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

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

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

4. T-cell fate after TCR engagement Sharpe & Freeman, 2002 Alegre et al., 2001

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

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

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 Rudd, 2008 SHP2  LAT and ERK dephosphorylation PP2A  AKT dephosphorylation CBL-B: E3 ligase (ubiquitylation pathway)

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

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

12. Could extrinsic-factors also be responsible for CTLA-4 function? CTLA- 4 +/+ CTLA-4 -/- Rag2 -/- WTCTLA-4 -/- Bone marrow adoptivelly transfered LiverHeart 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 Egen et al., 2002 Nature immunology Tumor cell Autoimmune diseases

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. Fixing the TCRβ chain prolonged but did not eliminate the disease in Ctla4−/− mice. 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 Was compared Ctla4−/− mice with Ctla4−/− mice expressing the DO11.10 TCRβ chain (DOβCtla4−/− mice) surviving 4 weeks of age 7 to 10 weeks of age

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

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

22. Tissue-infiltrating T cells from Ctla4−/− mice are antigen specificity Pattern of migration and Population expansion CD4+ T cells (5 × 10 5 cells) isolated from various tissues Recipient mice Rag2−/− DOβCtla4+/+ DOβCtla4+/− DOβCtla4−/− 3 weeks 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.

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

24. 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. DOβ Ctla4−/− CD4+ CYTOMETRY Analysis of lymphoid nonlymphoid tissues CD4+

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

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

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

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. DO11.10 Rag2−/−Ctla4+/+ DO11.10 Rag2−/−Ctla4−/− Retrovirus infected 29TCRα Thy-1.1 PDIA2-specific Ctla4−/− T cells infiltrate the pancreas. 3 weeks Rag2−/− CD4+ T cells (1 × 10 6 ) CYTOMETRY Thy-1.1+ Inguinal lymph nodes Pancreatic lymph nodes Pancreas Lungs Have the pancreatic accumulation. Infiltration of the pancreas itself was greatly affected by the presence of CTLA-4 To examine how CTLA-4 regulates autoreactive T cells in vivo

30. 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 T cells diminishes pathogenicity by inhibiting their infiltration into target tissues. HISTOLOGY (HE) inguinal lymph nodes pancreatic lymph nodes pancreas lungs DO11.10 Rag2−/−Ctla4+/+ DO11.10 Rag2−/−Ctla4−/− Retrovirus infected 29TCRα Thy-1.1 3 weeks Rag2−/− CD4+ T cells (1 × 10 6 ) Cell-intrinsic mechanism

31. Test if CTLA-4 expression by Treg cells is required for their suppressive activity for self antigen–specific T cells Rag2−/− Ctla4−/− 29TCRα+ DO11.10 cells CD4+CD62LhiCD25+ Treg cells (Ctla4+/+ or Ctla4−/−) CYTOMETRY Thy 1.1+ Measured PDIA2-specific T cells Pancreatic lymph nodes Pancreatic 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? CHO CTLA-4 + CHO CD86-GFP Flow cytometry BafA 3h Confocal Microscopy CHO CTLA-4 + - Blue CHO CD86 – Green (GFP)

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

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

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

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

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

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

43. CONCLUSION