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Daiani Alves Patrícia Assis Tiago Medina

New intrinsic- and extrinsic factors of CTLA-4 regulation. Daiani Alves Patrícia Assis Tiago Medina. CTLA-4 forms generated by splicing. Teff et al., 2006. CTLA-4: a negative regulator of T cell activation. WT. CTLA-4 -/-. After 4 weeks. Lymphonodes and spleen were removed. CTLA-4 -/-.

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Daiani Alves Patrícia Assis Tiago Medina

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  1. New intrinsic- and extrinsic factors of CTLA-4 regulation Daiani Alves Patrícia Assis Tiago Medina

  2. CTLA-4 forms generated by splicing Teff et al., 2006

  3. CTLA-4: a negative regulator of T cell activation WT CTLA-4-/- After 4 weeks Lymphonodes and spleen were removed CTLA-4-/- Organs weight and number of lymphocytes were determined D: Thymus E: Spleen F: Myocardium G: Lung Waterhouse et al., 1995

  4. T-cellfateafter TCR engagement Alegre et al., 2001 Sharpe & Freeman, 2002

  5. Ligand-independent CTLA-4 function Stimulation Inhibition Stimulation Chikuma & Bluestone, 2003.

  6. Inhibition of T cells by CTLA-4 regulation Nature immunology Egen et al., 2002

  7. CTLA-4 surface expression and its internalization Rudd et al., 2009

  8. CTLA-4 inducing prosurvival signaling pathways Rudd et al., 2009

  9. Cell-intrinsic factors of CLTA-4 regulation SHP2  LAT and ERK dephosphorylation PP2A  AKT dephosphorylation CBL-B: E3 ligase (ubiquitylation pathway) Rudd, 2008

  10. Cell-intrinsic factors of CLTA-4 regulation ↓ TCR ζ‑chain ↓ LAT, SLP76 and GADS adaptors Rudd, 2008

  11. Inhibition of T-cell raft signaling Chikuma & Bluestone, 2003.

  12. Could extrinsic-factors also be responsible for CTLA-4 function? CTLA-4+/+ WT CTLA-4-/- CTLA-4-/- Bone marrow adoptivelly transfered Rag2-/- Rag2-/- Liver Heart Backman et al., 1999

  13. Cell-extrinsic factors of CLTA-4 regulation Rudd, 2008

  14. The reverse stop-signal model for CTLA-4 Rudd, 2008

  15. Daiani Alves

  16. Patrícia Assis

  17. CTLA-4: clinical application Tumor cell Autoimmune diseases Nature immunology Egen et al., 2002

  18. Shows that hyperproliferative and destructive T cell populations in CTLA-4-deficient mice are not on autopilot but require specific signals provided by autoantigens to cause tissue damage

  19. Was compared Ctla4−/− mice with Ctla4−/− mice expressing the DO11.10 TCRβ chain (DOβCtla4−/− mice) surviving 7 to 10 weeks of age 4 weeks of age DO11.10 TCRβ - β Chain do TCR specific for OVA, presented by the MHC class II molecule and is expressed in 80% to 90% of T cells in the thymus of transgenic animals Fixing the TCRβ chain prolonged but did not eliminate the disease in Ctla4−/− mice.

  20. Examine the characteristics specificity of CD4+ T cells CYTOMETRY Naive T CD4+ cells (CD62L) Activated-memory T CD4+ cells (CD44) DOβCtla4+/+ DOβCtla4+/− DOβCtla4−/− (6-week-old) Splenic CD4+ T cells CD4+ T cells in DOβCtla4−/− mice had an activated surface phenotype

  21. Examine the tissue specificity of CD4+ T cells DOβCtla4+/+ DOβCtla4+/− DOβCtla4−/− (6-week-old) Histology HE Normal tissue histology Lymphocytic infiltration Fixation of the TCRβ chain in Ctla4−/− mice did not alter the multiorgan nature of disease in Ctla4−/− mice

  22. Tissue-infiltrating T cells from Ctla4−/− mice are antigen specificity DOβCtla4+/+ DOβCtla4+/− DOβCtla4−/− Splenic CD4+ T cell populations isolated from DOβCtla4−/− mice, showed expansion in vivo and migrated into many organs. T cells isolated from peripheral organs of DOβCtla4−/− mice accumulated selectively in their organ of origin. CD4+ T cells (5 × 105 cells) isolated from various tissues Recipient mice Rag2−/− 3 weeks Pattern of migration and Population expansion

  23. Selective migration of CD4+ T cells isolated from DOβCtla4−/− mice was associated histologically with the induction of tissue pathology DOβCtla4+/+ DOβCtla4+/– DOβCtla4−/− Spleen Lungs Pancreas CD4+ T cells (5 × 105 cells) purified intense tissue- destructive infiltration Rag2−/− perivascular infiltration and epithelial changes in the lungs 3 weeks tissue-destructive lesions of the exocrine pancreas HISTOLOGY (HE) Spleen Lungs Pancreas Tissue-infiltrating T cells from DOβCtla4−/− mice cause tissue-specific inflammation

  24. CD4+ DOβ Ctla4−/− CD4+ CYTOMETRY Analysis of lymphoid nonlymphoid tissues To distinguish if the tissue-specific accumulation of DOβCtla4−/− T cells could have been due to either reactivity to tissue-specific antigens or to selective homing properties ‘imprinted’ after tissue entry TCRα chains derived from pancreas-infiltrating of Ctla4−/− T cells confer selective pancreatic accumulation.

  25. minimal pancreatic disease exocrine-specific tissue destruction Infiltating of antigen-specific T cells cause tissue injury in the absence of CTLA-4.

  26. Nonobese diabetic mice deficient in the regulator AIRE show immune cell reactivity to pancreatic acinar cells DOβCtla4−/− mice showed acinar tissue–restricted autoimmunity... To determine if PDIA2 is an autoantigen in Ctla4−/− mice. PDIA2 (10 µM/ 24 h) + irradiated splenocytes (5 × 105 cells) ELISA IL-2 Concentrations in supernatants T cells (1 × 105 cells) pancreatic lymph nodes Activated the cells in vitro Ctla4+/+ Ctla4−/− 20-day-old ELISA anti-PDIA2 titers Serum PDIA2 (protein disulfide isomerase–associated 2), an acinar-specific enzyme PDIA2 seems to be an authentic autoantigen in Ctla4−/− mice.

  27. Isolation of PDIA2-specific TCRs from TCRα library. DOβCtla4−/− CD4+ T cell hybridoma Hybridomas expressing the TCRα library responded to anti-CD3 but not OVA CONTROL Was examined CD3+ hybridoma cells for TCR reactivity to various antigens • Cultured for 20 h • - medium alone • - anti-CD3 (1 µg/ml) • OVA(323–339) (0.3 µM) • + • Irradiated splenocytes. hybridomas expressing the TCRα library reacted to PDIA2 Induction of GFP

  28. To enriched PDIA2 reactivity, was isolated these hybridoma populations by sorting GFP+ cells after stimulation with PDIA2 Expression of the 29TCRα chain in the DOβ+ hybridomas regenerated PDIA2-specific reactivity (auto-antigen) in Ctla4−/− mice.

  29. To examine how CTLA-4 regulates autoreactive T cells in vivo PDIA2-specific Ctla4−/− T cells infiltrate the pancreas. CYTOMETRY Thy-1.1+ Inguinal lymph nodes Pancreatic lymph nodes Pancreas Lungs Retrovirus infected 29TCRα Thy-1.1 DO11.10 Rag2−/−Ctla4+/+ DO11.10 Rag2−/−Ctla4−/− CD4+ T cells (1 × 106) 3 weeks Rag2−/− Have the pancreatic accumulation. Infiltration of the pancreas itself was greatly affected by the presence of CTLA-4

  30. HISTOLOGY (HE) inguinal lymph nodes pancreatic lymph nodes pancreas lungs Retrovirus infected 29TCRα Thy-1.1 DO11.10 Rag2−/−Ctla4+/+ DO11.10 Rag2−/−Ctla4−/− CD4+ T cells (1 × 106) 3 weeks Rag2−/− The pancreatic infiltration was exocrine specific and was not present in the heart or lungs Together these results suggest that CTLA-4 on autoantigen-specific effector Tcells diminishes pathogenicity by inhibiting their infiltration into target tissues. Cell-intrinsic mechanism

  31. Test if CTLA-4 expression by Treg cells is required for their suppressive activity for self antigen–specific T cells Ctla4−/− 29TCRα+ DO11.10 cells CYTOMETRY Thy 1.1+ Measured PDIA2-specific T cells Pancreatic lymph nodes Pancreatic Rag2−/− CD4+CD62LhiCD25+ Treg cells (Ctla4+/+ or Ctla4−/−) Cotransfer of Ctla4+/+ Treg cells resulted in the infiltration of significantly fewer PDIA2-specific T cells into the pancreas

  32. Cotransfer of Ctla4+/+ Treg cells prevented the destruction of pancreatic tissue by Ctla4−/− PDIA2-specific T cells These results demonstrate that autoimmune responses by tissue-specific Ctla4−/− T cells can be regulated by CTLA-4-expressing Treg cells. Cell-extrinsic mechanism

  33. Conclusion

  34. Set a molecular explanation to CTLA-4 function compatible with a cell-extrinsic mechanism

  35. CTLA-4 could potentially deplete its ligands CD86? BafA Confocal Microscopy + Flow cytometry 3h CHO CTLA-4+ CHO CD86-GFP CHO CTLA-4+ - Blue CHO CD86 –Green (GFP)

  36. Time course of CD86 acquisition… BafA + CHO CTLA-4+ CHO CD86-GFP It's suggested transfer and degradation of CD86 into CTLA-4+ cells.

  37. C-terminus of CTLA-4 is required for endocytosis? Confocal Microscopy + BafA Flow cytometry + CFSE Anti-CD3 2h 5 days CHO CTLA-4+ CHO CTLA-4+ del36 CHO CD86-GFP CD4+ CD25- T cell By trans-endocytosis, CTLA-4 removes CD86 from neighboring cells, resulting in impaired T cell proliferation

  38. CTLA-4 in human CD4+ T cell are also able to capture CD86? Confocal Microscopy + Anti-CD3 PBMC 72 h CD4+CD25- T cell CTLA-4 transfected CD4+CD25- T cell CTLA-4 mediated trans-endocytosis was specific to CD80 and CD86

  39. Does TCR stimulation enhances the CD86 acquisition? PBMC Confocal Microscopy + staphylococcal enterotoxin B (SEB) + 72 h 6 days Dcs SEB pulsed cells SEB CD4+ T cell TCR stimulation increased the acquisition of CD86

  40. CD4+ CD25+ T cells (Treg cells) are able to acquire CD86 from APCs? Anti-CTLA-4 Confocal Microscopy + Anti-CD3 PBMC + + CFSE Flow cytometry CD4+CD25+ T cell CD4+CD25- T cell CD4+CD25- T cell CD4+CD25+ T cell Depletion of co-stimulatory molecules by CTLA-4 has functional consequences

  41. The removal and degradation of CD80 and CD86 from APCs by CTLA-4 also take place in vivo? Rag2-/- CD86-GFP OVA OVA chloroquine Confocal Microscopy 6h Rag2-/- Rag2-/- DO11.10 T cell DO11.10 T cell CTLA-4 +/+ CTLA-4 -/-

  42. CONCLUSION CTLA-4 CD86/ CD80

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