Dendritic Cells in T Cell Migration and Tolerance Induction. Dimitris Skokos The Rockefeller University

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1 Dendritic Cells in T Cell Migration and Tolerance Induction Dimitris Skokos The Rockefeller University

2 infection mature DCs an epithilium with dendritic cells Immature DCs steady state Regulatory T cells, T cell deletion, anergy Steinman & Nussenzweig, PNAS, 99, 2002, 351

3 Microbial stimuli Immature DC Capture of antigens MHC II lysosomes Mature DC Stimulation of T cell immunity absorvative uptake, eg, DEC-205, FcR macropinocytosis phagocytosis: microbes,dying cells CD40, CD86 CCR7 IL-12 High-MHC - peptide Trombetta and Mellman, Annu. Rev. Immunol. 2005, 23,975

4 T cell tolerance induction upon αdec-ova targeting recombinant antibody production αdec Ab Ova T cell deletion by αdec-ova -1d OTII T cell Transfer (CFSE) PBS αdec-ova αdec-ova + αcd40 d3 B10.BR d0 αdec-ova +/-αcd40 Injection d3/7 CD4, Vα2 FACS-Analysis Vα2 CFSE gated: CD4 + d7 Hawiger D. et al. JEM 2001 Shakhar G. et al. Nat Immunol 2005

5 Have DCs the same or different effect on T cell migration under tolerogenic versus immunogenic conditions? In vivo imaging set-up and analysis

6 Selection criteria: blood flow, DCs and T cell-internal control motility CD11c-EYFP WT T-CFP Blood-qDots QuickTime and a Video decompressor are needed to see this picture.

7 Intra-vital imaging protocol αdec-ova +/- αcd40 OTII T-GFP WT-CFP d d h 6h 12h Intravital TPLSM

8 Early antigen-dependent T cell Arrest αdec-ova +/- αcd40,1-6h EYFP-DC CFP-T GFP-OTII QuickTime and a MPEG-4 Video decompressor are needed to see this picture.

9 Antigen - dependent T cell arrest on dendritic cells of both primed and tolerized T Cells * p<0.001

10 Summary (I) Dendritic cells induce T cell tolerance under steady state conditions. In contrast, immunity ensues if a maturation stimulus is given to the antigentargeted mice Upon encountering high affinity peptide-mhc complexes (pmhc) in vivo, T cells arrest and maintain contact with DCs for several hours in both tolerogenic and immunogenic conditions

11 What is the mechanism that controls T cell stopping and is it related to T cell tolerance induction?

12 Different factors play a role in determining the fate of a T cell response DC MHCII TCR T activation state of T cell antigen dose peptide presence or absence of co-stimulatory molecules duration of TCR triggering MHC:Peptide (pmhc)-tcr affinity (potency)

13 Model: moth cytochrome c (mcc) NH 2 - V F A G L K K A N E R A D L I A Y L K Q A T K -COOH (Schwartz R.H, JI 1987, Davis M.M, JI 1994) altered peptide ligand (APL) synthesis of APLs sharing similar affinity for the I-E k molecule and different affinity for the TCR. (Croft M, JI 1998, Bottomly K, JI 1997) System: establishing an in vivo model to study the role of antigen-tcr affinity by targeting DCs in situ with the different peptide variants (APLs) recombinant antibody production αdec-apl mcc

14 Effect of different APL on T cell activation Protocol Co-culture: Irradiated CD11c + DCs + AND CD4 + T cells + variant dose of APL Cpm (x 10 3 ) Proliferation assay WT L98A T102L Y97K Number of spot / CD4 + T INF-γ producing cells Elispot assay WT L98A T102L Y97K antigen peptide dose (µg/ml)

15 Activation of AND T cells in vivo by α-dec-apls -1d AND T cell Transfer (CFSE) B10.BR d0 αdec-apls Injection d3/7, 3h CD4, Vα11, Vβ3 FACS-Analysis αdec/iso-mcc αdec-ova Low Medium High activation markers proliferation 3h 72h d3 d7 CD62LCD69 CD62L CFSE

16 Antigen specific T cells are not deleted, do they become anergic? Peptide re-stimulation protocol AND CD4 + T cells AND CD4 + T cells - - αdec-apls CFA/mcc PBS d0 group 1 group 2 d0 αdec-apls d3/7 T cell isolation CD11c + + peptide re-stimulation d7 CFA/mcc d10 T cell isolation CD11c + + peptide re-stimulation

17 AND T cells become anergic Proliferation assay Day 3 Day 7 Day 10 PBS PBS Low Medium Native mcc High CFA/mcc Elispot assay Proliferation index Re-challenge with CFA/mcc PBS PBS Low Medium Native mcc High CFA/mcc INF-γ production /10 6 CD4 + T cells group 1 Re-challenge with CFA/mcc group 2

18 Summary (II) established an in vivo model to study the role of antigen-tcr affinity interaction in T cell migration and tolerance by targeting DCs in situ with the different peptide variants (APLs) under steady state conditions a peptide analogue that is unable to stimulate clonal proliferation or cytokine production can induce profound T cell unresponsiveness to systemic rechallenge with the immunogenic peptide (mcc) in CFA thus, proliferation is not required to induce T cell anergy in equilibrium conditions

19 How pmhc-tcr affinity governs T cell migration in vivo? In vivo T cell migration includes: trafficking between lymph nodes T cell movement within T cell zones JG Cyster Annu Rev. Immunol. 2005, :127

20 High and low affinity pmhc induce transient retention of antigenreactive T cells within Lymph nodes in vivo T cell retention in the lymph node -5h AND(CFSE) WT (CMRA) T cell Transfer B10.BR d0 αdec-apls Injection Blood collection every 24h FACS-Analysis Ratio of AND/WT CD4+ T cells in the blood PBS αdec-t102l αdec-y97k αdec-l98a PBS αdec-t102l αdec-y97k αdec-l98a (h) 24h 48h 72h CD69

21 Intra-vital imaging protocol αdec-apls AND T-GFP WT-CFP h h h 6h 12h Intravital TPLSM

22 TCR-pMHC affinity control dynamic interactions in vivo Speed (percent of control) Displacement rate (µm/min) Low medium High Low medium high 1-6h 6-12h Speed (percent of control) Confinement index Low medium high Low medium high Low medium high 1-6h 6-12h 1-6h 6-12h Low medium High no specific (WT-CFP) specific (AND-GFP)

23 Summary (III) high, medium and low affinity pmhc induce transient retention of antigen-reactive T cells within Lymph nodes Only high affinity peptide-tcr interaction dictates early-antigen T cell arrest in vivo. Thus, TCR-peptide affinity controls T cell arrest in vivo

24 What is the mechanism that controls T cell arrest? examine the relationship between TCR affinity, Ca 2+ signaling and T cell motility Protocol time image analysis Glass supported lipid bilayers containing: pmhc + ICAM molecules Ca 2+ measurement, export of statistics and analysis (excel) AND T cell blasts loaded with Fura-2 (ratio-metric Ca 2+ dye) Lipid bilayer ICAM I-E k :APL (pmhc)

25 Potency-dependent T cell arrest is mediated by Ca 2+ flux High pmhc affinity High Ca 2+ Low Ca 2+ Low pmhc affinity QuickTime and a Animation decompressor are needed to see this picture. QuickTime and a Animation decompressor are needed to see this picture. QuickTime and a Animation decompressor are needed to see this picture. QuickTime and a Animation decompressor are needed to see this picture.

26 Summary (IV) High Ca 2+ flux is not required to induce T cell retention within the lymph nodes Antigen specific T cells exposed to high affinity peptide in vitro increase calcium flux and become immobile. Low potency peptide-tcr interaction do not cause any calcium flux or cessation of T cell movement Is Ca 2+ signal enough to control T cell arrest?

27 Protocol for transmigration assay Filter coated with ICAM and a dose range of MHC-APL molecules Trans-well plate 2h incubation The number of AND T cells at the bottom of the well was measured by facs I-E k :APL ICAM AND T cell blasts

28 Transmigration Assay Inhibition of transmigration (%) High Medium Low β2m MSA Inhibition of transmigration (%) I-E k /L98A (High) (70 ng/ml) I-E k /peptide (ng/ml) 0 - K + Ba Ba+K +

29 Summary (V) Cytoplasmic calcium elevation is necessary for T cells to stop migrating upon encounter of high affinity MHC:peptide Ca 2+ can come from either cytoplsamic stores or influx from the extracellular space

30 Intra-vital Ca 2+ imaging protocol αdec-apls AND T (CMRA) Fluo 4-loaded, Ca sensitive dye h d 5min 90min Confocal

31 Visualizing Ca +2 flux upon antigen-tcr affinity interaction AND-CMRA Ca+2-Fluo4 Dextran-A647 Medium Low QuickTime and a Cinepak decompressor are needed to see this picture. QuickTime and a Cinepak decompressor are needed to see this picture. QuickTime and a Cinepak decompressor are needed to see this picture. * HEV e cells, have been counted

32 Summary (VI) only AND T cells from mice injected with high affinity pmhc exhibited an instantaneous increase in intracellular Ca 2+ a significant reduction in average velocity of AND T cells in mice injected with high affinity pmhc is correlated with Ca 2+ increase

33 Is there a Ca +2 -independent pathway of anergy induction? low affinity peptide induces anergy without T cell arrest in vivo and without increasing cytoplasmic Ca 2+ in vitro or in vivo anergy induction mechanisms utilize Ca 2+ and NFAT mediated signaling (R. Schwartz; A. Rao; V.M. Dixit).

34 Is there a Ca +2 -independent pathway of anergy induction? -5h AND T cell Transfer L B10.BR d0 αdec-apls Injection +/- CsA or L CsA d7 T cell isolation peptide re-stimulation Immunol. Rev. 2003, 192, 161

35 Anergy induction by low potency peptide is CsA resistant Day 7 CsA L PBS + + High + * + Medium + + * Low + + * Proliferation (cpm x 1000)

36 Conclusion high affinity pmhc induce Ca 2+ -dependent T cell arrest on DCs while inducing calcineurin dependent anergy lower affinity pmhc induces T cell retention within the lymph node without perturbing T cell dynamics. In addition, low affinity pmhc induces anergy via a biochemically distinct, calcineurin independent, process

37 Acknowledgements The Rockefeller University Skirball Insitute, NYU Michel C. Nussenzweig Randy Lindquist Daniel Hawiger Revati Masilamani Tanja Schwickert Michael L. Dustin Guy Shakhar Janelle Waite Rajat Varma Tom Cameron Ralph M. Steinman Sponsors: