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Distinct functions of CTLA-4 at different stages of immunity

Distinct functions of CTLA-4 at different stages of immunity. Lab meeting 09/22/09 Melanie Stumpf. Working hypothesis. different portions of the receptor have distinct functions in regulating homeostasis vs T cell activation/tolerance. Background. CTLA-4 function:

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Distinct functions of CTLA-4 at different stages of immunity

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  1. Distinct functions of CTLA-4 at different stages of immunity • Lab meeting • 09/22/09 • Melanie Stumpf

  2. Working hypothesis different portions of the receptor have distinct functions in regulating homeostasis vs T cell activation/tolerance Background • CTLA-4 function: • - negative regulator that terminates TCR signaling upon antigen stimulation • - mediated by engagement with its ligands B7-1/2 • - based on dephosphorylation of TCR via phosphatases associated with ITEM motif • - primary receptor controlling T cell tolerance • - important for homeostatic proliferation of naive T cells • CTLA-4 isoforms: • Full length: - expressed in Tregs • - highly upregulated in all T cell subsets upon activation • liCTLA-4: - constitutively expressed in naïve T cells; diminishes during T-cell activation • - “expressed in memory/regulatory cell populations (CD45RB low)” • - linked to T1D in NOD mice, express only 20% compared to B6 Novel CTLA-4 mutant mice: 1) NOD. CTLA-4KO.BAC-Tg: B6.CTLA-4 floxed Ex2 2) B6. CTLA-4-Ex4-KI (Y201V)

  3. CD4 CTLA-4 CTLA-4 FoxP3 CD44 CD44 NOD. CTLA-4KO.BAC-Tg mice do not die, indicating successful transgene expression Primary total LN ( males ~ 4 month) CTLA-4WT BAC Tg- CD62L CTLA-4 KO BAC Tg+ NOD.CTLA-4KO.BAC-Tg • Chikuma et al. 2005: Tetracyclin induced expression of a transgenic PYAA mutant form • - delayed LPD lethality • - reduced T cell proliferation, IFN-g production, TCR-mediated ERK phosphorylation • Role of liCTLA-4 in.. • ... LPD development • ... Tregs • ... T1D in NOD mice

  4. - Expectation: reduction to ~ 50% (like in Idd5.1 congenics) compared to 80% in NOD - but BAC-Tg contains 20kb vs Idd5.1 locus with 2000kb Diabetes incidence in NOD.BAC-Tg mice

  5. ligand independent CTLA-4 full length CTLA-4 EX2 EX3 EX4 EX1 EX3 EX4 EX1 Expression profile of fl and li CTLA-4 isoforms

  6. Expression profile of fl and li CTLA-4 isoforms CTLA-4 protein expression 1 2 3 4 1 2 3 4 1: NOD SP+LN (d3 activated ) 37kDa 2: NOD T1D SP+LN GAPDH 3: CTLA-4KO_B7.1-2KO SP+LN (d3 activated ) Full length CTLA-4 25kDa 4: empty 10kDa Ligand independent CTLA-4 Mouse a-GAPDH Santa Cruz Rabbit a-CTLA4 C-term “HANA” Shunsuke

  7. Analysis of B6.CTLA-4-Ex4KI (Y201V) mice • CD2/LCK driven transgene expressing a Y201V mutant (Masteller et al. 2000, Yi et al. 2004) • - higher CTLA-4 surface expression • - Th2 phenotype • - slightly activated phenotype of CD4 T cells • - delayed LPD • - no effect on Treg suppressive function in vitro

  8. CTLA-4 CTLA-4 CD4 CTLA-4 Foxp3 Primary SP cells ( mice 5 month) surface intracellular CTLA-4WT CTLA-4 HET CTLA-4 KI Analysis of B6.CTLA-4-Ex4KI (Y201V) mice

  9. CTLA-4 CTLA-4 CD4 Primary SP cells ( mice 3 month) surface intracellular CTLA-4WT CTLA-4 KI Analysis of B6.CTLA-4-Ex4KI (Y201V) mice

  10. CD4+ at d7 CD4+ at d3 CD8+ at d3 w/o stim. with stim. w/o stim. CD8+ at d7 with stim. with stim. with stim. 26.99 20.35 33.68 3.86 83.54 89.82 CTLA-4WT 21.87 14.07 18.77 3.16 60.98 53.03 CTLA-4 HET 11.14 8.98 12.75 32.49 42.94 2.09 CTLA-4 KI PKH PKH PKH Analysis of B6.CTLA-4-Ex4KI (Y201V) mice Total SP and LN cells: - PKH labeling - w or w/o 2 µM a-CD3 + 200U IL2 - assay proliferation at d3 and d7

  11. IFN-g IFN-g IL4 ELISA Purified CD4+ T cells: - 2 µM a-CD28/a-CD3 + 200U IL2 At d3: - PMA/Ionomycin/Monensin 4.5h 82.82 CTLA-4WT 10.32 81.6 CTLA-4 KI 18.2 Analysis of B6.CTLA-4-Ex4KI (Y201V) mice

  12. Spontaneous disease onset in EAE model B6.CTLA-4-Ex4KI. Vb11-Tg mice

  13. Teff Treg CTLA-4 WT Teff Treg Teff CTLA-4 Y201V Treg CTLA-4 Y201V MOG-Tg

  14. Analyze Treg function in Y201V mice: Do Tregs require the IC portion of the receptor to suppress? Phospho-flow to address TCR downstream signaling Analyze Treg function in EAE model: Treatment with MOG peptide in combination with antibody therapy to deplete Tregs • Adoptive transfer into B6.RAG-KO: WT Teff + WT Tregs • WT Teff + KI Tregs • KI Teff + WT Tregs • KI Teff + KI Tregs Ongoing and future perspectives Treatment with a-CD69, a-CD8 or a-CD25 Abs to selectively deplete Teff or Tregs

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