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 Organs weight and number of lymphocytes were determined CTLA-4 -/ D: Thymus E: Spleen F: Myocardium G: Lung Waterhouse et al., 1995

T-cell fate after TCR engagement

Alegre et al., 2001 Sharpe & Freeman, 2002

Ligand-independent CTLA-4 function

Inhibition Stimulation Stimulation Chikuma & Bluestone, 2003.

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

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

Cell-intrinsic factors of CLTA-4 regulation

↓ TCR ζ-chain ↓ LAT, SLP76 and GADS adaptors Rudd, 2008

Inhibition of T-cell raft signaling

Chikuma & Bluestone, 2003.

Could extrinsic-factors also be responsible for CTLA-4 function?

WT CTLA-4 -/ CTLA 4 +/+ CTLA-4 -/ Bone marrow adoptivelly transfered Rag2 -/ Rag2 -/-

Liver Heart

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

Tumor cell

Autoimmune diseases Egen et al., 2002 Nature immunology

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

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.

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

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

Tissue infiltrating T cells from Ctla4−/− mice are antigen specificity DOβCtla4+/+ DOβCtla4+/− DOβCtla4−/− CD4+ T cells (5 × 10 5 cells) isolated from various tissues Recipient mice Rag2−/− 3 weeks Pattern of migration and Population expansion 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 DOβCtla4+/+ DOβCtla4+/– DOβCtla4−/− Spleen Lungs Pancreas CD4+ T cells (5 × 105 cells) purified Rag2−/− 3 weeks HISTOLOGY (HE) Spleen Lungs Pancreas intense tissue destructive infiltration perivascular infiltration and epithelial changes in the lungs tissue-destructive lesions of the exocrine pancreas Tissue infiltrating T cells from DOβCtla4−/− mice cause tissue-specific inflammation

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

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

TCRα chains derived from pancreas-infiltrating of Ctla4−/− T cells confer selective pancreatic accumulation.

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

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.

Ctla4+/+ Ctla4−/− 20-day-old T cells (1 × 10 5 cells) pancreatic lymph nodes Activated the cells

in vitro

Serum ELISA anti-PDIA2 titers PDIA2 (protein disulfide isomerase –associated 2), an acinar-specific enzyme PDIA2 (10 µM/ 24 h) + irradiated splenocytes (5 × 10 5 cells) ELISA IL-2 Concentrations in supernatants PDIA2 seems to be an authentic autoantigen in Ctla4−/− mice.

Isolation of PDIA2 specific TCRs from TCRα library.

DOβCtla4−/− CD4+ T cell hybridoma CONTROL

Hybridomas expressing the TCRα library responded to anti-CD3 but not OVA 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.

Induction of GFP 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.

To examine how CTLA-4 regulates autoreactive T cells in vivo PDIA2 specific Ctla4−/− T cells infiltrate the pancreas.

DO11.10 Rag2−/−Ctla4+/+ DO11.10 Rag2−/−Ctla4−/− Retrovirus infected 29TCRα Thy-1.1

CD4+ T cells (1 × 10 6 ) Rag2−/− 3 weeks 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

DO11.10 Rag2−/−Ctla4+/+ DO11.10 Rag2−/−Ctla4−/− Retrovirus infected 29TCRα Thy-1.1

CD4+ T cells (1 × 10 6 ) Rag2−/− 3 weeks HISTOLOGY (HE) inguinal lymph nodes pancreatic lymph nodes pancreas lungs 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.

Cell-intrinsic mechanism

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 CD4+CD62LhiCD25+ Treg cells (Ctla4+/+ or Ctla4−/−) Rag2−/− 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?

BafA

CHO CTLA-4 + CHO CD86-GFP 3h Confocal Microscopy CHO CTLA-4 + CHO CD86 – -

Blue Green (GFP)

Time course of CD86 acquisition… BafA

CHO CTLA-4 + CHO CD86-GFP

It's suggested transfer and degradation of CD86 into CTLA-4 + cells.

C-terminus of CTLA-4 is required for endocytosis?

BafA

CHO CTLA-4 + CHO CTLA-4 + del36 CHO CD86-GFP 2h Confocal Microscopy CD4 + CD25 T cell 5 days CFSE

By trans-endocytosis, CTLA-4 removes CD86 from neighboring cells, resulting in impaired T cell proliferation

CTLA-4 in human CD4 + T cell are also able to capture CD86?

PBMC CD4 + CD25 T cell CTLA-4 transfected

Anti-CD3

72 h Confocal Microscopy

CTLA-4 mediated trans-endocytosis was specific to CD80 and CD86

Does TCR stimulation enhances the CD86 acquisition?

PBMC

staphylococcal enterotoxin B (SEB)

6 days SEB CD4 + T cell Dcs SEB pulsed cells 72 h Confocal Microscopy

TCR stimulation increased the acquisition of CD86

CD4 + CD25 + T cells (T reg cells) are able to acquire CD86 from APCs?

Anti-CTLA-4

PBMC

CD4 + CD25 + T cell

CD4 + CD25 -

CD4 + CD25 +

T cell

T cell Anti-CD3

Confocal Microscopy CD4 + CD25 T cell CFSE Flow cytometry

Depletion of co-stimulatory molecules by CTLA-4 has functional consequences

The removal and degradation of CD80 and CD86 from APCs by CTLA-4 also take place in vivo?

Rag2 -/ CD86-GFP OVA OVA chloroquine 6h Confocal Microscopy Rag2 -/ Rag2 -/ DO11.10 T cell CTLA-4 +/+ CTLA-4 -/-

CONCLUSION