Lymphocyte recirculation P. Naquet [email protected]

Centre d’Immunologie de Marseille-Luminy

Leukocyte trafficking Bone marrow

Precursor cell

Production: cytokines

Blood

Mature cell

Recruitment: chemokines

Post capillary veinule Effector cell Dying cell

Spleen

REGULATION

Effector phase

Inflamed tissue

Naive lymphocytes Spleen MATURE STAGE

Bone marrow

2,5x1011 / day

Thoracic duct

Blood

T1/2 = 30 min 5x1011 / day

0,3x1011 / day

Lymph nodes

High endothelial veinule

DEVELOPMENTAL STAGE

Primary Lymphoid organs

Effector lymphocytes Spleen

Bone marrow

Inflamed Lymph nodes

2,5x1011 / day

Thoracic duct 0,3x1011 / day

Lymph nodes

Blood

T1/2 = 30 min 5x1011 / day

Inflamed tissue Post capillary veinule

Tonsils Peyer’s patches

Lung Liver Others

Memory lymphocytes Spleen

Bone marrow

Inflamed Lymph nodes

Blood

Inflamed tissue

2,5x1011 / day

Thoracic duct 0,3x1011 / day High

T1/2 = 30 min 5x1011 / day

endothelial veinule

Lymph nodes

Tonsils Peyer’s patches

TCM T CENTRAL MEMORY CELLS

Post capillary veinule

Lung Liver Others

TEM T EFFECTOR MEMORY CELLS

EXITING BLOOD 1. WHERE and HOW ? - adhesion to vessels - transmigration 2. TO GO WHERE ? - chemoattraction and guidance

Multistep model of leucocyteendothelium interaction Tethering Rolling Binding Firm arrest Crawling 4000 mM/s -> 40 mM/s

Transmigration

Initial contact Selectins, then integrins

Integrin activation via chemokines

Integrin binding then cross talk

Regulation occurs at multiple steps Selectin expression

Vessel permeability

Chemokine receptor expression

Chemokine production and GAG binding

Integrin activation and crosstalk

Integrin ligands expression

Tissue/ECM remodeling

Tethering and rolling

Capturing leukocytes Tethering

Rolling

4000 mM/s -> 40 mM/s

Selectin expression

Rolling on Selectins CD62-L, -P, -E Selectins

Selectin ligands

Function

L-selectin : most leucocytes

HEV: GlyCAM1 (PNad), CD34, podocalixin, …

Node homing

P and E selectins: activated endothelium

Activated leucocytes: PSGL1, ESL1, CD44

Tissue homing in inflammation

Tissue specific or Inflammation specific

Endothelial selectins P selectin Homodiameric sialomucin Resting endothelium -> stored in secretory granules (Weibel-Palade) Found in α granules in platelets Mobilized rapidly to the cell surface by histamine, TNFa, LPS, thrombin, C5a Predominant leukocyte rolling receptor on acutely inflamed endothelial cells E selectin Not constitutively expressed by vessels (except skin) Requires transcriptional induction by inflammatory mediators Non fully overlapping function P selectin

- predominant in rolling

E selectin

- involved in transition from slow rolling to firm arrest - cooperates with CXCR2

Selectin binding to ligands

Mechanism

Structure:

Low affinity interaction Localization on microvilli Catch bond (shear stress) Cleavage

Protein core modified by glycosylation (fucosylation), sialylation, sulfatation Ex: Fucosyl transferase deficiency = no lymph nodes

PSGL1 6-sulfo sialyl Lewis X (sLex)

FucT-VII upregulation in T cells

Capturing leukocytes A multicellular process Tethering

Leukocytes Circulating microparticles P selectin Chemoattractants (CCL5, CXCL4, CXCL5)

Platelets

Mast cells

Chemoattraction

Chemokines activate integrins and direct migration Rolling

Firm arrest

Chemokine classes Structure: 67-127 aa, only two membrane bound Classification according to N-terminal residues CXC mainly leucocyte chemokines CC predominantly lymphocyte chemokines XCL1 (lymphotactin) and XCL2 CX3CL1 fractalkine Conservation of the N-terminal region : 2 sites - receptor binding (ELR motif for CXCR1 and CXCR2 binding - cell/matrix adhesion site : binding to GAGs

Chemokine functions At the systemic level - Homeostatic: - developmentally controlled secretion, - control normal cell flow in lymphoid organs - low redundancy - Inflammatory: - inducible chemokines - recruit cells to inflammatory sites - high promiscuity - Dual function - relatively lymphocyte-specific

At the cell level - leukocyte chemoattraction - cell survival, effector responses, coactivation

Chemokines and their receptors

Specificity

Promiscuity

Chemokine recruitment TIME Leukocytes

Monocytes

Effector lymphocytes

IL-8, RANTES, MIP1a, MCP1

GAG synthesis (heparin, HS, DS, CS)

Inflammation

Regulation of chemokine activity Chemokine secretion is regulated by inflammatory stimuli: cytokines, TLR, Stress,… Chemokine half life and concentration are locally regulated by binding to cellular or ECM GAG which permit the establishment of relatively fixed concentration gradients Proteolytic degradation of chemokines can affect their receptor binding potential, generate antagonist peptides. Proteases such as CD26, MMPs or cathepsins have this property Natural chemokines may exert both agonist or antagonist effects Ex: CXCR3 (Th1 cells) agonists (CXCL9, 10, 11) are CCR3 (Th2 cells) antagonists

Chemokine signaling G Protein Coupled Receptor family (GPCR) - heterotrimeric G proteins - activated G proteins stimulate PI3K, PLCb, Src Kinases and regulate the recruitment of PH domain containing cytoskeletal proteins Desensitisation corresponds to receptor internalization followed either by degradation or recycling -> this process determines the « sensing » property of each receptor Receptor sensitivity to gradients within a narrow range of chemokine concentration

PI3K/PIP3 -> recruitment of effectors to the plasma membrane

Chemokine sensing in neutrophils Rear edge

Leading edge Integrin and Chemokine Receptor redistribution

Amplification loop Via GTPAses

PTEN

PIP3

Chemokine Concentration gradient Cell motion

PI3K

PI3Kg or PTEN mutant cells loose directional control PI3K PI(4,5)P2

PIP3 PTEN

Navigation is probably context dependent

Distinct Adaptor Modules ?

Chemokine secretion patterns Allergic asthma model

eosinophils lymphocytes

Temporal specificity

BAL

monocytes 8 MCP1,5 MDC MIP1a

15

21

Eotaxin RANTES

Days post OVA pulse

Chemokine interplay Ligand induced down regulation Promiscuous binding to receptors Redundancy: high with proinflammatory chemokines Control of cell directionality

Navigation in complex chemokine’s gradient Uniform concentration: - initially: maintain orientation - later: change orientation

Complex gradients: - stop at disorienting concentration of 1 gradient -change direction for a second agonist gradient

Opposing gradient: - cells goes against an attracting gradient

Chemokines and integrins Chemokines bound to endothelial proteoglycans (ex GROα) or secreted in blood (ex MCP1) GROa -> CXCR2 => conversion of rolling into firm arrest under flow MCP1 -> CCR2 => shape change and diapedesis on stimulated endothelium Chemokine signalling: Differential effects on integrins, further completed by integrin cross talk Inside out signalling: conformational changes and integrin clustering -> avidity changes Migration towards the interendothelial junction and diapedesis Multiple integrins ligands αLβ2 (LFA-1) -> ICAM-1 then JAM1 α4β1 -> VCAM-1 then fibronectin (splice variant)

Integrins ICAM-1 JAM

E cadherin

MADCAM1 VCAM-1

Affinity and avidity

Rolling on integrins α4β1, α4β7, αLβ2 • Required for transition from fast to slow rolling • Intermediate affinity state ? • Ligands on endothelial cells

α4β1 α4β7 αLβ2

VCAM1 MadCAM1 ICAM1

Natalizumab Efalizumab

Leukocyte adhesion deficiency • LADI: deficit in expression / function of beta2 integrin • LADII: deficit in sialyl Lewisx expression (GDP fucose transporter) • LADIII: deficit in integrin activation

Transmigration

Mac1/ICAM1 dependent crawling

Adhesive haptotactic gradient -> further cell or signal specificity

Different modes of migration • Mesenchymal migration: leading edge, polarized adhesion and proteolysis (UPA), cell contraction (myosin) • Cluster/cohort migration: clustered cells bind via cadherins and communicate via GAP junctions -> mesenchymal migration • Ameboid migration: less dependent on integrin, no proteolysis, cell shape dependent

Specificity A combinatorial code

Chemokine receptors

Intra-thymic migration Thymocyte migration CCL25

DP

CCR9+ CCL21

SP

CCR7+

CD40 CD80

Entering lymphoid organs Homeostatic behavior

Lymph node overview Antigen Dendritic cells

Cytokines Chemokines Afferent Lymph vessels

Zone B

Zone B

Zone B

HEV T cell zone

medulla

Subcapsular sinus

Zone B

Blood vessel

T cell entry in node : HEV

Lymph node homing

L-selectin

CCR7

BLOOD CD34 CCL21

HEV NODE

CCL19

Chemokine’s expression in lymph nodes

Dual receptor signalling by CCL19 and CCL21

T ly m phom a

Binding Desensitization

CCL19

CCL21

+++

+++

+++

+/-

DC lines CCL19

CCL21

chem otax is

+++

+++

Dendrites form ation

+++

Desensitization

-

*

+/+

•Concentration where chemotaxis is impaired

DC’s dendrites, like tentacle catch T cells

Benvenuti et al. Science 2004, 305:1150

Antigen Dendritic cells

Cytokines Chemokines

Afferent Lymph vessels

Zone B

Zone B

Tolerant DC Subcapsular sinus What determine B homing of DCs Zone Specific T cells

T cell zone

Non specific Functional consequence for theT Tcells cells

Zone B

Blood vessel

medulla

T and B cells homing into LN B zone B zone CCR7

CXCR4

CXCL13

B CXCR5 HEV

T CCR7 CXCR4

CCR7 -> CCL21/19 CXCR4 -> CXCL12 CXCR5 -> CXCL13

T zone CCL21/19

Temporal requirements in lymph node Antigen Dendritic cells

Cytokines Chemokines Afferent Lymph vessels

Zone B

Zone B

HEV Meeting point: Zone B - Information on immunological status T cell zone of non lymphoid tissues - T/DC interactions - T/B cell interactions - staying time: 72h medulla

Subcapsular sinus

Zone B

Homeostasis: - survival signals - staying time: 12-24h Blood vessel

Effector cells Relative specialization of CR expression Th1, Tc1: CXCR3, CXCR6, CCR5 Th2, Tc2: CCR3, CCR4, CCR8 Inflammatory chemokines (such as CCL5 / RANTES) attract both Th1 and Th2 cells via CCR5 and CCR3 Fostering specialized responses via selective recruitment Ex: eotaxin attracts eosinophils and Th2 cells on allergic sites

Homeostatic chemokine receptors in lymphocyte differentiation

Chemokine’s receptor modulation following activation CCR7 CXCR4

CXCR3 CCR5

Naive: CCR7 CXCR4

D=1-2: CCR7 CXCR4 CXCR3

IL12

CCR4

IL4 CCR7 CXCR4

CXCR3

CCR7 CXCR4 CXCR3 CXCR5

CCR4

Exiting lymphoid organs HOW ?

The immunosuppressive agent FTY720

- Identified in skin allograft rejection rat models - Known to specifically sequester lymphocytes from blood - Normal antigen-specific responses - Active to prevent collagen-induced arthritis, nephrotic syndrom in Lpr mice, and extrinsic allergic encephalitis

Lysophospholipids

Plateletderived

Non hydrolysable analog

S1P • Present in plasma (100-300 nM) • Bound to albumin and other proteins (HDL) • Affect different cell functions but predominant effect on chemotaxis • Bind GPCR expressed by hematopoietic cells • Concentration gradients (3-30 nM) optimally chemotactic, promote cell emigration; inhibitory at higher doses

Lysophospholipid receptors

- Expression by endothelial cells, smooth muscle cells, myocardium Putative markers of neoplasia - Activity of PTX-sensitive GPCR

Cellular GPCRs for immunoregulatory LPLs

FTY720 is a prodrug Pharmacokinetics

Cell activity

Sphingosin-1P analog

FTY720 is lymphopenic

FTY720 is active on the efferent draining lymph

=> Active sequestration in lymph nodes, Peyer’s patches, but not in spleen

Control animals

Medullary Lymph node sinuses

T / B zones

FTY720-treated animals

Lymphocyte egress

Lymphocyte retention

FTY720 effect on node lymphocyte egress Science 2002

FTY720 effect on antigen driven T cell activation Lymph nodes

Blood

Division rate

Egress rate

Biological effect of FTY720

Tissue specific homing

A combinatorial code

Gut homing Naive > memory L-selectin α4β7 CCR7

CCR9

BLOOD MadCAM-1 CCL21

HEV

CCL19 CCL25

PEYER’s PATCH

IEL αEβ7

Gut DC E-cadherin

Imprinting α4β7 CCR9 (TECK signalling)

CD8

Homing to gut Peyer’s patch DC

Pathways to gut mucosa

Territorial behavior: gut versus skin

CCL27, CCL17

Redundancy of chemokine receptor binding may have biological importance

CCL17

CCL22

Sticking

++++

++++

Rolling

++

++++

Ca flux

++

++++

desensitization downmodulation

+

++++

Dissociation

slow

fast

T CCR4 CCL17 Endothelium Skin

CCL22

D’Ambrosio et al. JI 2002 169:2303

Lymphoid organogenesis Deficiency in CXCR5 or CXCL13 -> lack of peripheral lymph nodes and Peyer’s patches -> disorganized splenic architecture (no T / B zones) Similar to lymphotoxin (LT) or LT-R deficiency

Peyer’s Patch organogenesis α4β1 α4β1

BLOOD

Amplification loop

RORγ α4β7

CXCR5

MadCAM-1

CXCL13 CCL19

IL-7R IL-7

PEYER’s PATCH ANLAGEN

α4β1

LT

VCAM-1 RelB

LT-R

Lymph node organo- and neogenesis

LT

RANK-L

CXCL13 CCL19 CCL21 LT-R

Neogenesis Of lymph nodes In ectopic tissues By transgenic Chemokine expression

RANK

Relevance in autoimmunity And cancer

Metastasis - Uncontrolled growth - Uncontrolled tissue organisation (loss of boundaries, tumor cell plasticity) - Neo vascularisation (ischemia) - Extravasation - Domiciliation (seed and soil hypothesis) -> specific adhesion / chemoattraction -> privileged growth microenvironment -> enhanced migration (MMP) - Premetastatic niche

Figure 1. Top row, HPCs from bone marrow niche to premetastatic niche

Kaplan, R. N. et al. Cancer Res 2006;66:11089-11093

Copyright ©2006 American Association for Cancer Research

Tissue homeostasis: who are you and where are you ? Maintenance (cell polarity, adhesion, tightness)

Ephrins

Cadherins

Migration

Stem Segregation

Integrins

MMP

Chemokines

Tumor cell plasticity Epithelial -> Mesenchymal phenotypes Cadherin / Wnt / MMP

Vascular transmigration K

Acquisition of molecular triggers: chemokine R, CAM

Vascular mimicry ? Acquisition of molecular adresses Ephrins, VE-cadherins, …

Chemokines can affect cell growth CXCR2 homologous to Karposi’s sarcoma herpes virus-GPCR Constitutive signalling of KSHV-GPCR, augmented by CXCL8 Overexpression leads to sarcoma-like lesion Mutation of CXCR2 induces cell transformation Autocrine function via CXCR2 in some cell types

Chemokines in breast cancer Overexpression of CXCR4 and CCR7 by breast cancer CXCL12 (CXCR4-L) and CCL21 (CCR7-L) highly expressed in favorite metastatic sites Signalling via CXCR4 and CCR7 triggers cancer cell motility Neutralization of CXCL12-CXCR4 interaction blocks metastatic homing Similar situation with CCR10 expression by melanoma confering attraction to CCL27-secreting skin

Chemokines: Not just leukocyte chemoattractants in the promotion of cancer Strieter, R. M. Nature Immunology 2, 285 - 286 (2001)

Chemokines and anti-tumor responses Inflammation: neovascularisation and tissue remodelling versus attraction of immune cells Specific chemokine release -> attraction of dendritic cells and lymphocytes Combined immunotherapy: chemokine + cytokine + tumor antigen

Myeloma + transfected fibroblast (XCL1 + IL-2) -> tumor regression Breast K + Ad Virus delivery of XCL1 + IL-12 -> LT, NK, DC

From lymphocytes to lymphomas • T lymphomas derived from memory T cells – Intestinal T cell lymphoma (α4β7, αEβ7) – Cutaneous T cell lymphoma (CLA+, CCR4+) -> skin • Mycosis fungoides: skin restricted (CLA, CCR4) • Sezary syndrome: skin and node (L sel, CLA, CCR7, CCR4)

• B lymphomas – Mantle cell lymphomas (naive B derived) – Marginal zone B cell lymphomas of MALT (chronic activation) -> α4β7 – Follicular lymphomas – Myelomas -> bone marrow