Distinct functions of CTLA-4 at different stages of immunity Joint lab meeting 06/02/09 Melanie Stumpf

Background CTLA-4 molecule: - described as a negative regulator upon T cell activation (Walnut et al., 1994) - terminates TCR signaling by inhibiting cytokine production and cell proliferation CTLA-4 and disease: - Mutations in humans are associated with: T1D, RA, Graves’ disease, SLE, MS (general susceptibility to autoimmune diseases) - Deficiency in mice: severe LPD and multiorgan autoimmunity (die ~ 3-5 weeks after birth) Expression: Full length: - intracellular in Tregs; upregulated in all T cell subsets upon activation liCTLA-4: - high intracellular levels in naïve T cells; diminishes during T-cell activation - “expressed in memory/regulatory cell populations (CD45RB low)”

Goals of the project and working hypotheses Role of CTLA-4 in T-cell homeostasis vs immune activation and tolerance induction controlled by distinct extrinsic vs intrinsic functions of the different CTLA-4 isoforms 1) CTLA-4 acts upon initial T cell signaling as homeostatic regulator on naive T cells - controlling tonic stimulation by self-MHC---raising the activation threshold Preferentially due to liCTLA-4 expression? Can liCTLA-4 prevent KO mice from LPD? Can liCTLA-4 prevent NOD mice form T1D? - liCTLA-4 Tg expression in KO background delays LPD - T cells only expressing liCTLA-4 display a higher activation threshold - Hph-1-ctCTLA-4 prevents/decreases T cell activation in allergic asthma and RA models - liCTLA-4 is genetically linked to T1D in NOD mice (SNPs in Idd5 locus) - blocking B7 binding: 1) exacerbates immunity after disease onset and/or reverses tolerance - engagement of CTLA-4 terminates response - in EAE and Colitis model---anti-CTLA-4 increases disease severity based on inhibition of Treg function - 2) CTLA-4 acts in terminating ongoing Ag-driven immune responses - depends on B7 engagement ---mediated by flCTLA-4 Differences in signal transduction between the B7-binding vs non-binding setting Do fl- and liCTLA-4 interact with different TCR complex components/associated proteins? What role plays the ICD in the context of homeostasis vs activation and tolerance induction? 3) CTLA-4 on regulatory cells limits autoreactivity and maintains tolerance - linked to flCTLA-4 ----receptor ligation is required for Treg function Examine Treg for expression of liCTLA-4 Do Tregs expressing only liCTLA-4 have supressive activity? Is there an intrinsic function of CTLA-4 in Tregs?

CTLA-4 transgenic/KI mouse models NOD. CTLA-4KO.BAC-Tg B6.CTLA-4 floxed Ex2 EX1 EX4 EX3 EX2 LoxP NEO B6. CTLA-4-Ex4-KI (Y201V)

NOD.BDC2.5+.CTLA-4KO.Ex2fl-BAC-Tg - liCTLA-4 Tg expression in KO background delays LPD - T cells only expressing liCTLA-4 display a higher activation threshold - Hph-1-ctCTLA-4 prevents/decreases T cell activation in allergic asthma and RA models - liCTLA-4 is genetically linked to T1D in NOD mice (SNPs in Idd5 locus) NOD. BAC-Tgs express both B6 fl and liCTLA-4 --> diabetes incidence of female NOD vs NOD.BAC-Tg+ Primary total LN ( males ~ 4 month) CTLA-4 KO BAC Tg+ CTLA-4WT BAC Tg- CD4 B220 CD8 CTLA-4 FoxP3 CD44 CD62L

Cre-mediated deletion of floxed EX2 in vitro CD4+ LN cells d0 antiCD28 antiCD3 IL2 d2 Ctrl-GFP CRE-GFP d4 FACS Sort GFP+ + IL2 only d7 CTLA-4 KO BAC Tg+ CTLA-4WT BAC Tg- GFP CTLA-4 Ctrl-GFP CRE-GFP day 4

FACS analysis of liCTLA-4 expressing activated T cells CD4+ LN cells d0 antiCD28 antiCD3 IL2 d2 Ctrl-GFP CRE-GFP d4 FACS Sort GFP+ + IL2 only d7 day 0 CTLA-4 KO BAC Tg+ CTLA-4WT BAC Tg- CD44 CD62L day 4 GFP+/CD4+ CD44 CD62L day 7 CD62L CTLA-4 GFP+/CD4+ injected into NOD.BDC2.5- 1x106 3x106

Ongoing and future perspectives Diabetes incidence in NOD.BAC-Tg mice: - Expectation: reduction to ~ 50% (like in Idd5.1 congenics) compared to 80% in NOD - but BAC-Tg contains only CTLA-4 and not ICOS (200kb vs Idd5.1 locus with 2000kb) - breeding BAC-Tg mice to FoxP3-Cre strain In vivo deletion of Ex2 in NOD.CTLA-4 KO: - breeding BAC-Tg mice to VAV-Cre strain (expressed in hematopoietic progenitors) Assays based on RV-Cre deletion of Ex2: - Cytokine production - secondary response - memory cell differentiation - Treg function using suppression assays - AT of activated CD4+ T cells together with p31-SP coupled APCs into NOD Biochemical and molecular-biology studies: - discriminate specific binding partners of the two isoforms - expression profile analysis (microarray)

B6. R26-YFP.OX40-Cre.CTLA4-Ex4-KI (Y201V) 2 publications using transgene with Lck-prom. and CD2 enhancer: 1) - Full rescue of CTLA-4 KO in Y201V mutant mice - Th2 biased phenotype - higher surface expression of Y201V-CTLA-4 2) - No full rescue of CTLA-4 KO in Y201V mice - mice develop LPD between 4-5 month of age CTLA-4 CD4 CTLA-4WT CTLA-4 Y201V Primary SP cells ( mice~ 5 month) CFSE CD69 CD62L CD44 CD25 pregated on CD4+

Ongoing and future perspectives Reproducing results of 5 month old mice: - PMA/Ionomycine stimulation ---> cytokine FACS/ELISA - anti-CD3+ IL2 ---> CFSE dilution assay Analyze younger mice in regard to identified phenotype Analyze Treg function AT or mixed BM chimera B71_2KO settings Biochemical approaches to identify changes that might affect downstream signaling

Thanks