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

2. 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)”

3. 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 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?

4. 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)

5. 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

6. 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

7. 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

8. 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)

9. 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+

10. 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

11. Thanks