Requirement of Treg-intrinsic CTLA4/PKCη signaling pathway for suppressing tumor immunity
Christophe Pedros, … , Kok-Fai Kong, Amnon Altman
Christophe Pedros, … , Kok-Fai Kong, Amnon Altman
Published December 7, 2017
Citation Information: JCI Insight. 2017;2(23):e95692. https://doi.org/10.1172/jci.insight.95692.
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Research Article Immunology

Requirement of Treg-intrinsic CTLA4/PKCη signaling pathway for suppressing tumor immunity

Abstract

The ability of Tregs to control the development of immune responses is essential for maintaining immune system homeostasis. However, Tregs also inhibit the development of efficient antitumor responses. Here, we explored the characteristics and mechanistic basis of the Treg-intrinsic CTLA4/PKCη signaling pathway that we recently found to be required for contact-dependent Treg-mediated suppression. We show that PKCη is required for the Treg-mediated suppression of tumor immunity in vivo. The presence of PKCη-deficient (Prkch–/–) Tregs in the tumor microenvironment was associated with a significantly increased expression of the costimulatory molecule CD86 on intratumoral CD103+ DCs, enhanced priming of antigen-specific CD8+ T cells, and greater levels of effector cytokines produced by these cells. Similar to mouse Tregs, the GIT/PAK/PIX complex also operated downstream of CTLA4 and PKCη in human Tregs, and GIT2 knockdown in Tregs promoted antitumor immunity. Collectively, our data suggest that targeting the CTLA4/PKCη/GIT/PAK/PIX signaling pathway in Tregs could represent a novel immunotherapeutic strategy to alleviate the negative impact of Tregs on antitumor immune responses.

Authors

Christophe Pedros, Ann J. Canonigo-Balancio, Kok-Fai Kong, Amnon Altman

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Figure 1

The CTLA4/PKCη pathway controls human Treg-suppressive activity.

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The CTLA4/PKCη pathway controls human Treg-suppressive activity. (A) CD4...
(A) CD4+CD25+CD127lo Tregs from human blood were left unstimulated (ns) or were stimulated (stim) for 5 minutes with anti-CD3 and anti-CTLA4 antibodies. CTLA4 immunoprecipitates and whole cell lysates (WCL) were immunoblotted with anti-PKCη and anti-CTLA4 antibodies. Data representative of 2 independent experiments are shown. (B–D) Human Tregs were retrovirally transduced with irrelevant shRNA (ShControl) or 2 different shRNA targeting Prkch (shPrkch-1 and shPrkch-2). PKCη expression in purified transduced Tregs was assessed by immunoblotting (B) and quantitated as the percentage of expression in the ShControl group (C). Foxp3 expression in transduced Tregs was assessed by intracellular staining (D). (E and F) Suppressive activity of the transduced Tregs was analyzed by coculture at different ratios with CTV-prelabeled PBMCs stimulated with anti-CD3. (E) Representative histograms of CTV dilution in gated CD4+ responder cells. (F) Cumulative data expressed as the percentage inhibition of responder CD4+ cell proliferation (mean ± SEM of 5 independent experiments). Statistical significance of differences between groups was determined by 1-way ANOVA and Tukey’s multiple comparisons test. ns, P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ****P ≤ 0.0001. Statistical significance levels are shown against the + ShControl Treg group.