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
WT
CTLA-4-/-
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
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.,
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
CTLA4+/+
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)
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
Serum
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
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 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)
Rag2−/−
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)
Rag2−/−
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
Conclusion
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 CD86-GFP
CHO CTLA-4+ - Blue
CHO CD86 – 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
2h
CTLA-4+
CHO
CHO CTLA-4+ del36
CHO CD86-GFP
Confocal
Flow cytometry
Microscopy
Anti-CD3
5 days
CFSE
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?
Anti-CD3
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
OVA
chloroquine
6h
Rag2-/DO11.10 T cell
CTLA-4 +/+
CTLA-4 -/-
Rag2-/-
Confocal
Microscopy
CONCLUSION