January 29, 2018 | Author: Anonymous | Category: Science, Health Science, Immunology
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New intrinsic- and extrinsic factors of CTLA-4 regulation

Daiani Alves Patrícia Assis Tiago

CTLA-4 forms generated by splicing

Teff et al., 2006

CTLA-4: a negative regulator of T cell activation



After 4 weeks

Lymphonodes and spleen were removed

CTLA-4-/Organs weight and number of lymphocytes were determined

Waterhouse et al., 1995

D: Thymus E: Spleen F: Myocardium G: Lung

T-cell fate after TCR engagement

Alegre et al., 2001 Sharpe & Freeman, 2002

Ligand-independent CTLA-4 function



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.,

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




Bone marrow adoptivelly transfered Rag2-/-



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)

CYTOMETRY Splenic CD4+ T cells

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 × 105 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 tissuedestructive 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 × 105 cells) pancreatic lymph nodes


Activated the cells in vitro

PDIA2 (10 µM/ 24 h) + irradiated splenocytes (5 × 105 cells)

ELISA IL-2 Concentrations in supernatants

ELISA anti-PDIA2 titers

PDIA2 (protein disulfide isomerase–associated 2), an acinar-specific enzyme

PDIA2 seems to be an authentic autoantigen in Ctla4−/− mice.

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

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 PDIA2specific 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. CYTOMETRY Thy-1.1+ DO11.10 Rag2−/−Ctla4+/+ DO11.10 Rag2−/−Ctla4−/−

Retrovirus infected 29TCRα Thy-1.1

CD4+ T cells (1 × 106)


3 weeks

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

HISTOLOGY (HE) DO11.10 Rag2−/−Ctla4+/+ DO11.10 Rag2−/−Ctla4−/−

Retrovirus infected 29TCRα Thy-1.1

CD4+ T cells (1 × 106)


3 weeks

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 Rag2−/−

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


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

CTLA-4 could potentially deplete its ligands CD86? BafA

3h CHO CTLA-4+

Confocal Flow cytometry Microscopy


CHO CTLA-4+ - Blue CHO CD86 – Green (GFP)

Time course of CD86 acquisition… BafA



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

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

2h CTLA-4+

CHO CHO CTLA-4+ del36


Confocal Flow cytometry Microscopy Anti-CD3

5 days


CD4+ CD25- T cell

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?


72 h

Confocal Microscopy

PBMC + CD4 CD4+CD25 CD25- TT cell cell CTLA-4 transfected

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

Does TCR stimulation enhances the CD86 acquisition? PBMC

staphylococcal enterotoxin B (SEB)

72 h

6 days SEB CD4+ T cell

Dcs SEB pulsed cells

Confocal Microscopy

TCR stimulation increased the acquisition of CD86

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

Anti-CTLA-4 Anti-CD3 PBMC CD4+CD25+ T cell + CD4 CD25- T cell CD4+CD25+ T cell

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 chloroquine

6h Rag2-/DO11.10 T cell CTLA-4 +/+ CTLA-4 -/-


Confocal Microscopy


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