Daiani AlvesPatrícia AssisTiago 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

WT CTLA-4-/-

After 4 weeks

Lymphonodes and spleen were removed

Organs weight and number of

lymphocytes were determined

D: ThymusE: SpleenF: MyocardiumG: 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, 2003.

StimulationInhibition Stimulation

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-/- Rag2-/-

WT CTLA-4-/-

Bone marrow adoptivelly transfered

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

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

HistologyHE

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 × 105 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 × 105 cells) pancreatic lymph nodes

Activated the cells in vitro

PDIA2 (10 µM/ 24 h)+ irradiated splenocytes

(5 × 105 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 hybridomaCD4+ 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 × 106)

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-1.1 3 weeks Rag2−/−

CD4+ T cells (1 × 106)

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-4+ - 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+ del36CHO CD86-GFP

BafA

2h

Confocal MicroscopyFlow cytometry

CD4+ CD25- T cell

Anti-CD35 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 cellCTLA-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 hstaphylococcal enterotoxin B

(SEB) SEB CD4+ T cell6 days

Confocal Microscopy

TCR stimulation increased the acquisition of CD86

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

CD4+CD25- T cellCD4+CD25+ T cell

Anti-CD3 Confocal Microscopy

Flow cytometryPBMC

CD4+CD25+ T cell

Anti-CTLA-4

CFSECD4+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-/-

Rag2-/-

CD86-GFP OVAchloroquine

Confocal Microscopy6h

DO11.10 T cellDO11.10 T cellCTLA-4 +/+ CTLA-4 -/-

CONCLUSION