy

vii-ix

Clinical Correlation: Reading List

x

Team Based Learning Exercise - Integrative Exercises

xi-xii

Lectures & Clinical Correlations

(Begins at Page Number 1)

OVERVIEW AND ELEMENTS OF THE IMMUNE SYSTEM Medical Importance of the Immune System / How the Immune System Works Cells and Organs of the Immune System Innate Immunity/Inflammation

Syllabus Page 1 13 22

ANTIGENS, ANTIBODIES AND T CELL RECEPTORS - STRUCTURE AND ACTIVITIES Immunogens & Antigens Antibody Structure and Function I+II

38 49

COMPLEMENT Complement

73

ANTIBODY, T CELL RECEPTORS, AND MHC – STRUCTURE AND ACTIVITIES Genetic Basis of Ab Structure Role of MHC in the Immune Response The T Cell Receptor: Structure and Genetic Basis Adaptive Immune Response: I+II

92 101 114 123

CELLULAR ACTIVITIES AND IMMUNE MEDIATION Antigen-Antibody Interactions - ImmunoAssays Antibody-Mediated Reactions Cell-Mediated Reactions

145 162 173

IMMUNE SYSTEM AND INFECTIOUS DISEASE Immunology of HIV Infection Infection and Immunity

182 194

MEDICAL APPLICATIONS OF IMMUNOLOGY (Immunopathology) Immune Regulation & Tolerance Autoimmunity Clinical Scenarios Immunology of Cancer Immunoprophylaxis (Vaccines) & Immunotherapy Disorders of the Immune Response Transplantation Team Based Learning Exercise Evolution of the Immune System

206 219 226 227 228 239 251 254 255

Timeline of Immunology

(located at end of syllabus) (located at end of syllabus)

APPENDIX: Resource Information i

Cover Description: Principles of Modern Immunobiology. B.H. Park and R.A. Good. 1974. Lea & Febiger, Henry Kimpton Publishers, Philadelphia. p54.

The purpose of the Immunology course is to provide a basic knowledge of the immune response and its involvement in health and disease. A series of lectures cover course components; additional materials are presented through clinical correlations that focus on clinically applied immunological concepts. An effort has been made to increase clinical relevance and problem-solving skills through an essay assignment and through a team-learning exercise.

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SCHEDULE - IMMUNOLOGY 2013 Session Date Time Instructor OVERVIEW AND ELEMENTS OF THE IMMUNE SYSTEM Jeffrey Actor 1 1/8/2013 10:00-10:50 2 1/8/2013 11:00-11:50 Jeffrey Actor 3 1/11/2013 11:00-11:50 Jeffrey Actor ANTIGENS AND ANTIBODIES 4 1/15/2013 10:00-10:50 5 1/15/2013 11:00-11:50 6 1/18/2013 9:00-9:50 COMPLEMENT 7 1/22/2013 10:00-10:50

MEDIC web site Topic Medical Importance of the Immune System Cells and Organs of the Immune Sytstem Innate Immunity/Inflammation

Immunogens & Antigens Antibody Structure and Function I Antibody Structure and Function II

Sudhir Paul Keri Smith Keri Smith

Complement

Rick Wetsel

ANTIBODIES, T CELL RECEPTORS, AND MHC - STRUCTURE AND ACTIVITIES Steven Norris Genetic Basis of Ab Structure 8 1/22/2013 11:00-11:50 9 1/24/2013 9:00-9:50 Jeffrey Actor Role of MHC in the Immune Response 10 1/24/2013 10:00-10:50 Jeffrey Actor The T Cell Receptor: Structure and Genetic Basis 11 1/24/2013 11:00-11:50 Jeffrey Actor Adaptive Immune Response 1 12 1/25/2013 10:00-10:50 Jeffrey Actor Adaptive Immune Response 2 13 1/25/2013 11:00-11:50 Antigen-Antibody Interactions Keri Smith 1/31/2013 1:00-3:00 1:00-3:00 Midterm Exam CELLULAR ACTIVITIES AND IMMUNE MEDIATION Steven Norris 14 2/5/2013 9:00-9:50 15 2/7/2013 10:00-10:50 Steven Norris

Antibody-Mediated Reactions Cell-Mediated Reactions

IMMUNE SYSTEM AND INFECTIOUS DISEASE Steven Norris 16 2/8/2013 10:00-10:50 17 2/8/2013 11:00-11:50 Jeffrey Actor

Immunology of HIV Infection Infection and Immunity

MEDICAL APPLICATIONS OF IMMUNOLOGY (Immunopathology) Shen-An Hwang 18 2/12/2013 11:00-11:50 19 2/14/2013 10:00-10:50 Sandeep Agarwal Sandeep Agarwal and Jeffrey Actor 20 2/14/2013 11:00-11:50 21 2/18/2013 11:00-11:50 Priya Weerasinghe 22 2/19/2013 11:00-11:50 Semyon Risin 23 2/21/2013 11:00-11:50 William Shearer 24 2/25/2013 10:00-10:50 Wasim Dar 25 2/28/2013 8:00-9:50 Jeffrey Actor 26 2/28/2013 10:00-10:50 Adan Rios 3/7/2013 1:00-4:00 3/19/2013 Essay Assignment Due

1:00-4:00

Immune Regulation & Tolerance Autoimmunity Immunology: Clinical Scenarios Cancer Immunology Immunoprophylaxis (Vaccines) & Immunotherapy Disorders of the Immune Response Transplantation Team Based Learning Evolution of the Immune System

Final Exam Essay must be submitted prior to 5:00pm

MEDICAL SCHOOL IMMUNOLOGY - 2013 COURSE DESCRIPTION Course Director:

Jeffrey K. Actor, Ph.D.

Office Hours:

Fridays at 1:00-2:00p in MSB 2.214; or by appointment

LECTURERS

OFFICE

TELEPHONE

email

Jeffrey K. Actor, Ph.D. Sandeep K. Agarwal, M.D., Ph.D. Wasim A. Dar, M.D., Ph.D. Shen-An Hwang, Ph.D. Steven J. Norris, Ph.D. Sudhir Paul, Ph.D. Adan Rios, M.D. Semyon A. Risin, M.D., Ph.D. William T. Shearer, M.D., Ph.D. Keri C. Smith, Ph.D. Priya Weerasinghe, Ph.D. Rick A. Wetsel, M.D., Ph.D.

MSB 2.214 BCM MSB 6.256 MSB 2.2221E MSB 2.278 MSB 2.230A UPB-830 MSB 2.022 Texas Children's MSB 2.248 MSB 2.408 SRB 430A

713-500-5344 713-798-3390 713-500-7400 713-500-5265 713-500-5338 713-500-5347 713-500-7766 713-500-5294 832-824-1274 713-500-2250 713-500-5275 713-500-2412

[email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected]

IMMUNOLOGY WEB PAGE: http://www.uth.tmc.edu/pathology/medic/immunology/immuntbl.htm

1) COURSE ORGANIZATION The purpose of the Immunology course is to provide a basic knowledge of the immune response and its involvement in health and disease. All lectures will be presented in MSB 2.006. An effort has been made to increase clinical relevance and problem-solving skills through an essay assignment and facultypresented clinical correlations, and a team based learning exercise. Any questions on the lecture material should be addressed to Dr. Actor or directly to that lecturer. If you have general problems or comments regarding the course, your grades, or the faculty, please contact the course director. If the problem is not resolved, you should make an appointment to see Dr. Robert L. Hunter (Chairman of Pathology) at MSB 2.136 (500-5301) or, finally, Dr. Patricia Butler (Assoc. Dean for Educational Programs) or Dr. Margaret McNeese (Assoc. Dean for Student Affairs). 2) COURSE MATERIALS a)

Lectures. The student is responsible for all material covered in lectures and faculty presented clinical correlations, as well as for any additional handouts or assignments (whether provided in this syllabus or at a later time). Immunology is a rapidly advancing area, so the lectures may contain new information not covered in the textbooks. Therefore you should make every effort to attend lecture and take complete and accurate notes. Tapes of the lectures are available from the Conference Operations Office (LRC) and can be used to verify your notes. Streaming video is available on-line through the UT Med School student web pages.

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b) Reading. Two textbooks are required for the course. Chapter assignments are listed directly in the syllabus chapter. Required Case Studies are listed separately in this syllabus. R. Coico and Sunshine, G. Immunology: A Short Course. (6th Ed) John Wiley & Sons, Inc., 2009. R. S. Geha and Notarangelo, L. Case Studies in Immunology: A Clinical Companion. (6th Ed) Garland Publishing, New York, 2012. The Coico et al. text was selected because it is well-organized, clearly and concisely written, and contains chapter summaries, study questions, and case studies. The lecture schedule has been loosely organized to match the Coico et al. book chapters. Knowledge of the assigned reading is required, even if the material is not covered in the lectures. Modifications of the study questions may be used in the exams. The Geha and Rosen text provides examples of the role of immunology in health and disease, and is used extensively in the Clinical Correlations and as ‘Clinical Vignettes’ in the lectures. Cases from Geha and Notarangelo presented (in part or in full) during lecture are considered required reading. Please see the list of required associated cases for each lecture, located under “Clinical Correlation Required Readings”. c) MEDIC IMMUNOLOGY Web Page. You are encouraged to make use of the MEDIC Immunology web site at: http://www.uth.tmc.edu/pathology/medic/immunology/immuntbl.htm . Materials are also on Backboard. The website is actively updated during the course to include links for lecture materials and information that will assist in understanding of course materials. Alternative recommended texts available in the bookstore:     

Actor, J.K. Elsevier’s Integrated Immunology and Microbiology (2nd Ed.), Mosby/Elsevier, Philadelphia, 2012. Parham, P. The Immune System. 3rd Edition. Garland Publishing, New York, 2009. A. K. Abbas and Lichtman, A. H. Basic Immunology – Functions and Disorders of the Immune System, 3rd Edition (updated edition). Saunders-Elsevier. Philadelphia, PA. 2011. Kindt, TJ, Goldsby, RA, Osborne, BA. Kuby Immunology (6th Ed.), W.H. Freeman and Company, New York, 2007. Murphy, K. Janeway’s Immunobiology (8th Ed.). Garland Publishing, New York, 2012.

Each of these texts may be found at the LRC or the HAM Library. d) Essay Assignment. Students must turn in one essay assignment worth 10 points. Students must attend one of the City-Wide Infectious Disease Rounds and provide a written review of one of the cases. The assignment and due date are described in detail elsewhere in the syllabus, as well as on Blackboard. e) Team Based Learning. There will be one team based learning exercise as a portion of the course. The TBL is detailed later in the syllabus. f) Clinical Scenarios. In addition to the regular lectures, we will have a Clinical Scenario session during the semester. Past experience has shown that immunology (or any other medical topic) is easier to learn and remember if it is presented as clinical cases involving 'real' patients. In each clinical correlation, cases relevant to immunology will be discussed by faculty. The correlate scenarios are related to those presented in the Geha and Notarangelo text, but may vary to accommodate additional learning materials. g) AIDS Education Project. Students who participate in the AIDS Education Project (i.e. undergo the training and participate according to guidelines) are eligible for two extra credit points. See your AIDS Education Project representative for details. h) Study Questions. Additional study questions are provided at the end of some lecture outlines. The v

purpose of these questions is to test your knowledge and extend your learning beyond rote memorization toward more 'cognitive' learning. The study questions will not be graded, but questions related to these assignments will appear in the examinations. Answers are posted on the Immunology web site. i) Streaming video/Video tapes. Lectures will be made available for viewing via streaming video over the internet. j) Office hours and other assistance. Students are encouraged to approach the lecturers if they need assistance in understanding the course material. Dr. Actor is also available at his office (MSB 2.214) by individual appointment or by phone or email. 3) GRADING a) Examinations. There will be two major exams consisting of multiple choice, matching, and national board format questions. The midterm exam will contain 60 questions (worth 40% of your grade), the final exam will have 80 questions (worth 40% of your grade). Exam answers will be posted according to accepted policies of the university. Sessions may be scheduled for question review after each exam. b) Essay Assignment. The essay assignment is required and will be worth a possible 10 points. Grading will be based on adherence to the format described in this syllabus, thoroughness, and application of your budding medical knowledge and logic; you are not expected to 'know it all' at this point. The due date is listed in the Essay instruction page in the syllabus, and posted on Blackboard. Essays may be turned in early. Assignments turned in late will only receive a maximum of half credit (no exceptions). c) Team Based Learning. Questions answered for the TBL session will be worth a total of 10 points. d) Extra credit. One opportunity for extra credit is available. The extra credit option is voluntary, and is not a course requirement. Participation in AIDS Education Project

2 points

e) Overall grade. The total possible points and grade assignments are given below. The total value of points for the course is 100 points, plus 2 points possible through completion of the extra credit option. Midterm Final Essay Assignment Team Based Learning

40 points 40 points 10 points 10 points

(60 questions) (80 questions)

Extra Credit

2 possible additional points

f) Final grade assignment. The final grade is based on percentage of points earned (max of 102 points) as related to total possible points (max of 100). Honors High pass Pass Marginal Performance Fail

90-100 % 85.5-89.99 % 69.5-85.49 % 65.5-69.49 % 65.49 % or below

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IMMUNOLOGY ESSAY ASSIGNMENT The purpose of this assignment is to encourage you to explore an important form of information in medicine: grand rounds presentations. You are not expected to master the analysis of the information presented, but you should demonstrate that you have made an honest attempt to understand and interpret it. The assignment is worth a possible 10 points (maximum). The assignment is due any time on, or before, 5:00pm on March 19, 2013. Assignments turned in late will receive a maximum of only half credit. Completed assignments are to be turned into the Health Education Office (MSB 2.120). Assignments are not accepted via e-mail. YOU MUST TYPE YOUR ESSAY. THE ASSIGNMENT SHOULD BE APPROXIMATELY TWO-THREE PAGES FOR YOUR ANSWER. THIS IS NOT A SHARED ASSIGNMENT; DUPLICATE ESSAYS (TURNED IN BY MORE THAN ONE STUDENT) WILL NOT BE ACCEPTED. Examples of the essay assignment can be found posted at: http://www.uth.tmc.edu/pathology/medic/immunology/Immuno/Ex1.Essay.Assignment.pdf or on Blackboard. ______________________________________________________________________________ ASSIGNMENT: Grand Rounds Review. Attend one of the City-Wide Infectious Disease Conferences held every Wednesday at Noon in the auditorium behind the elevators on the ground floor of the DeBakey Building, Baylor College of Medicine (BCM Rm M112). (This building is the white building next to the new Baylor Graduate School Building and across the street from the Jones Library). Usually three cases are presented as unknowns, a differential diagnosis is made, and the outcome and ramifications of the case are discussed. The presenters often provide handouts for the case, but you may wish to take notes of the conference to help you glean out the information. Note that the infectious disease and microbiology aspects are generally covered in detail, whereas the immunology is often not discussed. Your job is to investigate the immunologic aspects of the disease and incorporate them into your interpretation and case description. This is an Immunology assignment; limit your discussion of Microbiology to pertinent information only. It is not permitted to record or capture pictures/slides of presented materials. No picture taking is allowed (due to HIPAA regulations, patient confidentiality, and proprietary information). Choose only ONE of the cases presented. The case must have an immunologic implication. (e.g. The infection was cleared by immune mechanisms, or the patient was immunodeficient and thus developed an unusual infection). a) Briefly describe the case, concentrating on the clinical manifestations (patient's symptoms + findings from examination and tests) that are most relevant. Use medical terms where you can, but define them in a few words. Include the diagnosis, treatment, and outcome (if presented). USE YOUR OWN WORDS. Include a copy of the handout for the case, if one was provided (see scoring note below*). b) Using your microbiology and immunology texts, describe the organism(s) which caused the infection in this case. What is the normal course of disease, and how did they differ in this case? What treatments are generally effective, and were they effective in this patient? vii

c) The major portion of the essay should be devoted towards discussion of the immunologic implications and principles of the case. Describe in as much detail as possible the normal immune mechanisms to combat this infectious agent and how they affect the course of infection (e.g. Macrophages phagocytose and process the antigen and present antigen fragments in association with MHC Class II proteins to antigen-specific CD4+ helper T cells, role of complement, cell phenotypes involved, etc.). Be specific and included details! How was the immune response of this patient different than normal (if this is applicable)? Did the patient have an underlying condition that contributed to the development of this infection? Did the patient have cancer, AIDS, hereditary immunodeficiency or some other condition affecting the immune response? How did the immune response (or lack thereof) affect the outcome of this case? Did the immune response contribute to the pathogenesis of disease (i.e. is immunopathology involved)? Describe immunization or other immunologic procedures (such as passive transfer of antibodies) used in the prevention or treatment of this disease. d) Cite references used in your analysis of the case. You will need to refer to published journal articles to obtain specific background information or methods needed to comprehend the case. Points will be subtracted if relevant citations are absent. You must include at least 2 primary publications (meaning: journal articles) published within the past 3 years. Web pages are not considered as primary references. You may also include syllabus chapters as references, but must also include additional references that demonstrate you have expanded your discussion to materials outside the course lecture presented materials. Syllabus chapters are NOT primary references. Up to 1 point is subtracted if the references are missing, incomplete, or inadequate to support your discussion. Recommended: use PubMed to find related articles for the report. e) *You must include a copy of the handout from the Grand Rounds session. 1 point is subtracted if the handout is missing. Therefore, chose a case with a handout whenever possible. f) The length of the essay should not exceed 3 pages (including references). 1 point is subtracted for going over the set page limit. In summary, make sure you:  Describe clinical manifestations of the case.  Discuss immunological aspects of case.  Give full citations to cite your ideas, including use of current references from journal articles.  Attach a copy of the Grand Rounds handout for the case.  Turn in your assignment on time. Essays may be submitted anytime prior to the stated deadline. Late submitted essays will automatically receive a 5 point deduction.

ESSAY GRADES: Essays will be returned to students as quickly as grading allows. Inquiries regarding essay assignment grading must be submitted within one week after receipt of returned assignments. Requests for review of essays past the one week period will be denied.

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Policy on Exam Grading (MS1 and MS2 courses) and Exam Review Sessions Recommended by the Educational Policy Subcommittee: August 12, 2010 Approved by the Curriculum Committee: August 25, 2010 Revised by Educational Programs: August 31, 2010 Approved by the Curriculum Committee: September 15, 2010 Revised and Revisions Approved by the Curriculum Committee: May 18, 2011 The following policy delineates procedures related to exam grading and review/protest sessions to be followed by all first- and second-year courses. 1. Course directors will score examinations through LXR and post results on MSGradebook as soon as possible. 2. Course directors will use item statistics generated by LXR to identify problematic questions. Upon review, if a course director determines that a question was written incorrectly (e.g. had more than one or no correct answer), then the director will give all students credit for that particular question. 3. Large group post-exam review sessions may be held to provide feedback on difficult examination topics, in a manner deemed appropriate by the course director. Copies of the examinations will not be returned to the students during these sessions. a. Course directors may meet with individual students to review examinations. The format of these sessions, which may involve reviewing specific examination questions, will be determined by the course director on a case by case basis.

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CLINICAL CORRELATIONS 2013 Required Readings 2013 Immunology Clinical Correlation Required Readings: R. S. Geha and Notarangelo, L. Case Studies in Immunology: A Clinical Companion. (6th Ed) Garland Publishing, New York, 2012. Reading for Lecture: Required Readings Assigned to lecture: Cells and Organs 30. Congenital Asplenia Assigned to lecture: Innate Immunity 15. Chediak-Higashi Syndrome 25. Neutropenia 26. Chronic Granulomatous Disease 27. Leukocyte Adhesion Deficiency Assigned to lecture: Antibody Structure and Multiple Myeloma (Blackboard file) Function 46. Hemolytic Disease of the Newborn Assigned to lecture: Complement 32. Factor I Deficiency 33. Deficiency of C8 Assigned to lecture: MHC 8. MHC Class II Deficiency 12. MHC Class I Deficiency Assigned to lecture: T cell receptor 7. Omenn Syndrome T-Cell Lymphoma (Blackboard file) Assigned to lecture: Adaptive Immune Response 47. Toxic Shock Syndrome Assigned to lecture: Antibody-Mediated Reactions 41. Autoimmune Hemolytic Anemia 50. Allergic Asthma 52. Drug-Induced Serum Sickness Assigned to lecture: T cell 45. Acute Infectious Mononucleosis Mediated Reactions 53. Contact Hypersensitivity to Poison Ivy Assigned to lecture: Infection and Immunity 28. Recurrent Herpes Simplex Encephalitis 48. Lepromatous Leprosy Assigned to lecture: Immunology of HIV Infections 10. Acquired Immune Deficiency Syndrome (AIDS) Assigned to lecture: Autoimmunity 36. Rheumatoid Arthritis 35. Systemic-Onset JID 40. Multiple Sclerosis 42. Myasthenia Gravis Assigned to lecture: Disorders of the Immune 1. X-linked Agammaglobulinemia Response 4. CVID 9. DiGeorge Syndrome 16. Wiskott-Aldrich Syndrome Assigned to lecture: Transplantation Kidney Graft Complications (Blackboard file) 11. Graft-Versus-Host Disease Clin. Corr. Class

Date 2/14

Time 11:00-11:50 AM

TBL

2/28

8:00-9:50 AM

  

Case Readings 36. Rheumatoid Arthritis 37. Systemic Lupus Erythematosus Distributed Reading: Inflammatory Bowel Diseases (Crohn’s Disease, Ulcerative Colitis, and Celiac) 39. Crohn’s Disease 44. Celiac Disease

Required readings complement lectures and presented materials. It is highly encouraged to view these clinical cases. Case materials may not be covered in full during lectures, however, all required case study readings contain material that may be tested on exams. Assigned readings may be discussed in multiple lectures, in addition to the “assigned” lectures.

*Note: Most cases also appear in the previous edition: Geha and Rosen. Case Studies in Immunology. Garland Publishing, New York, NY. 5th edition, 2007. Listed titles have different case numbers between book editions. Cases not included in the newer edition are posted on Blackboard, or can be found in the LRC. x

Spring Semester, 2013

Team Based Learning Exercise The Immunology course will have one Team Based Learning exercise where students will be required to address a clinically based scenario and provide answers to related questions. Students will be assigned specific reading prior to the session, which will assist in mastering of the material so as to allow participation in the group activities. Materials will include new material in Immunology, as well as materials already mastered in other courses. The format will be similar to the Clinical Applications course. The Team Based Learning Exercise is mandatory. The Team Based Learning Exercise encompasses a graded set of exercises related to multiple integrated aspects of a clinical scenario. The exercise is worth a maximum of 10 points towards your overall Immunology grade. The session will utilize clinical scenario(s) to present problem(s). Students are divided into teams; utilizing the groups already in place for the Clinical Applications course. Approximately 5 problem questions arising from the clinical scenario are crafted to foster discussion within the teams; each team is required to come to a consensus as to the solution to the problem. Written justification may be required for the team solution, to be prepared and handed in for grading at the end of the session. Team Based Learning Exercise: February Immunology 28th

8:00-9:50 a.m.

Persons missing the session must provide written notice explaining circumstances for not attending. Written approval must be obtained from the Office of Educational/Student Affairs prior to consideration for any makeup session or alternate assignment.

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Spring Semester, 2013

Clinical Applications: Integrative Exercises There will be a series of Clinical Applications (Integrative Exercises) throughout the first year, up to three of which are scheduled for the Spring 2013 semester. These exercises are designed to integrate content from the basic science courses and the ICM course and to help students develop reasoning skills they will utilize in their clinical years. The administration of these Exercises is held separate from the Immunology course, but material from the exercises will be subject to assessment in all of the first year courses. Attendance is required at these sessions and will be monitored. The dates of the Clinical Applications Integrative Exercises during the Spring semester are as follows; see Blackboard to confirm times and room assignments: Students will be assigned to work in small groups of four to six students. These groups will remain together for all seven of the Integrative Exercises throughout the year. During the Integrative Exercises, each group will discuss a clinical problem that integrates material from the current basic science courses and will develop a team answer to a question regarding that clinical problem. The teams will then prepare a written justification for their answer for one of these problems. These justifications will be handed in for grading. Pre-reading and pretests may be posted to Blackboard as necessary for each exercise. You will receive email notifications regarding any prereading or pretest assignments. The graded responses from all of the sessions will contribute to the final grade in the Integrative Exercise course. Each of the group members will receive the same score. Students who have unexcused absences will receive a score of 0 for all responses for that Integrative Exercise session.

Information presented within any Clinical Application Exercise throughout the year is a potential source of testable material for exams in any MSI class.

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MEDICAL IMPORTANCE OF THE IMMUNE SYSTEM [HOW THE IMMUNE SYSTEM WORKS] Jeffrey K. Actor, Ph.D. MSB 2.214, 713-500-5344 (Special thanks to Gailen D. Marshall, Jr., MD, PhD, and Steven J. Norris, PhD) Objectives 1. To appreciate the components of the human immune response that work together to protect the host 2. To understand the concept of immune-based diseases as either a deficiency of components or excess activity as hypersensitivity 3. To introduce the 7 main concepts of the course KEYWORDS

immunodeficiency, hypersensitivity

Required Knowledge: Hypersensitivity Chart Reading: Coico and Sunshine. Immunology: A Short Course. John Wiley & Sons, Inc, New York, NY. 6th edition, 2009. Chapter 1. Web Resource: http://www.uth.tmc.edu/pathology/medic/immunology/Immuno/How2.htm The chief function of immunity is to discriminate between self and non-self. The immune cells and organs of the body comprise the primary defense system against invasion by microorganisms and foreign pathogens. A functional immune system confers a state of health through effective elimination of infectious agents (bacteria, viruses, fungi, and parasites) and through control of malignancies by protective immune surveillance. In essence, the process is based in functional discernment between self and non-self, a process which begins in utero and continues through adult life. Immune responses are designed to interact with the environment to protect the host against pathogenic invaders. The goal of these chapters is to appreciate the components of the human immune response that work together to protect the host. Furthermore, we will strive to present a working clinical understanding of the concept of immune-based diseases resulting from either immune system component deficiencies or excess activity. Immunological memory as the basis for vaccine efficacy Continued discrimination for health depends upon immunological memory where the adaptive immune system can more efficiently respond to previously encountered antigen. This results in resistance to repeated infection with pathogens and the resulting clinical syndrome. This principle accounts for the clinical utility of vaccines which have done more to improve mortality rates worldwide than any other medical discovery in recorded history.

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The clinical immunologist is a physician who has specialized in the diagnosis and treatment of disorders of the immune system. Many other clinical specialties (such as oncology, hematology, infectious diseases, transplant surgery, etc.) also deal with immunologicallybased diseases in their area of specialization. Much of the work of modern clinical immunologists revolves around refining diagnostic techniques for greater clinical utility and evaluating new therapeutic modalities such as recombinant cytokines and cytokine modulators. Protection against foreign pathogens Normal physiologic functions of the immune system include the ability to discern self from non-self, and recognition of foreign pathogens. This represents recognition of environmental challenges in an attempt to preserve homeostasis while responding to pathogenic agents. The goal is to respond with specificity, allowing sufficient intensity and duration to protect the host without causing damage to self. The Immune system protects against foreign pathogens, of which four major classes can be defined. These include (1) Extracellular bacteria, parasites and fungi; (2) Intracellular bacteria and parasites; (3) Viruses (intracellular); and (4) Parasitic worms (extracellular). Immunodeficiency and dysfunction as the basis of disease Immunological diseases can be grouped into two large categories – deficiency and dysfunction. Immunodeficiency diseases occur as the result of the absence (congenital or acquired) of one or more elements of the immune system. Immune dysfunction occurs when a particular immune response occurs that is detrimental to the host. This response may be against a foreign antigen or self antigen. It may also be an inappropriate regulation of an effector response resulting in the absence of a protective response. Notwithstanding, the host is adversely affected. A healthy immune system occurs as a result of balance between innate and adaptive immunity, cellular and humoral immunity, inflammatory and regulatory networks and small biochemical mediators (cytokines). Because specific mechanism affects prognosis as well as therapeutic approaches, Gel and Coombs classified these dysfunctional immune responses into hypersensitivity diseases. Hypersensitivities will be discussed throughout the course, and in much greater detail in a later chapter. There is considerable overlap in underlying mechanisms that contribute to the hypersensitive responses. The major mechanisms are outlined on the following page.

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Type I (also called immediate hypersensitivity) is due to aberrant production and activity of IgE against normally nonpathogenic antigens (commonly called allergens). The IgE binds to mast cells via high affinity IgE receptors. Subsequent antigen exposure results in crosslinking of mast cell bound IgE with activation of mast cells that release preformed mediators (eg. histamine, leukotrienes, etc.) and synthesize new mediators (i.e. chemotaxins, cytokines). These mediators are responsible for the signs and symptoms of allergic diseases. [A = Allergic] Type II is due to antibody directed against cell membrane-associated antigen that results in cytolysis. The mechanism may involve complement (cytotoxic antibody) or effector lymphocytes that bind to target cell-associated antibody and effect cytolysis via a complement independent pathway (Antibody dependent cellular cytotoxicity, ADCC). Cytotoxic antibodies mediate many immunologically-based hemolytic anemias while ADCC may be involved in the pathophysiology of certain virus-induced immunological diseases. [C = Cytotoxic] Type III results from soluble antigen-antibody immune complexes that activate complement. The antigens may be self or foreign (i.e. microbial). Such complexes are deposited on membrane surfaces of various organs (i.e. kidney, lung, synovium, etc). The byproducts of complement activation (C3a, C5a) are chemotaxins for acute inflammatory cells. These result in the inflammatory injury seen in diseases such as rheumatoid arthritis, systemic lupus erythematosus, postinfectious arthritis, etc). I = Immune Comples] Type IV (also called Delayed Type Hypersensitivity, DTH) involves macrophage-T cellantigen interactions that cause activation, cytokine secretion and potential granuloma formation. Diseases such as tuberculosis, leprosy and sarcoidosis as well as contact dermatitis are all clinical examples where the tissue injury is primarily due to the vigorous immune response rather than the inciting pathogen itself. D = DTH] EXPANDED FIGURES OF HYPERSENSITIVITIES INCLUDED IN APPENDIX.

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Clinical suspicion for immunodeficiency may be made when patients present with chronic infection or chronic inflammatory status, poor wound healing, constant fatigue and malaise, or when unresponsive to vaccine administration. Certain infections with organisms may be suggestive of deficiency in an immune related component. Alternatively, disruptions in homeostasis may lead to immunodeficiency, such as those induced inadvertently by a physician through medical treatment (iatrogenic). The mechanisms for clinical immunodeficiency are varied, and will be examined (in part) throughout the remainder of the course. Therapeutic intervention for immune based diseases Therapy for these diseases has historically been nonspecific, centering on repair of the damaged tissues and inhibition of the aberrant immune responses with immunosuppressive drugs. Recent work using such cutting edge techniques as recombinant DNA technology, gene therapy, and stem-cell research have opened up an entire new avenue to address these diseases by providing diagnostic and therapeutic modalities not previously available. For immunodeficiency states, we have developed the g ability to replace elements through marrow transplants, recombinant immune molecule administration and, soon, gene therapy.

Introduction to 7 Main Concepts towards Understanding Medical Immunology 1.

The chief function of the immune system is to distinguish between self and non-self. Health – effective elimination or control of health-threatening agents Infectious agents – bacteria, viruses, fungi, parasites Tumors – the immune system also plays an important role in the control of malignant cells through a mechanism called immune surveillance Hyporeactivity – inability to recognize and control health-threatening agents (immunodeficiency) Congenital immunodeficiency – immune defects due to genetic defects Acquired immunodeficiency – caused by multiple agents, including Human Immunodeficiency Virus and tumors Malnutrition – severe malnutrition compromises the immune system Young/Old Age – increased susceptibility to infection Hyperreactivity – aberrant immune responses Systemic autoimmunity – e.g. systemic lupus erythematosus Organ-Specific autoimmunity – e.g. thyroiditis

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Allergies and Asthma – aberrant immune response to environmental allergens or chemicals Immunopathology – general term for damage to normal tissue due to the immune reaction to infectious agents or other antigens (e.g. rheumatic fever, leprosy)

2.

Figure: Immune based diseases can be caused by lack of specific functions (immune deficiency) or excessive activity (hypersensitivity). The immune system consists of two overlapping compartments: the innate immune system and the adaptive immune system. Innate immune system  Most primitive type of immune system; found in virtually all multicellular animals (arguably also in plants!)  Always present and active, constitutively expressed  Nonspecific; not specifically directed against any particular infectious agent or tumor  Same every time; no ‘memory’ as found in the adaptive immune system  First line of defense against infection  Includes: o Physical barriers (skin, mucus lining of gastrointestinal, respiratory and genitourinary tracts) o Phagocytic cells – neutrophils, macrophages o Protective chemicals – acid pH of stomach, lipids on skin surface o Enzymes – lysozyme in saliva, intestinal secretions; digests cell walls of bacteria o Alternate complement pathway – cascade of serum proteins that are activated by bacterial cell wall components

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Adaptive or acquired immune system  Found only in vertebrates (fish, amphibians, birds and mammals)  Must be induced to be active against infections or tumors  Antigen-specific – adaptive immune responses recognize antigens, which can be proteins, carbohydrates, lipids and nucleic acids.  Memory – response against a given antigen is much stronger after the first (primary) response. This heightened reactivity is called secondary responses, and is due to increased numbers of memory B and T cells to that antigen  Regulation – discriminates between self and non-self, prevents autoimmune reactions in most individuals o B lymphocytes – differentiate into plasma cells that produce antibodies o T lymphocytes – subdivided into CD4+ and CD8+ populations  Helper activity – help other lymphocytes respond to antigen (mostly CD4+ T cells, subdivided into phenotypic responders)  Delayed type hypersensitivity – activate macrophages to phagocytose, kill pathogens (mostly CD4+ T cells)  T cell-mediated cytotoxicity – cytotoxic T cells (mostly CD8+ T cells) bind to and kill target cells (e.g. virus-infected cells and tumor cells)  Suppressor T cells/Treg cells – down-regulate the responses of other lymphocytes

Table: Elements of Innate and Acquired Immune Responses Innate Adaptive Rapid response (minutes to hours) Slow Response (days to weeks) PMNs and Phagocytes B cells and T cells Preformed effectors with limited variability B cell and T cell receptors with highly selective Pattern Recognition Molecules recognizing specificities structural motifs Soluble activators Antibodies (humoral) Proinflammatory mediators Cytokines (cellular) Non-specific Specific No memory, no increase in response upon Memory, maturation of secondary response secondary exposure

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3.

The antigenic specificity of the adaptive immune system is due to antigenspecific receptors. 

Immunoglobulins (also called antibodies) – produced by B cell lineage o IgM, IgD, IgG, IgA, and IgE subtypes o Surface immunoglobulin (Ig) – antigen-specific receptor of B lymphocytes o Secreted immunoglobulin (Ig) – Ig molecules secreted by plasma cells



T cell receptor (TCR) – antigen-specific receptor of T lymphocytes o  and  TCR subtypes

Coico and Sunshine, 2009. Fig. 1.3.



The basic reaction in immunology is the binding of antigen to an antigen-specific receptor. The affinity of this interaction is similar to that of an enzyme binding to its substrate. Ag + Ab AgAb



Typically, each antibody or T cell receptor molecule recognizes a single epitope, a small region (e.g. 6-10 amino acids) of an antigen. In a given B- or T-cell, the antigen-specific receptors of all are identical. o Exception – IgM and IgD can be coexpressed on certain B cells Each B cell and T cell has its own antigenic specificity, determined by the amino acid sequence of its surface Ig or TCR. The region of the Ig or TCR that binds to the antigen is called the paratope. In each person, there are ~106 to 108 different Igs and TCRs, giving rise to an almost endless supply of antigenic specificities. This is called diversity.

  

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4.

The generation of antigen-binding diversity occurs prior to antigen exposure through a DNA rearrangement process called VDJ joining.



The “business end” of an antibody or TCR is the variable region. This region contains the antigen-binding site that binds to the epitope (meaning: the conformational shape recognized). Variable Region Coico and Sunshine, 2009. Fig. 1.2.

   

 

The variable region is formed during B and T cell development. This process occurs prior to exposure to a given antigen. The DNA encoding the variable region is subdivided into V, D, and J gene segments. There are multiple V, D, and J gene segments in the Ig and TCR genetic loci. In most cells, these gene segments are spread out, so that all the V segments are together, all the D segments are together, and all the J segments are together. This is called the germline configuration, because it is the arrangement seen in sperm and ova. The V, D, and J gene segments are brought together to form a contiguous exon encoding the variable region. The V, D, and J segments are selected randomly in each cell, giving rise to combinatorial diversity. This is similar to the “Pick 5” game in Texas lotto, in which a large number of different number combinations exist. The light chain gene locus (and some TCR genes) has only V and J regions. There are several other mechanisms for generating diversity, as will be discussed in a later lecture.

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5.

To generate an active immune response against a certain antigen, a small number of B and T cell clones that bind to the antigen with high affinity undergo activation, proliferation, and differentiation into plasma cells (for B cells) or activated T cells. This process is called ‘clonal selection’. 



  

B and T cells are resting cells that lack functional activity until they undergo activation, proliferation, and differentiation into plasma cells or activated T cells. This process takes several days, which explains the lag between being exposed to an infectious agent and eventually getting better when the immune response ‘kicks in’. Of the millions of different specificities of B and T cells produced, only a few will have surface Ig or TCRs that bind the antigen with high affinity. However, we produce B and T cells that will react with virtually any antigen, including those that are man-made and are not found in nature (e.g. di-nitrophenol). In nearly all cases, activation of a B or T cell requires two signals: binding of the antigen-specific receptor to the antigen, and exposure to proteins called cytokines expressed by helper T cells. The blast cells resulting from activation undergo proliferation, resulting in a ~100fold expansion of the number of cells reactive to the antigen. Some of these cells become effector cells (plasma cells and activated T cells that express activities that help to eliminate the pathogen. Others become memory cells that can give rise to secondary responses as described below.

…(106-108 clones)

Coico and Sunshine, 2009. Fig. 1.1.

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6.

  

 

The adaptive immune system has memory, meaning that the response against an antigen is much greater after the first exposure. The first response to an antigen is called the primary response, and responses thereafter are called secondary responses. The different properties of secondary responses are due to memory cells generated during the primary responses. Secondary responses have o Higher antibody levels o Increased proportion of IgG and other immunoglobulin isotypes o Shorter lag period o Higher affinity for antigen Vaccination is effective because it primes the immune system to provide secondary responses when the individual is exposed to an infectious agent. Each exposure to an antigen tends to increase the secondary response. This is why booster immunizations are often used in vaccinations.

Coico and Sunshine, 2009. Fig. 4.12.

Figure: Primary and secondary antibody responses. The adaptive immune system has memory, allowing for maturation of a rapid secondary immune response with higher specificity and magnitude directed against foreign substances.

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7.  



The immune system is tightly regulated. Self-reactive B and T cell clones are generated as a natural part of the random VDJ recombination process. The adaptive immune system has developed several mechanisms to eliminate or inhibit self-reactive B and T cells. o Elimination of self-reactive cells during their development through apoptosis. o Permanent inactivation of self-reactive cells through a process called clonal anergy. o Inhibition of self-reactive cells by suppressor cells, inhibitory cytokines, and other factors Each immune response requires a combination of multiple factors, thereby limiting the number of spurious responses.

SUMMARY – MEDICAL IMPORTANCE OF THE IMMUNE SYSTEM AND HOW THE IMMUNE SYSTEM WORKS Thus it can be said that the healthy immune system occurs as a result of balance – between innate and adaptive immunity, cellular and humoral immunity, inflammatory and regulatory networks and even cytokine modulators. Disease occurs when the balance is altered either by deficiency or dysfunction. Current and future research efforts center about defining exact hypersensitivity and/or immunodeficiency mechanisms in specific diseases, developing diagnostic assays that have individual patient relevance and finding more specific agents that can regulate or eliminate aberrant immune responses while leaving the rest of the system intact. Research opportunities abound in the broadening area of clinical immunology. SUMMARY 

The immune response is designed to interact with the environment to protect the host against pathogenic invaders.



Immune-based diseases are either because of a lack of specific elements (immune deficiency) or excess activity (hypersensitivity).



Hypersensitivity Chart: Know the differences between types of hypersensitivity.

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1. The chief function of the immune system is to distinguish between self and nonself. 2. The immune system consists of two overlapping compartments: the innate immune system and the adaptive immune system. 3. The antigenic specificity of the adaptive immune system is due to antigen-specific receptors. 4. The generation of antigen-binding diversity occurs prior to antigen exposure through a DNA rearrangement process called VDJ joining. 5. To generate an active immune response against a certain antigen, a small number of B and T cell clones that bind to the antigen with high affinity undergo activation, proliferation, and differentiation into plasma cells (for B cells) or activated T cells. This process is called ‘clonal selection’. 6. The adaptive immune system has memory, meaning that the response against an antigen is much greater after the first exposure. 7. The immune system is tightly regulated.

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CELLS AND ORGANS OF THE IMMUNE SYSTEM Jeffrey K. Actor, Ph.D. 713-500-5344 Objectives: (1) Identify cell types involved in specific and non-specific immune responses. (2) Present the developmental pathway of immune system cells. (3) Understand structure and function of primary and secondary lymphoid organs. Keywords: Reticuloendothelial System, Leukocytes, Myeloid Cells, Lymphocytes, Antigen Presenting Cells (APC), GALT, MALT, BALT, Cluster of Differentiation (CD). Reading: Coico and Sunshine. Immunology: A Short Course. John Wiley & Sons, Inc, New York, NY. 6th edition, 2009. Chapter 2; Geha and Notarangelo. Case Studies in Immunology. Garland Publishing, New York, NY. 6th edition, 2012. Case 30: Congenital Asplenia. Web Resource: http://www.uth.tmc.edu/pathology/medic/immunology/Immuno/cellsimmsys.html Immune system cells are derived from pluripotent hematopoietic stem cells in the bone marrow. These cells can be functionally divided into groups that are involved in two major categories of immune responses: innate (natural) and acquired. Innate immunity is present from birth and consists of non-specific components. Acquired immunity by definition requires recognition specificity to foreign (non-self) substances. The major properties of the acquired immune response are specificity, memory, adaptiveness, and discrimination between self and non-self. The acquired immune response is subdivided into humoral and cellular immunity, based on participation of two major cell types. In Humoral Immunity, B lymphocytes synthesize and secrete antibodies. Cellular Immunity (CMI) involves effector T lymphocytes which secrete immunoregulatory factors following interaction with antigen presenting cells (APCs).

Figure. The developmental pathway of pluripotent bone marrow stem cells. Coico and Sunshine, 2009. Fig. 2.1.

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Figure. The interrelationship between innate and acquired immunity. An intricate communication system allows components of innate and acquired immunity to work in concert to combat infectious disease. Coico and Sunshine, 2009. Fig. 2.12.

Cluster of Differentiation (CD): Cell surface molecules are identifiable by monoclonal antibodies. In humans, these molecules have been given number designations. The acronym CD describes the cluster of antigens with which the antibody reacts; the number describes the order in which it was discovered. As of 2010, the list of determinants officially identified 350 individual and unique markers (link to Human CD Molecules). Surface expression of a particular CD molecule may not be specific for just one cell or even a cell lineage. However, many are useful for characterization of cells. CD-specific monoclonal antibodies have been useful for 1) determining the functions of CD proteins; 2) identifying the distribution of CD proteins in different cell populations in normal individuals; 3) measuring changes in the proportion of cells carrying these markers in patients with disease (e.g. decrease in CD4+ T cells is a hallmark of HIV infection); 4) developing therapeutic measures for increasing or decreasing the numbers or activities of certain cell populations.

Figure. Nomenclature of Inflammatory Cells.

Reticuloendothelial System Cells of the RES provide natural immunity against microorganisms by 1) a coupled process of phagocytosis and intracellular killing, 2) recruiting other inflammatory cells through the production of cytokines, and 3) presenting peptide antigens to lymphocytes for the production of antigen-specific immunity. The RES consists of 1) circulating monocytes; 2) resident macrophages in the liver, spleen, lymph nodes, thymus, submucosal tissues of the respiratory and 14

alimentary tracts, bone marrow, and connective tissues; and 3) macrophage-like cells including dendritic cells in lymph nodes, Langerhans cells in skin, and glial cells in the central nervous system. Leukocytes Leukocytes provide either innate or specific adaptive immunity. These cells are derived from myeloid or lymphoid lineage. Myeloid cells include highly phagocytic, motile neutrophils, monocytes, and macrophages that provide a first line of defense against most pathogens. The other myeloid cells, including eosinophils, basophils, and their tissue counterparts, mast cells, are involved in defense against parasites and in the genesis of allergic reactions. Cells from the lymphoid lineage are responsible for humoral or cell mediated immunity. Myeloid Cells Neutrophils: Neutrophils are the most highly adherent, motile, phagocytic leukocytes and are the first cells recruited to acute inflammatory sites. They ingest, kill, and digest pathogens, with their functions dependent upon special proteins, such as adherence molecules, or via biochemical pathways (respiratory burst). Eosinophils: Eosinophils defend against parasites and participate in hypersensitivity reactions via cytotoxicity. Their cytotoxicity is mediated by large cytoplasmic granules, which contain eosinophilic basic and cationic proteins. Basophils/Mast cells: Basophils, and their tissue counterpart mast cells, produce cytokines that help defend against parasites, and also cause allergic inflammation. These cells display high affinity surface membrane receptors for IgE antibodies, and have many cytoplasmic granules containing heparin and histamine. The cells degranulate when cell-bound IgE antibodies are crosslinked by antigens, and produce low-molecular weight vasoactive mediators (e.g. histamine). Monocytes/Macrophages: Monocytes and macrophages are involved in phagocytosis and intracellular killing of microorganisms. Macrophages are differentiated monocytes, which are one of the principal cells found to reside for long periods in the RES. These monocytes/macrophages are highly adherent, motile and phagocytic; they marshal and regulate other cells of the immune system, such as T lymphocytes; they serve as antigen processing-presenting cells. Dendritic Cells: Dendritic cells provide a link between innate and adaptive immunity by interacting with T cells in a manner to deliver strong signals for development of memory responses. Dendritic cells recognize foreign agents and pathogens through a series of pattern recognition receptors (non-specific), and are able to present antigen to both T helper and T cytotoxic cells to allow those lymphocytes to mature towards functionality. Lymphoid Cells Lymphoid cells provide efficient, specific and long-lasting immunity against microbes/pathogens and are responsible for acquired immunity. Lymphocytes differentiate into three separate lines: (1) thymic-dependent cells or T lymphocytes that operate in cellular and humoral immunity; (2) B lymphocytes that differentiate into plasma cells to secrete antibodies; and (3) natural killer (NK) 15

cells. T and B lymphocytes produce and express specific receptors for antigens while NK cells do not. B Lymphocytes: B lymphocytes differentiate into plasma cells to secrete antibodies. The genesis of mature B cells from pre-B cells is antigen-independent. The activation of B cells into antibody producing/secreting cells (plasma cells) is antigen-dependent. Mature B cells can have 1-1.5 x 105 receptors for antigen embedded within their plasma membrane. Once specific antigen binds to surface Ig molecule, the B cells differentiate into plasma cells that produce and secrete antibodies of the same antigen-binding specificity. If B cells also interact with T helper cells, they proliferate and switch the isotype (class) of immunoglobulin that is produced, while retaining the same antigen-binding specificity. T helper cells are thought to be required for switching from IgM to IgG, IgA, or IgE isotypes. In addition to antibody formation, B cells also process and present protein antigens. T Lymphocytes: T lymphocytes are involved in the regulation of the immune response and in cell mediated immunity, and help B cells to produce antibody. Mature T cells express antigen-specific T cell receptors (TCR). Every mature T cell also expresses the CD3 molecule, which is associated with the TCR. In addition mature T cells usually display one of two accessory molecules, CD4 or CD8, which define whether a T cell will be a helper T lymphocyte, or a cytotoxic T lymphocyte (CTL). The TCR/CD3 complex recognizes antigens associated with the major histocompatibility complex (MHC) molecules on target cells (e.g. virus-infected cell). Development of T lymphocytes During differentiation in the thymus, immature T cells undergo rearrangement of their TCR  and  genes to generate a diverse set of clonotypic TCRs. Immature thymocytes are selected for further maturation only if their TCRs do not interact with self-peptides presented in the context of self-major histocompatibility complex (MHC) molecules on antigen presenting cells. T Helper Cells: T helper cells (Th) are the primary regulators of T cell- and B cell-mediated responses. They 1) aid antigen-stimulated subsets of B lymphocytes to proliferate and differentiate toward antibody-producing cells; 2) express the CD4 molecule; 3) recognize foreign antigen complexed with MHC class II molecules on B cells, macrophages or other antigen-presenting cells; and 4) aid effector T lymphocytes in cell-mediated immunity. Currently, it is believed that there are two main functional subsets of Th cells, plus multiple other helper subsets of importance. T helper 1 (Th1) cells aid in the regulation of cellular immunity, and T helper 2 (Th2) cells aid B cells to produce certain classes of antibodies (e.g., IgA and IgE). The functions of these subsets of Th cells depend upon the specific types of cytokines that are generated, for example interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) by Th1 cells; IL4, IL-6 and IL-10 by Th2 cells. Two other classes of T helper cells are thought to be involved in oral tolerance and serve as regulators for immune function. Th17 cells, characterized by IL-17 secretion, are thought to be involved as effector cells for autoimmune disease progression, and protect surfaces (skin, gut) from extracellular bacteria. Tfh cells (follicular helper T cells) also provide help to B cells enabling them to develop into antibody-secreting plasma cells. They function inside of follicular areas of lymph nodes. Finally, although no longer prevalent in the literature, a subclass called Th3 cells were historically identified as secreting IL-4 and TGF- to provide help for IgA production; they were thought to be suppressive for Th1 and Th2 cells. 16

T Cytotoxic Cells: T cytotoxic cells (CTLs) are cytotoxic against tumor cells and host cells infected with intracellular pathogens. These cells 1) usually express CD8, and, 2) destroy infected cells in an antigen-specific manner that is dependent upon the expression of MHC class I molecules on antigen presenting cells. T Suppressor/ T Regulatory Cells: T suppressor cells suppress the T and B cell responses and express CD8 molecules. T regulatory cells (Tregs) also affect T cell response, with many cells characterized as CD4+CD25+, TGF- secretors. Tregs regulate/suppress other T cell activities, and help prevent development of autoimmunity. Natural Killer T Cells: Natural killer T cells (NKT) are a heterogeneous group of T cells that share properties of both T cells and natural killer (NK) cells. These cells recognize an antigenpresenting molecule (CD1d) that binds self- and foreign lipids and glycolipids. They constitute only 0.2% of all peripheral blood T cells. The term “NK T cells” was first used in mice to define a subset of T cells that expressed the natural killer (NK) cell-associated marker NK1.1 (CD161). It is now generally accepted that the term “NKT cells” refers to CD1d-restricted T cells coexpressing a heavily biased, semi-invariant T cell receptor (TCR) and NK cell markers. Natural killer T (NKT) cells should not be confused with natural killer (NK) cells. Natural Killer Cells: NK cells are large granular “innate” lymphocytes that nonspecifically kill certain types of tumor cells and virus-infected cells. NK cells share many surface molecules with T lymphocytes. These circulating large granular lymphocytes are able to kill “self” in the absence of antigen-specific receptors. NK cells are especially effective against viral infected cells, and keep the expansion of virus in check until adaptive immunity kicks in. In this regard, they also secrete interferon-gamma, which is an effective immunoregulator. NK cells can also kill via antibody-dependent cellular cytotoxic mechanisms (ADCC) via their Fc receptors. NK cells 17

express a large number of receptors that deliver either activating or inhibitory signals, and the relative balance of these signals controls NK cell activity. Antigen Presenting Cells (APCs) are found primarily in the skin, lymph nodes, spleen and thymus. They may also be present throughout the diffuse lymphoid system. Their main role is to present antigens to antigen-sensitive lymphoid cells. APCs may be characterized by their ability to phagocytose antigens, location in body, and expression of Major Histocompatibility Complex (MHC) related molecules. Two main types of APCs are Dendritic Cells and Macrophages. Of note, B cells are a special class of APCs; because they have antigen-specific antibody receptors they are enabled to internalize and process targeted antigens.

Lymphoid Organs The lymphatic organs are tissues in which lymphocytes mature, differentiate and proliferate. Lymphoid organs are comprised of epithelial and stromal cells arranged either into discretely capsulated organs or accumulations of diffuse lymphoid tissue. The primary (central) lymphoid organs are the major sites of lymphopoiesis, where B and T lymphocytes differentiate from stem cells into mature antigen recognizing cells. The secondary lymphoid organs, therefore, are those tissues in which antigen-driven proliferation and differentiation take place. Historically, the primary lymphoid organ was first discovered in birds, in which B cells undergo maturation in the bursa of Fabricius, an organ situated near the cloaca. Humans do not have a cloaca, nor do they possess a bursa of Fabricius. In embryonic life, B cells mature and differentiate from hematopoietic stem cells in the fetal liver. After birth, B cells differentiate in the bone marrow. Maturation of T cells occurs in a different manner. Progenitor cells from the bone marrow migrate to the thymus where they differentiate into T lymphocytes. The T lymphocytes continue to differentiate after leaving the thymus, and are driven to do so by encounter with specific antigen in the secondary lymphoid organs. 18

Primary Lymphoid Organs Thymus Gland: The lymphoid organ in which T lymphocytes are educated, mature and multiply. It is a lymphoepithelial organ composed of stroma (thymic epithelium) and lymphocytes, almost entirely of the T-cell lineage. This is where T lymphocytes learn to recognize self antigens as self, and where these cells differentiate and express specific receptors for antigen. Only 5-10% of maturing lymphocytes survive and leave the thymus. Fetal Liver and Adult Bone Marrow: Islands of hematopoietic cells in the fetal liver and in the adult bone marrow give rise directly to B lymphocytes. Secondary Lymphoid Organs The spleen and lymph nodes are the major secondary lymphoid organs. Additional secondary lymphoid organs include the tonsils, appendix, and Peyer’s patches. Aggregates of cells in the lamina propria of the digestive tract lining may also be included in this category, as well as any tissue described as MALT (mucosa-associated lymphoid tissue), GALT (gut-associated lymphoid tissue) or BALT bronchus-associated lymphoid tissue). Last but not least, the bone marrow can serve as an important secondary lymphoid organ. In addition to being a site of B cell generation, the bone marrow contains many mature T cells and plasma cells. Figure. Distribution of lymphoid tissues in the body. Actor, Elsevier’s Integrated Immunology and Microbiology. 2012.

Lymph Node: Lymph nodes form part of the network which filters antigen from tissue fluid or lymph during its passage from the periphery to the thoracic duct. Histologically, the lymph node is composed of a B cell cortex containing primary and secondary follicles, a T cell paracortex, and a central medulla which contains cords of lymphoid tissue. Spleen: The spleen is a filter for blood, and is actively involved in the removal of dying and dead erythrocytes. There are two main types of tissue; red pulp and white pulp. The white pulp contains the lymphoid tissue, arranged around a central arteriole as a periarteriolar lymphoid sheath (PALS). The PALS is composed of T and B cell areas, and contains germinal centers. Dendritic reticular cells and phagocytic macrophages can be found in germinal centers where they work to present antigen to lymphocytes.

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Clinical Vignette - Congenital Asplenia (Case 30 in Geha and Notarangelo): Mr. and Mrs. Vanderveer had five children. Their 10 month old daughter developed a cold, followed by upper respiratory infection. The child became feverish, convulsive and died; the causative agent was Haemophilus influenza which was isolated from the throat and cerebrospinal fluid. At autopsy she was found to have no spleen. How does the lack of a spleen affect B cell function, and what implications does this have towards immune responses to infective agents? In adults? In children?

Review your histology chapters dealing with Hematopoiesis and the Immune System! Table. Myeloid Leukocytes and Their Properties Phenotype Morphology Circulating Differential Count* PMN Neutrophil granulocyte 2-7.5x109/L PMN Eosinophil granulocyte 0.04-0.44x109/L PMN Basophil granulocyte 0-0.1x109/L PMN Mast Cell granulocyte Tissue Specific Monocytes monocytic 0.2-0.8x109/L Macrophag e monocytic Tissue Specific Dendritic Cell monocytic Tissue Specific * Normal range for 95% of population, +/- 2 standard deviations

Table. Lymphoid Leukocytes and Their Properties Total Lymphocytes 1.3-3.5x109/L B Cell monocytic Adaptive Plasma Cell T Cell Natural Killer T Cell (NKT) Natural Killer Cell (NK)

Effector Function Phagocytosis and digestion of microbes Immediate hypersensitivity (allergic) reactions; defense against helminths Immediate hypersensitivity (allergic) reactions Immediate hypersensitivity (allergic) reactions Circulating macrophage precursor Phagocytosis and digestion of microbes; antigen presentation to T cells Antigen presentation to naïve T cells; initiation of adaptive responses

monocytic monocytic

Adaptive Adaptive

Effector Function Humoral immunity Terminally differentiated, antibody secreting B cell Cell-mediated immunity

monocytic (rare) monocytic

Adaptive Innate

Cell-mediated immunity (lipids) Innate response to microbial or infection

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Summary: Cells of the Immune System Immune system cells are derived from pluripotent hematopoietic stem cells. Immune responses by these cells are divided into innate (natural) and acquired categories. Acquired immunity requires recognition specificity to foreign antigens, and is subdivided, based on participation B lymphocytes (humoral) and T lymphocytes (CMI). Surface molecules on human cells may be defined according to designation of Cluster of Differentiation (CD) antigens, which are useful for identifying different cell populations. Cells of the RES provide natural immunity against microorganisms via phagocytosis and intracellular killing, recruitment of other inflammatory cells, and presentation of antigens. Leukocytes provide innate or specific adaptive immunity, and are derived from myeloid or lymphoid lineage. Myeloid cells include highly phagocytic, motile neutrophils, monocytes, and macrophages that provide a first line of defense against most pathogens. The other myeloid cells, including eosinophils, basophils, and their tissue counterparts, mast cells, are involved in defense against parasites and in the genesis of allergic reactions. Cells from the lymphoid lineage are responsible for humoral or cell mediated immunity. The major properties of the acquired immune response are specificity, memory, adaptiveness, and discrimination between self and non-self. Lymphoid cells in these categories include T and B lymphocytes and NK cells. T and B cells produce and express specific receptors for antigens while NK cells do not. Receptor specificity is related to gene rearrangement of variable region components during development, according to essential features for clonal selection. B lymphocytes secrete antibodies; their activation is antigen-dependent following which they differentiate into plasma cells. Upon interaction with T helper cells, they proliferate and switch the isotype (class) of immunoglobulin produced, while retaining the same antigen-binding specificity. B cells also process and present protein antigens; they have specific surface antigens (CD molecules) necessary for response to foreign antigens. T lymphocytes are involved in regulation of immune response and in cell mediated immunity. During thymic differentiation, immature T cells undergo rearrangement of their TCR genes to generate a diverse set of clonotypic TCRs. Immature thymocytes are selected for further maturation only if they recognize foreign antigens in the context of "self" molecules. Mature T cells usually display one of two accessory molecules. CD4+ T helper cells are the primary regulators of T cell- and B cell-mediated responses, and are further subdivided into subsets dependent upon cytokines secreted. CD8+ T cytotoxic cells (CTLs) are cytotoxic against tumor cells and host cells infected with intracellular pathogens. T suppressor cells suppress the T and B cell responses and express CD8 molecules. T regulatory cells (Treg) are helper cells that suppress other T cell activity and help prevent autoimmunity. Natural Killer cells (NK) are large granular lymphocytes that nonspecifically kill certain types of tumor cells and virus-infected cells. The NK cells are able to kill “self” in the absence of antigenspecific receptors. They kill via antibody-dependent cellular cytotoxic mechanisms (ADCC) via their Fc receptors.

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INNATE IMMUNITY and INFLAMMATION Jeffrey K. Actor, Ph.D. 713-500-5344 Objectives: (1) Introduce innate immune defense mechanisms. (2) Define chemical mediators involved in inflammation. (3) Review cell types involved in innate immune responses, and their role in inflammation. (4) Define ADCC, chemokines, and Pattern-recognition receptors. Keywords: Innate Immunity, Innate Defense Barriers, Neutrophils, Monocytes, Macrophages, Natural Killer (NK) cells, Phagocytosis, APC, ADCC, Chemokines, Complement, PRRs, Inflammasome. Reading: Coico and Sunshine. Immunology: A Short Course. John Wiley & Sons, Inc, New York, NY. 6th edition, 2009. Chapters 2, 10 and 11; Geha and Notarangelo. Case Studies in Immunology. Garland Publishing, New York, NY. 6th edition, 2012. Case 15. Chediak-Higashi Syndrome; Case 25. Neutropenia; Case 26. Chronic Granulomatous Disease; Case 27. Leukocyte Adhesion Deficiency. Web Resource: http://www.uth.tmc.edu/pathology/medic/immunology/Immuno/Innate.html Innate immune mechanisms provide the first line of defense from infectious disease. The innate immune system is comprised of components which are present prior to the onset of infection and constitute a set of mechanisms that are not specific for a particular organism. Rather, the innate components recognize classes of molecules frequently encountered on invading pathogens, so as to allow defensive measures while the specific immune response is either generated or upregulated. Innate immune components are present from birth and consist of non-specific components. The innate defensive barriers can be divided into four major categories: 1. Anatomic - skin, mucous membranes 2. Physiologic - temperature, low pH, chemical mediators 3. Phagocytic and Endocytic - phagocytose to kill and digest microorganisms 4. Inflammatory - induction of vascular fluid leakage to area of tissue damage Anatomic Barrier. The skin and mucous membranes provide an effective barrier against microorganisms. The skin has the thin outer epidermis and the thicker underlying dermis to impede entry, as well as sebaceous glands to produce sebum. Sebum is made of lactic acid and fatty acids, which effectively reduce skin pH to between 3 and 5 to inhibit organism growth. Mucous membranes are covered by cilia which trap organisms in mucous and propel them out of the body. Physiologic Barrier. The physiologic barrier includes factors such as temperature, low pH, and chemical mediators. Many organisms can not survive or multiply in elevated body temperature. Soluble proteins such as lysozymes, interferons and complement components play a major role in innate immunity. Lysozmes can interact with bacterial cell walls; interferons alpha and beta are natural inhibitors of viral growth; complement components use both specific and non-specific immune components to convert inactive forms to active components that damage membranes of pathogens. Low pH in the stomach discourages growth. 22

Phagocytic and Endocytic Barriers. Blood monocytes, tissue macrophages and neutrophils phagocytose and kill microorganisms via multiple complex digestion mechanisms. Bacteria become attached to cell membranes and are ingested into phagocytic vesicles. Phagosomes fuse with lysosomes where lysosomal enzymes digest captured organisms. Inflammatory Barriers. Invading organisms cause localized tissue damage leading to complex inflammatory responses. In 1600 BCE, Celsus described the four cardinal signs of inflammation as rubor (redness), tumor (swelling), calor (heat), and dolor (pain). Later, Galen (2nd century) a fifth sign was added; functio laesa (loss of function). Inflammatory responses lead to (1) Vasodilation causing erythema (redness) and increased temperature; (2) increased capillary permeability which allows exudates (fluid) to accumulate leading to tissue swelling (edema); and (3) influx of cells to site of tissue damage. Once cells enter area of damage, they release further chemotactic factors to call in additional cells to damaged area, leading to Chemotaxis, Activation, Margination, Diapedesis (extravasation), and finally recognition and attachment of these cells to the damaged site. -------------------------Chemical Mediators of Inflammation

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Hageman factor: Plasma globulin (110 kD), blood clotting factor XII, which is activated by contact with surfaces to form Factor XIIa, that in turn activates factor XI. Factor XIIa also generates plasmin from plasminogen and kallikrein from prekallikrein. Both plasmin and kallikrein activate the complement cascade. Hagemann factor is important both in clotting and activation of the inflammatory process.

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Thrombin: Protease (34 kD) generated in blood clotting that acts on fibrinogen to produce fibrin. Consists of two chains, A and B, linked by a disulphide bond. Thrombin is produced from prothrombin by the action either of the extrinsic system (tissue factor + phospholipid) or, more importantly, the intrinsic system (contact of blood with a foreign surface or connective tissue). Both extrinsic and intrinsic systems activate plasma factor X to form factor Xa which then, in conjunction with phospholipid (tissue derived or platelet factor 3) and factor V, catalyses the conversion.

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Kallikrein: Plasma serine proteases normally present as inactive prekallikreins which are activated by Hageman factor. Act on kininogens to produce kinins, to mediate vascular reactions and pain.

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Plasmin: Trypsin like serine protease that is responsible for digesting fibrin in blood clots. Generated from plasminogen by the action of another protease, plasminogen activator. The enzyme catalyses the hydrolysis of peptide bonds at the carbonyl end of lysine or arginine residues. It also acts on activated Hageman factor and on complement.

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Bradykinin: Vasoactive nonapeptide (RPPGFSPFR) formed by action of proteases on kininogens. Very similar to kallidin (which has the same sequence but with an additional N terminal lysine). Bradykinin is a very potent vasodilator and increases permeability of post capillary venules, it acts on endothelial cells to activate phospholipase A2. It is also spasmogenic for some smooth muscle and will cause pain.

Arachidonic Acid Metabolites: Inflammatory Role

Cell Types Involved in Innate Immunity The cell types involved in innate immune responses include the polymorphonuclear cells (neutrophils), monocytes and macrophages, eosinophils, and Natural Killer (NK) cells. Some of these cells are capable of killing target cells via nonspecific (non-MHC dependent) through release of lytic enzymes, perforin or TNF. Others are involved in phagocytic mechanisms that kill via intracellular processes. Neutrophils. Neutrophils are typically the first infiltrating cell type to site of inflammation. Endothelial cells increase expression of E-selectin and P-selectin which are recognized by neutrophil surface mucins (PSGL-1 or sialyl Lewisx). Chemoattractants (IL-8) trigger adhesion and subsequent diapedesis. Multiple complement components (e.g. C5a) are chemotactic for neutrophils, along with fibrinopeptides and leukotrienes. Activated neutrophils express high Fc receptors and complement receptors to allow increased phagocytosis of invading organisms. Activation of neutrophils leads to respiratory burst producing reactive oxygen and nitrogen intermediates, as well as release of primary and secondary granules containing proteases, 25

phospholipases, elastases and collagenases, and lactoferrin. Pus, a yellowish white opaque creamy matter produced by the process of suppuration consists of innumerable neutrophils (some dead and dying) and tissue debris.

Figure. Cell membrane adhesion molecules and cytokine activation events associated with neutrophil transendothelial migration. Left: Weak binding of selectin ligands on the neutrophil to Eselectin on the endothelial cells. Middle: IL-1 and TNF- upregulation of E-selectin, which facilitates stronger binding. Right: The activation effects of IL-8 on neutrophils cause a conformational change in the integrins (e.g., LFA-1) to allow them to bind ICAM-1. Coico and Sunshine, 2009. Fig 11.4.

Mononuclear Cells and Macrophages. Mediators such as MIP-1 and MIP-1 attract monocytes to the site of pathogenic infection. The monocytes express surface ligands which recognize ligands (VCAM-1) on endothelial cells, leading to diapedesis. Activated tissue macrophages secrete IL-1, IL-6 and TNF- which further increase expression of adhesion molecules on endothelial cells to recruit neutrophils and more monocytes. These molecules also increase release of acute-phase proteins from the liver to assist in events leading to body temperature increase. Monocytes and macrophages ingest and destroy bacteria. Multiple factors assist in preparing the particulate for engulfment and targeting for destruction, including various opsonins comprised of complement components. Phagocytes bear several different receptors that recognize microbial components and induce phagocytosis. Five such receptors on macrophages are: CD14, Toll-like receptors (such as TLR-4), the macrophage mannose receptor, the scavenger receptor, and the glucan receptor. All 5 receptors bind bacterial carbohydrates. CD14 and CR3 are specific for bacterial lipopolysaccharide (LPS). In addition, complement receptors assist in this process. Figure. Endocytosis and phagocytosis by macrophages.

Phagocytosed organisms are subjected to killing by 26

lysosomal enzymes in phagolysosomes. Killing of phagocytosed microbes is sone via ROS and NO mediated mechanisms. These same substances can also be released to kill extracellular microbes.

Figure. Important cytokines secreted by macrophages in response to bacteria and bacterial products include IL-1, IL-6, CXCL8 (IL-8), IL-12, and TNF-a. TNF-a is an inducer of a local inflammatory response that helps to contain infections. CXCL8 is also involved in the local inflammatory response, helping to attract neutrophils to the site of infection. IL-1, IL-6, and TNF-a have a critical role in inducing the acutephase response in the liver and induce fever, which favors effective host defense in several ways. IL-12 may also activate natural killer (NK) cells.

The Inflammasome: Assembly and activation of the inflammasome is an essential process in innate immune defense. The inflammasome is a cytosolic, multiprotein platform that allows activation of pro-inflammatory caspases that cleave the precursor of interleukin-1β (pro-IL-1β) into the active form. Secretion of active IL-1β helps to initiate a potent inflammatory response. Antigen Presentation. Phagocytosed or pinocytosed antigens may then be presented to the adaptive immune system cells. Dendritic cells, macrophages and monocytes are specifically good at presenting antigens to T lymphocytes. In addition, B cells, are also extremely good APCs. Some of the critical molecules which play a role in antigenic presentation by APCs to T cells are given in the accompanying figure.

Figure from Immunology (6th ed). 2006. Goldsby, Kindt, Osborn and Kuby. WH Freeman Publisher.

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NK Cells. NK cells are large granular lymphocytes that nonspecifically kill certain types of tumor cells and virus-infected cells, and function as both cytolytic effectors and regulators of immune responses. NK cells express a large number of receptors that deliver either activating or inhibitory signals; the relative balance of these signals controls NK cell activity. NK cells are activated upon detection of abnormalities in target cells such as the loss of antigen presentation molecules (MHC class I expression) or up-regulation of stress-induced ligands. A variety of receptors trigger the NK cytolytic activity directed toward certain tumor targets, virally infected cells, and even normal immune system constituents such as immature dendritic cells. NK cells are also important regulators of the adaptive immune system via their ability to secrete a number of cytokines in response to immune activation.

Antibody-Dependent, Cell-Mediated Cytotoxicity (ADCC). ADCC is a phenomenon in which target cells coated with antibody are destroyed by specialized killer cells. Among the cells that mediate ADCC are NK cells, macrophages, monocytes, neutrophils and eosinophils. The killing cells express receptors for the Fc portion of antibody coated targets. Recognition of antibody coated target leads to release of lytic enzymes at the site of Fc mediated contact. In the case of NK cells and eosinophils, target cell killing may involve perforin-mediated membrane damage. Coico amd Sunshine, 2009. Fig.15.1

Clinical Relevance Clinical Vignette – Case 15. Chediak-Higashi Syndrome: Chediak-Higashi syndrome is a rare inherited disorder in which a severe immunological deficiency has been linked to deficits in NK cell function and to deficiency in chemotactic and bactericidal function for neutrophils. Thus, these individuals are more susceptible to bacterial infections. These individuals have characteristic giant lysosomes within neutrophils. Bone marrow transplantation is the only effective therapy.

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Chemokines. Chemokines are specialized cytokines that are chemotactic for leukocytes. They are small polypeptides that are synthesized by a wide variety of cell types. They act through receptors that are members of the G-protein coupled signal transducing family. All chemokines are related in amino acid sequence and their receptors are integral membrane proteins that are characterized by containing seven membrane-spanning helices. Chemokines fall mainly into two distinct groups. The CC chemokines have two adjacent cysteine residues (hence the name "CC"). The CXC chemokines have an amino acid between two cysteine residues. Each chemokine reacts with one or more receptors, and can affect multiple cell types. Chemokines and their functions will be covered again in greater depth in the Adaptive Immunity chapter. Properties of selected chemokines. Chemokine CCL2 (MCP-1) CCL3 (MIP-1) CCL5 (Rantes) CCL11 (Eotaxin) CXCL8 (IL-8)

Major Cell Source Monocytes and Macrophages, Fibroblasts Monocytes, T cells, Fibroblasts, Mast cells T cells, Endothelium Monocytes and Macrophages, Endothelium and Epithelium Monocytes and Macrophages, Fibroblasts, Endothelial cells

Cell Type Attracted Chemoattractant for monocytes Chemoattractant for neutrophilic granulocytes Chemoattractant for Eosinophils and Basophils, Monocytes and Dendritic cells, and T cells Chemoattractant for Eosinophils Chemoattractant for Neutrophils

Additional Chemokines are listed in the Appendix.

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Complement Components. The activation of complement is an important component of innate immunity. This will be discussed in detail in further lecture. A brief introduction to complement follows:

Activation of the complement system results in the production of several different polypeptide cleavage fragments that are involved in five primary biological functions of inflammation and immunity. 1. Direct Cytolysis of foreign organisms (e.g. bacteria): Antibodies recognizing pathogenic determinants form the basis of a physical structure to which complement components interact. Specifically, complement component C1 interacts with the Fc portion of IgM and IgG (except IgG4) binding to the surface of bacteria. The binding of C1 initiates a cascade of events whereby a membrane attack complex (MAC) is built upon the cellular surface. Synthesis of the MAC structure culminates in assembly of a pore channel in the lipid bilayer, causing osmotic lysis of the cell. MAC formation requires prior activation by either the classical or alternative pathways, and utilizes the proteins C5b, C6, C7, C8, and C9. 2. Opsonization of foreign organisms. Complement components (e.g. C3b or inactivated C3b; iC3b) bind to pathogens. Interaction with receptors (CR1, CR2, CR3, and CR4) on the surface of macrophages, monocytes, and neutrophils leads to enhanced phagocytosis and targeted destruction of organisms. 3. Activation and directed migration of leukocytes. Proteolytic degradation of C3 and C5 leads to production of leukocytes chemotactic anaphylatoxin. For example, C3a is chemotactic for eosinophils. C5a is a much more potent chemokine, attracting neutrophils, monocytes and macrophages, and eosinophils. Interaction of C3a, C4a or C5a with mast cells and basophils leads to release of histamine, serotonin, and other vasoactive amines, resulting in increased vascular permeability, causing inflammation and smooth muscle contraction.

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4. Solubilization and clearance of immune complexes. One of the major roles complement plays is the solubilization and clearance of immune complexes from the circulation. First, C3b and C4b can covalently bind to the Fc region of insoluble immune complexes, disrupting the lattice, and making them soluble. C3b and C4b bound to the immune complex are recognized by the CR1 receptor on erythrocytes facilitating their transport to the liver and spleen. In the liver and spleen the immune complexes are removed and phagocytosed by macrophage-like cells. The RBCs are returned to the circulation. 5. Enhancement of humoral immune response. Coating of antigens with C3d (a breakdown product of C3) facilitates their delivery to germinal centers rich in B and follicular dendritic cells.

Clinical Relevance Clinical Vignette – Factor I Deficiency (Case 32, Geha and Notarangelo): The alternative pathway of complement activation is important in innate immunity. Deficiency in Factor I (as well as deficiency in Factor H) affects cleavage of C3b, and therefore leads to reduced C3bi. The nonproduction of iC3b results in defective opsonization, which is critical for removing and destroying invading bacteria.

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Receptors of the Innate Immune System. Receptors of the innate immune system recognize broad structural motifs that are highly conserved within microbial species [ called PathogenAssociated Molecular Patterns (PAMPs)]. Such receptors are referred to as Pattern-Recognition Receptors (PRRs). In a similar manner, Damage/Danger-Associated Molecular Pattern molecules (DAMPs) initiate immune activity as part of the noninfectious inflammatory response. Engagement of any of these receptors leads to triggering of signal pathways that promote inflammation. Receptor (location)

Target (source)

Effect of Recognition

Receptors of the Innate Immune System. [Table adapted from Immunology (2002) by Goldsby, Kindt, Osborne and Kuby - W.H.Freeman, et al., NY.]

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FIGURE 2.6. (A) Pattern recognition receptors called TLRs binding to molecules with specific pattern motifs expressed by various pathogens. (Coico, 2009)

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Link Between Innate and Adaptive (Acquired) Immunity  Recognition of pathogen via Pattern Recognition Receptors (PRR) or Toll-Like Receptors (TLR) leads to activation and maturation of APCs.  APCs process antigen and present to naïve T cells. Specifically, dendritic cells form a major bridge between cells of the innate and adaptive immune responses.  Presentation is accompanied by secretion of cytokines to assist development of T cell response (e.g. Th1 maturation via presence of IL-12).  Absence of internal activation signals can leads to Th2 development (MyD88 regulated).

Figure 6-4. Link between innate and adaptive (acquired) immunity. Pathogen recognition through pattern recognition receptors is an important bridge between innate and adaptive immune function. Recognition leads to activation and maturation of the presenting cell. Here, dendritic cells are depicted as primary presenting cells, which assist in dictating subsequent responses. Processed antigen is presented to naive T cells, accompanied by secretion of cytokines to assist development and maturation of T-cell phenotypic response (e.g., T helper cell-1 maturation via presence of interleukin-12). Inset box shows important Toll-like receptors and specific ligands involved in pathogen recognition. At least 15 different Toll-like receptors have been identified. A more complete list of Toll-like receptors and ligands is provided in the Appendix. 34

Summary: Innate Immunity Immune system cells are derived from pluripotent hematopoietic stem cells. Immune responses of the innate immune system provide natural immunity against microorganisms via phagocytosis and intracellular killing, recruitment of other inflammatory cells, and presentation of antigens. Leukocytes that provide innate immunity are derived from myeloid lineage. These cells include highly phagocytic, motile neutrophils, monocytes and tissue macrophages, eosinophils, and Natural Killer (NK) cells. These cells provide a first line of defense against most pathogens. Innate defense barriers include (1) anatomic barriers, (2) physiologic barriers, (3) Phagocytic barriers, and (4) inflammatory barriers. Damage to tissue caused by invading pathogens can lead to rubor, tumor, calor, dolor, and functio laesa. Tissue damage leads to an influx of inflammatory cells through chemotaxis, activation, margination and diapedesis. The inflammatory process is initiated and controlled via multiple chemical mediators. Neutrophils are usually the first cell type to arrive at the site of tissue damage. Activation leads to respiratory bursts and release of granules to control bacterial growth. Mononuclear cells and macrophages engulf organisms via multiple mechanisms, leading to control and destruction within intracellular phagosomes. NK cells are large granular lymphocytes that kill targets via ADCC or through lysis using perforin-induced mechanisms. Chemokines and complement components are critical for activation of innate immune functions. Defects may lead to severe clinical complications. Pattern Recognition Receptors present on innate immune system cells assist in the recognition of bacteria and virions. Recognition by PRRs of PAMPs leads to activation of multiple facets of cellular response. In a similar manner, damage/danger associated DAMPS can function to elicit innate inflammatory functions in non-infectious situations.

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A general summary chart of innate components, effectors and function: Component Effectors General Function Anatomic and Skin and Mucous Membranes - Physical barriers to limit entry, spread Physiologic Temperature, Acidic pH, Lactic acid and replication of pathogens Barriers Chemical Mediators Inflammatory Mediators

Hageman factor Thrombin Kallikrein Bradykinin Leukotrienes and Prostaglandins Complement Cytokines and Interferons Lysozymes Acute Phase Proteins and Lactoferrin

- Clotting, activation of inflammation - Protease acting to produce fibrin - Mediating vascular reactions and pain - Vasoactive nonapeptide; spasmogenic for some smooth muscle and will cause pain - Vasodilation and increased vascular permeability - Direct lysis of pathogen or infected cells - Activation/Mediation of other immune components - Bacterial cell wall destruction - Mediation of response

Inflammatory Mediators

Complement Cytokines and Interferons Lysozymes Acute Phase Proteins and Lactoferrin Leukotrienes and Prostaglandins

- Direct lysis of pathogen or infected cells - Activation of other immune components - Bacterial cell wall destruction - Mediation of response - Vasodilation and increased vascular permeability

Cellular Components

Polymorphonuclear Cells  Neutrophils, Eosinophils  Basophils, Mast Cells Phagocytic-Endocytic Cells  Monocytes and Macrophages  Dendritic Cells Other Cells  NK cells

- Phagocytosis and intracellular destruction of microorganisms

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- Presentation of foreign antigen to lymphocytes - ADCC

IMMUNOGENS AND ANTIGENS Sudhir Paul, PhD OBJECTIVES To learn the molecular attributes and properties of compounds which render them immunogenic and antigenic. KEYWORDS Immunogen, antigen, hapten, epitope, adjuvant. READING Chapter 3 of the Coico, et al textbook. Geha and Notalangelo, 2012. Case Studies in Immunology, 6th Ed., 46. Hemolytic Disease of the Newborn. Multiple Myeloma (On file on Blackboard/LRC). Web Resource: http://www.uth.tmc.edu/pathology/medic/immunology/Immuno/immantigen.html ANTIGEN OR IMMUNOGEN? IMMUNOGEN - Agent capable of binding immune receptors AND inducing an immune response by B cells and T cells ANTIGENS - Agent that binds with varying degrees of specificity to immune receptors (antibodies on B cells; T cell receptor on T cells) All immunogens are antigens, but not all antigens are immunogens. IMPORTANCE OF IMMUNOGENICITY  Germfree colostrum-deprived piglets are immunologically "virgin" and extremely susceptible to microbial infection due to lack of passive maternal immunity. They are, however, highly immunologically competent as determined by their excellent immune response to various immunogens. An immunogen is the inducer of specific antibody formation. The initial step in the primary immune response is priming of multipotent uncommitted cells ("virgin" X cells) to committed monopotent cells (Y cells). Y cells proliferate and differentiate into antibodyforming cells (Z cells). Adapted from Y.-B. Kim 1975  Vaccines are the cornerstone of eradicating microbial disease – many available vaccines. See slide.  New vaccines are needed for emerging diseases. See slide. EPITOPES RECOGNIZED BY T OR B CELLS Epitopes are the three dimensional arrangements of atoms (sites) on the surface of an antigen that bind to the paratope of an antibody OR the linear peptides that bind the MHC molecules/T cell receptor. Epitopes recognized by B cells generally differ from those recognized by T cells.

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B cells can mount specific antibody responses without or with help from T cells (Tindependent or T dependent cells).

PHYSICOCHEMICAL FORCES INVOLVED IN ANTIGEN-IMMUNE RECEPTOR BINDING

Antibody binding to antigen does not involve covalent chemical bonds. Instead, several weaker types of molecular interactions are utilized. Thus, the reactions are reversible. There are four kinds of forces that stabilize antigen-antibody interactions: 1. Electrostatic interactions. Usually due to the attraction between the charged amino acid residues in proteins such as lysine, arginine, glutamic acid and aspartic acid, for instance. The number of such interactions will enhance the affinity of the interaction dramatically. 2. Hydrogen bonding. Electrostatic binding with covalent character. Example, -H atom shared by electronegative N and O atoms.

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3. Van der Waal’s forces. Attractive and repulsive forces between induced oscillating dipoles in the electron clouds of two adjacent atoms. The force is proportional to the 7th power of the distance separating two molecules. This is a weak force, but is additive, and there are many van der Waal’s contacts in antibodyantigen complexes. 4. Hydrophobic bonding. Usually involves non-polar amino acids, e.g., leucine, isoleucine. MAJOR CLASSES OF ANTIGENS/IMMUNOGENS The following major chemical classes of compounds may be antigenic/immunogenic: 1. Proteins or glycoproteins. Most proteins or glycoproteins are excellent antigens. The greater the complexity and molecular weight, the better it is as an antigen. 2. Carbohydrates or polysaccharides. Bacterial capsules (i.e. pneumococci) are powerful antigens. The ABO blood group epitopes are carbohydrates. 3. Lipids. Are not routinely antigenic, but if used as a hapten, immune responses can be elicited, i.e. sphingolipids. 4. Nucleic Acids. Are poorly immunogenic themselves, but as haptens are good antigens. Antibodies to DNA are important in patients with systemic lupus erythematosus. SEQUENTIAL AND CONFORMATIONAL EPITOPES Two general classes of epitopes can be distinguished. They are best described as they exist on protein antigens, but other classes of antigens (i.e. carbohydrates and nucleic acids) can also express antigenic/immunogenic epitopes under some circumstances. 1. Conformational (Non-Sequential) Epitopes Conformational epitopes require the native 3-dimensional configuration of the molecule to be intact for their expression. Denaturation of the molecule destroys these kinds of epitopes and antibodies specific for conformational epitopes will not bind denatured antigens. Conversely, denaturing the molecule prior to injection of the animal (cooking an egg) will alter the conformation of the molecule and the antibodies elicited that are specific for antigens on the denatured form will not react with the undenatured form of the molecule. 2. Sequential Epitopes Sequential epitopes are short stretches of amino acids (4-7 in length) which can be recognized MHC molecules in short peptides or by antibodies in short peptide

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regions within larger antigens. Thus, the only requirement is that the right sequence of amino acids is expressed.

EXAMPLE OF CLINICAL RELEVANCE Two cases in Geha & Notarangelo, 6th edition, emphasize the influence of genetic factors in immunogenicity of infectious agents and immunogenicity of administered vaccines. Case 12—MHC Class I Deficiency—This case describes the consequences of a failure of antigen processing for protection from infectious agents. Tatiana Islayev, age 17, had been chronically ill since age 4. She had a history of repeated sinus, lung, and middle ear infections due to a variety of respiratory viruses. Her 7-year old brother Alexander had a similar history. The parents and 3 other children were healthy. Tatiana and Alexander had received oral polio vaccinations as well as DPT and BCG vaccinations and tolerated them well. WBC analysis showed a profound deficiency of CD8 T cells. Further studies showed that both Tatiana and Alexander had a nonsense mutation in their TAP-2 genes, a gene coding for a protein that transports peptide fragments into the lumen of the endoplasmic reticulum where it binds to MHC class I molecules and this complex is then transported to the surface of the cell to be recognized by a CD8 T cell. Case 8—MHC Class II Deficiency—This case illustrates a genetically acquired susceptibility to pyogenic and opportunistic infections. Helen Burns was the second child born to her parents. She had received routine polio and DPT vaccinations at 2 months of age. At 6 months of age she developed pneumonia in both lungs, accompanied by a severe cough and fever. Pneumocystis carinii was isolated from a tracheal aspirate and she was treated with pentamidine and seemed to recover fully. Since P. carinii was found (an opportunistic pathogen), severe combined immunodeficiency (SCID) was suspected. Her T cells were found incapable of responding to tetanus toxoid and her serum Ig concentrations were very low. Her CD4 T cells were very low but her CD8 cell count was normal (ruling out a diagnosis of SCID). While working up her sib and parents for possible bone marrow donation, it was found that Helen’s B cells did not express MHC Class II molecules. Her mother was selected as the best donor of bone marrow. The graft was successful and normal immune function was restored. 41

REQUIREMENTS FOR IMMUNOGENICITY Four characteristics that contribute to the immunogenicity of a substance: 1. Size, dose, route Usually, compounds of less than 1,000 daltons are non-immunogenic. Compounds between 1,000 and 6,000 daltons may or may not be immunogenic. Those greater than 6,000 daltons are generally immunogenic. Intermediate dose is most immunogenic. Immunogenicity is also a function of route of administration. 2. Chemical Composition Physicochemical complexity is usually necessary for a compound to be immunogenic. Homopolymers of amino acids usually are not immunogenic (i.e. B. anthracis poly-gamma-D-glutamic acid, 50,000 daltons). 3. Foreignness Foreignness was once considered to be an absolute requirement for immunogenicity. It is now clear that certain self-components can be immunogenic to the individual. Foreignness is an excellent general guideline as to whether something might be immunogenic, but it is not a definitive requirement for immunogenicity. Particulate and denatured antigens are often more immunogenic. 4. Adjuvants/Degradability Adjuvants enhance immune responses by inducing cytokine release or antigen processing. T-dependent immunogens must be enzymatically degraded in order to be immunogenic. Peptides of D-amino acids are non-immunogenic whereas their L-isomers usually are immunogenic. Genes mapping to the Major Histocompatibility Complex (MHC) can profoundly affect the degree of immunogenicity of any substance. HAPTENS Haptens are low molecular weight compounds that are non-immunogenic by themselves but become immunogenic after conjugation to high molecular weight carrier substances that are immunogenic. The figure below illustrates coupling non-immunogenic p-aminoarsonic acid to a carrier to make it immunogenic. Clinical Relevance—The hapten concept has been adapted to modern vaccine technology with great success. There are several vaccines licensed that are based on covalently coupling isolated epitopes to carrier molecules, usually tetanus toxoid. Hapten-type vaccines for pneumococcus and for Haemophilus are currently available. Several others are in development. Such an approach provides a much safer type of vaccine compared to using whole immunogen molecules or killed viral preparations.

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MULTIVANT ANTIGENS Macromolecules usually have multiple unique or repeat epitopes. The former type of immunogens induce heterogenous immune responses (mixtures of antibodies or T cells directed to individual antigens). The latter type of immunogens are often T-independent. Both types of antigens can form large complexes with multiple antibodies, a phenomenon that can cause pathological immune complex deposition, particularly in the kidney. IMMUNOLOGIC SPECIFICITY AND CROSS-REACTIVITY The forces mediating antigen-antibody recognition allow for a high degree of specificity. That is, antibodies specific for one epitope or hapten can easily distinguish that epitope or hapten from other similar structures. However, this specificity is not absolute because antibodies specific for one epitope can bind with structurally similar, but non-identical epitopes although with a lower affinity. Specificity and cross-reactivity can be distinguished by inspecting the following table reporting antibody reactivity with various structurally defined carbohydrate epitopes:

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CROSS-REACTIVITY Cross reactivity refers to the situation where the cell receptor or antibody can react with two molecules because a) they share one or more identical epitopes or b) the epitope in question is similar enough in sequence or in shape to bind to the receptor with a weaker, yet functional, affinity. Examples: 1. Toxoids—Antibodies elicited with toxoids react with native toxins (Clinical Application—vaccination with tetanus toxoid and with diphtheria toxoid). 2. ABO Blood Group Antigens—Antibodies elicited by certain environmental carbohydrate antigens react with the human A or B blood group antigens. 3. There are 4 strains of the flavivirus that causes Dengue. The virus infects cells of the monocyte-macrophage lineage. Infection with one strain elicits antibodies reactive with a common epitope on all 4 strains. Upon infection with a different strain, the antibody to the common epitope reacts by cross-reaction and facilitates phagocytosis by macrophages, thus helping the virus gain entry and the second infection is typically much more severe than the first due to this cross-reactivity. 4. Yet another example of cross-reactivity is the ability of antibodies to bacterial antigens to attack host tissues, causing autoimmune disease.

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ADJUVANTS Definition: An adjuvant is a substance, which when mixed with an immunogen, enhances the immune response against the immunogen. The adjuvant itself is not usually immunogenic. Examples of Immunologic Adjuvants 1. Freund’s Complete Adjuvant. This is a mixture of a petroleum based oil, an emulsifying agent and killed Mycobacteria. A water-inoil emulsion is formed with microdroplets of antigen solution surrounded by the oil. This works by slowly releasing antigen over a long period of time while inducing a delayed hypersensitivity reaction. It is used experimentally, but not in humans. 2. Lipopolysaccharide (LPS). Is experimental 3. Muramyldipeptide. Is experimental 4. Synthetic Polynucleotide (Poly AU). Is experimental 5. Aluminum Hydroxide (alum precipitate). Is used in humans. Functions to enhance the ingestion and eventual processing of antigen. 6. Cytokines. Are experimental Currently, adjuvant research is a high priority research area for enhancing the immunogenicity of the new genetically engineered vaccines. Aluminum hydroxide is currently the only FDA licensed adjuvant in the US. There are several new adjuvants in phase III clinical trials but the FDA has not yet licensed any of these. Some other adjuvants are licensed in other countries, but are not available in the US. Adjuvant MF59 is licensed in Europe, but not in the US. MF59 consists of stable droplets ( IgG1> IgG2 -IgG4, IgA, IgD, and IgE do not activate Activation of the classical pathway requires the local reaction of antibodies with two or more antigenic sites. These Ag-Ab complexes may consist of a single IgM molecule bound to two or more antigenic sites, or two or more human IgG molecules (IgG1, IgG2, or IgG3) bound to epitopes. Such a complex could (for example) occur on a bacterial cell surface or in an aggregate of antibodies with soluble antigens. Ag-Ab reaction causes conformational changes in CH2 of IgG and CH3 of IgM, permitting the binding of C1q. Binding of two or more arms of C1q causes conformational changes that lead to cleavage and activation of the bound zymogens C1r and C1s. FIGURE 2. Bridging of two membrane-bound IgG molecules by the C1 component. Binding distorts the C1 molecule and triggers activation.

Activated C1s can cleave C4 and C2 into large (C4b and C2b) and small (C4a and C2a) fragments. C4 is cleaved first, and approximately 1% binds to a nearby surface via a covalent linkage. C2 can complex with surface bound C4b and can be cleaved by C1s. The resulting C4bC2b complex is the classical pathway C3 convertase, and has the ability to specifically cleave C3 into large (C3b) and small (C3a anapylatoxin) fragments. C3 is the most abundant complement protein and plays a pivotal role in complement activation. Many molecules can be cleaved into C3b and C3a. Cleavage results in exposure of the labile thiolester bond in C3b, permitting some to bind covalently to proteins and carbohydrates on cell surfaces. The C3a anaphylatoxin is released into the blood and mediates many important inflammatory activities that will be discussed

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later. Some of the C3b binds to C4bC2b to form C4b2b3b, which is the C5 convertase. This complex by the C2b protease subunit will cleave C5 into big (C5b) and small (C5a anaphylatoxin) subunits. C5a is released into the blood and as C3a mediates many important inflammatory activities. C5a on a molar basis is 100 times more potent than C3a. FIGURE 3. The classical pathway of complement activation generates a C3 convertase that deposits large numbers of C3b molecules on the surface of the pathogen.

FIGURE 4. Cleavage of C3 and C4 exposes a thiolester bond that causes the resulting large fragments, C3b and C4b, to bind covalently to nearby molecules on bacterial or viral surfaces.

The classical pathway of complement can also be activated by the serum mannose binding lectin complex (MBL-MASP). This complex is structurally similar to the C1 complex. However, instead of binding to immune complexes it binds to directly to polysaccharides on gram-negative bacteria. The mannose binding lectin is C1q-like in structure and the MBL associated proteases (MASP) are similar to C1r and C1s. MBLMASP on binding bacterial surfaces can cleave C4 and C2 thereby activating the remainder of the classical pathway.

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ALTERNATIVE PATHWAY ACTIVATION The main difference between the classical and alternative pathways is that the initiation of the classical requires an activating substance. The alternative pathway, by contrast, runs continuously and spontaneously at low levels in the blood plasma. The alternative pathway activation occurs when C3b binds to a surface that lacks inhibitors that block complement activity, such as most bacterial cell surfaces. Certain plastic surfaces, like those initially used in heart-lung machines and dialysis machines, also activate the alternative pathway with obvious deleterious effects. Because antibody is not necessary, the alternative pathway represents an innate immune response and can react as soon as bacteria enter the body. The alternative pathway is also important in amplifying reactions initiated by the classical pathway. The low level activation of C3 that allows the alternative pathway to be activated is called the tick-over model. The thiolester bond in C3 is spontaneously hydrolyze at low rates yielding a C3b-like [C3(H2O)] molecule that now has a binding site for factor B exposed. The bound factor B is attacked by factor D, which cleaves it into Ba and Bb fragments. The Ba fragment is released, while the Bb fragment remains noncovalently associated with C3(H2O), forming an initial C3 convertase. The Bb subunit of this convertase has serine protease activity specific that can now specifically cleave additional C3 molecules into C3a and C3b fragments. If a activator surface is nearby, such as a bacterial surface, then the newly formed C3b molecule can covalently attach and bind factor B. The bound factor B is cleaved by factor D and the surface-bound C3 convertase (C3bBb) then attacks another native C3 molecule and so on. The activating surface (bacteria) thus accelerates a reaction that in its absence occurs at a slow rate. FIGURE 5. THE TICK-OVER MODEL

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LYTIC PATHWAY-FORMATION OF THE MEMBRANE ATTACK COMPLEX (MAC) After the C3 convertase cleaves C3 to generate C3b, the next step in either the classical, lectin, or alternative pathways is the binding of C3b to the C3 convertase complex, changing it to a C5 convertase, which catalyzes the proteolytic cleavage of the C5 protein. Cleavage of C5 to C5a and C5b represents the first step of the lytic pathway. The small C5a fragment is released into the blood and is the most potent complement anaphylatoxin. The large C5b molecule binds proteins C6 and C7. The complex C5b67 has hydrophobic regions that permit it to insert into the lipid bilayer nearby cell membranes. Subsequent binding of C8 permits some leakage of cell contents, causing slow lysis. This process is accelerated by binding of multiple C9 molecules, which assemble to form a protein channel through the membrane. C9 is analogous to perforins produced by cytolytic T cell and NK cells. C5b6789 is called the Membrane Attack Complex (MAC). MAC formation is an important mechanism for eliminating bacteria resistant to intracellular killing by phagocytes, such as Neisseria species. FIGURE 6. FORMATION AND REGULATION OF THE MEMBRANE ATTACK COMPLEX (MAC)

REGULATION OF COMPLEMENT ACTIVATION Complement activation is a tightly regulated series of reactions, that without control would result in the inappropriate activation on normal host cells. This would result in excess inflammatory mediators and by direct lysis of host cellular membranes by MAC.

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This does not normally happen because there exist several complement inhibitors in serum as well as on the surface of host cells.

C1-inhibitor (C1INH)

serum protein binds to activated C1r,C1s, removing it from C1q

C4-binding protein (C4BP)

serum protein that binds C4b displacing C2b; co-factor for C4b cleavage by factor I

Complement Receptor 1 (CR1;CD 35) Binds C4b displacing C2b, or C3b displacing Bb; cofactor for I Factor H (H) serum protein binds C3b displacing Bb; cofactor for I Decay Accelerating Factor (DAF;CD55)

Membrane protein displaces Bb from C3b and C2b from C4b

Membrane Cofactor Protein (MCP;CD46)

membrane protein that promotes C3b and C4b inactivation by I

CD59 (Protectin)

Prevents formation of MAC on homologous cells. Widely expressed on membranes

S Protein (Vitronectin)

serum protein binds C5b-7 prevents insertion into membrane

Clusterin (SP-40-40)

serum protein binds C5b-7 prevents insertion into membrane

Complement Receptors There are several characterized complement receptors that are involved in binding complement activation and degradation products. They are expressed on various cell

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types and are involved in mediating many of the biological functions attributed to complement. TABLE II DISTRIBUTION AND FUNCTION OF RECEPTORS FOR COMPLEMENT PROTEINS ON SURFACE OF CELLS

C5a Receptor (C5aR;CD88) is a seven transmembrane G-protein coupled receptor expressed primarily on neutrophils and macrophages. Also found on hepatocytes and various tissue epithelial cells. Causes smooth muscle contraction, histamine release from mast cells and vasodilation. Will modulate the hepatic acute phase response. It also is a potent chemoattractant for neutrophils, monocytes, macrophages, and eosinophils. C3a Receptor (C3aR) also seven transmembrane receptor. Tissue distribution currently being worked out. Causes smooth muscle contraction, histamine release from mast cells, and vasodilation. Chemoattractant for eosinophils but not neutrophils.

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BIOLOGICAL FUNCTIONS OF COMPLEMENT 1. CYTOLYSIS OF FOREIGN ORGANISM BY C5B-9 MAC COMPLEX 2. OPSONIZATION AND PHAGOCYTOSIS C3b, C3bi is coated on microorganisms (opsonization) Receptors for C3b (CR1) and C3bi (CR3) on macrophages and neutrophils can then bind the complement coated bacteria facilitating the phagocytosis reaction

3. ACTIVATION OF INFLAMMATION AND CHEMOTAXIS OF LEUKOCYTES BY COMPLEMENT ANAPHYLATOXINS (C3A, C4A, C5A)

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All three peptides mediate: 1. smooth muscle contraction 2. histamine release from mast cells and 3. increase vascular permeability. C5a on binding C5aR mediates chemoattraction of neutrophils, monocytes, macrophages, and eosinophils. C3a is a chemoattractant for eosinophils but not neutrophils.

4. SOLUBILIZATION AND CLEARANCE OF IMMUNE COMPLEXES One of the major roles complement plays is the solubilization and clearance of immune complexes from the circulation. First, C3b and C4b can covalently bind to the Fc region of insoluble immune complexes, disrupting the lattice, and making them soluble. C3b and C4b bound to the immune complex is recognized by the CR1 receptor on erythrocytes facilitating their transport to the liver and spleen. In the liver and spleen the immune complexes are removed and phagocytized by macrophage-like cells. The RBCs are returned to the circulation. Individuals deficient in the early complement components cannot make C3b and C4b. They are therefore predisposed to immune complex diseases such as systemic lupus erythematosus.

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5. ENHANCEMENT OF THE IMMUNE RESPONSE CR2 (CD21) is expressed on B-cells and follicular dendritic cells. This receptor binds the C3d fragment of C3. C3b coated on antigens will be broken down eventually to C3d and C3c fragments by factor I. The C3c fragment is released into the blood with no know function. The C3d fragment remains covalently bound to the antigen. Coating of antigens with C3d facilitates their delivery to germinal centers rich in B and follicular dendritic cells. CR2 also is part of the B-cell coreceptor complex. Binding of C3d coated antigens to CR2 leads to signaling through CD19. Animals deficient in C3 have an impaired immune response to T dependent antigens.

COMPLEMENT DEFICIENCIES AND ASSOCIATED ABNORMALITIES Human deficiencies in many of the complement proteins have been described. These deficiencies are usually attributable to inherited mutated genes. Genetic deficiencies in classical and alternative pathway components, including C1q, C1r, C4, C2, properdin, and factor D have all been described. C2 deficiency is the most common of the complement deficiencies. Deficiencies in the early classical pathway proteins predispose individuals to the development of systemic lupus erythematosus (SLE). The reason for this is not completely clear, but it is at least partly do to the inability of these individuals to clear immune complexes readily. Because of its central importance in killing bacteria, homozygous C3 deficiency can be lethal, especially in young children if it is not diagnosed. Deficiencies in the terminal components predispose these individuals to recurrent bacterial infections with Neisserial species.

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Table III. Complement Deficiencies and Associated Diseases

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Dear Immunology students, There is confusion between designation of the correct term used for the complement C3 convertase, with discrepancies between the lecturer and what is printed in the Coico, 2009 text. We will use the nomenclature provided by the lecturer and use what is listed in the syllabus. Here is our understanding. The nomenclature for complement has undergone revision so that the large, target-bound fragment is consistently given the 'b' designation, while the small, soluble fragment is called 'a'. Thus over the last 10 years texts have begun to reverse 2a and 2b, which were initially named incorrectly by this convention. According to the current (but not universally accepted) nomenclature, the C3 convertase is C4b2b and C4a and C2a are the released fragments; Coico, et al. apparently have not yet adapted this change. You will see other opinions in nomenclature, primarily from older texts that have not adopted the new naming structure. Therefore, due to a change in nomenclature in order to maintain the a=smaller and b=larger scheme, the correct C3 Convertase is C4b2b.

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SUMMARY 1.

The complement system is a group of over 30 serum proteins and cell surface molecules that act as an important part of the overall immune system.

2.

The activities of complement include: 1) cytolysis of foreign organisms, 2) opsonization and phagocytosis of foreign organisms, 3) activation of inflammation and directed migration of leukocytes, 4) solubilization and clearance of immune complexes, and 5) enhancement of humoral immune response.

3.

The complement cascade is a series of reactions involving complement proteins. It can be divided into two phases: activation and lysis (MAC formation). Activation involves protein-protein interactions and proteolytic cleavage, whereas the lytic pathway involves protein-protein interactions.

4.

There are three activation pathways: the classical, lectin, and alternative. Classical pathway activation requires antigen-antibody complexes (containing IgM or certain IgG subclasses). The Lectin pathway is a newly described pathway that activates the classical pathway independent of antibody. The Mannose Binding Lectin complex is substituted in place of C1 and recognizes polysaccharides on bacterial surfaces. The alternative pathway is an innate system in which complement components react directly with foreign substances. Classical pathway activation involves the proteins C1,C4,C2, and C3, and the alternative pathway utilizes the proteins C3, B, D, and P.

5.

Complement activation results in the release of anaphylatoxins (C3a,C4a, and C5a). They are important mediators of inflammation, causing recruitment and activation of neutrophils, macrophages, and other cell types. Activation also produces cleavage products (C3b, C3bi, and C4b) which serve as opsonins, enhancing phagocytosis. The C3d cleavage fragment is involved in enhancing the immune response.

6.

MAC formation produces a channel in the cytoplasmic membrane of bacteria or other cells, leading to cell lysis. It requires prior activation by either the classical or alternative pathways, and utilizes the proteins C5b, C6, C7, C8, and C9.

7.

The complement system is regulated by several inhibitors, including C1 inhibitor, Factor H, C4 binding protein, CD 59, and Decay Accelerating Factor.

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8.

Deficiencies in complement components result in increased susceptibility to bacterial infections and can lead to autoimmune diseases, including systemic lupus erythematosus.

STUDY PROBLEMS 1.

Watch the Complement Video in the Learning Resource Center (VT 803).

2.

From memory, draw the classical, lectin, and alternative pathways of complement.

3.

Understand how each is activated.

4.

Name 5 biological functions mediated by complement activation.

5.

Name the complement activation products that mediate each function.

6.

What are the three complement anaphylatoxin peptides?

7.

How does complement clear immune complexes from the circulation?

8.

What complement activation products bind covalently to cell surfaces?

9.

Know how the complement activation pathways are affected if a certain component is missing.

10.

If a patient is missing a particular complement protein, what disease(s) are they predisposed?

11.

On the figure shown on the next page, assign the activation fragments responsible for that function.

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Study Problem 11.

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Complement Component Table I

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Actor. 2012. Figure 6.6. Activation of complement through the classic pathway (antigen-antibody complexes), the alternative pathway (recognition of foreign cell surfaces), or the lectin pathway (or mannose-binding pathway) promotes activation of C3 and C5, leading to construction of the membrane attack complex..

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GENERATION OF ANTIBODY DIVERSITY Steven J. Norris, Ph.D. Recommended Reading: Actor, 2012, Chapter 3. WebResource: http://www.uth.tmc.edu/pathology/medic/immunology/Immuno/geneab.html

I. General principles 1. Ag-binding diversity results from differences in the Variable domains due to –  Multiple V gene segments  V(D)J joining  Random assortment of H, L chains  Junctional/insertional diversity  Somatic mutation 2. Functional diversity is due to differences in the Constant domains  IgM  C’ fixation  IgG  C’ fixation, opsonization through Fc receptors  IgA  secreted Ig  IgE  allergic reactions Changes in these Ig isotypes occur through isotype switching 3. Generation of Ag-binding diversity occurs during B or T cell development before exposure to antigens; isotype switching occurs in B cells after antigen exposure. 4. Both the generation of Ag-binding diversity and isotype switching involve DNA rearrangement. 5. Each B cell and all of its progeny produce only one type of heavy and light chain V region, and thus have a single antigen specificity. This specificity can be ‘fine tuned’ by point mutations (so-called somatic hypermutation) that occurs after antigen exposure.

Development of Ag-binding Diversity (VDJ rearrangement) (IgM/IgD producing B cells)  Exposure to Ag + T cell cytokines results in isotype switching (B cells change expression from IgM to other isotypes) 92

II. The heavy chain Ig locus and VDJ rearrangement 1. The heavy chain Ig locus is on chromosome 14 in humans. It encodes the IgM and IgD heavy chains ( and , respectively) that are expressed by B cells initially, as well as the other heavy chain isotypes (1, 2, 3, 4, 1, 2, and ) that are expressed after antigen exposure. In all cells except B cells, it is found in the so-called germline configuration, i.e. the same arrangement that is found in ova and spermatozoa; this germline form of the locus is hence passed from one generation to the next. 2. In the germline form, the Ig heavy chain locus contains multiple V, D, J , and C gene segments: Abbreviation: V D J C

Meaning: “Variable” “Diversity” “Joining” “Constant”

Number: ~50 ~20 ~6 9*

Size: Function: ~95 aa ~3-6 aa Form part of Variable Domain ~13 aa ~110 aa Form the Constant Regions per Domain *One for each heavy chain isotype; each constant region contains 3 or 4 C domains (see figure on proceeding page)

3. Stem cells in the bone marrow or fetal liver are stimulated to differentiate into B cells. These B cell precursors (pro-B cells) are then stimulated to undergo VDJ rearrangement. The pro-B cells begin to express Rag-1 and Rag-2, which direct the recombination of the Ig genes in B cell precursors and the TCR genes in T cell precursors. In B cells, the first step is D-J joining, in which the DNA between randomly selected D and J regions is looped out and the intervening sequence is deleted (see diagram). (This process involves the recognition of 7-bp and 9-bp sequences next to the D and J regions; the same type of recognition occurs in all Ig and TCR VDJ rearrangements.)

4. Following D-J joining, a similar looping out and deletion mechanism occurs between the V and D regions, resulting in V-D joining. The resulting contiguous V,D, and J gene segments have no intervening introns and form the Variable Region Exon. If the 93

14 undergoes rearrangement. If this recombination is unsuccessful, the cell undergoes apoptosis and dies.

Coico et al., 2009 Fig. 6.3. V(D)J rearrangement. V-J joining in the V locus is shown

5. After successful rearrangement, the  chain is transported to the surface of the cell along with the surrogate light chain proteins, VpreB and 5. The presence of this complex on pre-B cells triggers the initiation of light chain rearrangement. Coico., 2009, Fig. 7.2. (A) IgM-like receptor on Pre-B cells; (B) Surface IgM on mature B cells.

6. In mature B cells, the  (IgM) and  (IgD) heavy chains can both be expressed on the same cell by the alternative splicing of RNA as shown in Fig. 6.4 (preceding page). Clinical vignette – B cell maturation, from Geha and Notarangelo, “Case Studies in Immunology”. Case 1

III.

X-linked Agammaglobulinemia – medical student Bill Grignard has normal T cell function, but has almost no B cells or antibodies due to a defect in signaling at the pre-B cell stage.

Light chain rearrangement.

1. In humans and most other mammals, there are two light chain loci called kappa () and lambda (). These are located on two different chromosomes. The germline arrangement is similar to that of the heavy chain locus, except there are no D gene segments. Also, for the kappa locus there is only one constant region, whereas the lambda locus has multiple constant regions, each with its own J gene segment. 2. Once successful heavy chain rearrangement occurs, the pre-B cell proceeds with kappa gene rearrangement. In this case, randomly selected V and J segments in one chromosome join together to form the Variable Region Exon; no D segments are involved. 3. If a functional kappa chain is produced, V(D)J rearrangement stops and the cell becomes a immature B cell that expresses only IgM on its surface. The surface IgM will be anchored by a hydrophobic ‘tail’, and will look like the molecule shown in Fig. 94

7.2 (see above). Later, the cell can coexpress both IgM and IgD and thus become a mature B cell. 4. If the first rearrangement does not produce a kappa chain, then the second ‘sister’ chromosome will undergo rearrangement. If that is unsuccessful, then the two lambda loci will rearrange one after the other. If those are nonproductive, the cell will undergo apoptosis and die, as was the case for the heavy chain locus.

5. The end result of this process is an immature B cell expressing only IgM with either kappa or lambda light chains. As the cell leaves the bone marrow, it begins to express both IgM and IgD and thus becomes a mature B cell. This difference is important because immature B cells are more readily tolerized (made nonresponsive to antigen) than are mature B cells. 6. The steps of B cell development are summarized in the following figure (Coico, 2009, Fig. 7.1). B cell tumors may be ‘frozen’ at different stages of this maturation process; the less mature tumor types tend to be more aggressive and to have a poorer prognosis.

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IV.

Mechanisms of Ag-binding diversity

It is estimated that each individual is capable of producing B and T cells with 1015 to 1018 different antigen binding sites, each with a different (but perhaps overlapping) antigen specificity! As a result, there are very few compounds that will not induce an immune response, even if they are synthetic and have never been found in nature previously. Equally amazing is that most of the diversity is generated during V(D)J rearrangement, prior to antigen exposure. Thus each individual randomly produces a huge “repertoire” of B and T cells, only a small proportion of which (10,000 higher rate of mutation than ‘regular’ DNA. This somatic hypermutation occurs only after antigen stimulation. Some of these mutations increase the affinity of antibody for antigen, and those B cells expressing antibody with higher affinity will be selectively stimulated, increasing the proportion of high affinity antibody in secondary responses. This process is called affinity maturation. Altogether, these mechanisms produce almost an endless variety of antibody specificities. V.

Isotype switching

Isotype switching (also called class switching) results in the changing of the isotype of antibody expressed by a given B cell, e.g. from IgM to IgG3. Here are some features of isotype switching. 1. The constant region gene expressed is always the one immediately downstream of the V region (exception: both IgM and IgD can be expressed by ‘niave’ B cells that have not been stimulated by antigen). 2. The V region does not change during isotype switching; therefore the same antigenic specificity is retained. 3. Isotype switching results when antigen-stimulated B cells receive a cytokine signal from T helper cells. For example, IL-4 stimulates B cells to switch to IgE or IgG1. 4. Switching involves the deletion of intervening DNA between specific recombination sites called switch regions (see figure below). Because the intervening DNA is lost, the B cell cannot ‘switch back’ to an isotype that has already been deleted. 5. The V region and C regions are transcribed together, and RNA splicing and translation results in expression of the ‘new’ isotype.

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VI.

Membrane vs. secreted Ig expression

1. B cells express only membrane-bound immunoglobulins. 2. Plasma cells express only secreted immunoglobulins. 3. The difference between membrane vs. secreted Ig is the presence or absence of a hydrophobic ‘tail’ at the carboxy terminus of each heavy chain. 4. Regulation of membrane vs. secreted Ig expression is due to alternative splicing of the RNA transcript. 1. Membrane-bound form – RNA splicing results in retention of the M exons, which encode the hydrophobic amino acid region that anchors the Ig in the membrane. 2. Secreted form – the RNA transcript is cleaved before the M exons, resulting in a readily secreted, hydrophilic form of the heavy chain. 5. Differentiation of a B cell into a plasma cell results in this change. VII.

Regulation of Ig expression.

1. After VDJ joining, the promoter 5’ to the V region is brought within a few thousand base pairs of the enhancer element between the J region and the constant region. The enhancer greatly increases the rate of transcription, increasing Ig production. 2. Ig production is further increased in the differentiation of B cells into plasma cells. VIII. The immunoglobulin gene superfamily How did this fantastic mechanism evolve in the first place? 1. Antibodies are present in some form in all vertebrates, but are not found in invertebrate animals. However, vertebrates and invertebrates express a large number of closely related cell surface proteins, which are collectively called the immunoglobulin gene superfamily. It is thought that antibodies and T cell receptors evolved from cell surface receptors used for other functions, such as cell-cell interactions. 98

2. Domain structure. All members of the immunoglobulin (Ig) gene superfamily contain structures called Ig domains. An example of the two domains found in Ig light chains is shown below. Domains have the following features:  Primary amino acid sequence similarity (often only 20-30 percent)  About 100-110 amino acids in length  Beta-pleated sheet structure  Commonly have an intrachain disulfide bond

3. Members of the Ig gene superfamily are important in both immunologic and nonimmunologic systems, and include the following:  Immunoglobulins  T cell receptors  Major histocompatibility class I and II proteins  Many receptors specific for leukocytes (e.g. CD3, CD4, and CD8)  Many additional cell-surface receptors not exclusively involved in the immune system (e.g. intercellular adhesion molecule 1 [ICAM1], neuro-cellular adhesion molecule [NCAM], and chorioembryonic antigen [CEA]) Coico et al., Fig. 4.14 4. The ‘Big Bang’ theory – John Marchalonis at University of Arizona and others have proposed that the genes encoding recombinase proteins RAG1 and RAG2 were obtained by horizontal transfer of DNA from fungi or bacteria, resulting in the sudden acquisition of the antibody system in early vertebrates.

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SUMMARY – GENERATION OF ANTIBODY DIVERSITY 1. Generation of antigen-binding diversity results from V(D)J recombination during B cell development and somatic mutation after antigenic stimulation. Functional diversity of antibodies results from isotype switching, allowing expression of the 9 different Ig isotypes. Each B cell and all of its progeny express only one heavy chain and one light chain V region sequence, and thus all have the same antigenic specificity. 2. During B cell development, rearrangement of the heavy chain locus occurs first. DJ recombination is followed by V-D recombination, resulting in formation of the V domain exon comprised of V, D, and J gene segments. This process requires pairing of 7-bp and 9-bp sequences, after which the intervening DNA is ‘looped out’ and deleted permanently from the chromosome. 3. Light chain rearrangement occurs through a similar process, in which randomly selected V and J gene segments in the kappa or lambda light chain loci are joined. 4. V(D)J recombination proceeds through a hierarchy of heavy chain locus  kappa locus  lambda locus. Functional heavy and light chains must be produced, or the developing B cell undergoes apoptosis and dies. 5. There are five sources of antibody diversity: 1) presence of multiple V gene segments; 2) Combinatorial diversity, resulting from random recombination of V, D, and J segment combinations; 3) junctional and insertional diversity, resulting in changes in the V-D and D-J junctions; 4) co-expression of different H and L chain pairs; and 5) somatic hypermutation. 6. Isotype switching occurs after antigenic stimulation and requires cytokines produced by T cells. DNA is deleted between switching regions, so that a different constant region gene is juxtaposed close to the V domain exon. Expression of different Ig isotypes results. 7. B cells express only the membrane-bound form of Ig, whereas plasma cells express only the secreted form. This results from differential termination of heavy chain transcription. 8. Ig gene expression is upregulated by enhancer elements and other factors; expression by plasma cells is much higher than in B cells. 9. Immunoglobulins and T cell receptors are members of the immunoglobulin gene superfamily, which apparently first evolved a large set of cell surface receptors.

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THE ROLE OF THE MHC IN THE IMMUNE RESPONSE Jeffrey K. Actor, Ph.D. 713-500-5344 Objectives: (1) Understand genetic organization of the major histocompatibility complex. (2) Present an overview of differential processing of antigens in the MHC class I and class II pathways. (3) Discuss MHC restriction as related to presentation of antigens. (4) Present an overview of disease association with MHC type. (5) Introduce CD1 non-peptide lipid presentation. Keywords: HLA, H-2, Class I, Class II, 2-microglobulin, APC, Polymorphism, CD1 Reading: Coico and Sunshine. Immunology: A Short Course. John Wiley & Sons, Inc, New York, NY. 6th edition, 2009. Chapter 8; Geha and Notarangelo. Case Studies in Immunology. Garland Publishing, New York, NY.6th edition, 2012. Case 8: MHC Class II Deficiency. Web Resource: http://www.uth.tmc.edu/pathology/medic/immunology/Immuno/MHC.html The Major Histocompatibility Complex (MHC) is a locus on a chromosome comprised of multiple genes encoding histocompatibility antigens that are cell surface glycoproteins. MHC genes encode both class I and class II MHC antigens. These antigens play critical roles in interactions among immune system cells; class I participates in antigen presentation by macrophages to CD8+ lymphocytes (CTL), class II molecules participates in antigen presentation by macrophages to CD4+ lymphocytes (T helper). MHC genes are very polymorphic. The locus also encodes a third category of MHC genes, those of the class III type. The class III MHC molecules include complement proteins, tumor necrosis factor, and lymphotoxin. In man, the MHC locus is designated as HLA (Human Leukocyte Antigen). MHC molecules gain their name because they were first identified as the targets for rejection of grafts between individuals. When organs are transplanted across MHC locus differences between donor and recipient, graft rejection is prompt. In 1980 the Nobel Prize was awarded to Baruj Benacerraf, Jean Dausset and George D. Snell, for their work involving the major histocompatibility complex and rejection of skin grafts using inbred strains of lab mice. In mice, the MHC locus is designated as H-2. It has since been determined that the function of the MHC is the presentation of antigen fragments (epitopes) to T cells.

Organization and Structure of the MHC Genes and Gene Products MHC molecules are organized into 3 classes. Class I molecules are found on all nucleated cells. The class II molecules are found on B-cells and macrophages. Class III genes encode for various soluble proteins that include certain complement components. Human MHC: The HLA locus in humans is found on the short arm of chromosome 6. The class I region consists of HLA-A, HLA-B, and HLA-C loci and the class II region consists of the D region which is subdivided into HLA-DP, HLA-DQ, and HLA-DR subregions. Class I molecules are important in presentation of intracellular antigen to CD8+ t cells as well as for effector functions of target cells. Class II molecules are important in the induction of an immune response, since antigen-presenting cells must complex an antigen with the class II molecules to present it in 101

the presence of cytokines to CD4+ lymphocytes. Class III molecules encoded by genes located between those that encode class I and class II molecules include complement components.

Coico and Sunshine, 2009. Fig. 8.1

Polymorphism of Class I and Class II MHC genes Each chromosome 6 encodes three class I molecules B, C and A and three class II proteins DR, DP and DQ. All six of these MHC molecules show a high level of allotypic polymorphism, i.e. certain regions of the molecules differ from one person to another. The chance of two unrelated people having the same allotypes at all twelve sets of genes that encode MHC molecules is very small. There is a 25% chance of having identical genes that encode MHC molecules with each sibling, but only six of the twelve sets of genes that encode MHC molecules are inherited from each parent. MHC class I and class II molecules that are not possessed by an individual are seen as foreign antigens upon transplantation and are dealt with by the recipient's immune system accordingly. The highly polymorphic class I and class II MHC products are central to the ability of T cells to recognize foreign antigen and the ability to discriminate "self" from "non-self". The Class I MHC molecules are each somewhat different from one another with respect to aminoacid sequence, and all three are co-dominantly expressed in the membrane of every nucleated cell in an animal - but, depending on the organ involved, at different levels of expression (as high as 5 x 105 molecules per cell on lymphocytes). The term "co-dominantly expressed" means that each gene encoding these proteins on each parental chromosome of the diploid cells is expressed. MHC class I molecules expressed on progeny (F1) cells match maternal or paternal class I molecules since only the  subunit genes exhibit species-specific polymorphisms. MHC class II molecules expressed on F1 cells include homologous and heterologous  dimer mixtures since both  and  subunit genes exhibit species-specific polymorphism. Homologous dimers match class II molecules expressed on either parental cell type while heterologous dimers are unique to the F1 genotype and are functionally non-equivalent to parental class II molecules.

Structure of the MHC Class I Molecule Each class I locus codes for a transmembrane polypeptide of molecular weight approximately 45 kDa, containing three extracellular domains (1, 2, 3). The molecule is expressed at the cell surface in a noncovalent association with an invariant polypeptide called 2-microglobulin (2M) 102

of 12 kDa. 2M is a member of the Ig superfamily, the complex of class I and 2M appears as a four-domained molecule with the 2M and 3 domain of class I juxtaposed near the cell surface membrane.

Figure. View of MHC class I showing how a T cell receptor interacts with the class I molecule/2-M with peptide bound in the peptide binding groove.

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Figure. Schematic representation of an intact class I antigen in the plasma membrane. MHC class I showing the association of a class I molecule with 2M.

Structure of the MHC Class II Molecule

Class II molecules have 2 transmembrane polypeptide chains ( and , 30-34 and 26-29 kDa respectively); the peptide-binding site is shared by the two domains furthest from the cell membrane. The overall structure of the peptide-binding site is very similar for both class I and class II MHC molecules; the base is made of -pleated sheet, as in an immunoglobulin domain – the sides of the groove that holds the peptide are -helices. Peptides bind within the allele specific pockets defined by the 2 transmembrane polypeptide chains, where they are presented to the TCR for recognition. The extracellular domain shows variability in amino acid sequences, yielding grooves with different shapes. These grooves cradle the processed antigen for interaction with the T cell receptor. The CD4 molecule assists in the recognition process, and binds to the invariant portion of the MHC class II molecules. Like class I genes, class II genes also exhibit polymorphism with multiple allelic forms expressed. In humans, allelic forms are designated different from the mouse. For examples, human class II genes are given numbers such as HLA-D4 or HLA-D7.

Figure. View of MHC class II showing how a T cell receptor interacts with the class II molecule with peptide bound in the peptide binding groove.

Figure. Schematic representation of an intact class II antigen in the plasma membrane. MHC class II showing the two chain class II molecule.

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MHC Class III Molecules: Class III HLA genes encode complement components that show no structural similarity to either class I or class II molecules. These genes, along with genes encoding tumor necrosis factor (TNF), separate HLA class II and class I genes on the chromosome.

MHC and Antigen Presentation There are two major classes of presented antigen (Ag) called endogenous and exogenous Ag. MHC class I presents endogenous Ag epitopes to CD8+ T cells and MHC class II present exogenous Ag epitopes to CD4+ T cells. All nucleated cells are capable of presenting MHC class I, but only specialized cells present Ag epitopes on MHC class II. These are macrophages, dendritic cells and B cells. When exogenous Ag enters the body it is phagocytosed, digested and the resulting fragments are presented on MHC class II. When the CD4+ T cell receptor binds Ag-MHC class II it is activated to proliferate and secrete cytokines which in turn activate the other immune competent cells to generate humoral and/or cellular immunity. When CD8+ T Cell (CTL) receptor binds Ag-MHC class I it is activated to produce and secrete toxin that kills the cell to which it is bound. The cells that ingest, digest and present exogenous Ag epitopes on MHC class II are called antigen presenting cells (APCs), and the process of ingestion, digestion and presentation is called antigen processing and presentation. All nucleated cells can display MHC class I, but only APCs display MHC class II.  

CD8+ T Cell recognize Ag-MHC class I and CD4+ T Cell recognize Ag-MHC class II. Antigen is recognized in conjunction with proteins of the major histocompatibility complex (MHC). Different antigen degradation and processing pathways produce MHC-peptide complexes where "endogenous" peptides associate with class I molecules and "exogenous" peptides associate with class II molecules.

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Endogenous (cytoplasmic) antigen processing and MHC class I presentation: MHC class I molecules bind peptide fragments derived from proteolytically degraded proteins endogenously synthesized by a cell. Small peptides are transported into the endoplasmic reticulum where they associate with nascent MHC class I molecules before being routed through the Golgi apparatus and displayed on the surface for recognition by cytotoxic T lymphocytes. MHC class I molecules bind small antigenic peptides that are 8-10 amino acid residues in length.

Coico and Sunshine, 2009. Fig. 8.7.

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Exogenous (endosomal) antigen processing and MHC class II presentation: MHC class II molecules bind peptide fragments derived from proteolytically degraded proteins exogenously internalized by "antigen presenting cells," including macrophages, dendritic cells, and B cells. The resulting peptide fragments are compartmentalized in the endosome where they will associate with MHC class II molecules before being routed to the cell surface for recognition by helper T lymphocytes. MHC class II molecules bind larger antigenic peptides usually 13-18 amino acid residues in length (but may be longer). Like class I molecules, class II MHC molecules are synthesized in the RER. The class II  and  chains reside there as a complex with an additional polypeptide called the invariant chain (Ii). The invariant chain blocks the groove of the class II molecule and prevents endogenous antigens from binding there. The MHC/invariant chain complex is transported to an acidic endosomal or lysosomal compartment that contains a degraded antigen peptide. The invariant chain comes off the complex, exposes the groove of the class II molecule, and allows the antigen peptide to slip into the groove. The class II/antigen peptide complex is then transported to the surface of the APC where it is available for interaction with CD4 TH cells.

FIGURE 8.5. Processing of exogenous antigen in MHC class II pathway, (Ii = invariant chain; CLIP = fragment of Ii bound to MHC class II groove.) Coico and Sunshine, 2009. Figure 8.5.

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Coico and Sunshine, 2009. Table 8.1.

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The role of the MHC in Thymic Education The education process by which T cells in the thymus learn to recognize antigenic peptides in the context of self-MHC molecules is a two-step process involving both positive and negative selection. 

Step 1: Initially, immature thymocytes within the thymic cortex express low levels of TCR, but high levels of both CD4 and CD8 (double-positive cells). They interact with thymic epithelial cells that express high levels of both class I and class II MHC molecules. Thymocytes with moderate affinities for these selfMHC molecules are allowed to develop further, while thymocytes with affinities too high or too low for self-MHC are induced to die by apoptosis. The thymocytes that survive are said to have been "positively selected" through their interaction with self-MHC.



Step 2: The positively selected thymocytes then begin to express high levels of TCR, some of which recognize self components other than self-MHC. These cells must be deleted to prevent autoimmune destruction of healthy host tissues. Negative selection is the elimination of T cells reactive with self components other than the MHC. Negative selection occurs in the deeper cortex, at the corticomedullary junction, and in the medulla of the thymus. The thymocytes interact with antigen processed and presented by interdigitating cells and macrophages. Only thymocytes that fail to recognize self antigens are allowed to survive and proceed along the maturation process, with the remainder undergoing apoptosis. Eventually, T cells that survive the negative selection process lose either CD4 or CD8, becoming "single positive" cells. Fewer than 5% of thymocytes survive selection and leave the thymus to take up residence in the secondary lymphoid organs.

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Role of MHC in activation of T lymphocytes The binding between the TCR and the MHC/antigen peptide complex is highly specific and acts as the first signal to induce T cell activation. T cells do not respond to either self-MHC alone or to free peptide. Activated T cells differ from resting T cells in that they proliferate and secrete lymphokines and/or lytic substances. The affinity of the TCR for the MHC/antigen complex is often too low to fully activate the T cell; there are numerous accessory molecules that increase avidity between the T cell and APC by performing an adhesive function. Cytotoxic T lymphocytes: CTLs are able to kill target cells directly by inducing apoptosis. Nucleases and other enzymes activated in the apoptotic process may help destroy the viral genome, thus preventing the assembly of virions and potential infection of other cells. CTL induce apoptosis only in the target cell; neighboring tissue cells are not affected. Two mechanisms for induction of apoptosis have been identified:  

Preformed perforins are released at the target cell surface which generate transmembrane pores in the target cell, through which a second protein, granzyme, can gain entry to the cytosol and induce the apoptotic series of events. Apoptitic signaling via membrane-bound molecules can occur via Fas on the target cell surface and Fas ligand on the CTL surface. The processes of antigen recognition, CTL activation and delivery of apoptotic signals to the target cell can be accomplished within 10 minutes. The apoptotic process in the targeted cell may take 4 hours or more and continues long after the CTL has moved on to interact with other tissue cells.

T helper lymphocytes: The initial interaction between T lymphocytes and the APC is mediated by adhesion molecules. Interactions occur between LFA-1, CD-2 and ICAM-3 on the T-cell, and with ICAM-1, ICAM-2, LFA-1 and LFA-3 on the APC. These molecules synergize in binding of lymphocytes to the APCs. This transient binding allows the T-cell to sample the large numbers of MHC molecules on the surface of the APCs for their specific peptide. If a T-cell recognizes its peptide ligand bound to MHC, signaling via the T-cell receptor complex is induces more conformational changes, eventually leading to the production of T-cell cytokines. T cells require co-stimulation through binding of the CD-28 ligand with the CD-80/CD-86 ligand of the APC. T-dependent B cell activation: B cells can also specifically take up antigen via binding through their surface Ig. This is internalized, broken down to peptides and the peptides are presented on the B cell surface held in the peptide binding grooves of MHC class II molecules. If this B cell interacts with a primed T cell that recognizes the peptide/class II complex on the B cell then the T cell may transiently express accessory ligands. This results in cells going into cell cycle and the secretion of cytokines. For the B cell, this action, in concert with correctly released T cell cytokines, will drive isotype switching as well as maturation of the B lymphocyte into a plasma cell.

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Association of Disease with MHC Haplotype Particular MHC alleles are associated with better protection against certain infections. Certain alleles are associated with a greater chance of developing autoimmunity. Some diseases are distinctly more common in individuals with a particular MHC allele or MHC haplotype. Diseases with a strong association with certain MHC alleles include insulindependent diabetes and Graves' disease. Expression of HLA-DR4 is associated with rheumatoid arthritis. Nearly 90% of people with ankylosing spondylitis carry the HLAB27 allele. Expression of HLA-DR2 is associated with multiple sclerosis. The Association of HLA serotype with susceptibility to autoimmune disease will be covered in the AutoImmunity Lecture. An updated list of genetic polymorphisms associated with increased susceptibility to disease will be presented during that lecture. It is hypothesized that in some cases MHC molecules serve as receptors for the attachment and entry of pathogens into the cell; this makes individuals with a certain HLA type more susceptible to infection by a particular intracellular pathogen using that HLA molecule as a receptor. Alternatively, an infectious agent might possess antigenic determinants that resemble MHC molecules (molecular mimicry). Such resemblance might allow the pathogen to escape immune detection because it is seen as "self," or it may induce an autoimmune reaction. Because MHC molecules differ in their ability to accommodate different peptides, some individuals who express certain MHC genes may lack the ability to present microbial epitopes capable of inducing protective T cell responses. Finally, there may simply be no T cells capable of recognizing a particular MHC/antigen combination, leading to a "hole in the T cell repertoire."

Coico and Sunshine, 2009. Figure 8.6.



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Clinical Vignette - The case of Helen Burns (Case 8 in Geha and Notarangelo): Helen Burns was 6 months old when she developed pneumonia, caused by the opportunistic pathogen Pneumocystis carinii. Helen was tested for severe combined immunodeficiency; it was found that Helen's T cells could be stimulated with mitogen (phytohemagglutinin), but could not respond to specific antigenic stimuli. It was further established that Helen had low overall immunoglobulin levels and decreased CD4 cells. Her CD8 cell counts were within normal range. Helen's white blood cells were examined for expression of MHC Class I and Class II molecules. She was diagnosed with MHC Class II deficiency. A bone marrow transplant was performed using Helen's mother as a donor. The graft was successful and immune function was restored. How does MHC Class II deficiency selectively affect CD4 T cell function, and what implications does this have towards immune responses to infective agents?

Geha and Notarangelo, 2012. Figure 8.3.

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Summary: Role of HLA in the Immune System MHC molecules are organized into 3 classes. Class I molecules are found on all nucleated cells. The class II molecules are found on B-cells and macrophages. Class III genes encode for various soluble proteins that include certain complement components. Human class I region genes consists of HLA-A, HLA-B, and HLA-C loci and the class II region genes consists of the D region which is subdivided into HLA-DP, HLA-DQ, and HLA-DR subregions. All MHC molecules show high allotypic polymorphism. Class I molecules are important in presentation of Ag epitopes to CD8+ T cells as well as for effector functions of target cells. Each class I locus codes for a transmembrane polypeptide containing three extracellular domains (1, 2, 3), which is expressed at the cell surface in a noncovalent association with 2-microglobulin (2M). All nucleated cells express Class I molecules. Class II molecules present antigen in the presence of cytokines to CD4+ lymphocytes. Class II molecules have 2 transmembrane polypeptide chains. Peptides bind within the allele specific pockets defined by the 2 transmembrane polypeptide chains, where they are presented to the TCR for recognition. Different antigen degradation and processing pathways produce MHC-peptide complexes where "endogenous" peptides associate with class I molecules and "exogenous" peptides associate with class II molecules. MHC class I molecules bind small antigenic peptides that are 8-10 amino acid residues in length; MHC class II molecules present slightly larger peptides. T cells in the thymus learn to recognize antigenic peptides in the context of self-MHC molecules by a two-step process involving both positive and negative selection. The binding between the TCR and the MHC/antigen peptide complex acts as a signal to induce T cell activation. T cells do not respond to either self-MHC alone or to free peptide. Accessory molecules increase avidity between the T cell and APC by performing an adhesive function. CTLs recognize Ag in the context of MHC class I, and kill target cells directly by inducing apoptosis. They release preformed perforins at the target cell surface to generate transmembrane pores in the target cell, through which a second protein, granzyme, gains entry to the cytosol to initiate an apoptotic series of events. CTLs can also deliver apoptitic signals via surface bound molecules. T helper lymphocytes recognize Ag and MHC class II on the APC in a manner mediated by adhesion molecules. The recognition is specific and requires co-stimulation through ligand interactions on the APC. Activation of the T helper cell leads to specific cytokine release. B cells are good antigen presenters to T cells. Certain MHC alleles are associated with a greater chance of protective immune responses to pathogens, as well as towards developing autoimmunity. Some diseases are distinctly more common in individuals with a particular MHC allele or MHC haplotype. Possible reasons for this are discussed.

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T CELL RECEPTOR: Structure and Genetic Basis Jeffrey K. Actor, Ph.D. 713-500-5344 Objectives: (1) Present an overview of the T receptor structure and organization of the gene loci encoding for the T cell receptor chains; (2) explain mechanisms underlying generation of T cell receptor diversity; (3) examine the stages in thymic selection of T lymphocytes; and (4) compare and contrast the T cell receptor with the B cell receptor. Keywords: T cell receptor (TCR). Reading: Coico and Sunshine. Immunology: A Short Course. John Wiley & Sons, Inc, New York, NY. 6th edition, 2009. Chapter 9; Geha and Notarangelo. Case Studies in Immunology. Garland Publishing, New York, NY. 6th edition, 2012. Case 7: Omenn Syndrome. Web Resource: http://www.uth.tmc.edu/pathology/medic/immunology/Immuno/TCR.html The acquired immune response is subdivided, based on participation of two major cell types. B lymphocytes originate in the bone marrow, and synthesize/secrete antibodies. This is termed humoral immunity. T lymphocytes mature in the thymus, and secrete immunoregulatory factors following interaction with antigen presenting cells; this is termed cellular immunity (CMI). Lymphocyte Biology Lymphoid cells provide efficient, specific and long-lasting immunity against microbes/pathogens and are responsible for acquired immunity. This lecture will primarily examine the biology of two classes of lymphocytes: (1) thymic-dependent cells or T lymphocytes that operate in cellular and humoral immunity; and (2) B lymphocytes that differentiate into plasma cells to secrete antibodies. T and B lymphocytes produce and express specific receptors for antigens. The major properties of the acquired immune response are specificity, memory, adaptiveness, and discrimination between self and non-self. All of these properties are related to the random selection of variable region components during the development of both B cells and T cells. The lymphatic organs are tissues in which lymphocytes mature, differentiate and proliferate. The primary (central) lymphoid organs are those in which B and T lymphocytes mature into antigen recognizing cells. In embryonic life, B cells mature and differentiate from hematopoietic stem cells in the fetal liver. After birth, B cells differentiate in the bone marrow. Maturation of T cells occurs in a different manner. Progenitor cells from the bone marrow migrate to the thymus where they differentiate into T lymphocytes. The T lymphocytes continue to differentiate after leaving the thymus, and are driven to do so by encounter with specific antigen in the secondary lymphoid organs. The secondary lymphoid organs are those tissues in which antigen-driven proliferation and differentiation take place. The spleen and lymph nodes are the major secondary lymphoid organs. 114

Additional secondary lymphoid organs include the tonsils, appendix, and Peyer’s patches. Aggregates of cells in the lamina propria of the digestive tract lining may also be included in this category, as well as any tissue described as MALT (mucosa-associated lymphoid tissue), GALT (gut-associated lymphoid tissue) or BALT bronchus-associated lymphoid tissue). T Lymphocytes: T lymphocytes are involved in regulation of immune response and cell mediated immunity. They provide necessary factors to help B cells produce antibody. Mature T cells express antigen-specific T cell receptors (TCR). Every mature T cell expresses the CD3 molecule, which is associated with the TCR. The TCR/CD3 complex recognizes antigens associated with the major histocompatibility complex (MHC) molecules on target cells (e.g. virus-infected cell). The TCR is also expressed on the cell surface in association with co-receptor or accessory molecules (CD4 or CD8).

The structure of the T-cell receptor (TCR) complex showing the predominant form of the antigen-binding chains,  and , and the associated signal transduction complex, CD3 (, , and  chains) plus  (zeta) or eta) or  (theta. (-) and (+) represent electrostatic interactions.

T Cell Receptor: The TCR is a transmembrane heterodimer composed of two disulfide-linked polypeptide chains. T lymphocytes of all antigenic specificities exist prior to contact with antigen. Each lymphocyte carries a TCR of only a single specificity. T Lymphocytes can be stimulated by antigen to give rise to progeny with identical antigenic specificity. Lymphocytes reactive with “self” are deleted or inactivated to ensure that no immune response is mounted against self components. The vast majority of T lymphocytes express alpha [] and beta [] chains on their surface. Cells that express gamma [] and delta [] chains comprise only 5% of the normal circulating T cell population in healthy adults. Each chain (, or ) represents a distinct protein with approximate molecular weight of 45 kDa. An individual T cell can express either an  or a  heterodimer as its receptor, but never both. The TCR recognizes antigen in the form of peptides which are bound in the groove on MHC molecules (reviewed in detail in lecture: Role of MHC in Immune Response). The interactions between heterodimers create three hypervariable regions called complementarity determining regions (CDRs 1, 2, and 3). 115

The interaction of TCR, MHC, and peptide. The complementarity determining regions (CDRs) of the TCR V regions and peptide bound in the peptide-binding groove of an MHC class I molecule are depicted. [Based on the crystal structure described by K. C. Garcia et al. (1998): Science 279: 1166.]

The T cell receptor genes are closely related members of the immunoglobulin gene superfamily. Each chain consists of a constant (C) and a variable (V) region, and is formed by a gene-sorting mechanism similar to that found in antibody formation. The repertoire is generated by combinatorial joining of variable (V), joining (J), and diversity (D) genes, and by N region diversification (nucleotides inserted by the enzyme deoxynucleotidyl-transferase). Unlike immunoglobulin genes, genes encoding TCR do not undergo somatic mutation. Thus there is no change in the affinity of the TCR during activation, differentiation, and expansion. 116

TCR-CD3-complex: The TCR heterodimer is tightly associated with six independently encoded CD3 subunits (, , , ,  and ) required for efficient transport to the cell surface. CD3 subunits possess long intracellular tails and are responsible for transducing signals upon TCR engagement. Genes Coding for T-Cell Receptors: Genes which code for the T cell receptor and the mechanisms used to generate TCR diversity are similar to those of immunoglobulins.  The TCR V, D, and J genes are mixed together in a more complicated manner than found for immunoglobulin genes.   and  uses only V and J gene segments.   and  use V, D, and J gene segments.  There are many more V and V genes (50-100) than V and V genes (5-10) present in germ line.  The  and  chain genes are mixed together in one locus. The genes encoding the  chain are entirely located between the cluster of V and J gene segments. The top and bottom rows show germline arrangement of the variable (V), diversity (D), joining (J), and constant (C) gene segments at the T-cell receptor  and  loci. During T- cell development, a V-region sequence for each chain is assembled by DNA recombination. For the chain (top), a V gene segment rearranges to a J gene segment to create a functional gene encoding the V domain. For the  chain (bottom), rearrangement of a D, a J, and a V gene segment creates the functional V-domain exon. Geha and Notarangelo, 2012. Figure 7.1.

Order of TCR Gene Rearrangement:  The earliest cell entering the thymus has its TCR genes in the germ line configuration (unrearranged).  Both  and  chain genes then begin to rearrange, more or less simultaneously.  If the  chain genes rearrange successfully, then  chain genes also start to rearrange. If both  and  genes rearrange functionally, no further gene rearrangement takes place and the cell remains a  T cell.  If  and/or  rearrangements are not functional, then  gene rearrangement continues followed by  gene rearrangement. In this manner, a  product appears, and the cell becomes an  T cell. 117

The Process of Recombination: Recombination of V, D, and J gene segments is coordinated by recombinase-activating genes RAG-1 and RAG-2. The enzymes recognize specific DNA signal sequences consisting of a heptamer, followed a spacer of 12 or 23 bases, and then a nonamer. If either RAG gene is impaired or missing, homologous recombination events are abolished. This gives rise to severe combined immunodeficiency (SCID). Mutations which result in partial enzymatic activity can also occur, and can give rise to immunodeficiency diseases. An example of such disorder is Omenn Syndrome, discussed in detail in the Case Studies in Immunology (Geha and Notarangelo, chapter 7) text.

Generation of T-Cell Receptor Diversity: The overall level of diversity is greater for T cell receptors than that for immunoglobulins. This is primarily due to additional junctional diversity in possible TCR gene rearrangements. Most of the variability in the TCR occurs within junctional regions encoded by D, J and N nucleotides. This is 118

the region that corresponds to the CDR3 loops that form the center of the antigen binding sites. So, while the center of the binding site is highly variable, the remaining portion of the heterodimer is subject to relatively little variation. Number of V gene pairs Junctional diversity Total Diversity

Immunoglobulins ~2 - 3.4 x 106 ~3 x 107 ~1014

T cell : Receptors 5.8 x 106 ~2 x 1011 ~1018

Development of T lymphocytes During differentiation in the thymus, immature T cells undergo rearrangement of their TCR  and  genes to generate a diverse set of clonotypic TCRs. Immature thymocytes are selected for further maturation only if their TCRs do not interact with selfpeptides presented in the context of self-major histocompatibility complex (MHC) molecules on antigen presenting cells. Different signals lead to the alternate developmental outcomes of maturation or apoptosis (positive versus negative selection). Positively selected thymocytes undergo alternate commitment to either the T killer or T helper lineages, which correlate precisely with a cell's TCR specificity towards MHC class I or II molecules, respectively. Lineage commitment is marked phenotypically by the loss of expression of one of the co-receptor molecules, CD8 or CD4. Immature thymocytes express both co-receptors (double positive), while T killer or T helper cells express only CD8 or CD4, respectively (single positive CD8+ or CD4+). Figure. Changes in surface molecules of thymocytes at different stages of maturation.

The majority of peripheral blood T lymphocytes express the  and  form of the TCR. In healthy adults, less than 5% express a heterodimer comprised of the  and  chains. Virtually all the cells that express the TCR- are CD4+CD8- (T helper) or CD4-CD8+ (T cytotoxic or T suppressor). Almost all cells expressing TCR- are CD4-CD8- (double negative). While the TCR- expressing lymphocytes are known to function as helper and cytotoxic cells, the function of the TCR- cells is not well understood.

Figure. Main stages in the development of a T lymphocyte. 119

++, CD4+CD8+ cell interacts with Thymic epithelial cell Interaction = Pos Selection

No interaction =

MHC + self

CD4+CD8+ cell DEATH

MHC + non-self

++, CD4+CD8+ cell interacts with interdigitating cell High affinity interaction =

DELETION

Low affinity interaction =

SURVIVAL Commitment CD4+ or CD8+

Figure. Main stages in Thymic Selection.

T Helper Cells: T helper cells (Th) are the primary regulators of T cell- and B cell-mediated responses. They 1) aid antigen-stimulated subsets of B lymphocytes to proliferate and differentiate toward antibody-producing cells; 2) express the CD4 molecule; 3) recognize foreign antigen complexed with MHC class II molecules on B cells, macrophages or other antigen-presenting cells; and 4) aid effector T lymphocytes in cell-mediated immunity. Currently, it is believed that there are two main functional subsets of Th cells, plus other helper subsets of importance. T helper 1 (Th1) cells aid in the regulation of cellular immunity, and T helper 2 (Th2) cells aid B cells to produce certain classes of antibodies (e.g., IgA and IgE). The functions of these subsets of Th cells depend upon the specific types of cytokines that are generated, for example interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) by Th1 cells; IL4, IL-6 and IL-10 by Th2 cells. Two other classes of T helper cells are thought to be involved in oral tolerance and serve as regulators for immune function. Th3 cells secrete IL-4 and TGF- and provide help for IgA production, and have suppressive properties for Th1 and Th2 cells. Th17 cells, characterized by IL-17 secretion, are thought to be involved as effector cells for autoimmune disease progression. T Cytotoxic Cells: T cytotoxic cells (CTLs) are cytotoxic against tumor cells and host cells infected with intracellular pathogens. These cells 1) usually express CD8, and, 2) destroy infected cells in an antigen-specific manner that is dependent upon the expression of MHC class I molecules on antigen presenting cells. T Suppressor/ T Regulatory Cells: T suppressor cells suppress the T and B cell responses and express CD8 molecules. T regulatory cells also affect T cell response, with many cells characterized as CD4+CD25+, TGF- secretors.

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 T Cells: Not all T cells express  TCRs. An alternative is to express  chains of the TCR. Generally,  cells lack CD4, although some  cells do express CD8. The functions of  cells are not well understood.  T cells can function in the absence of MHC molecules. They home to the lamina propria of the gut, and are thought to assist in protection against microorganisms entering through epithelium at mucosal surfaces. Their range of response to antigens is limited.  expressing cells have been found to be active towards mycobacterial antigens and heat shock proteins. They have the ability to secrete cytokines like their  counterparts. Natural Killer T Cells: Natural killer T cells (NKT) are a heterogeneous group of T cells that share properties of both T cells and natural killer (NK) cells. hese cells recognize an antigenpresenting molecule (CD1d) that binds self- and foreign lipids and glycolipids. They constitute only 0.2% of all peripheral blood T cells. The term “NK T cells” was first used in mice to define a subset of T cells that expressed the natural killer (NK) cell-associated marker NK1.1 (CD161). It is now generally accepted that the term “NKT cells” refers to CD1d-restricted T cells coexpressing a heavily biased, semi-invariant T cell receptor (TCR) and NK cell markers. Natural killer T (NKT) cells should not be confused with natural killer (NK) cells. -------------------------Comparison of the B cell and T cell receptors: Both BCRs and TCRs share these properties:  they are integral membrane proteins  they are present in thousands of identical copies exposed at the cell surface  they are made before the cell ever encounters an antigen  they are encoded by genes assembled by the recombination of segments of DNA  allelic exclusion ensures only one receptor with a single antigenic specificity  they demonstrate N region addition during gene rearrangement  they have a unique binding site  this site binds to a portion of the antigen called an antigenic determinant or epitope  the binding, like that between an enzyme and its substrate depends on complementarity of the surface of the receptor and the surface of the epitope  the binding occurs by non-covalent forces (again, like an enzyme binding to its substrate)  successful binding of the antigen receptor to the epitope, if accompanied by additional "signals", results in: 1. stimulation of the cell to leave G0 and enter the cell cycle 2. repeated mitosis leads to the development of a clone of cells bearing the same antigen receptor; that is, a clone of cells of the identical specificity.       

BCRs and TCRs differ in: their structure the genes that encode them the type of epitope to which they bind TCRs do not somatically mutate TCRs do not undergo isotype switching TCR gene recombination exhibits far greater junctional diversity than Ig genes TCRs are never secreted from the T cell

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Clinical Vignette - Omenn Syndrome (Case 7 in Geha and Notarangelo): Patients with Omenn syndrome demonstrate severe immunodeficiency characterized by the presence of activated, anergic, oligoclonal T cells, hypereosinophilia, and high IgE levels. There is a body of evidence to indicate that the immunodeficiency manifested in patients with Omenn syndrome arises from mutations that decrease the efficiency of V(D)J recombination. These individuals bear missense mutations in either the RAG-1 or RAG-2 genes that result in partial activity of the two proteins. In many cases, amino acid substitutions map within the RAG-1 homeodomain and decrease DNA binding activity, while others lower the efficiency of RAG-1/RAG-2 interaction.

Summary: T Lymphocytes T lymphocytes are involved in regulation of immune response and in cell mediated immunity. Every mature T cell expresses CD3, which is associated with the TCR. During thymic differentiation, immature T cells undergo rearrangement of their TCR  and  genes to generate a diverse set of clonotypic TCRs. Immature thymocytes are selected for further maturation only if they recognize foreign antigens in the context of MHC molecules. Mature T cells usually display one of two accessory molecules. CD4+ T helper cells are the primary regulators of T cell- and B cell-mediated responses, and are further subdivided into functional subsets dependent upon cytokines secreted. CD8+ T cytotoxic cells (CTLs) are cytotoxic against tumor cells and host cells infected with intracellular pathogens. T suppressor cells suppress the T and B cell responses and express CD8 molecules. Summary: T Cell Receptor: Structure and Genetic Basis Mature T cells express antigen-specific TCR in a complex with CD3 molecules. The TCR is a disulfide-linked heterodimer composed of either  or  chains. T cells express either  or  chain heterodimers, but never both. T cell receptor genes are closely related members of the immunoglobulin gene superfamily and derive part of their structural diversity form recombination of different V, D, and J gene segments. The mechanisms for T cell receptor gene switching are similar to those of immunoglobulin genes, but T cell receptor genes do not have somatic mutations.  chains of the TCR have only V and J segments, and join to  chains.  chains of the TCR have genes for V, D, and J segments. The process of recombination is coordinated by recombinase-activating genes RAG-1 and RAG-2. If  rearrangements are unsuccessful on both chromosomes,  chains join to  chains to give  phenotypic T cells.  chains have only V and J segments; chains have V, D, and J segments.

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ADAPTIVE IMMUNE RESPONSE I and II Jeffrey K. Actor, Ph.D. MSB 2.214, 713-500-5344 Required Reading: Coico and Sunshine, 2009. Chapters 7, 9, 10, 11. Geha and Notarangelo. Case Studies in Immunology. Garland Publishing, New York, NY. 6th edition, 2012. Case 47. Toxic Shock Syndrome.

Web Resource: http://www.uth.tmc.edu/pathology/medic/immunology/Immuno/Biologybt.html OBJECTIVES 1. Distinguish between innate and adaptive immune responses on the basis of antigen specificity, HLA restriction and memory. 2. Understand role of immune mediators including cytokines, chemokines, costimulatory and adhesion molecules in the development of adaptive immune responses. 3. To describe the various effector and regulatory functions of T and B cells. 4. To demonstrate the molecular events associated with T cell and B cell activation. 5. Compare and contrast effector cells in cytotoxic mediated immunity. 6. To develop a practical understanding of mechanisms and clinical relevance of T dependent and - independent antibody responses. KEY WORDS

cytokine, T cell receptor, B cell receptor, helper T cell, cytotoxic T lymphocyte, NK cell, NKT cell, T-dependent antibody, Tindependent antibody response.

I. OVERVIEW OF THE IMMUNE RESPONSE The acquired immune response is subdivided, based on participation of two major cell types. B lymphocytes originate in the bone marrow, and synthesize/secrete antibodies. This is termed humoral immunity. T lymphocytes mature in the thymus, and secrete immunoregulatory factors following interaction with antigen presenting cells; this is termed cellular immunity (CMI). A.

Purpose – maintain homeostasis

B.    C.

Discriminates Self from nonself (foreign, effete) Pathogenicity – ability to cause disease Intracellular vs. extracellular pathogen Begins in utero

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D.

Remembers previous encounters

E.

Goal is proper specificity, intensity, and duration

The major properties of the acquired immune response are specificity, memory, adaptiveness, and discrimination between self and non-self. All of these properties are related to the random selection of variable region components during the development of B cells and T cells. The essential features for clonal selection of these cells include:    

B and T lymphocytes of all antigenic specificities exist prior to contact with antigen. Each lymphocyte carries specific surface molecules (immunoglobulin or T cell receptor) of only a single specificity. Lymphocytes can be stimulated by antigen under appropriate conditions to give rise to progeny with identical antigenic specificity. Lymphocytes potentially reactive with “self” are deleted or inactivated to ensure that no immune response is mounted against self components.

II. MOLECULAR COMPONENTS A.

Cytokines

When confronted with above challenge, the host immune response will determine appropriate degree of antigen-specific cell mediated vs. humoral response. In order to accomplish this, various regulatory networks controlled by cytokines are activated. 1.

Physical and Biological Properties 

small mol weight peptides and glycopeptides



produced by a variety of cell types accessory cells leukocytes somatic cells – endothelium, fibroblasts, etc.



short plasma half lives (makes determining levels clinically difficult)



modulate immune/inflammatory responses by stimulating/inhibiting various cell populations (inflammatory, epithelial, fibroblast)



one cell type can make multiple cytokines and a single cytokine can be made by a variety of cell types

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2.



redundancy – multiple cytokines can have the same biological activity



pleomorphism/pleotropic – the same cytokine can have multiple activities depending upon the target cell, concentration and/or presence of other cytokines



action can be endocrine, paracrine and/or autocrine

Classification – Biological Activity  Interferons (,, – interfere with viral replication but also have immunomodulatory properties 

Colony Stimulation Factors – support growth of WBC elements of bone marrow; mediate various inflammatory reactions



Tumor Necrosis Factors – produce hemorrhagic necrosis of tumors in mice; major mediator of inflammation and is elevated in sepsis syndrome



Chemokines – groups of molecules that mediate chemotaxis of various inflammatory cells



Interleukins – various immunoregulatory functions between (inter) various leukocyte (leukin) populations

AN EXPANDED LIST OF CYTOKINES, INTERFERONS CHEMOKINES ARE INCLUDED IN THE APPENDIX.

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AND

III. T LYMPHOCYTES T lymphocytes are involved in the regulation of the immune response and in cell mediated immunity, and help B cells to produce antibody. Mature T cells express antigen-specific T cell receptors (TCR). Every mature T cell expresses the CD3 molecule, which is associated with the TCR. In addition mature T cells usually display one of two accessory molecules, CD4 or CD8. The TCR/CD3 complex recognizes antigens associated with the major histocompatibility complex (MHC) molecules on target cells (e.g. virus-infected cell). Important T cell markers

Surface Markers of T cells. Additional markers include: CD45RO, Leukocyte common antigen for memory T cells (activated). CD45RA, Leukocyte common antigen for naive T cells (resting). Coico and Sunshine, 2009. Figure 9.4.

TCR-CD3-complex: The TCR heterodimer is tightly associated with the CD3 coreceptor made up of independently encoded subunits (, , , and two  chains). The CD3 complex is required for efficient transport of the TCR to the cell surface. CD3 subunits possess long intracellular tails and are responsible for transducing signals upon TCR engagement with MHC presented antigen. A. T Helper cells T helper cells (Th) are the primary regulators of T cell- and B cell-mediated responses. They 1) aid antigen-stimulated subsets of B lymphocytes to proliferate and differentiate 126

toward antibody-producing cells; 2) express the CD4 molecule; 3) recognize foreign antigen complexed with MHC class II molecules on B cells, macrophages or other antigen-presenting cells; and 4) aid effector T lymphocytes in cell-mediated immunity. 1.

Paired Interactions between the APC and CD4+ T cell (Immunological synapse)   

Antigen receptor  MHC II : TCR - antigen binding  MHC II : CD4 molecule - the co-receptor Costimulatory pairs - second signals  CD40:CD40L (CD154)  CD28/CTLA-4:B7 (CD80, CD86) Adhesion molecules  CD58(LFA-3):CD2  CD54(ICAM-1):CD11a/CD18 (LFA-1)

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2.

The TH1/TH2 paradigm

The TH1/TH2 paradigm was first proposed by Mossman and Coffman to explain the differential effects of T cell help – i.e. T cells helping B cells and T cells helping other T cells. These cells were distinguished functionally rather than morphologically by the differences in cytokine patterns that they produced. 

Differential production of specific cytokine patterns by subpopulations of CD4+ cells



TH1 help other T cells develop immunity (mostly T cell-mediated)



TH2 help B cells (and other WBC) develop immunity against extracellular pathogens (mostly through IgE, mast cells and eosinophils

against intracellular pathogens

Dendritic Cell

Currently, it is believed that there are multiple functional subsets of Th cells. In addition to the ones mentioned above: Th17 cells, characterized by IL-17 secretion, are thought to be involved as effector cells for autoimmune disease progression, and protect surfaces (skin, gut) from extracellular bacteria. Tfh cells (follicular helper T cells) provide help to B cells in germinal areas enabling them to develop into antibody-secreting plasma cells; they function inside of follicular areas of lymph nodes. The functions of all the subsets of Th cells depend upon the specific types of cytokines that are generated, for example interferon-gamma (IFN-gamma) by Th1 cells and IL-4 by Th2 cells, and IL-17 by Th17 cells.

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Regulatory T cells (Treg) represent subpopulations of T helper cells that trigger suppressive activities following engagement of their T cell receptor with presenting antigen occupying the MHC on an antigen presenting cell. They typically secrete molecules such as TGF-β, which function to suppress other T helper cell type activity. They usually express CD4 and CD25 on their cell surface, and express the transcription factor Foxp3. T-cell Regulation: Treg receive a signal via CTLA-4 which induces their suppressive activity. Treg may also receive a signal triggering their suppressive activity following interaction with an MHC class II molecule. Treg may then suppress the activation of CD4+ T cells by secreting TGF-  (and IL-10). FIGURE 12.4. From Coico, 2009.

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3. Intracellular Events – receptor mediated transcription of cytokine genes by a sequence of molecular events

FIGURE. Intracellular events in CD4+ T-cell activation. The result of activation events is enhanced transcription and increased stabilization of IL2 mRNA. Coico, 2009.

          

MHCII/peptide binds TCR TCR activates CD3 CD3 transduces activation signal across membrane Tyrosine kinases (Fyn, Lck) activated by CD45 Fyn, Lck cluster with ITAMs and phosphorolate them ZAP-70 (another tyrosine kinase) binds to ITAMs Activated ZAP-70 binds to phospholipase c- (PLC-) PLC- splits PIP2 into DAG and IP3  DAG activates PKC >>>>> NF- (transcription factor)  IP3 increases iCa++ >>> activated calcineurin >>> NF-AT Transcription factors enter nucleus and bind to chromosomes Upregulate T cell activation genes (cytokine, cytokine receptors) Upregulate adhesion molecules on surface to promote further activation events.

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4.

Other events of T cell activation  



Decreased expression of selectins molecules to allow homing to lymph nodes Requirement for multiple signals from APC to activate cell

Function of costimulatory pairs – promote the T cell activation process  CD40: CD154(CD40L)  CD80/CD86 (B7.1,B7.2):CD28  CD80 binding upregulates TH1  CD86 binding upregulates TH2  CD28 binding upregulates IL-2 production  Lack of CD28 binding induces tolerance  CD80/86:CTLA-4  downregulates IL-2 production  negative activation signal – tolerance  induces memory cell formation

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B. Role of Cytotoxic Cell-Mediated Immunity in Host Defense Host defenses against extracellular infectious agents (e.g., bacteria, protozoa, worms, fungi) typically utilize (1) Antibody, (2) Complement, and/or (3) activated Phagocytes. However, these mechanisms are not adequate for defense against intracellular infectious agents (an infectious agent that invades a host cell). Therefore a different defense system is required. The mechanisms used are those referred to as cytotoxic cell mediated immunity. Induction of helper function for cytotoxic cell mediated immunity. In many cases, first CTL encounter with antigen must have help from Helper T cells. The helper cells must recognize antigen presented by MHC Class II molecules on an APC (antigen presenting cell) (dendritic cell or macrophage). The activated Th1 cell secretes IL-2 and IFN-, which activates CTLs. Activation of Th1 cells also triggers the activation of NK cells and macrophages which then target specific cells.

Generation of CD8+ T cells effector cells and target cell killing. (A) dendritic cells activate CD8+ T cells directly. (B) One pathway for CD4+ T cells to activate CD8+ T cells. (C) Target cell killing by a CD8+ effector T cell. Coico and Sunshine, 2009. Fig 10.10.

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Effector cells in Cytotoxic Cell Mediated Immunity. Both innate and adaptive cells play a role in cytotoxic cell mediated immunity. The major cell players and their properties are listed and summarized in the table below. 

CTLs – Antigen specific and MHC Class I restricted. i. CTLs express CD8. ii. CTLs kill their targets by using Perforin, Granzymes, Cytokines, Fas and Fas ligand.



NK cells - nonspecific (they do not use a T cell receptor). i. Morphologically large granular lymphocytes (LGLs); ii. Non-T and non-B lymphocytes lacking surface CD3, CD4, CD8 and CD19. They do not express immunoglobulins or TCRs. iii. NK cells express CD16 and CD56. iv. NK cells kill by releasing perforin, granzymes and cytokines (IFN- and TNF).



Lymphokine activated killer cells (LAK cells) are i. Morphologically LGLs. ii. Non-T non-B lymphocytes. iii. Reaction –nonspecific.



NK-ADCC i. antibody-dependent cellular cytoxicity (ADCC). ii. Have Fc receptors (CD16) that recognize Fc portion of IgG.

Table 1: Effector Cells in Cytotoxic Cell Mediated Immunity Effector Cell CD markers Effector MHC Molecules recognition CTL TCR,CD3,CD8,CD2 Perforin, required cytokines (TNFClass I β, IFN-) NK cell CD16,CD56, CD2 Perforin, no cytokines (TNFβ, IFN-) NK cell CD16,CD56, CD2 Perforin, no ADCC cytokines (TNFβ, IFN-) LAK cell CD16,CD56, CD2 Perforin, no cytokines (TNFβ, IFN-) Macrophage CD14 TNF-α, no enzymes, NO, O radicals

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Antigen recognition specific TCR nonspecific specific IgG nonspecific nonspecific

Cytotoxic cells (CTLs) directly kill tumor cells and host cells infected with intracellular pathogens. These cells 1) usually express CD8, and, 2) destroy infected cells in an antigen-specific manner that is dependent upon the expression of MHC class I molecules on antigen presenting cells.

1.    2.  

3.  

General Considerations Adaptive host defense against intracellular pathogens CD8+ CTL is MHC I restricted Is affected by TH1 cells which are also antigen-specific but MHC II restricted Development of CTL TCR interacts with MHC I – antigen complex o In association with CD8 o Also involves costimulatory molecules IL-2R upregulated o IL-2 from Th cells cause clonal proliferation o IFN causes activation of CTL Killing of Target cells by CTL IFNupregulates perforin formation o Perforins form transmembrane channels that kill target o Similar to complement-mediated lysis IFNupregulates granzyme formation o Serine proteases 136

o Pass to target through the perforin-induced channels o Activate target cell apoptosis    

Fas/FasL (CD95/95L) interaction o FasL expression on T cell upregulated in activated CTL o Initiates apoptosis in target through formation of capsases CTL releases “doomed” target to kill more target cells if available As response is regulated, CTLs themselves undergo apoptosis Remnant is antigen-specific memory CTL

Table 2: Cytotoxic Products of Activated CTLs Cytotoxic Product Perforins

TNF- Fas ligand Nucleases Serine proteases

Effect on Target cell - Polymerize in the membrane of the target cell to form poly-perforin channels that allow cytosol to leak out and toxic molecules to enter the cell. - Degrades proteins in cell membrane - Initiates apoptosis - Degrades DNA and RNA in the cell - Degrade proteins in the cell membrane

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C. Recognition of “Different” Antigens by T cell receptors 1. Presentation of Lipids and Glycolipids Natural Killer T Cells: Natural killer T cells (NKT) are a heterogeneous group of T cells that share properties of both T cells and natural killer (NK) cells. These cells recognize an antigen-presenting molecule (CD1d) that binds self- and foreign lipids and glycolipids. They constitute only 0.2% of all peripheral blood T cells. The term “NK T cells” was first used in mice to define a subset of T cells that expressed the natural killer (NK) cell-associated marker NK1.1 (CD161). It is now generally accepted that the term “NKT cells” refers to CD1d-restricted T cells co-expressing a heavily biased, semiinvariant T cell receptor (TCR) and NK cell markers. Natural killer T (NKT) cells should not be confused with natural killer (NK) cells. Upon activation, NK T cells are able to produce large quantities of interferon-gamma, IL4, and granulocyte-macrophage colony-stimulating factor, as well as multiple other cytokines and chemokines (such as IL-2 and TNF-alpha). NKT cells seem to be essential for several aspects of immunity because their dysfunction or deficiency has been shown to lead to the development of autoimmune diseases (such as diabetes or atherosclerosis) and cancers. NKT cells have recently been implicated in the disease progression of human asthma. The clinical potential of NKT cells lies in the rapid release of cytokines (such as IL-2, IFN-, TNF- α, and IL-4) that promote or suppress different immune responses.   

 

CD1- antigen presenting molecules present lipid and glycolipids derived from microbial antigens to T cells. These molecules are non-MHC restricted and nonpolymorphic. They are distinct from MHC class I and II. Similar structure to MHC class I, having three extracellular domains and expressed in association with 2 microglobulin on APC. Binds hydrophobic region of lipid with polar bound by TCR. Binds to a variety of T cells including NK1.1 (CD4+) cells. (NKT cells). o Induces NK1.1 to secrete large amounts of IL-4 o May be important in generating TH2 activities

 Overall, the CD1 molecules bind antigen in a deep, narrow hydrophobic pocket, with ligands interacting via hydrophobic interactions rather than hydrogen bonding.The role of CD1 in pathogenesis has not yet been fully determined. 138

2. Superantigens  Activate T cells expressing a specific Vsegment as part of TCR  Presented by Class II molecules on MHC but not in peptide groove  Several organisms have components that function as superantigens o Staphylococcus o Rabies virus  Activate large numbers of T cells (possible mechanism for toxic shock syndrome) SUPERANTIGENS

•Superantigens bind directly to T-cell receptors and MHC, without processing. •Usually involves direct interaction to V region of TCR.

V VD

V VD

J

J

J

J

C

C

C

C

3. Mitogens  Polyclonal activators of T cells by activating widespread mitosis  Derived from plant lectins  Phytohemagglutinin (PHA)  concanavalin A (conA)  pokeweed mitogen (PWM)  Other mitogens  Endotoxin (lipopolysaccharide) – mouse B cells, monocytes/macrophages  AntiCD3 – polyclonal T cell activator

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human

VI.

B CELL ACTIVATION AND FUNCTION

B Lymphocytes: The genesis of µ and delta chainpositive, mature B cells from pre-B cells is antigenindependent. B cell development is characterized by recombinations of immunoglobulin H and L chain genes and expression of specific surface monomeric IgM molecules. At this stage of development, B cells are highly susceptible to the induction of tolerance. Cells bearing only monomeric IgM are referred to as immature. These cells may undergo deletion (death by apoptosis), anergy (long term inactivation, or receptor editing (reactivation via V-D-J gene recombination). Once these cells acquire IgD molecules on their surface, they become mature B cells that are able to differentiate after exposure to antigen into antibody-producing plasma cells. Mature B cells can have 1-1.5 x 105 receptors for antigen embedded within their plasma membrane. The activation of B cells into antibody producing/secreting cells (plasma cells) is antigen-dependent. Once specific antigen binds to surface Ig molecule, the B cells differentiate into plasma cells that produce and secrete antibodies of the same antigenbinding specificity. If B cells also interact with T helper cells, they proliferate and switch the isotype (class) of immunoglobulin that is produced, while retaining the same antigen-binding specificity. This occurs as a result of recombination of the same Ig VDJ genes (the variable region of the Ig) with a different constant (C) region gene such as IgG. T helper 2 cells are thought to be required for switching from IgM to IgG, IgA, or IgE isotypes. The generation of memory B cells is associated with class switching; this process occurs in the spleen or lymph node. In addition to antibody formation, B cells also process and present protein antigens. After the antigen is internalized it is digested into fragments, some of which are complexed with MHC class II molecules and then presented on the cell surface to CD4+ T cells. B cells secrete antibody upon antigenic stimulation, a multi-step process involving interactions with T cells. B cells express many surface molecules which assist in the process of antibody production through delivery of various activation signals. Some of these costimulatory molecules are depicted in the figure below. Fc receptors are important in "feedback" mechanisms to deliver negative signals to the cell.

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Surface Markers of human and murine peripheral B cells. Remember that B cells carry the HLA-D (and I-A/I-E), class II restricted major histocompatability marker, as well as have specific receptors for complement receptors. Coico and Sunshine, 2009. Figure 7.7.

A. T cell - B cell cooperation 



T dependent antigens  Require CD4+ help for B cells to make antibody  Must be to same antigen but different epitopes (linked recognition)  B cell epitope - hapten  T cell epitope - carrier T – B interactions  For primary response, requires APC (dendritic cell the best)  For secondary response, no APC necessary  Requires cytokines for B cell growth (IL-4), proliferation (IL-6)  Isotype switch from IgM to o IgG (IFN o IgA (IL-5) o IgE (IL-4,13)  External Ag on B cell bound by surface IgM  Internalized, processed and presented via MCH II to TCR in association with CD4 molecule  Costimulation (CD40:CD154; CD28:CD80/86)  Adhesion (CD58:CD2; ICAM-1:LFA-1; CD72:CD5)  Result is cytokine production by T cell that binds via receptor to B cell

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Coico and Sunshine. 2009. Figure 10.9. B.

T independent Responses    

Do not need T cell help to make antibody Antigen is typically polymerized molecules (such as polysaccharides) Only generate IgM responses Do not generate memory

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C.     

B Cell Activation Pathways Surface IgM is crosslinked CD19,21,81 are coreceptors for BCR Tyrosine kinases activated (Lyn, Fyn, Blk, Lck) Phosphorylates the ITAMs of Ig/Ig molecules associated with surface Ig Syk then activated   

Activated Syk activates PLC-which splits PIP2 into DAG and IP3  DAG>>PKC>>>multiple kinases  IP3 >>calcineurin Both pathways activate transcription factors – NF-, NF-AT Result in nucleus is upregulation of cytokine receptor and Ig genes

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SUMMARY 

The major properties of the acquired immune response are specificity, memory, adaptiveness, and discrimination between self and non-self.



Lymphoid cells in these categories include T and B lymphocytes. T and B cells produce and express specific receptors for antigens. Receptor specificity is related to gene rearrangement of variable region components during development, according to essential features for clonal selection.



Cytokines are small molecular weight glycopeptides with a variety of cellular origins and functions, both effector and regulatory.



Helper T cells (TH) provide assistance to B cells to make antibody and other T cells to become cytotoxic by production of specific cytokines, expression of co stimulatory and adhesion molecules molecular mechanisms involving transcription factors



Cytotoxic T lymphocytes (CTL) are for host defense against intracellular pathogens and induce death of the target cells by various mechanisms



T cell antigen receptors can be activated by a variety of molecules such as proteins, lipids/glycolipids, superantigens and mitogens



B cells make antibodies, the quantity and isotype of which is dependent upon whether the T cell is involved (T –dependent) or not (T-independent) and relates to both the nature of the antigen and the underlying immunological capabilities of the host

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IMMUNOLOGY ANTIGEN-ANTIBODY INTERACTIONS, IMMUNE ASSAYS, EXPERIMENTAL SYSTEMS Dr. Keri C. Smith OBJECTIVES The objective of these lectures is to learn how the exquisite specificity of antibodies can be used in the clinical laboratory for diagnostic assays that measure either antibodies or antigens and review experimental systems that will be discussed later in the course. KEYWORDS Affinity, agglutination, prozone, zeta potential, precipitation, immunoelectrophoresis, radial immunodiffusion, nephelometry, radioimmunoassay, ELISA. READING Chapter 5 of the Coico et al textbook, 2009. Case 46 in Case Studies in Immunology, 6th Ed. Web Resource: http://www.uth.tmc.edu/pathology/medic/immunology/Immuno/antibodies.html INTRODUCTION It is clear that antibodies play a major role in protection from a variety of diseases, toxins, viruses, parasites, etc. In addition, once antibodies have been made, they can be used for a variety of diagnostic assays in the laboratory to detect the presence of absence of a particular antigen or bacterium or virus in a sample. The use of antibodies specific for red blood cell antigens has made routine transfusions possible and opened up the whole area of transplantation. The reaction of antigen with its homologous antibody is a two-stage phenomenon. The initial or primary binding reaction can occur invisibly. The secondary manifestation of that interaction is dependent on several factors such as: a) Isotype of the antibody b) Valence of antigen c) Form (particulate or soluble) of the antigen The type of assay used depends vitally on these factors. For example, determination of a patient’s red blood cell type is done using intact red cells and so the assay called agglutination is used. The kinds of assays used to detect soluble antigens such as growth hormone cannot be used for red cell typing because of the particulate nature of the red cell. Review of Figure 5.1 on page 60 in the textbook will demonstrate many of the features of antigens and of antibodies and fragments of antibodies that can dictate design of specific assays. It is clear that valency of both antigen and antibody can be important. Please review this figure thoroughly.

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PRIMARY INTERACTIONS BETWEEN ANTIBODY AND ANTIGEN Antigens and antibodies interact as the result of multiple weak, non-covalent reactions. You should now review these interactions from the “Immunogens and Antigens” lecture. Due to the relative weakness of these forces, Ab-Ag reactions can be readily dissociated by: a) low or high pH b) by high salt concentrations c) by chaotropic ions. ASSOCIATION CONSTANT The strength of the primary interaction between one paratope and its epitope can be precisely measured by using the law of mass action since the reaction is noncovalent. The binding of an antigen univalent epitope such as a free hapten (H) to a paratope can be represented by the equation: Ab + H  AbH The association constant is then defined by the expression: K= [AbH]/[Ab][H] The K value represents the intrinsic association constant or the Affinity for monoclonal antibodies and will represent an average association constant for polyclonal antibodies AFFINITY AND AVIDITY Definition: The intrinsic association constant, the reaction between a single paratope and its epitope, is termed the affinity. Affinity measurements cannot account for the overall efficiency of binding because having more paratopes/molecule will enhance the overall efficiency of binding since each paratope on each molecule is identical in its affinity. Thus, antigen multivalency enhances antibody-binding efficiency. This enhancement due to antigen multivalency is called avidity. Affinity and avidity are illustrated in the following figure:

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SECONDARY INTERACTIONS BETWEEN ANTIBODY AND ANTIGEN. AGGLUTINATION REACTIONS Definition: The term agglutination infers aggregation of insoluble particles. Aggregation of red blood cells or bacterial cells is routinely used for estimation of the concentration of antibodies in a serum taken from a patient or experimental animal. Definition: The term titer is used to describe the highest dilution of that serum that will agglutinate a standard amount of the cells (i.e. 50 ul of a 1% suspension).

Clinical Wisdom A set of sera can be compared for their titers, but the titer determined by another laboratory or another technician on the same set of sera could vary significantly from those found earlier. This happens routinely in blood banking, but the results reported by the same technician within a given laboratory over time are usually internally consistent. It is imperative to understand that titers of sera or of monoclonal antibodies are not quantitative measurements. PROZONE-Agglutination reactions can sometimes exhibit the phenomenon of prozone. This occurs because very high concentrations of antibodies can totally saturate 147

all epitopes on each cell added so that no cross linking occurs. As the concentration of antibodies is lowered by dilution in succeeding tubes, the numbers of cellular epitopes and antibodies then reach a ratio where effective agglutination occurs. ZETA POTENTIAL-An electrical potential between two like charged particles prevents them from physically associating. The short distance between Fab arms of IgG molecules may not overcome this repulsion, but the larger IgM molecule might be sufficiently large to overcome zeta potential. The high sialic acid density on the surface of red cells is difficult to overcome and the size, coupled with the multivalency, of IgM makes it more efficient as an agglutinator of red cells. COOMBS’ TEST-The Coombs’ test can overcome zeta potential by using a second layer of antibodies to bridge cells. If the red cell is coated with IgG antibodies, an antiglobulin antiserum can be added (Definition: a serum containing antibodies specific for the Fc region of IgG) and it can then cross-link the IgG antibodies previously bound to the cell thereby agglutinating the red cells. This assay is described in Figure 5.2 in the textbook. Direct Coombs’ Test In this assay, patient blood that is suspected of having antibodies already bound to the red cell (i.e. blood from a baby at risk for Erythroblastosis fetalis) is mixed with the antiglobulin serum and positive agglutination is diagnostic for the presence of anti-Rh antibodies bound to the red cells.

Indirect Coombs’ Test This is to detect the presence in serum of a non-agglutinating antibody. For example, serum from a pregnant patient suspected of having circulating IgG antiRh antibodies is mixed with Rh+ red cells, then the antiglobulin is added. Positive agglutination is then diagnostic for the presence of anti-Rh in patient serum, indicating that the fetus is at risk for erythroblastosis fetalis.

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Clinical Vignette Review Case 46—Hemolytic Disease of the Newborn. Indirect Coomb’s titers were used as a principal diagnostic tool in this case.

PASSIVE AGGLUTINATION-Passive agglutination is a way to use the extraordinary sensitivity of agglutination assays to detect antibodies specific for soluble antigens such as thyroglobulin to help diagnose Hashimoto’s disease, for example. In this assay, purified soluble thyroglobulin is attached to something particulate such as micro-latex beads or red cells. Then sera containing suspected antibodies specific for thyroglobulin can be titered in a standard agglutination format. If red blood cells are used as the particle, the assay is usually called passive hemagglutination to acknowledge the red cell as the carrier of the antigen. PRECIPITATION REACTIONS Precipitation Reaction in Solution (Fluid Phase Reactions) Antigen-Antibody reactions that result in the formation of visible precipitation of the reactants are classed as secondary manifestations of Ag-Ab reactions. This reaction provided the first quantitative assay (NOT Qualitative, as written on page 63 of your

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textbook) for antibody, but is rarely used today. However, understanding the Ab-Ag interactions that lead to this reaction is important, as the immune complexes formed are also found in vivo. In this reaction, various amounts of soluble antigen are added to a fixed amount of serum containing antibody. As illustrated in the figure below, when small amounts of Ag are added, Ab-Ag complexes are formed with excess Ab, and each molecule of Ag is bound by Ab and cross-linked to other Ab molecules. When enough Ag is added, ALL of the antigen and antibody complex and fall out as precipitate (the zone of equivalence). When an excess of Ag is added only small Ag-Ab complexes form (no crosslinking) and the precipitate is reduced. This reaction is affected by the number of binding stes that each Ab has for antigen, and the maximum number of Abs that can be bound by an antigen or particle at one time. This is defined as the valence of the antigen or antibody (see figure below) and valence of Ab and Ag has to be > 2 or precipitation will not occur. It is important to note that the valence of an Ag is almost always less than the number of epitopes on an Ag, since steric considerations limit the number of distinct antibody molecules that can bind to a single antigen at any one time.

PRECIPITATION REACTIONS IN GELS Often, precipitation reactions are used for analysis in situations where quantitation is important but in other situations only a qualitative answer is necessary. These latter reactions can be done in a gel matrix that slows down the rate of diffusion of reactants and holds the precipitate in the gel web so that it is effectively immobilized for visualization either directly or with the aid of various staining methods. Several qualitative and quantitative methods are in wide use in medicine today for analysis of numerous hormones, enzymes, toxins, and for analysis of the products of the immune system itself. All methods described below will be designated as qualitative or quantitative analysis methods. OUCHTERLONY DOUBLE DIFFUSION ASSAY The Ouchterlony Assay was developed by Orjan Ouchterlony in the 1950’s and is still in widespread use. It has two important features. a) it is inexpensive to use. 150

b) it can be used to compare the relatedness of two antigens (Antigenically, are they totally different, are they the same, or only similar?). The assay is called a Double Diffusion assay because both the antigen and antibodies are diffusing. It is a qualitative assay. Format: Reagents are put in wells made in a thin layer of agar or agarose made up in physiological buffer. The molecules in each well then diffuse slowly into the agar in a radial fashion (diffusion in a circular fashion with an ever-increasing radius). Thus, antigen and antibody slowly diffuse toward one another. A positive result will be that a thin opaque precipitate line or band will form in the agar at right angles to a line connecting the centers of the two wells and it will usually be symmetrical, extending the same distance either side of the line connecting the well centers. The presence of a line is a qualitative assay for the presence of either antibody in the antiserum (using a standard antigen solution) or for the presence of antigen (using a standard antiserum). See Figure 5.5 in the Textbook. The most widespread use of the Ouchterlony technique is for comparison of antigens. It has also been used in forensic medicine and in a variety of diagnostic assays. Study note: The three patterns of reactions (identity, non-identity, and partial identity) described in Fig. 5.5 on pg. 64 are important to understand RADIAL IMMUNODIFFUSION Radial immunodiffusion is a type of agar gel precipitation technique that is quantitative. Once again, it relies on having a standardized antiserum or standard antigen on hand in order to analyze the unknown sample. Technique: The technique of doing radial immunodiffusion and some typical results are described in Fig. 5.6, pg. 65 in the textbook. IMMUNOELECTROPHORESIS Immunoelectrophoresis is a variation of the Ouchterlony double diffusion in gel technique. It is designed to analyze complex protein mixtures containing many different antigens. The method and typical results are shown in Fig. 5.7, pg. 65 in the textbook. Clinical Relevance: The medical diagnostic use of immunoelectrophoresis is for diagnosis of conditions where certain proteins are suspected of being absent (e.g. hypogammaglobulinemia) or of being overproduced (e.g. Multiple Myeloma). It is usually used as a first screening test, followed by quantitative tests.

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Immunoelectrophoresis is a qualitative assay. It is also used in medical research for following the different steps of a purification protocol to show the disappearance of unwanted proteins when purification of one component from a mixture is desired. NEPHELOMETRY Nephelometry is a widely used methodology for accurately measuring quantities of the Ig classes in serum. Obviously, dramatic increases or decreases in quantities of these could contribute to diagnosis of numerous diseases. In this assay, proteins in the sample react with specific antibody (ie an anti-IgE antibody). The mixture is placed in a tube and inserted into the Nephelometer. When light passes through the suspension that contains aggregated particles, a portion of the light is scattered. The scattered light is measured and compared with stored standards. Thus, this is a quantitative method using liquid-phase precipitation principles. It can be applied to measuring any soluble substance provided specific antisera are available. WESTERN BLOTS Also called immunoblotting-a mixture of antigens is usually separated by electrophoresis on a gel, transferred onto a medium such as nitrocellulose that binds proteins tightly and then antibodies that have an enzyme covalently attached are poured on the nitrocellulose. Substrate for the enzyme is added, turns colors when enzyme is present, and the colored line shows that the antigen was present. See textbook, Fig. 5.8. Clinical Correlation—HIV infections are frequently diagnosed by doing Western Blots of patient’s serum for content of antibodies specific for various HIV antigens. See Figure 5.8 in the Coico book. See Case 10—Acquired Immune Deficiency Syndrome (AIDS).

IMMUNOASSAYS DIRECT BINDING IMMUNOASSAYS The principle of radioimmunoassays is diagrammed in Figures 5.9 & 5.10 in the textbook. In Fig. 5.9 radioactive antigen is reacted with a limited amount of antibody. In the second step, Figure 5.10, unlabelled test antigen is mixed with the labeled antigen prior to addition of the antibody. The amount of labeled antigen bound to antibody is then reduced by a factor related to the ratio of labeled to unlabeled antigen in the mixture. The unlabeled antigen effectively competes for available antibody since it is identical immunologically, but unlabeled. A standard curve of inhibition can be generated using precisely known amounts of unlabeled antigen and then test samples containing unknown concentrations of antigen can be analyzed and simply read off the standard curve to find the concentration of antigen in the unknown.

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SOLID-PHASE IMMUNOASSAYS There are a group of assays in which the antigen or the antibody is coated on the surface of a plastic microplate and sensitive indicators such as radioactivity or enzymatic action are used to detect the presence of Ag or of Ab. There are 5 of these assays classified in two groups according to the types of antigens being analyzed: soluble or cellular. A. SOLUBLE ANTIGENS RADIOIMMUNOASSAY--There are many different formats for doing radioimmunoassay (RIA). Only one will be described here, that is to detect antigen. In this format the following steps are done: a) free antigen is first coated onto the surface of plastic plates and the excess is removed by rinsing out the wells with buffer solutions. b) the remaining plastic surface is then blocked by adding an irrelevant protein solution and washing c) antiserum is added to the plate, incubated and then washed out. This leaves the plate with Ab bound to the Ag that is, in turn, bound (noncovalently) to the plastic. d) a radioactive indicator is added which recognizes the Ab but not the Ag. (There is a protein called Protein A derived from Staphylococcus aureus that reacts specifically with the Fc region of most vertebrate IgG molecules. The Protein A can be radiolabeled. The overall procedure is shown in the following diagram: Clinical Relevance: Current Laboratory RIA Assays The specific assays are to quantitate the amounts of specified antigens in body fluids such as blood or urine. Assays are available for measuring Renin, Gastrin, Parathyroid Hormone, Growth Hormone, Urine Microalbumin, Vitamin B12 and Folate. MemorialHermann Hospital currently is phasing out the radioimmunoassay laboratory.

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ENZYME LINKED IMMUNOADSORBENT ASSAY (ELISA) The ELISA assay is quite similar to the RIA except that the indicator reagent used in ELISA is not radioactive. Instead, the indicator (Protein A) is coupled to an enzyme molecule that converts added substrates to a colored product that can be detected spectrophotometrically due to the color change. The assay is done as diagrammed below:

Clinical Relevance: There are a large number of commercial ELISA kits available for diagnostic purposes. Currently the Memorial-Hermann Hospital Laboratories offer ELISA assays for Hepatitis antigen, HIV and HTLV antigens. Specific assays are also available for detection of antibodies in patient’s serum for Hepatitis A virus, Hepatitis B surface antigen, Hepatitis B core antigen, Hepatitis C virus, cardiolipin, H. pylori, and for HIV types 1 and 2. Another ELISA assay is available for detection of antibodies to Human T-lymphotropic virus type I.

Clinical Vignette This is the case of a woman who contracted the AIDS virus from a blood transfusion and transmitted it to her fetus later. Antibodies specific for the gp120 HIV antigen were measured in the infant using an ELISA. The mother and father were also tested and were found to have anti-gp120 antibodies by ELISA.

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ELISPOT assays Variation of the ELISA method. Incubate with cells instead of soluble antibody. The # of spots after addition of detection antibody and precipitable substrate = the number of cells secreting a specific antibody, thus can be used to determine the frequency of antigen specific B cells. Also used for T cell assays (e.g. the number of T cells producing a cytokine, as illustrated below). Used in biomedical research.

B. CELLULAR ANTIGENS IMMUNOFLUORESCENCE It is sometimes of diagnostic value to determine if a particular antigen is found on or in the cells of a particular tissue. In this case, assays are needed that can be performed directly on biopsies of tissue and seen using a microscope. The method originally developed by Albert Coons and his colleagues at Harvard involves covalent attachment of fluorescent organic compounds to specific antibodies that then can be used to detect the antigen in question. The fluorescent compounds excite at different wavelengths. This is a highly sensitive and specific assay, and cells individual cells can be stained with up to 12 different compounds.

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1. Direct Immunofluorescence-The antibody specific for the antigen in question is directly labeled with the fluorophor and used to identify the antigen. 2. Indirect Immunofluorescence-This is similar to the Coombs’ reaction discussed earlier (review that if necessary). It is a two step method in which the unlabeled antibody specific for the antigen in question is reacted first with the tissue and the excess antibody is washed away. Then the slide is flooded with a fluorescent anti-Ig (preferably Fc specific). This method has the advantage that it is significantly more sensitive than the Direct method. Clinical Relevance: Immunofluorescence, using the indirect format, is used in clinical laboratories for screening patient’s sera for anti-DNA antibodies in suspected cases of systemic lupus erythematosus. IMMUNOHISTOCHEMISTRY is a similar technique. Instead of fluorescent labels, the detection antibodies are labeled with enzymes such as horseradish peroxidase or alkaline phosphatase (these are also used in ELISA). Addition of substrate then colors the membranes of the cells expressing the antigen of interest. FLUORESCENCE ACTIVATED CELL SORTING (FACS) ANALYSIS FACS analysis is used to identify, and sometimes purify, one cell subset from a mixture of cells. The technique and a diagram of the instrument are on page 69, Fig. 5.12. This is an extremely effective tool to identify and/or isolate specific cell subsets. The organic fluorescent compounds attached to the detection antibodies are excited by different fluorescent wavelengths, and all emit at different wavelengths as well, allowing for specific detection of the markers. Current instrumentation can detect up to 15different antigens on one cell (though most investigators use 4 colors at most). Sorting of cells can also be accomplished using antibodies coupled to magnetic beads (magnetic activated cell sorting, or MACS). The cells are then placed over a magnetized column, and any cells with labeled antibody bound to them can be isolated from the unbound population.

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Clinical Relevance: FACS can measure CD4+ cell numbers in AIDS patients to follow disease progression. FACS was used in Case 5—MHC Class I Deficiency to measure peripheral blood lymphocytes

LYMPHOCYTE FUNCTION ASSAYS Lymphocyte function can be compromised in certain diseases or can occur as a result of a genetic abnormality. A diagnosis can be confirmed in many cases if it is known whether or not the B or T cells are normal, if the existing B cells can make antibodies, or if the T cells can produce the correct cytokines. Mitogen Activation—Lipopolysaccharides can cause polyclonal stimulation of B cells in vitro. This activation is accurately measured by incorporation of radioactive nucleosides. Several lectins, including concanavalin A and phytohemagglutinin are effective T cell mitogens. Pokeweed mitogen stimulates polyclonal activation of both B and T cells. Numerous assays can measure antibody production by stimulated B cells (ie ELISA). Cytotoxicity assays measure the ability of cytotoxic T cells or NK cells to kill radioactive target cells that express a specific antigen for which the cytotoxic T cells may be sensitive. MONOCLONAL ANTIBODIES AND T CELL HYBRIDOMAS Due to cross reactivity of antibodies and the need for more controllable assays it is sometimes of great advantage to have a homogeneous antibody preparation that is specific for only a single epitope and with high affinity. Since polyclonal antibody mixtures consist of a multitude of antibodies specific for different epitopes on even simple antigens like tetanus toxoid, and the fact that there are an array of different subpopulations of antibodies with different affinities even in the subset specific for a single epitope, significant cross reactions can occur when using polyclonal antibodies for analytical assays. This can lead to misinterpretation of results occasionally. Kohler and Milstein developed a method for making murine antibodies that are monoclonal, that is, all antibodies are derived from a single precursor plasma cell so that all the antibodies in the preparation are identical and derived from the same original clone. The method is outlined in Figure 5.13 in the textbook as well as in the following:

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The specific details of the hybridoma technology are covered in the textbook. (See Figure 5.13, Coico and Sunshine, 2009). T CELL HYBRIDOMAS The general method is also used for making T cell hybridomas. Clinical Relevance: T cell hybridomas are valuable for the large-scale production of several T cell-derived lymphokines that are used as antigen in diagnostic kits and are also used therapeutically. GENETICALLY ENGINEERED ANTIBODIES Attempts to develop human hybridoma technology have not been very successful. To adapt the murine system for making human antibodies, recombinant DNA methodologies have been developed to “humanize” murine antibodies. The methods usually use murine V-region sequences coupled to human C-region sequences. There are many variations on this theme and the methodologies are also applicable to engineering receptors (for cytokines, etc.) into cell lines in which they are not normally expressed. MICROARRAYS TO ASSESS GENE EXPRESSION Levels of expression of thousands of genes can be measured simultaneously using a technology called gene chips or microarrays. Briefly, thousands of short cDNA representing genes from all parts of the genome are attached to a slide. Samples of mRNA from cells in culture are used and reverse transcribed into cDNA and by labeling 158

this cDNA from different sources (ie normal cells and tumor cells) with different fluorochromes, the differential expression of distinct sets of genes can be measured. By scanning with a laser, different spots can have different colors depending on the success of binding by the two different cDNA’s. This methodology has great potential in fields such as clinical diagnosis of lymphoid tumors. EXPERIMENTAL ANIMAL MODELS Note: These will be covered very briefly in the lecture but students should review these models for general knowledge as they will be mentioned later in the course and will likely be mentioned in other courses. Numerous strains of mice exist that mimic specific human diseases. These became available due to the development of inbred strains of mice that freely accept cell and tissue grafts from other members of the strain. Since the Major Histocompatibility Complex antigens are identical in all members, grafts are accepted. One type of graft that is useful is to do Adoptive Transfer of lymphoid cells from an antigen primed mouse to one that was not immunized to study the function of various cell subsets. This is a type of Passive Immunization. The SCID mouse (Severe Combined Immunodeficiency Disease) mouse has no T or B cells. It is useful for studying human hematopoietic stem cells since they do not reject the foreign human cells. Thymectomized and congenically athymic mice (Nude mice) are useful for studying T cell function and T cell subsets. Neonatal thymectomy coupled with irradiation (to destroy B cells and lymphoid precursors) coupled with reconstitution with bone marrow (to repopulate with B cells) is widely used for T cell study. Nude mice are a mutant strain that fail to develop a thymus. They can be reconstituted using syngeneic adult thymic epithelial tissue. TRANSGENIC MICE Transgenic mice are made by injecting a cloned gene into fertilized mouse egs. The eggs are injected into pseudopregnant mice. If the transgene is constructed with a specific promoter region, it is possible to control the gene’s expression in certain tissues. Care must be used in interpreting results with transgenic mice since the transgenes frequently are grossly overexpressed. KNOCKOUT MICE Replacing a given gene with one that has been mutated or has been changed so it is not expressed in the adult creates Knockout mice. These are widely used to study the function of specific cytokines and MHC molecules.

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SUMMARY 1. In addition to functioning in vivo, antibodies are used in numerous diagnostic formats in the clinical laboratory. 2. The primary binding reaction of antibody with antigen follows the rules of the Law of Mass Action and an Association Constant (antibody affinity) can be accurately measured while functional avidity is defined as the affinity enhancement due to multivalency. 3. Secondary Ag-Ab reactions include the agglutination assay used in blood banking. A prozone in agglutination assays is due to a huge excess of antibody molecules. Zeta potential is an electrical repulsion of like-charged particles. Coombs tests utilize anti-Ig reagents. 4. Precipitation reactions between antibodies and soluble antigens occur regularly in vivo. The degree of precipitation depends on valency of antigen, ratio of antibody to antigen and the classes/subclasses of antibodies that predominate. Usually IgG is the only effective antibody class mediating precipitation. 5. Several precipitation reactions in gel media are widely used for different purposes. The Ouchterlony double diffusion assay is a qualitative assay for measuring antigen presence and comparing antigens. Immunoelectrophoresis is a qualitative method for measuring the numbers of components in mixtures and Radial immunodiffusion is a quantitative method. 6. Nephelometry is a widely used method for measuring Ig concentrations. 7. Radioimmunoassays and ELISA (Enzyme Linked ImmunoSorbent Assay) are the two most widely used immunoassays used in US clinical laboratories although radioimmunoassays are slowly being phased out in favor of ELISA

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STUDY QUESTIONS - ANTIGEN-ANTIBODY REACTIONS Study questions for Antigen-Antibody Interactions 1. What is the classic example where a Coomb’s type agglutination assay would be more sensitive than the direct agglutination method. 2. Describe the steps in setting up a quantitative precipitation reaction. What does the experiment tell you? 3. Make a list of all the immunoassays in this chapter and categorize them as a) Quantitative or as b) Qualitative. 4. Describe a situation where you would order an Ig class quantitation measurement done on a patient’s serum. What instrument would the lab use to do this? 5. Describe the steps in developing an enzyme linked immunosorbent assay. How would you make it quantitative? 6. Write the two equations that together define antigen-antibody Affinity.

Answers to study questions may be found at: http://www.uth.tmc.edu/pathology/medic/immunology/Immuno/antibodies.html

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IMMUNE EFFECTOR MECHANISMS I: ANTIBODY-MEDIATED REACTIONS Steven J. Norris, Ph.D. Recommended Reading: Actor, 2012, Chapters 7 and 10. Web Resource: http://www.uth.tmc.edu/pathology/medic/immunology/Immuno/geneab.html

I. INTRODUCTION The immune system cannot be understood in isolation from infectious diseases. All living organisms exist in a hostile environment and are continually used as hosts and energy sources by other organisms. Since there are many different foreign invaders which can infect humans, we must have many different ways to defend ourselves. To provide the versatility required, the two major effector arms of specific immunity: antibody (humoral) and cellular, employ an incredible variety of accessory mechanisms. These lectures will introduce you to some of these mechanisms. In defense against infections, antibody is generally operative against extracellular bacteria or bacterial products, whereas cell mediated immunity (CMI) primarily operates against intracellular viral and bacterial infections, as well as fungal infections. The killing effects of immune reactions are extremely efficient and, when specifically directed to a given infection, are able to eliminate large number of organisms in a short period of time. The immune response is a double-edged sword. In most cases, the immune system is protective, providing life-saving defenses against infectious diseases and tumors. However, it can also be destructive, causing immunopathology, defined as tissue damage resulting from the immune response. These destructive responses result in some of the adverse effects of infections, in allergies or hypersensitivity reactions (antibody- or T cell-mediated reactions to environmental or administered antigens), and in distinct autoimmune disorders (antibody- or T-cell mediated reactions to self-antigens). The seven immune mechanisms listed below are active in both immunoprotective and immunopathologic reactions. II. SEVEN IMMUNE MECHANISMS Until the 1960s, immune reactions where not classified according to mechanism, but were presented as a bewildering list of lesions with peculiar names. The first working classification of Type I to Type IV immune mechanisms as introduced be Gell and Coombs was a major advance in understanding immunopathologic reactions; seven mechanisms are presented in this handout. The terms Type I - Type IV reactions, although out of date, are still used in some textbooks. TABLE 1: Classification of Immune Mechanisms Handout

Gell and Coombs (1963)

General Properties

Antibody-Mediated Inactivation or Activation Cytotoxic or Cytolytic Immune Complex Atopic or Anaphylactic

-Type II Type III Type I

Toxin, virus inactivation Opsonization, ADCC, C’-mediated lysis Ag-Ab complex formation in tissue IgE mediated allergic reactions

Cell-Mediated T-cell Cytotoxic (TCTL) Delayed Hypersensitivity (TDTH)

-Type IV

Lysis of virus-infected cells; contact hypersensitivity CD4+ T cell-mediated activation of macrophages

Either Granulomatous Reactions

--

Chronic reaction to poorly degradable antigens Page 162

These immune mechanisms are similar in many ways to antibody- or cell-mediated reactions observed in vitro. Primary reactions consist of the formation of Ag-Ab complexes or Ag-TCR reactions, secondary reactions the effects of this interaction that can be demonstrated in vitro, and tertiary reactions the corresponding in vivo manifestations (see figure).

Factors affecting the induction of different forms of immunity •

Type of infectious agent or antigen.

•

Route of infection/exposure.

•

Activation of Th1 vs. Th2 cells.

•

Location/cell type involved in antigen presentation.

•

Cytokines expressed by antigen presenting cells and T cells.

•

Genetic factors.

•

Non-genetic factors. (e.g. age and nutritional status)

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ANTIBODY-MEDIATED IMMUNE MECHANISMS 1. INACTIVATION (NEUTRALIZATION) REACTIONS A. Definition - binding of antibody to an epitope (toxin, virus, cell receptor, etc.) resulting in inactivation (loss of function), neutralization (loss of infectivity), or abnormal activation. B. Mechanisms 1. Binding of antibodies to a protein can stearically inhibit its binding to substrate, or alter its conformation, resulting in loss of activity. 2. Antibody binding to viral receptor proteins can interfere with binding to cells, alter viral structure, or mediate Ab- or C’-mediated opsonization and clearance 3. In some cases, antibodies against hormone or neurotransmitter receptors can either block or activate the receptor. C. Medical Aspects - examples 1. Protective a. Immunization of individuals with diphtheria toxoid or tetanus toxoid results in expression of antibodies. These preformed antibodies do not prevent colonization by C. diphtheriae or C. tetani, but bind to the toxins and prevent them from interacting with the corresponding host cell receptors, thus preventing disease. b. Infection or immunization with viruses (including polio, influenza, measles, mumps or rubella) results in expression of antibodies that bind to viral receptors and prevent infection upon subsequent exposures. 2. Immunopathologic a. Myasthenia gravis - autoimmune antibodies bind to acetylcholine receptors at the neuromuscular junction, causing their internalization and downregulation. The synaptic folds also become decreased or ‘simplified’, reducing interaction with the neurotransmitter and inhibiting skeletal muscle contraction. (Aristotle Onassis had this disease.) b. Graves disease - antibodies against the TSH receptor bind to thyroid cells and result in activation and abnormally high production of thyroxines. (George and Barbara Bush and dog Millie) c. Pernicious anemia - antibodies against intrinsic factor interfere with its binding of vitamin B12 in the GI tract, resulting in B12 deficiency and anemia.

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Clinical Vignette – Inactivation Reactions (Geha and Notarangelo, “Case Studies in Immunology”, 6th edition) Case 42

Myasthenia Gravis – binding of anti-AchR antibodies results in skeletal muscle weakness

2. CYTOTOXIC REACTIONS A. Definition - reaction of antibodies with cell surface antigens may result in destruction of cells by opsonization, complement activation, or AntibodyDependent Cellular Cytotoxicity (ADCC). Also called Type II hypersensitivity. B. Mechanisms 1. Complement activation may lyse bacteria directly through formation of the membrane attack complex (MAC). A single IgM molecule or 2 or more IgG molecules complexed to surface antigens are sufficient to activate the classical pathway. 2. Phagocytosis of infectious agents by macrophages or neutrophils can be enhanced through antibody binding (interaction with Fc receptors) or fixation of C3b (interaction with complement receptors). 3. ADCC results from IgG-mediated binding of null lymphocytes (and in some cases macrophages) to target cells via Fc receptors, and direct killing of the target cell through cytolytic mechanisms (see below). 4. In parasitic infections, IgE-mediated binding of eosinophils to helminths results in eosinophil degranulation and damage to the worm tegument (surface). C. Medical Aspects (Examples) 1. Protective a. Many bacteria (particularly Gram positive bacteria) are susceptible to C’mediated killing and/or opsonization. This is particularly true of pyogenic bacteria (such as Staph and Strep) that result in massive accumulations of neutrophils (see Immune Complex reactions below). b. Ab and C’-mediated MAC formation and opsonization are active against some protozoal infections, including Plasmodium and Trypanosoma. Page 165

c. ADCC may be active against virally-infected cells, tumor cells, protozoa, and helminths. 2. Immunopathologic a. Transfusion reactions - ABO mismatches result in rapid lysis of transfused cells due to anti-A or anti-B isohemagglutinins, naturally occurring IgM antibodies that bind to the transfused erythrocytes and activate complement. b. Rh reactions - birth of an Rh+ infant to a previously sensitized Rh- mother may result in binding of maternal anti-Rh antibodies to the infant’s erythrocytes, causing opsonization and phagocytosis  hemolytic disease of the newborn. c. Hemolytic anemia - autoantibodies can cause erythrocyte lysis, anemia. d. Goodpasture’s syndrome - autoantibodies to basement membrane components and complement are bound in an even, ribbon-like pattern to glomeruli and other tissues. (Contrast with lumpy-bumpy appearance of immune complex disease; see below). Clinical Vignettes – Cytotoxic Reactions (Geha and Notarangelo, “Case Studies in Immunology”, 6th ed., 2012) Case 46 Case 41

Hemolytic Disease of Newborn– maternal anti-Rh antibodies cause hemolysis in Rh+ newborn (Cynthia Waymarsh) Autoimmune Hemolytic Anemia – patient Gwendolyn Fairfax develops hemolytic autoantibodies following a mycoplasma infection

3. IMMUNE COMPLEX REACTIONS A. Definition - formation of soluble or insoluble Ag-Ab complexes that can be deposited in tissue, leading to attraction of PMNs, inflammatory changes, and tissue damage. Also called Type III hypersensitivity. B. Mechanisms 1. As increasing concentrations of antigen-specific antibodies (particularly IgM and IgG) are expressed, any remaining antigen will form Ag-Ab complexes or socalled immune complexes. 2. The size of immune complexes formed in vivo will depend on the degree of cross-linking as it relates to antigen excess, equivalence, and antibody excess, similar to quantitative precipitation and agar double diffusion assays in vitro (see figure). 3. Depending on their size, immune complexes can fix complement, resulting in binding of C3b and release of the anaphylotoxins C3a and C5a. These cause local mast cell degranulation and attraction of neutrophils, leading to inflammation. Page 166

1. Large immune complexes are typically phagocytosed and destroyed by phagocytic cells (such as resident macrophages of the reticuloendothelial system). Smaller complexes can become lodged in the walls of venules, in joints, and in glomeruli. Deposition of immune complexes causes complement activation, attraction of neutrophils, and release of lysosomal contents (“frustrated phagocytosis”), resulting in vasculitis, reactive arthritis, and glomerulonephritis. 2. The uneven distribution of immune complexes, complement components, and lysosomal contents results in the formation of lumpybumpy membrane deposits detectable by binding the anti-Ig or anti-C3 antibodies. 3. Injection of an antigen in a previously immunized individual can result in an Arthus reaction due to deposition of Ag-Ab complexes, complement activation, and resulting erythema, edema, and attraction of neutrophils. An Arthus reaction typically takes 2 to 6 hours to develop.

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C.

MEDICAL ASPECTS (EXAMPLES) 1. Protective In pyogenic infections (e.g. Staphylococcus aureus), immune complexes attract neutrophils which marginate on the endothelial cells and enter the tissue. A predominance of neutrophils constitutes an acute inflammatory response (occurs within a few days). Bacteria are killed through phagocytosis and release of lysosomal contents. Accumulation of dead bacteria, neutrophils and other cells killed by bacterial toxins or lysosomal contents, and fibrin accumulate, forming pus. This reaction may wall off the infection. 1. Immunopathologic a) Serum sickness - In the early 1900’s, serum from horses immunized with rabiesvirus or other agents was used for passive immunization. Administration of horse serum elicited antibodies against horse serum proteins in the patient, so that subsequent injections yielded immune complexes. These could cause severe muscle and joint pain and fever, as well as glomerulonephritis. Use of hyperimmune human serum antibodies for passive immunization has virtually eliminated this problem. b) Systemic lupus erythematosus (SLE) and related autoimmune diseases (e.g. Sjogren’s syndrome and scleroderma) are caused by antibodies against DNA and other normal cell components. The accumulation of immune complexes results in skin rashes, glomerulonephritis, and pericarditis. c) Rheumatic fever - infection with Streptococcus pyogenes can result in formation of antibodies cross reactive with heart antigens (cytotoxic reaction) and circulating immune complexes (immune complex disease). These cause heart and kidney damage and vasculitis in other tissues.

Clinical Vignettes – Immune Complex Reactions (Geha and Notarangelo, “Case Studies in Immunology”, 6th ed. 2012) Case 37 Case 52

Systemic Lupus Erythematosus – Nicole Chawner, age 16, butterfly rash after sun exposure. Immune complexes due to antibodies against DNA and other nuclear components cause tissue damage Drug-Induced Serum Sickness – Gregory Barnes, antibodies against penicillin cause vasculitis, hemorrhage

4. ANAPHYLACTIC OR ATOPIC REACTIONS A. Definition - IgE-mediated activation of mast cells and other cells types and its effects. Also called allergic reactions, immediate type hypersensitivity and Type I hypersensitivity. The term anaphylactic (literally “away from protection”) arose from the recognition that immunization and subsequent challenge with some antigens lead to adverse reactions rather than protective effects (prophylaxis).

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B. Mechanisms 1. Requires the production of antigen-specific IgE, also called reagin or reaginic antibody. Isotype switching to IgE during formation of memory cells requires Th2 expression of IL-4. IL-6 further enhances the production of IgE. An individual having significant levels of IgE against a certain antigen is said to be sensitized. Individuals vary greatly in levels of IgE production; those expressing high levels are called atopic patients. 2. Very little IgE is found in the circulation. Rather, most is bound to the surface of mast cells present in tissue around blood vessels, or basophils found in the circulation or tissue. IgE binds specifically to the FcR1 receptor, and can persist for weeks to months on the surface of mast cells. 3. Crosslinking of antigen-specific bound IgE by antigen causes a decrease in cyclic AMP levels and mast cell activation, resulting in rapid degranulation and de novo synthesis of arachidonic acid, which is subsequently converted to leukotrienes, prostaglandins, and thromboxanes. 4. Within seconds to minutes, the preformed contents of mast cell granules act locally to produce a typical wheal and flare reaction (in cutaneous exposures) or hayfever symptoms (in CROSS LINKING respiratory tract IgE exposures). ALLERGEN Histamine + bind MAST s to CELL tissu LEUKOTRIENES PROSTAGLANDINS DEGRANULATION e HISTAMINE RECEPTORS hista AND ALLERGIC REACTIONS INFLAMMATORY EFFECTS min H2 RECEPTORS - DILATION SMOOTH MUSCLE DILATION (INCREASED BLOOD FLOW) VASCULAR = SHOCK e NEUTROPHIL rece H1 RECEPTORS - CONSTRICTION EOSINOPHIL INFILTRATE LUNG = ASTHMA ptor ENDOTHELIAL CONTRACTION GI = DIARRHEA (INCREASED VASCULAR s H1 GU = URINATION PERMEABILITY) (ind VASCULAR ENDO = EDEMA uces smooth muscle contraction, endothelial cell separation and leakiness  vascular permeability) and H2 (mucus secretion, vasodilation). Eosinophil chemotactic factor (ECF-A) - attracts eosinophils (present in latephase or chronic anaphylactic reactions) Neutrophil-chemotactic factors (NCF) - attract neutrophils (late-phase) Heparin - anti-coagulant, not directly involved in anaphylaxis Wheal and flare - local erythema (due to vasodilation), edema (due to increased vascular permeability Hayfever - increased mucus secretion, mucosal swelling Prausnitz-Kustner reaction - passive cutaneous anaphylaxis, caused by experimental injection of IgE and antigen into skin. 5. In severe cases, systemic effects can cause shock (vascular collapse, loss of blood pressure) and/or airway obstruction (laryngeal edema, bronchoconstriction and mucus production resulting in suffocation). 6. Leukotrienes (formerly known as Slow-Reactive Substance A) cause long-term smooth muscle contraction which is not alleviated by antihistamines. Cause some Page 169

manifestations of asthma. Prostaglandins also promote bronchoconstriction, vasodilation, and chemotaxis of granulocytes. 7. Eosinophils attracted to the area also have bound IgE which can be crosslinked to cause release of granule contents: Major Basic Protein - damages parasites, may provide some protection in parasitic diseases. Also causes damage to host epithelium cells, contributes to asthma. Eosinophil Cationic Protein - also toxic to helminths, neurotoxin Platelet Activating Factor - yet another bronchoconstrictor. 8. Long-acting cells and substances contribute to late-phase reactions, including asthma. 9. Anaphylactic reactions can be reduced by a) avoidance of allergens; b) drugs such as cromolyn sodium (inhibits mast cell degranulation), corticosteroids (block arachidonic acid metabolism, inflammation); antihistamines (block binding of histamine to receptors); and epinephrine (reverses bronchoconstriction, decreases vascular permeability); c) hyposensitization - long-term injection of antigen to stimulate production of blocking IgG to reduce allergy symptoms; and d) desensitization - short-term injection of small quantities of antigen to deplete IgE, desensitize mast cells (e.g. desensitization with penicillin prior to administration of therapeutic doses).

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C. Medical Aspects (Examples) 1. Protective a) Helminth (worm) infections. IgE-mediated responses are thought to aid in the expulsion or killing of parasitic worms. In the GI tract, increased mucus secretion, intestinal mobility, and release of inflammatory products may result in dislodgement of intestinal worms such as Ascaris lumbridicoides. In addition, release of MBP and other products by eosinophils and mast cells damage schistosomes and trichinella parasites. Other parasites that also cause chronic inflammation against nematode associated antigens (Wuchereria bancrofti / Brugia malayi) may cause lymphatic obstruction and elephantiasis. 2. Immunopathologic a) Hay fever - allergic reactions to pollen and other allergens, causing increased nasal secretions, watery eyes. b) Asthma - a more severe respiratory reaction causing bronchoconstriction, increased mucus secretion. May be life-threatening. c) Cutaneous anaphylaxis - insect bites or exposure of skin to other allergens may cause a rapid anaphylactic reaction. Distinct from contact hypersensitivity (see next lecture). d) Food allergies - IgE-mediated reactions to seafood, nuts and other foods may cause severe anaphylactic reactions. e) Systemic anaphylaxis - hypersensitive individuals may develop vascular shock and respiratory failure as the result of exposure to an allergen (e.g. bee stings). Can be reversed by rapid administration of epinephrine. Clinical Vignettes – Anaphylactic Reactions (Geha and Notarangelo, “Case Studies in Immunology”, 6th Ed., 2012) Case 50 Case 49

Allergic Asthma –14 yo Frank Morgan rhinitis and persistent wheezing Acute Systemic Anaphylaxis – toddler John Mason has a near-fatal allergic reaction after repeated exposure to cookies containing peanut butter

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SUMMARY -- ANTIBODY-MEDIATED REACTIONS 1. The immune response is a double-edged sword, in that it can be both protective and destructive. The immune mechanisms involved in both protective and destructive immune reactions are the same. 2. Immune mechanisms can be subdivided into antibody-mediated and cell-mediated reactions. The antibody-mediated reactions include inactivation or activation, cytotoxic or cytolytic, immune complex, and atopic or anaphylactic reactions. The cell-mediated reactions include Tcell cytotoxicity and delayed-type hypersensitivity. Granulomatous reactions can be caused by either humoral or cellular responses, but typically result from chronic reactions to poorly degradable antigens. 3. The type of response that occurs is dependent on several factors, including the type of agent or antigen, the route of infection or antigen exposure, the relative activation of Th1 or Th2 subpopulations, the cell type involved in antigen presentation, host genetic factors (such as HLA type), and other factors such as age and nutritional status. Cytokines produced by Th1 and Th2 cells play a central role in what type of responses occur. 4. Responses to a given infectious agent or antigen are rarely, if ever, of a single type. Rather, there is a mixture of several responses, some of which may be protective and others destructive. 5. Inactivation (or neutralization) reactions are caused by direct inactivation of toxins or neutralization of viruses by the binding of antibody. Binding of antibodies to host receptors can cause abnormal blocking (as in myasthenia gravis or pernicious anemia) or activation (as in Graves disease). 6. Cytotoxic reactions result in cell damage or lysis due to antibody binding and complement activation. Cell lysis through formation of the complement membrane attack complex or opsonization by antibody or C3b derivatives are possible outcomes. Cytotoxic reactions are particularly effective against many bacterial and protozoal infections, and antibody-dependent cellular cytotoxicity can kill infected host cells or tumors. Immunopathologic effects include transfusion reactions, Rh reactions, hemolytic anemia, and Goodpasture's syndrome. 7. Immune complex reactions result from formation of antigen-antibody complexes that can lead to complement activation, attraction of PMNs, inflammatory changes and tissue damage. The size and location of the complex formation determines the pattern of disease. Although immune complex reactions can aid in the attraction of PMNs to a region of infection, we typically think of them as being destructive, as in glomerulonephritis, serum sickness, and rheumatic fever. 8. Anaphylactic or atopic reactions occur through IgE-mediated activation of mast cells and other cell types. Crosslinking of surface-bound IgE results in release of preformed granule contents (such as histamine and eosinophil and neutrophil chemotactic factors) as well as the de novo synthesis of arachidonic acid metabolites including leukotrienes and prostaglandins. Anaphylactic reactions may participate in protection against helminth infections, but are also wide-spread causes of hayfever, asthma, and other allergic reactions.

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IMMUNE EFFECTOR MECHANISMS II: CELL-MEDIATED REACTIONS Steven J. Norris, Ph.D. Recommended Reading: Actor, 2012, Chapters 7 and 10. Web Resource: http://www.uth.tmc.edu/pathology/medic/immunology/Immuno/geneab.html

Cell-mediated immunity (CMI) is defined as immune reactions in which T cells play a central role as effector cells (as opposed to regulatory cells). CMI includes T-cell cytotoxicity and delayed type hypersensitivity (DTH). Granulomatous responses usually result from DTH reactions to poorly degradable antigens, although antibody responses can also be involved. 5. T-CELL CYTOTOXICITY A. Definition - T mediated cellular cytotoxicity involving direct contact between the effector cell (CTL) and a target cell, resulting in target cell lysis or apoptosis. B. Mechanisms 1. In general, T-cell cytotoxicity involves CD8+ T cells. However CD4+ cytotoxic T cells also exist. 2. As in other effector mechanisms, naïve CD8+ cells must be activated by exposure to Ag-MHC I complexes and interleukins (e.g. IL-2) produced by helper T cells and must undergo proliferation and differentiation before becoming active Cytotoxic T Lymphocytes (CTLs). 3. The Ag-specific TCR of the cytotoxic T cell binds to the Ag-MHC-I complex on the surface of a target cell. In addition, a protein called Fas on the target cell binds to Fas ligand on the CTL. As in T-cell activation, other accessory proteins also form bridges between the cytotoxic cell and the target cell. 4. Binding of the TCR activates the release of granules containing perforin and granzymes by the CTL. The target cell is in close contact with the CTL, so most of the granule contents bind to the target cell. (Note: CTLs have mechanisms protecting themselves from self-destruction.) 5. Perforin forms a pore in the target cell, very similar to the pore formed by C9 in the complement pathway. If a sufficient number of pores are formed, the target cell can undergo rapid lysis. 6. Cytokines released by the CTL (including IFN-and TNF-may have cytotoxic effects on the target cell.

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7. Target cells can also undergo apoptosis or programmed cell death. In this case, killing is activated by two signals: the binding of Fas to the Fas ligand, and the leakage of granzymes into the target cell. 8. These two signals result the activation of two endogenous proteases in the target cell: JUN kinase and Caspase 8. These two enzymes act through a series of cytoplasmic and nuclear signals to start the irreversible process of apoptosis or cell death. Steps include nuclear condensation and fragmentation of nuclear DNA by endogenous Dnases. The process of cell death is complete in 1-2 days. 9. Once the target cell is ‘programmed’ to die, the CTL can detach and go on to kill many other target cells. 10. Null lymphocytes also generate lysis and apoptosis by similar mechanisms during natural killer (NK) activity and antibody-dependent cellular cytotoxicity (ADCC). However, T-cell receptor binding is obviously not involved in these activities. Apoptosis is also important in the elimination of self-reactive lymphocytes and the remodeling of tissues during development. 11. The protein Bcl-2 can block apoptosis by preventing the activation of caspases. It may be involved in the resistance of certain tumors to killing. A. Medical Aspects (Examples) 1. Protective a) Viral infections - T cell-mediated cytotoxicity appears to be the principal means of eliminating virally infected cells, although delayed type hypersensitivity must also play a role (see below). By killing cells expressing viral antigens on their surface, the host reduces virus production but may also destroy essential cells (e.g. neurons). b) Cancer - CTL along with DTH and NK activities are also thought to be important in eliminating malignant cells before they proliferate and become tumors. This process is called immune surveillance. Tumor cells often express so-called tumor-specific transplantation antigens or TSTAs. In virally-induced tumors, the TSTAs are often the same from one patient to the next, whereas chemical- or radiation-induced tumors usually express unique TSTAs. This complicates experimental strategies for specific immunotherapy, in which the subjects are vaccinated with TSTAs or given TSTA-specific T cells or antibodies to aid in tumor elimination. Page 174

c) Intracellular pathogens. Although less important than DTH, T cell cytotoxicity is also active in destroying intracellular pathogens. Most notably, CTL can destroy Plasmodium-infected hepatocytes during malaria. Also, this mechanism can lyse infected macrophages in tuberculosis, so that activated macrophages can then kill the released bacteria. 2. Immunopathologic a) Autoimmune diseases. Although it is often difficult to separate out T cell MALARIA ENDOGENOUS ANTIGEN PROCESSING AND T-CTL IMMUNITY INFECTION OF HEPATOCYTES

SPOROZOITES ENDOGENOUS PROCESSING

CLASS I MHC

IL-1

INDUCTION

IFN-g

T-CTL

T-CTL

T-CTL TL T -C

CIRCUMSPORATE ANTIGEN

EXPRESSION TL T -C

cytotoxicity and DTH, CTL almost certainly play a role in some autoimmune diseases. An example is insulin-dependent diabetes mellitus, in which the  cells in the islets of Langerhans are destroyed by autoreactive immune responses. Cytolytic T cells specific for  cells can be found at the scene in IDDM experimental models. Also, CTL are thought to be A. TISSUE CULTURE MONOLAYER responsible for thyroid cell DYING CELLS killing in Hashimoto’s thyroiditis (see figure). Reactive SPECIFIC T-CTL lymphocytes also surround target TISSUE CULTURE cells and separate them from TARGET CELLS neighboring cells and basement membranes, similar to what is B. AUTOIMMUNE THYROIDITIS THYROID seen in cell cultures. This DYING FOLLICULAR FOLLICULAR CELLS ‘disorientation’ also favors target CELLS cell death. b) Contact dermatitis. Again, T-CTL TO THYROID FOLLICULAR CELLS both CTL and TDTH are involved in contact dermatitis (described BASEMEMT MEMBRANE OF THYROID GLAND in more detail below). c) Viral exanthems. The eruptive lesions and fever characteristic of many viral infections are partially due to the host immune response. Tissue damage due to cytotoxic T cell responses may cause permanent loss of function. d) Graft rejection. Cytotoxic T cell (and DTH) responses are involved in acute graft rejection in transplant patients.

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Clinical Vignette –T-Cell Cytotoxicity (Geha and Notarangelo, “Case Studies in Immunology”, 6th Ed., 2012) Case 45

“Acute Infectious Mononucleosis” – 15 yo Emma Bovary had a severely sore throat, lymphadenopathy, and 2 weeks of fever, but eventually improves with supportive therapy.

Chromium release assay - measure of cytotoxic activity. Used to screen potential donor-recipient pairs in transplant patients. 1. Incubate virus-infected cell culture and normal cell culture with 51Cr to radiolabel cells. 2. Wash to remove excess radioactivity. 3. Incubate cell cultures with lymphocytes from virus-infected subject. 4. CTL activity will result in cell lysis and release of radioactivity into culture medium. 5. Determine radioactivity in supernatant, compare to control. Results are typically expressed as “percent specific killing”: % killing = cpm releasedexp - cpm releasedcontrol total cpm 6. What would be the percent specific killing in this example? 7. Another control would be to perform the same experiment with lymphocytes from an uninfected individual. What results would you expect?

51

Chromium Release Assay

Virus-infected cell monolayer 450 cpm

lysis, release of 51Cr

viral Ag-MHC complex 50 cpm

Normal cell monolayer 25 cpm No lysis, little release of 51Cr MHC 475 cpm

6. DELAYED TYPE HYPERSENSITIVITY (DTH) A. Definition - an in vivo reaction involving activation of macrophages by cytokines produced by lymphocytes (TDTH). Also called Type IV Hypersensitivity. B. Mechanisms 1. Naïve T cells are stimulated by specific interaction of their TCR with AgMHC II complexes on the surface of antigen presenting cells. They must undergo activation, proliferation and differentiation, as in other immune responses. 2. Upon restimulation with antigen (typically in the ‘target’ tissue such as skin, lung, or transplanted organs), the resulting memory TDTH cells (which have Th1 characteristics) express large quantities of cytokines including IL-2, macrophage chemotactic factor (MCF), IFN- and tumor necrosis factor  (TNF-.

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IFN

IT ! FO R

IL-2

GO

DTH

ETC. CTL

HELP!

ACTIVATION

AH! THANKS FOR THE GOODIES

5.

6.

7. 8.

3. IL-2 activates additional T cells, MCF attracts macrophages to the area, and IFN- activates macrophages, increasing their motility, phagocytic activity, and ability to kill intracellular bacteria (e.g. by oxidative mechanisms). TNF- can be cytotoxic. 4. Even in a sensitized individual, it takes 1-2 days for a sufficient number of T cells and macrophages to accumulate to cause a visible reaction (e.g.

erythema and induration EVOLUTION OF A DTH RESPONSE (SYPHILIS) [hardening] in a DAY 1 tuberculin skin test). DAY 3 DAY 7 DAY 12 That is why the reaction H is called delayed type H hypersensitivity. In H DAY 14 INDUCTIVE STAGE H contrast, anaphylactic reactions take minutes H H and immune complex H H REACTIVE STAGE reactions are maximal within ~6 hours after DAY 21 H exposure of sensitized individuals. TDTH cells have little or LATENT (HEALED) STAGE no direct effect on FIBROSIS pathogens or tissues. Their main activity is the recruitment and activation of macrophages. These guys do the dirty work of phagocytosing and killing pathogens or damaging tissue (in contact hypersensitivity, transplants, autoimmune reactions, etc.). Nonactivated macrophages are relatively quiescent; for example, they are incapable of killing M. tuberculosis and actually serve as hosts for its intracellular growth. The DTH activity of a patient can be tested by using antigens to which everyone is exposed, such as Candida albicans extracts. Patients who give negative skin test reactions to such antigens are considered to be anergic, i.e. deficient in cellular responses. DTH reactions can be inhibited by corticosteroids or blocked by cyclosporin and other immunosuppressive agents. These agents are commonly used to control autoimmune diseases and transplant rejection. Recent studies have shown that basophils may play a role in certain types of DTH reactions.

B. Medical aspects 1. Protective Page 177

a) Destruction of intracellular bacteria and other pathogens. DTH is the principal protection against mycobacterial infections and most parasitic and fungal infections. The importance of DTH is underscored in AIDS patients, who extremely susceptible to these organisms. b) Cancer - as mentioned above, DTH most likely plays a role in the immune surveillance for malignant cells. Unusual tumors occur at high frequency in patients with decreased CD4+ cell function (e.g. Kaposi’s sarcoma, lymphomas in AIDS patients). 2. Immunopathologic a) Contact hypersensitivity - skin reactivity to certain environmental agents, including poison oak/ivy, nickel, rubber products (including latex exam gloves!), PABA in suntan lotions, adhesives, and many other compounds. Typically the sensitizing agent is a hapten that binds to tissue proteins to form a hapten-carrier conjugate. These are processed and presented by Langerhans cells that are present in the skin and may migrate to lymph nodes. TDTH cells are sensitized and will react to subsequent exposures to the antigen. When exposed to irritants, keratinocytes often express MHC Class II proteins and cytokines, enhancing the hypersensitivity response. b) Autoimmune diseases - DTH reactions are involved in many autoimmune diseases, including multiple sclerosis, insulin dependent diabetes mellitus, Hashimoto’s thyroiditis, and rheumatoid arthritis. None of these appear to be ‘pure’ DTH responses, but rather involve a mixture of different effector mechanisms. c) Transplant rejection - DTH is active in acute allograft rejection, along with CTL reactions. In this type of reaction, activated macrophages cause tissue damage by release of lysosomal contents and oxygen radicals (rather than phagocytosis). Reactive T cells apparently recognize the allograft MHC proteins as “altered self”, and therefore are able to respond despite MHC restriction. An unusually high proportion of T cells (up to 10%) respond during allograft rejection. Clinical Vignette – DTH Reactions (Geha and Notarangelo, “Case Studies in Immunology”, 6th Ed., 2012) Case 51 Case 53 Case 48

Atopic Dermatitis – Tom Joad, 2 yo male with severe eczema Contact Hypersensitivity to Poison Ivy – 7 yo Paul Stein develops itchy eruptions after a hiking trip which responded to corticosteroids; the lesions ‘rebounded’ after the corticosteroids were stopped. Lepromatous Leprosy – Ursula Iguaran has leprosy, and develops disseminated lesions with large numbers of M. leprae due to a Th1-Th2 imbalance and a resulting poor DTH response.

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Blast transformation assays - in vitro measures of T cell reactivity. Proliferation

Add *Thymidine

*

Activation 1 day

2-5 days

* *

*

*

ConA Added * Thymidine Incorporated

1. Peripheral blood lymphocytes are incubated in the presence of: a) Mitogens - agents that cause nonspecific proliferation of certain populations of lymphocytes: Measure of the overall activity of that cell population. Concanavalin A (ConA) and phytohemagglutinin (PHA) - plant proteins that cause proliferation of T cells Lipopolysaccharide (LPS) - causes proliferation of B cells b) Antigens - provides information on the reactivity of the individual to specific antigens. Example: Mixed leukocyte culture - inactivated recipient cells mixed with donor lymphocytes. Shows whether CD4+ cells of recipient react to Class II MHC of donor.

Add mitogen or antigen

Donor lymphocytes added Nothing added

2. If reactive, lymphocytes begin to proliferate. 3HTime (days) thymidine is added, and the amount of radioactivity incorporated into DNA determined as a quantitative measure of proliferation. 3. High levels of incorporation relative to controls indicate a response. Why are responses to mitogens typically much higher than responses to specific antigens? 7. GRANULOMATOUS REACTIONS A. Definition - space-occupying lesion consisting of a predominantly mononuclear infiltrate (lymphocytes and macrophages) at the site of deposition of a poorly degradable antigen. B. Mechanisms 1. Usually caused by DTH reactions, but sometimes brought about by nonspecific reactions (e.g. silicosis) or antibody-mediated reactions. The archtypical example is the granuloma characteristic of tuberculosis. 2. CD4+ lymphocytes and macrophages accumulate at the site of the antigen in a typical DTH response. If the antigen (such as M. tuberculosis) continues to replicate or is not easily degraded, it will persist and cause continued accumulation of cells. The resulting granuloma can be up to several cm in diameter, and contains epithelioid cells (enlarged macrophages expressing TNF) and multinucleate giant cells (formed by the fusion of macrophages). In large granulomas, the center can become necrotic, forming a cavity. The granuloma can also displace normal tissue and cause fibrosis, decreasing tissue function (e.g. in the lung). 3. In inactive TB, granulomas containing viable M. tuberculosis can persist for decades without affecting health. However, breakdown of the granuloma or changes in the immune status of the individual may allow mycobacteria to grow out, resulting in active disease. 4. Persistence of immune complexes can also cause granuloma formation.

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GRANULOMATOUS REACTIONS INSOLUBLE ANTIGEN C1->C3b OPSONIZATION

MACROPHAGE

C3a, C5a, C5-7 CHEMOTAXIS

IgG ANTIBODY

+ LYMPHOKINES

T-DTH

ACTIVATED MACROPHAGES

SENSITIZED CELLS

CLINICAL CONDITIONS

GRANULOMA - SPACE OCCUPYING MASS

TUBERCULOSIS LEPROSY PARASITIC INFECTIONS SARCOIDOSIS GRANULOMATOSES

C. Medical aspects 1. Mycobacterial infections - as described above, granulomas are important in tuberculosis and leprosy. They can be detected in chest Xrays and are indicative of past or present active TB. 2. Parasitic infections - attempts to destroy or wall off parasites (such as worms) can result in granulomas. In extreme cases (eg. Roundworm Wuchereria bancrofti), these can occlude lymphatic vessels and cause elephantiasis. 3. Sarcoidosis - disease of unknown etiology that causes granulomas in multiple sites, including the lungs and skin. 4. Crohn’s disease - inflammatory disease of the bowel, in which granulomatous reactions can cause stricture (obstruction) and fistula formation. Etiology unknown.

Clinical Vignette – Granulomatous Disease, Geha and Notarangelo, “Case Studies in Immunology”, 6th Ed. Case 26

Chronic Granulomatous Disease – Randy Johnson develops granulomas and is unable to ward off Aspergillus and other opportunistic pathogens due to inability of his phagocytes to produce H2O2 and superoxide anion.

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SUMMARY -- CELL-MEDIATED REACTIONS 1. Cell-mediated reactions come about when T cells play a central role as effector cells. Another term is cell-mediated immunity or CMI. These reactions include T-cell cytotoxicity and delayed type hypersensitivity. 2. T-cell cytotoxicity occurs when an activated cytotoxic T cell (usually CD8+) binds directly to a target cell via a specific interaction of the TCR with Ag-MHC complexes on the target cell surface. Killing of the target cell occurs through two mechanisms. Release of granules containing perforins and granzymes result in formation of a pore in the target cell membrane, causing rapid lysis. A second major mechanism involves apoptosis, where programmed cell death is activated through a complex cascade involving Fas-Fas ligand interaction, activation of Jun kinase, Caspase 8, and other target cell signal transduction proteins, nucleus fragmentation, organelle destruction, and DNA cleavage. Cell death occurs over a 1-2 day period. T-cell cytotoxicity is protective against many viral infections, tumors, and intracellular pathogens, but is also involved in autoimmune diseases, contact dermatitis, viral rashes, and graft rejection. It can be quantitated through cell lysis assays, including the chromium release assay. 3. Delayed-type hypersensitivity (DTH) (also called Type IV hypersensitivity) is the activation of macrophages by cytokines produced by lymphocytes, typically Th1 cells. When Th1 cells are activated by exposure to antigen, they produce macrophage chemotactic factor, interferongamma, and tumor necrosis factor which attract and activate macrophages. These activated macrophages are much more effective in destroying intracellular pathogens and tumor cells. DTH is protective against many intracellular bacteria and protozoa, including mycobacteria and Pneumocystis carinii. Adverse effects include participation in contact hypersensitivity, autoimmune diseases, and transplant rejection. DTH responses can be measured indirectly by blast transformation assays or more directly by quantitation of cytokine production. 4. Granulomatous reactions are collections of lymphocytes and enlarged macrophages resulting from a chronic response to an antigen that is difficult to destroy. Persistent M. tuberculosis infection is an example of a disease process leading to granuloma formation. CD4+ Th1 cells attract macrophages to the area, but they continue to collect due to failure to eliminate the antigen. Granulomatous reactions are prominent in mycobacterial infections, some parasitic infections, sarcoidosis, and Crohn's disease.

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IMMUNOLOGY OF HIV INFECTION Steven J. Norris, Ph.D. Required Reading: Geha and Notarangelo, 6th edition (2012). Case Studies in Immunology. Garland Publishing, New York, NY. Case 10: AIDS. Web Resource: http://www.uth.tmc.edu/pathology/medic/immunology/Immuno/aids.html

I. HUMAN IMMUNODEFICIENCY VIRUS A. AIDS Related Retroviruses 1. Human Immunodeficiency Virus-I (HIV-1) is the type most commonly associated with HIV infection and AIDS in the United States and Europe. HIV-2, which shares ~50% nucleotide identity to HIV-1, is associated with a small number of cases in the U.S., but is prevalent in regions of Africa. 2. HIV-1 and HIV-2 are members of the retrovirus family, a group of viruses that have an RNA genome but form a DNA intermediate that is incorporated into the genome of the host cell. There are oncogenic (tumor causing) and cytolytic (cell-killing) subfamilies of retroviruses. HIV is part of a group called lentiviruses, slow-acting cytolytic retroviruses (lento means slow in music). An example of an oncogenic retrovirus is Human T Lymphocyte Virus (HTLV), which is associated with T cell lymphomas. Lentiviruses

.

Virus Human immunodeficiency virus Simian immunodeficiency virus Visna/maedi virus Equine infectious anemia virus Caprine arthritis/encephalitis virus

Disease Cause of human AIDS AIDS in monkeys Neurologic and lung disease in sheep Horse anemia Goat encephalitis

B. The HIV Genome and Structure 1. The HIV genome consists of a 9,000 bp segment of single-stranded RNA. It encodes a series of gene products that are cleaved by the HIV protease to form important structural and nonstructural proteins (see figure). 2. Proteins important in the immune response to HIV include: a) The envelope (env) glycoproteins gp120 and gp41, and their precursor gp160 b) The group antigen (gag) proteins p24 (major core protein) and p17 (protein that forms a scaffold during virion assembly) c) The pol proteins p66 and p51 (form reverse transcriptase), protease, and p32 (endonuclease) d) Regulatory proteins including tat (transactivator), rev (regulator of expression), vif (virion infectivity factor), and nef (negative factor). These regulate HIV virus gene expression and assembly.

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C. HIV Replication and Gene Expression (see figure) 1. Binding and internalization. gp120 on the surface of the virion binds with high affinity to CD4 on the surface of CD4+ T cells and some other cells types. Gp120 is removed, exposing gp41 underneath, which then promotes fusion between the cell membrane and viral membrane. As a result, the viral core is released into the cytoplasm of the cell. Alternatively, antibody or C3b bound to the surface of the virion can bind to Fc or complement receptors on macrophages and other cells, resulting in internalization (antibody dependent enhancement). Lastly, other “co-receptors” such as chemokine receptors (e.g. CXCR4 and CCR5) and the glycolipid galactosyl ceramide can interact with HIV, promoting infection of other cell types, albeit at much lower efficiency than CD4 binding. A 32-bp deletion in the CCR5 gene (ccr532) that eliminates CCR5 expression has been linked to resistance to HIV infection. 2. Reverse transcription and incorporation. The virion RNA is replicated by virus-associated reverse transcriptase, resulting in a double-stranded DNA copy of the viral genome. This DNA becomes circularized and then incorporated into host chromosome.

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3. Transcription and translation of viral genes. Host transcription factors along with viral factors such as tat activate transcription of the viral genes, resulting in protein expression. Large precursor proteins are cleaved into the final protein products by HIV protease. This step is blocked by drugs called protease inhibitors, thus inhibiting viral replication. 4. Assembly and budding. The viral core, including two copies of the RNA genome, assemble and bud through the cell membrane to form an infectious virion. 5. Latent infection vs. virus production. Resting CD4+ T cells typically exhibit latent infection, that is they contain HIV DNA but do not actively produce virus. Cell activation as indicated by expression of HLA-DR and other Class II MHC proteins is required for high level virus production. Enhanced viral production is linked to expression and activation of nuclear factor kappa B (NF-B) and other host cell transcription factors. During primary HIV or late symptomatic infection, 1 out of 10 peripheral blood CD4+ T cells may be latently infected, whereas only 1 out of 300 to 400 are actively producing virus. Tissue macrophages are an important reservoir of infection, in that they can become infected and produce low levels of virus without being killed. Many other cell types, including epithelial cells, can be infected. Macrophages and other cells can be latently infected for long periods and then express virus when activated by exposure to cytokines, viruses or other infectious agents, and other factors.

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II. CLINICAL COURSE OF HIV INFECTION HIV infection and AIDS are not equivalent. HIV infection means, quite literally, infection with HIV-1 or HIV-2. HIV+ patients can lead normal, healthy, productive lives. Unfortunately, HIV infection almost inevitably progresses to the profound immunodeficiency and opportunistic infections of Acquired Immunodeficiency Disease Syndrome (AIDS). This process usually takes 8 to 12 years for sexually transmitted infection, fewer years for blood transmission, and >>apoptosis b. Immature B cells (bone marrow) bind to self antigens on other BM cells >>>apoptosis or anergy

208

2.

Directing cell development towards regulatory lineage of selfreactive cells – natural T-regulatory cells a. Developed in the thymus b. Usually recognizes self antigens c. Controlled by multiple factors, including thymic DCs, Hassall’s corpuscles (epithelial cells), and “conditioned” DCs

209

3.

Tolerance induced by foreign antigens a. T-cell exposure of alloantigen in the thymus – similar development as observed for natural T-regulatory cells that recognize self antigens b. Immature B-cell, recognizing foreign antigens, development with regulatory functions in secondary lymph organs (e.g. spleen) i. Naïve B-cells encountering helper CD4+ T-cells expressing CD154 ii. Regulatory B-cells produces IL-10

c. This is part of self tolerance mechanism in utero i. Dizygotic twins sharing common placental blood supply ii. Neonatal tolerance induction in allogeneic animals  apparent apoptosis of allo-MHC clones  persistence of alloantigen needed for tolerance

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D.

Induction of Tolerance in Mature T and B Lymphocytes 1.

Major mechanisms a. Apoptosis  FAS/FASL interaction i. Autoimmune models – Lpr (Fas deficient) and Gld (FasL deficient)

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 Control of lymphocyte cell numbers i. Activation induced cell death (AICD) - autonomous vs. cell-cell contact ii. Lack of growth/proliferation factors (e.g. IL-2)  b. Anergy – unresponsive to subsequent antigen challenge  Lack of secondary signals for T and B cells (Ig and TNF superfamilies) i. CD28, ICOS/ICOSL (inducible co-stimulator) ii. CD40, OX40  Expression of suppressive secondary signals i. CTLA-4 (induced upon T-cell activation) ii. PD-1  Lack of helper cells i. Large antigen dose bypass T helper cell activation of B-cells ii. Usually CD40/40L interaction c. Immune regulation – cell types and function  T-cells i. T-regulatory cells (natural vs. inducible) ii. T-helper cells (TH1, TH2, TH9, TH17, etc) iii. Cytokine production (e.g. IL-10, TGF-1) iv. Cytokine sequestration (e.g. IL-2) v. Direct cell-cell contact (e.g. CTLA-4/CD80)  B-cells 212

i. B-regulatory cells ii. B-effector cells (Be-1 vs. Be-2) iii. Cytokine production (IL-10)  Dendritic cells i. Immature vs. mature ii. Tissue specific DCs (e.g. mucosal DCs) iii. Cytokine production (TGF-1, IL-10, IL-4, etc) iv. Alteration of surface marker expression (CD80/86, CD40, etc)

d. Antigen sequestration  Immune privilege sites  Exposure of privilege site antigens will result in autoimmunity (e.g. CNS)

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III.

PARAMETERS THAT INFLUENCE IMMUNE RESPONSES OF INDIVIDUALS A.

Age 1. Young a. Fetus makes IgM but not IgG until almost term b. Term babies immunocompetent but immature at birth c. Takes several months to develop full immunocompetence 2. Aged a. Immune senescence b. Deficiency vs. dysregulation c. Good memory response, poor naïve (primary) response d. Infectious deaths in elderly often occur with new organism strain

B.

Neurologic and Endocrine Factors 1. Psychoneuroimmunology – relationships between behavior (stress) neuroendocrine changes and immunity a. hypophyseal-pituitary-adrenal axis - corticosteroids b. sympathetic nervous system – catecholamines (epinephrine, norepinephrine) 2. Hypophyseal-pituitary-gonadal axis – particularly female hormones a. may explain female preponderance of immune-based diseases b. both TH1 and TH2 diseases noted 3. Nutritional Status a. trace mineral deficiency – zinc, selenium, etc. b. malnutrition – total calorie vs. protein – energy  infection activates TNF, IL-1, etc. (pyrogens)  fever produces anorexia, affecting appetite  poor appetite worsens malnutrition  cell mediated immunity affected first  c. leptin - associated with obesity - reverses starvation-induced immunosuppression d. Retinoic acid



C.

MHC Message Expression 1. Allotypes of MHC expression affects specific responses 2. Affects repertoire/magnitude of responses to vaccines 3. may also affect susceptibility to certain autoimmune responses

D.

Effects of Antigen

1.

Dose a. low and high doses tolergenic, mid dose immunogenic

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b. low dose tolerizes T cells only, high dose tolerizes both T and B cells

2.

2.

Route of Exposure  parenteral exposure is enhanced by adjuvant effects  IV exposure can tolerize naïve (but not memory) T cells a. IV antigen gets to spleen rapidly b. Picked up by resting B cells c. Resting B cells lack costimulatory molecules d. Result is anergy  Oral exposure a. activates muscosal T cells to secrete TGF-beta b. result is anergy Regulation by Antibody  Antibody feedback - antibody inhibits further specific antibody production  Antigen-antibody complex i. Binds BCR, CD32 simultaneously ii. Inhibits activation signal iii. Result is anergy

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Coico, et al., Fig 10.14. Antibody feedback inhibits B-cell activation, resulting in negative signal to the cell. 2009.

IV.

Tolerance in the clinical setting A.

Oral tolerance – a major environment for tolerance i. Dose of antigen 1. Low – favors regulatory T-cells 2. High – favors anergy/apoptosis ii. Mechanism 1. Dendritic cells – DCs that are CD103+ ( integrin) produces mainly TGF-b1 and induce activation of T-regulatory cells 2. Retinoic acid – Enhances TGF-b1 production and induction of T-regulatory cells 3. T-regulatory cells – Nearly all classes are induced by oral antigens (e.g. TH3, secretes TGF-1)

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B.

Pregnancy (tolerance of paternal antigens) i. The paradox 1. Fetal expression of paternal (foreign) antigens 2. Shedding of trophoblast into maternal circulation 3. Cellular debris from fetal tissue 4. Presence of fetal cells in maternal circulation ii. Mechanism 1. Immune stimulation by sperm exposure – Activation of effector T-cells also triggers T-regulatory cell activity, and overall consequence is expansion of T-reg populations (driven by TGF1 and prostaglandin E) 2. Extravillous trophoblast cells lack of expression of antigen presentation molecules (Class 1 and 2) but expresses nonclassical class I to evade killing by NK cells 3. T-regulatory cells – Maternal T-reg numbers peak during the window when implantation can occur and during pregnancy. Fetal T-regs are similar to adult T-regs

C.

Cancer (tolerance of tumor antigens) i. Cancer immunosurveillance hypothesis – immune response is efficient in controlling tumor growth and clinical disease ii. Cancer immuno-editing 1. Elimination – tumors detected and destroyed 2. Equilibrium – establishment of balance between tumor and immune response 3. Escape – immune control falters and tumor variants grow uncontrollably iii. Tumor microenvironment 1. Concomitant immunity (transient) – growth of tumor at one site despite rejection of subsequent introduction of the same tumor cells at a distant site iv. Mechanism 1. Direct immune suppression by tumor cells a. Expression of T-cell inhibitory molecules (e.g. PD-L1, B7H3, etc) b. Production of anti-inflammatory cytokines (e.g. IL-10, TGF-1, etc) c. Alters recruitment of “suppressor” cell types, both APCs and T-cells 2. T-regulatory cells – Population expands in cancer patients 3. Dendritic cells – IDO (indoleamine 2,3-dioxygenase) competent APCs activate mature T-regs

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Summary 

Immune response must be regulated to allow sufficient response to protect host with excessive or inappropriate responses that may create disease



Immunological tolerance is specific unresponsiveness to an antigen while allowing the rest of the host response to remain intact and capable of response



Tolerance can be at the level of B or T lymphocytes or both and can be accomplished by a variety of mechanisms including apoptosis, anergy, and suppressor T cell activity



Many factors influence the nature, intensity and duration of an immune response including age, neuroendocrine hormone levels, HLA allotypes, antigen dose, antigen access, and cytokine milieu.

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AUTOIMMUNITY AND AUTOIMMUNE DISEASES Sandeep K. Agarwal, M.D., Ph.D. Medicine-Immunology, Allergy & Rheumatology, BCM 713-798-3390 [email protected]

Objectives 1. Define and discuss autoimmunity. 2. Use autoimmune diseases to illustrate mechanisms of autoimmunity. 3. Provide you with clinical correlations and applications of the basic principles of immunology. Reading: Coico and Sunshine. Immunology: A Short Course. John Wiley & Sons, Inc, New York, NY. 6th edition, 2009. Chapter 12 (p190-204). R. S. Geha and Notarangelo, L. Case Studies in Immunology: A Clinical Companion. (6th Ed) Garland Publishing, New York, 2012. Chapter 36. Rheumatoid Arthritis; Chapter 40. Multiple Sclerosis; Chapter 41. Autoimmune Hemolytic Anemia. Web Resource: http://www.uth.tmc.edu/pathology/medic/immunology/Immuno/autoimmunity.html INTRODUCTION: The regulation of immune function and overall immuno-homeostasis is under control of multiple factors that include genetic and environmental components. HLA allotypes, antigen dose, and existing cytokine milieu can all influence responses to both pathogenic agents and self antigens.

It is highly recommended to review the reading materials PRIOR to lecture, as the lecture will primarily concentrate on clinical manifestations of autoimmune disorders.

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Autoimmune Lecture Outline Autoimmunity • Specific adaptive immune response mounted against a self-antigen – Loss of Self-tolerance to self-antigens – Loss of central and peripheral tolerance • Loss of central tolerance likely occurs all the time • May have a physiological role to clear defective or denatured molecules through the RE system • Normally kept in check by mechanisms of peripheral tolerance • May be triggered by infections or aging • May or may not cause disease Autoimmune Disease • Termed “horror autoxicus” by Paul Ehrlich • Tissue response and damage triggered by autoimmunity • Results from the dysregulation of immune processes and pathways that are involved in normal immunity Architecture of an Autoimmune Response • Innate and Adaptive components Autoimmune Disease and Clinical Phenotypes: AUTOIMMUNE DISEASE CLINCAL PHENOTYPE Systemic Lupus Erythematosus Rash; inflammation of joints and serosal linings; glomerulonephritis; hemolytic anemia, systemic symptoms Rheumatoid Arthritis Inflammation of synovium of diarthroidal joints, systemic inflammation Scleroderma Inflammation, dermal fibrosis, internal organ fibrosis, vasculopathy Ankylosing Spondylitis Inflammation of spine, joints, and tendon insertions; uveitis Multiple Sclerosis Demyelination, optic neuritis, neurological deficits Myasthenia Gravis Skeletal muscle weakness, diplopia, dysarthria, dysphagia Hashimoto’s Thyroiditis Hypothyroidism Graves Disease

Hyperthyroidism, opthalmopathy

Celiac Disease

Diarrhea and malabsoprtion

Autoimmune hemolytic anemia Anemia through lysis of red blood cells Type I diabetes

Failure of insulin production and glycemic control

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Genetic Susceptibility to Autoimmune Diseases Simple Genetic Traits Associated with Autoimmune Diseases Autoimmune Diseases and Concordance in Twins Common diseases: Multiple SNPs • Common diseases are believed to result from a combination of susceptibility alleles at multiple loci, environmental factors and stochastic events • Non-Mendelian Inheritance Patterns • Single nucleotide polymorphisms (SNPs) – Individual bases that exist as either of two alleles in the population Major Histocompatibility Complex – Association with Autoimmune Diseases Class I MHC Associations Ankylosing Spondylitis HLA-B27 Grave’s Disease HLA-B8 Class II MHC Associations Rheumatoid Arthritis Sjogren’s Syndrome Systemic Lupus Erythematosus Type I Diabetes Celiac Disease Myasthenia Gravis Multiple Sclerosis

HLA-DR4 HLA-DR3 HLA-DR3, DR2 HLA-DR3 HLA-DR3 HLA-DR3 HLA-DR2

HLA-B27and Autoimmune Disease HLA-DR4 and Rheumatoid Arthritis Single Nucleotide Polymorphisms in Autoimmune Diseases Mechanisms of Autoimmune Disease • Previous attempts to classify them as T-cell and B-cell mediated are outdated • Involve Innate and Adaptive Components • Classified based on the effector mechanisms that appear to be most responsible for organ damage: – Autoantibodies – T-cells

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Autoantibodies • Antibodies against to self-antigens • Can be found in normal, healthy individuals • Important effectors in autoimmune disease Autoimmune Hemolytic Anemia • Autoantibodies against RBC antigens – Warm autoantibodies • IgG, react with Rh antigen on RBC at 37degC • Result in opsonization of RBCs and macrophage phagocytosis – Cold autoantibodies (cold agglutinins) • IgM, react with I or i antigen on RBC when