Academical Year 2013-2014 Faculty Pharmaceutical, Biomedical and Veterinary Sciences

Biomedical Sciences

In vitro analysis of the alloreactive NK cell response against HIV patient CD4+ T-cells as a potential correlate of protection against HIV transmission By: Jef Hens

Master Thesis in partial fulfillment of the requirements for the degree Master in Biomedical Sciences

Promotor(en): Prof. Dr. Luc Kestens Copromotor: Dr. Wim Jennes

Institute of Tropical Medicine Departement of Biomedical sciences Laboratory of Immunology Nationalestraat 155 2000 Antwerpen

Table of contents Abstract ................................................................................................................................................... iv Samenvatting............................................................................................................................................v Abbreviation list ...................................................................................................................................... vi Introduction............................................................................................................................................. 1 1. Human immunodeficiency virus type 1 (HIV-1) .............................................................................. 1 1.1 HIV-1 Introduction ..................................................................................................................... 1 1.2 HIV-1 epidemiology ................................................................................................................... 1 1.3 HIV-1 structure and genome ..................................................................................................... 1 1.4 HIV-1 life cycle ........................................................................................................................... 2 1.5 HIV-1 pathogenesis.................................................................................................................... 3 1.6 Immune-related HIV-restrictive mechanisms ........................................................................... 4 2. Natural killer cells ............................................................................................................................ 6 2.1 Introduction of NK cells ............................................................................................................. 6 2.2 NK cell subsets ........................................................................................................................... 7 2.3 NK cell receptors........................................................................................................................ 7 2.4 NK cell activation mechanisms ................................................................................................ 10 3. NK cells and HIV-1 infection .......................................................................................................... 11 3.1 HIV-1 transmission .................................................................................................................. 11 3.2 HIV-1-NK cell interactions ....................................................................................................... 12 4. Alloreactive NK cells in haploidentical hematopoietic stem cell transplantation (haplo-HSCT)... 13 5.Preliminairy study........................................................................................................................... 14 6. objectives....................................................................................................................................... 15 Materials and Methods ......................................................................................................................... 16 1. Study population ........................................................................................................................... 16 2. NK cell cytotoxicity ........................................................................................................................ 16 2.1 Isolation and cultivation of NK cells ........................................................................................ 16 2.2 Isolation and cultivation of CD4+ T-cells .................................................................................. 16 2.3 Target cell death ...................................................................................................................... 17 2.4 NK cell degranulation/activation ............................................................................................. 17 3. HLA-Bw4, -C1 en -C2 genotyping of healthy blood donors and HIV-1 patients ............................ 17 4. Statistische analyse ....................................................................................................................... 17 Results ................................................................................................................................................... 18 1. Experimental set-up ...................................................................................................................... 18

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1.1 Weekly schedule...................................................................................................................... 18 1.2 Gating strategies...................................................................................................................... 19 2.Optimization of the experiments ................................................................................................... 23 2.1 NKG2A-PerCP antibodies ......................................................................................................... 23 2.2 Determining the volumes of NKG2A-PE .................................................................................. 23 3.Analysis of the different assays in the NK-CD4+ T-cell co-cultures................................................. 25 3.1 Target cell death ...................................................................................................................... 25 3.2 NK cell degranulation .............................................................................................................. 27 3.3 NK cell activation ..................................................................................................................... 29 3.4 Analyzing the KIR+/NKG2A+ NK cells in different conditions ................................................... 31 4. Optimizing the target cell death-assay, deciding the optimal gating strategy and NK-CD4+ T-cell co-culture .......................................................................................................................................... 32 5. Correlation between CD4+ T-cell death and NK cell degranulation and CD4+ T-cell death and NK cell activation .................................................................................................................................... 33 6. Alloreactive NK cell degranulation is limited to specific KIR+ NK cell subpopulations .................. 34 Discussion .............................................................................................................................................. 37 Acknowledgements ............................................................................................................................... 40 Attachements ........................................................................................................................................ 41 Protocols............................................................................................................................................ 41 1.Ficoll-Hypaque gradient centrifugation ..................................................................................... 41 2.Magnetic cell isolation of NK cells .............................................................................................. 42 3.Magnetic cell isolation of CD4+ T-lymphocytes .......................................................................... 43 4.NK-CD4 coculture ....................................................................................................................... 44 5.K562-culture maintenance ......................................................................................................... 45 Reference list: ........................................................................................................................................ 46

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Abstract Introduction: The cytotoxic potential of alloreactive NK cells was first described in allogeneic stem cell transplantation for the treatment of leukemia. In response to these results, our laboratory investigated the potential of alloreactive NK cells for establishing HIV-protection in HIV-discordant couples. HIV-protection in these HIV-discordant couples was related to an allogeneic KIR/HLA mismatch between partners, suggesting alloreactive NK cells kill allogeneic HIV-infected CD4+ T-cells in the absence of an inhibitory KIR-HLA interaction. The purpose of this study was to investigate alloreactive NK cell mediated CD4+ T-cell killing in vitro and analyse its dependence on inhibitory KIR interactions.

Methods: Blood samples of 26 healthy donors and 26 HIV-infected patients were used. NK cells and CD4+ T-cells were magnetically isolated by negative and positive selection, respectively. NK cells were stimulated with IL-2 for 3 days. Half of the CD4+ T-cells were stimulated with PHA and IL-2 for 1 day, mimicking the activation state of mucosal CD4+ T-cells, the other half were unstimulated. Allogeneic NK-CD4 co-cultures were set up to measure the % CD4+ T-cell death (7-AAD), NK cell degranulation (CD107a) and NK cell activation (CD69). 7-AAD-, CD107a- and CD69-expression was analyzed in NKCD4 co-cultures by flow cytometry. NK cells were stained for the inhibitory NK cell receptors KIR2DL1, KIR2DL2/3, KIR3DL1, and NKG2A. Using an algorithm, NK cells expressing one single KIR or NKG2A while lacking all other KIR or NKG2A were gated. Single-KIR+ NK subpopulations were studied to detect the presence of KIR-specific alloreactive NK cell responses. NK-K562 co-cultures were used as positive controls for all measurements. Results: Alloreactive NK cell mediated target cell death occurred in all of the co-cultures with K562 cells and in a large proportion of co-cultures with HIV patient derived CD4+ T-cells. Prestimulation of the CD4+ T-cells with PHA and IL-2 resulted in an increase of NK cell mediated CD4+ T-cell death. A CD4+ T-cell gating strategy that did not make use of a light scatter lymphocyte gate resulted in increased detection of CD4+ T-cell death. Importantly, CD4+ T-cell death correlated with NK cell degranulation in the NK-CD4+ T-cell co-cultures, suggesting that NK cell mediated CD4+ T-cell death was induced by degranulation of the cytolytic granules. The strongest correlation was seen when data of co-cultures with PHA-prestimulated-CD4+ T-cells was used, indicating that PHAprestimulation of the CD4+ T-cells generates more accurate and sensitive results. NK cell degranulation towards CD4+ T-cells was often induced by a single KIR+ NK cell subpopulation, whereas NK cell degranulation towards K562 cells was predominantly induced by the NKG2A+ NK cell subpopulation. Conclusion: Alloreactive NK cells are capable of inducing CD4+ T cell death in a large proportion of allogeneic NK-CD4+ T-cell co-cultures. PHA-stimulation and light scatter independent gating of CD4+ T cells consistently resulted in more accurate and sensitive results. Alloreactive NK cell mediated cell death correlated with NK cell degranulation, which we found was KIR-specific. Together, our data suggest that alloreactive NK cells are capable of killing allogeneic CD4+ T-cells by a KIR-specific NK cell degranulation mechanism. Future KIR/HLA genotyping will allow to study the exact contribution of KIR/HLA mismatches to the observed alloreactive NK cell responses.

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Samenvatting Inleiding: Het cytotoxische potentieel van alloreactieve NK cells werd voor het eerst beschreven in allogene stamcel transplantatie voor de behandeling van leukemia. Op basis van deze resultaten bestuurde ons labo het potentieel van alloreactieve NK cellen in het bekomen van HIV-immuniteit in HIV-discordante koppels. De HIV-immuniteit van deze HIV-discordante koppels bleek gerelateerd te zijn aan een allogene KIR/HLA mismatch, wat suggereerde dat alloreactieve NK cellen de allogene HIV-geïnfecteerde CD4+ T-cellen dode in de afwezigheid van een KIR-HLA interactie. In deze in vitro studie onderzoeken we deze CD4+ T-cel doding door alloreactieve NK cellen en de afhankelijkheid van inhiberende KIR interacties hiervan. Methodes: Bloedstalen van 26 gezonde donoren en 26 HIV-patiënten werden gebruikt. NK cellen en CD4+ T-cellen werden magnetisch geïsoleerd, respectievelijk door negatieve en positieve selectie. NK cellen werden gestimuleerd door IL-2 en geïncubeerd gedurende 3 dagen op 37°C. De helft van de CD4+ T-cellen werden gestimuleerd door PHA en IL-2 en geïncubeerd gedurende 1 dag op 37°C, de andere helft bleef ongestimuleerd en werd voor 1 dag geïncubeerd op 37°C. Allogene NK-CD4 coculturen werden gemeten op het % CD4+ T-celdood (7-AAD), NK cel degranulatie (CD107a) en NK cel activatie (CD69) en werd flowcytometrisch gemeten. NK cellen werden aangekleurd voor KIR3DL1, KIR2DL1, KIR2DL2/3 en NKG2A. Gebruikmakend van een algoritme werden NK cellen die slechts 1 KIR tot expressie brachten geïsoleerd in NK cel subpopulaties. Deze subpopulaties werden gebruikt om het KIR-specifieke effect van alloreactieve NK cellen te bestuderen. K562 cellen werden gebruikt als positieve controle. Resultaten: Alloreactieve NK cellen medieerde doelcel dood in elke co-cultuur met K562 cellen en in het merendeel van de co-culturen met CD4+ T-cellen. Prestimulatie van de CD4+ T-cellen met PHA zorgde een verhoogde CD4+ T-celdood. Het gebruik van een gating strategie die geen gebruik maakte van een light scatter lymfocyten gate zorgde voor een verhoogde detectie van CD4+ T-celdood. Een belangrijk resultaat was het verband tussen CD4+ T-celdood en NK cel degranulatie, wat suggereerde dat NK cellen via degranulatie van de cytolytische granules CD4+ T-celdood veroorzaakte. Het sterkste verband was zichtbaar wanneer data van PHA-gestimuleerde CD4+ T-cellen werd gebruikt, dit wijste aan dat het gebruik van PHA-gestimuleerde CD4+ T-cellen voor accuratere en sensitievere data zorgde. NK cel degranulatie tegenover CD4+ T-cellen was dikwijls geïnduceerd door een KIRspecifieke NK cel subpopulatie, waar dat NK cel degranulatie tegenover K562 cellen vooral werd geïnduceerd door de NKG2A+ NK cel subpopulatie. Conclusie: Alloreactive zijn in staat om in het merendeel van de allogene NK-CD4 co-culturen CD4+ Tceldood te induceren. PHA stimulatie en light scatter-onafhankelijke gating van CD4+ T-cellen zorgde voor accuratere en sensitievere data. Alloreactieve NK cellen zorgde via NK cel degranulatie voor de doding van CD4+ T-cellen, dit mechanisme bleek ook nog KIR-specifiek te zijn. Samen suggereren onze data dat NK cellen voor CD4+ T-cellen zorgen via KIR-specifieke NK cel degranulatie. Wanneer de KIR/HLA genotypes aanwezig zijn zal hun exacte bijdrage aan deze NK cel reacties ten volle bestudeerd kunnen worden.

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Abbreviation list 7-AAD ADCC AIDS AML APC APC-H7 APOBEC3 ART BSA BTC BV CAF CD cDC Cr CTL DC E:T FasL Fc FITC FSC GALT GM-CSF GTP GVHD GVL HAART HBSS HESN HIV-1 HLA HSCT IFN Ig IL ITAM TIM ITM LS LTNP KIR MHC MIC MS N-CAM NCR NK PBMC pDC

7-amino-actinomycine D Antibody dependent cell cytotoxicity Acquired immune-deficiency syndrome Acute myelogenous leukemia Allophycocyanine Allophycocyanine-H7 Apolipoprotein B editing complex 3 (A3) cytidine deaminases anti-retroviral treatment bovine serum albumin Bloedtransfusiecentrum Antwerpen Brilliant violet CD8+ antiviral factor Cluster of differentiation Conventional DC Chromium Cytotoxic T-lymphocyte Dendritic cell Effector-target cell ratio Fas-ligand Fragment crystallizable Fluoresceïne isothiocyanaat Forward scatter Gut associated lymphoid tissue Granulocyte macrophage-colony stimulating factor Guanosine-5'-triphosphate Graft versus host disease Graft versus leukemia Highly active-anti-retroviral treatment Hanks' balanced salt solution HIV-exposed seronegative Human immunodeficiency virus type 1 Human leukocyte antigen Hematopoietic stem cell transplantation Interferon Immunoglobine Interleukine Immunoreceptor tyrosine-based activation motif Immunoreceptor tyrosine-based inhibitory motif Institute of Tropical Medicine Large separation (column) Long-term non-progressor Killer-cell immunoglobulin-like receptor Major histocompatibility complex MHC class I chain-related gene Mini separation (column) Neural cell adhesion molecule Natural cytotoxic receptors Natural killer (cell) Peripheral blood mononuclear cell Plasmacytoidic DC vi

PE PE-Vio770 PerCP-Cy5.5 PRR SAMHD1 SH-2 SHP-1 SIV SCC RPMI Th TLR TNF TRAIL TRIM WHO ULBP

Phycoërythrine Phycoëryhtrine-Vio770 Peridinin chlorophyll protein-Cy5.5 Pattern recognition receptor sterile alpha motif and histidine/aspartic acid domain containing protein-1 Src homology region 2 domain Src homology region 2 domain-containing phosphatase-1 simian immunodeficiency virus side scatter Roswell Park Memorial Institute T helper (cell) toll-like receptor tumor necrosis factor (TNF)-related apoptosis-inducing ligand TRIpartite Motif world health organization UL16 binding protein

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Introduction 1. Human immunodeficiency virus type 1 (HIV-1) 1.1 HIV-1 Introduction HIV-1 is a lentivirus responsible for the development of the ‘acquired immune-deficiency syndrome’ (AIDS). HIV-1 originated in West-central Africa and can be transmitted via vertical and horizontal pathways. After transmission, HIV particles infect mucosal/peripheral cluster of differentiation (CD) 4+ T-cells, dendritic cells (DCs) or macrophages to replicate and spread out over the body. During infection, the CD4+ T-cell count decreases, weakening the host immune system. The diagnosis of AIDS is applied when the patient is infected by an opportunistic disease or a CD4+ T-cell count below 200 cells/mm3 blood. In 2012, 35,5 million people were infected around the world, making it world’s Number 1 tropical disease. 1.2 HIV-1 epidemiology HIV exists in two forms: HIV-1 and HIV-2. Both forms developed when the simian immunodeficiency virus (SIV) of (non-)human primates in West-central Africa transmitted to humans in the early 20th century (Sharp and Hahn, 2011). The HIV-1 strain is closely related to the SIVs found in chimpanzees of the subspecies Pan troglodytes troglodytes (Van Heuverswyn et al., 2006). The transmission of the SIVcpz virus strain gave rise to HIV-1, causing the HIV-1 pandemic. HIV-2 strains resemble the SIVsmm virus found in sooty mangabey (Cercocebus atys atys) (Santiago et al., 2005). HIV-1 is highly transmittable and can be found all over the world, whereas HIV-2 is less transmittable and resides within West-Africa. Since the discovery of the virus in 1983, four groups (M,N,O,P) of HIV-1 can be discriminated. Each group represents an independent transmission of SIVs onto humans (Sharp and Hahn, 2011). The largest group is M, representing 90% of all the HIV-1 infections. Group M can be divided into subgroups (A-K), but also recombinant forms between the subgroups can be seen (Burke, 1997). In September 2013, the world health organization (WHO) and UNAIDS released a new epidemiologic report about HIV infections in the world during 2012 (Fig.1). Globally, 35,3 million [32,2 million – 38,8 million] people are infected with HIV in 2012. The majority (25,0 million [23,5 million – 26,6 million]) of the infected people were found in Sub-Saharan Africa. New HIV infections in 2012 were estimated around 2,3 million [1,9 million – 2,7 million] infections, also mostly found in Sub-Saharan Africa (1,6 million [1,4 million – 1,8 million]). The amount of deaths caused by AIDS in 2012 was estimated around 1,6 million [1,4 million – 1,9 million] deaths. Every day in 2012, 6,300 people were infected by HIV. 95% of the newly infected people come from low- and middle-income countries. About 700 of 6,300 newly infected were children (