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Clinical Trial
. 2014 Feb;2(2):167-76.
doi: 10.1158/2326-6066.CIR-13-0155. Epub 2013 Nov 19.

CD4 T cells require ICOS-mediated PI3K signaling to increase T-Bet expression in the setting of anti-CTLA-4 therapy

Hong Chen  1 , Tihui Fu, Woong-Kyung Suh, Dimitra Tsavachidou, Sijin Wen, Jianjun Gao, Derek Ng Tang, Qiuming He, Jingjing Sun, Padmanee Sharma
Affiliations
Clinical Trial

CD4 T cells require ICOS-mediated PI3K signaling to increase T-Bet expression in the setting of anti-CTLA-4 therapy

Hong Chen et al. Cancer Immunol Res. 2014 Feb.

Abstract

The transcription factor T-bet controls the Th1 genetic program in T cells for effective antitumor responses. Anti-CTLA-4 immunotherapy elicits dramatic antitumor responses in mice and in human patients; however, factors that regulate T-bet expression during an antitumor response mediated by anti-CTLA-4 remain to be elucidated. We were the first to report that treatment with anti-CTLA-4 led to an increase in the frequency of T cells expressing inducible costimulator (ICOS). In both treated patients and mice, our data revealed that CD4(+)ICOS(hi) T cells can act as effector T cells, which produce the Th1 cytokine IFN-γ. We also showed in a small retrospective analysis that an increased frequency of CD4(+)ICOS(hi) T cells correlated with better clinical outcome and the absence of ICOS or its ligand (ICOSL) in mouse models led to impaired tumor rejection. Here, we show that CD4(+)ICOS(hi) T cells from anti-CTLA-4-treated patients had an increase in signaling via the phospoinositide-3-kinase (PI3K) pathway and an increase in expression of T-bet. An ICOS-specific siRNA transfected into human T cells led to diminished PI3K signaling and T-bet expression. Therefore, we hypothesized that ICOS, and specifically ICOS-mediated PI3K signaling, was required for T-bet expression. We conducted studies in ICOS-deficient and ICOS-YF mice, which have a single amino acid change that abrogates PI3K signaling by ICOS. We found that ICOS-mediated PI3K signaling is required for T-bet expression during an antitumor response elicited by anti-CTLA-4 therapy. Our data provide new insight into the regulation of T-bet expression and suggest that ICOS can be targeted to improve Th1 antitumor responses.

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Conflict of interest statement

The authors do not have any conflict of interest to declare related to this manuscript

Figures

Figure 1
Figure 1. ICOS expression correlates with increased PI3K-signaling, T-bet expression and Th1 cytokines
(A) RPPA data with unsupervised hierarchical clustering of 27 proteins that were significantly changed (p<0.05) in post-therapy CD4 T cells as compared to matched pre-therapy CD4 T cells. (B) Box plots to indicate changes in the expression levels of 7 proteins selected to represent PI3K-signaling in post-therapy (after) CD4 T cells as compared to pre-therapy (before) CD4 T cells. (C) Greater expression of proteins indicative of PI3K-signaling was detected in post-therapy CD4 T cells at weeks 3 and 7 as compared to pre-therapy CD4 T cells. (D) Greater expression of proteins indicative of PI3K-signaling was detected in post-therapy CD4+ICOShi T cells as compared to post-therapy CD4+ICOSlow T cells and pre-therapy CD4 T cells. (E) Greater expression of T-bet protein was detected in post-therapy CD4+ICOShi T cells as compared to post-therapy CD4+ICOSlow T cells and pre-therapy CD4 T cells. (F) Higher mRNA levels were observed for IFNγ and TNFα in post-therapy CD4+ICOShi T cells as compared to post-therapy CD4+ICOSlow T cells and pre-therapy CD4 T cells. *indicates p<0.05
Figure 2
Figure 2. ICOS-specific siRNA diminishes PI3K-signaling, T-bet expression and Th1 cytokines
(A) An ICOS-specific siRNA led to the decreased frequency of CD4+ICOShi T cells in post-therapy samples (Upper Panel, representative patient and Lower Panel, summary data from 3 different patients). (B) An ICOS-specific siRNA led to the decreased protein expression of T-bet and proteins indicative of PI3K-signaling (Upper Panel, representative experiment, and Lower Panel, summary data). Numerical values listed under each band represent calculated intensity for protein expression. (C) An ICOS-specific siRNA led to the decreased mRNA levels for ICOS, IFNγ, TNFα and T-bet (Lower panel). *indicates p<0.05; ns indicates no statistical significance.
Figure 3
Figure 3. ICOS expression is required for increased PI3K-signaling and T-bet expression during an in vivo antitumor immune response
(A) Greater expression of proteins indicative of PI3K-signaling in CD4 T cells from the spleen, draining lymph nodes (DLN) and tumor-infiltrating lymphocytes (TILs) of treated WT mice as compared to those in CD4 T cells from treated ICOS−/− mice (Upper Panel, representative experiment, and Lower Panel, summary data from 3 independent experiments with mean and sem). (B) Greater expression of proteins indicative of PI3K-signaling in CD4+ICOShi cells from treated WT mice as compared to CD4+ICOSlow cells from treated WT mice and CD4 T cells from treated ICOS−/− mice (Upper Panel, representative experiment, and Lower Panel, summary data from 3 independent experiments with mean and sem). (C) Greater expression of T-bet protein in CD4 T cells from the spleen, draining lymph nodes (DLN) and tumor-infiltrating lymphocytes (TILs) of treated WT mice as compared to those of treated ICOS−/− mice (Upper Panel, representative experiment, and Lower Panel, summary data from 3 independent experiments with mean and sem). (D) Greater expression of T-bet protein in CD4+ICOShi cells from treated WT mice as compared to T-bet protein in CD4+ICOSlow cells from treated WT mice and in CD4 cells from treated ICOS−/− mice (Upper Panel, representative experiment, and Lower Panel, summary data from 3 independent experiments with mean and sem).
Figure 4
Figure 4. T-bet expression requires ICOS-mediated PI3K-signaling during an in vivo antitumor immune response
(A) Tumor-bearing WT mice that received treatment with anti-CTLA-4 had an increased frequency of ICOS+ CD4 T cells, which was not found for CD4 T cells from ICOS−/− mice but was observed for CD4 T cells from ICOS-YF mice. (B) Treated WT mice had a significant increase in the frequency of T-bet+ CD4 T cells, which was not observed in the treated ICOS−/− and ICOS-YF mice (Upper Panel, representative experiment, and Lower Panel, summary data from 3 independent experiments with mean and sem). (C) Sorted CD4+ICOShi and CD4+ICOSlow T cells from WT and ICOS-YF mice revealed greater protein expression of T-bet in CD4+ICOShi cells from WT mice as compared to CD4+ICOSlow cells from WT mice or CD4+ICOShi and CD4+ICOSlow cells from ICOS-YF mice (Upper Panel, representative experiment and Lower Panel, Summary data from 3 independent experiments (mean and sem). UnTx indicates untreated; Tx indicates treated; *indicates p<0.05; ns indicates no statistical significance.
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