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

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

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

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

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

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

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

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

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

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

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

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

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

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

Daiani Alves

Patrícia Assis

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

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

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

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)

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

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.

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

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+

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

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

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

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.

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

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 weeks Rag2−/− CD4+ T cells (1 × 10 6 ) Cell-intrinsic mechanism

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

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

Conclusion

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

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

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

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

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

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

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

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 -/-

CONCLUSION