Tumor- antigens
CTLA-4 (CD152) impairs cytotoxic T-lymphocyte responses via
PDCD4 induction
H. Lingel
1, J. Wissing
2, F. Klawonn
2, A. Arra
1, M. Pierau
1, L. Jänsch
2, M. Brunner-Weinzierl
1A B S T R A C T
Inhibitory T-cell surface receptors like Cytotoxic T-lymphocyte-associated Protein-4 (CTLA-4) and Programmed cell death 1 (PD-1) play a crucial role in the termination of adaptive immune responses and promote the functionally impaired state of CD8
+T cell exhaustion. Their blockade is being used in immune-checkpoint therapy as a promising approach to restore effective T-cell responses against tumors. However, the intracellular pathways triggered by these receptors still remain incompletely understood. To determine target molecules downstream of CTLA-4, an accurate mass spectrometry analysis was performed. The dataset revealed that the engagement of CTLA-4 led to altered phosphorylation of proteins involved in T-cell signaling, DNA replication, RNA processing and microtubule polymerization. Beside other targets, a CTLA-4-induced expression of the translational inhibitor Programmed cell death 4 (PDCD4) could be revealed and characterized. This mechanism was responsible for the restriction of cytotoxic T-lymphocyte effector functions. Accordingly, the deficiency of PDCD4 led to superior control of melanoma growth in vivo. These findings point out that targeting of PDCD4 could provide a potent strategy to improve anti-tumor immunotherapy.
Tumor cell
APC
MHCI
Antigen
Cytotoxic T cell TCR
CD80 CTLA-4
IFN-γ PD-L1 PD-1
STOP
STOP
Immune system process
Response to stimulus
Biological adhesion
Metabolic process
Cellular component organization or biogenesis
-1 1
log2fold change
iTRAQ 117
P
Intensity
m/z
TCR CTLA-4 CD28 P
αCD3 agonistic
αCTLA-4
αCD28
B16-OVA tumor growth TRAMP-C1 tumor rejection
M E T H O D S
Micro- sphere
CD8+ T cell
Isolation of phosphoproteins
Labeling with isobaric tags Mass spectrometry
1 Otto-von-Guericke University, Magdeburg, Germany;
2 Helmholtz Centre for Infection Research, Braunschweig, Germany.
C O N C L U S I O N S
+ agon. αCTLA-4
0,04 0,08 0,06 0,31 0,13 0,58
24 h 48 h 72 h
αCD3 + αCD28
p-PDCD4S457
PDCD4
GAPDH
+ agon.αCTLA-4 αCD3 +αCD28
mRNA
P P
+ αCTLA-4 Glutaminase
PDCD4 KO WT PDCD4
GAPDH 4,2 1,8
Gls in situ
PDCD4
eIF4A Pdcd4
Gls-
transcript no
translation initiation FoxO1
impaired glutamine metabolism
CTLA-4
PDCD4-/- WT
+αCTLA-4 +αCTLA-4
IFN-γ
% Max
w/o Glu +Glu
PDCD4 FoxO1
CTLA-4 modulates the phosphoproteome in differentiating CD8+ T cells.
Phosphorylation profile of significantly regulated proteins in CD8+ T cells 48 h after differentiation with anti-CD3/CD28 and additional CTLA-4 engagement, acquired by iTRAQ mass spectrometry. Proteins were functionally clustered. Blue and red represent low and high relative phosphorylation, respectively.
iTRAQ mass spectrometry enabels simultaneous analysis of phosphoproteins.
Phosphorylated proteins were isolated from CD8+ T cells differentiated with anti- CD3/CD28 and additional CTLA-4 engagement or not. Comparative analysis of phosphoprtein abundance was performed using isobaric tags and LC-MS.
CTLA-4 induces PDCD4 expression in differentiating CD8+ T cells.
(left) Immunoblot analysis of phosphorylated and total PDCD4 in CD8+ T cells from 24 h to 72 h. (right) Pdcd4 mRNA expression profile after differentiation with or without additional agonistic αCTLA-4.
R E S U LT S
CTLA-4 impairs CD8+ T-cell glutamine metabolism and effector functions.
(left) CTLA-4 re-activates FoxO1 leading to PDCD4 expression. PDCD4 binds Gls mRNA inhibiting glutaminase protein translation. (right) Glutaminase expression (upper panel) and IFN- γ production in the presence or absence of exogenous glutamate (lower panel) in OT-I PDCD4-/- or OT-I PDCD4+/+ (WT) CD8+ T cells after activation with additional agonistic αCTLA-4.
PDCD4 deficiency leads to enhanced control of experimental tumors.
(left) Tumor volume of mice s.c. inoculated with OVA-expressing B16 melanoma, followed 5 days later by i.v. transfer of no T cells (crosses) or naïve TCR-transgenic CTLA-4 and PDCD4 wild-type (WT, white shaded) or CTLA-4- (light gray shaded) or PDCD4-deficient (dark gray shaded) OT-I CD8+ T cells. (right) Bioluminescence images of PDCD4 WT and deficient mice from day 5 or day 9 after subcutaneous transplantation of luciferase-expressing TRAMP-C1 prostate cancer cells.
