Requirement of Treg-intrinsic CTLA4/PKCη signaling pathway for suppressing tumor immunity
Christophe Pedros, Ann J. Canonigo-Balancio, Kok-Fai Kong, Amnon Altman
Christophe Pedros, Ann J. Canonigo-Balancio, Kok-Fai Kong, Amnon Altman
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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 5

GIT2 controls mouse and human Treg-suppressive activity.

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GIT2 controls mouse and human Treg-suppressive activity. (A) Rag1−/− mic...
(A) Rag1−/− mice received an adoptive transfer of CD25-depleted spleen cells with or without Tregs from WT or Git2–/– FIG mice and were implanted with B16-F10 melanoma cells, as in Figure 2. Tumor areas (mm2) were measured 3 times/week. Cumulative data of 4 independent experiments are shown (mean ± SEM). no Tregs, n = 11; + WT, Tregs, n = 14; + Prkch−/− Tregs, n = 12). (B–F). Human Tregs were retrovirally transduced with irrelevant shRNA (ShControl) or shRNAs targeting GIT2 (shGIT2) or PKCη (ShPrkch-2). Transduced Tregs were analyzed for GIT2 protein expression by immunoblotting (B) and for the expression of Foxp3 by intracellular staining (C). Suppressive activity of transduced Tregs was evaluated by coculture with CTV-labeled PBMCs stimulated with anti-CD3 (D). Data are expressed as inhibition of gated CD4+ Teff cell proliferation. (E and F) Analysis of CD86 expression by CD19+ APCs in suppression cocultures, showing histograms of a representative mouse in each group (E) and cumulative data of 3 experiments (mean ± SEM) (F). *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. Statistical significance was determined by repeated-measures 2-way ANOVA (A) or 1-way ANOVA (D and F) followed by Tukey’s multiple comparisons test. Significance against the shControl Tregs group is shown (D and F). (G and H) Human Tregs were left unstimulated or stimulated using anti-CD3, anti-CTLA4, or a combination of both antibodies. Activating phosphorylation of PAK1 (Ser144) and PAK2 (Ser141) and expression levels of PAK2 were assessed in cell lysates by immunoblotting. Representative data and quantification of signals in G are expressed as p-PAK2/PAK2 ratio (H). Cumulative data from 2 experiments (mean ± SEM). (I and J) Human Tregs transduced with ShControl or ShPrkch-2 were stimulated with an anti-CTLA4 antibody. Activating phosphorylation (Ser141) and total expression of PAK2 were assessed in cell lysates by immunoblotting (I). Signals in I were quantified by densitometry and expressed as p-PAK2/PAK2 ratio (J). Cumulative data from 2 experiments (mean ± SEM).