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Protein kinase C (PKC)-θ has been shown to be a critical TCR signaling molecule that promotes the activation and differentiation of naive T cells into inflammatory effector T cells. In this study, we demonstrate that PKC-θ–mediated signals inhibit inducible regulatory T cell (iTreg) differentiation via an AKT-Foxo1/3A pathway. TGF-β–induced iTreg differentiation was enhanced in PKC-θ−/− T cells or wild-type cells treated with a specific PKC-θ inhibitor, but was inhibited by the PKC-θ activator PMA, or by CD28 crosslinking, which enhances PKC-θ activation. PKC-θ−/− T cells had reduced activity of the AKT kinase, and the expression of a constitutively active form of AKT in PKC-θ−/− T cells restored the ability to inhibit iTreg differentiation. Furthermore, knockdown or overexpression of the AKT downstream targets Foxo1 and Foxo3a was found to inhibit or promote iTreg differentiation in PKC-θ−/− T cells accordingly, indicating that the AKT-Foxo1/3A pathway is responsible for the inhibition of iTreg differentiation of iTregs downstream of PKC-θ. We conclude that PKC-θ is able to control T cell-mediated immune responses by shifting the balance between the differentiation of effector T cells and inhibitory Tregs.

Survival of CD4+CD8+ double-positive (DP) thymocytes plays a critical role in shaping the peripheral T cell repertoire. However, the mechanisms responsible for the regulation of DP thymocyte lifespan remain poorly understood. In this work, we demonstrate that T cell factor (TCF)-1 regulates DP thymocyte survival by upregulating RORγt. Microarray analysis revealed that RORγt was significantly downregulated in TCF-1−/− thymocytes that underwent accelerated apoptosis, whereas RORγt was greatly upregulated in thymocytes that had enhanced survival due to transgenic expression of a stabilized β-catenin (β-catTg), a TCF-1 activator. Both TCF-1−/− and RORγt−/− DP thymocytes underwent similar accelerated apoptosis. Forced expression of RORγt successfully rescued TCF-1−/− DP thymocytes from apoptosis, whereas ectopically expressed TCF-1 was not able to rescue the defective T cell development because of the lack of RORγt-supported survival. Furthermore, activation of TCF-1 by stabilized β-catenin was able to enhance DP thymocyte survival only in the presence of RORγt, indicating that RORγt acts downstream of TCF-1 in the regulation of DP thymocyte survival. Moreover, β-catenin/TCF-1 directly interacted with the RORγt promoter region and stimulated its activity. Therefore, our data demonstrated that TCF-1 enhances DP thymocyte survival through transcriptional upregulation of RORγt, which we previously showed is an essential prosurvival molecule for DP thymocytes.

We demonstrate here that PKC-θ is a critical molecule to maintain immunological balance by shifting the ratio of inflammatory Th17 and inhibitory Treg cells. TGFβ-induced T regulatory cell (iTreg) differentiation was enhanced in naive PKC-θ -/- T cells or WT cells treated with a specific PKC-θ inhibitor. PKC-θ -/- T cells had reduced activity of the AKT kinase, and the expression of a constitutively active form of AKT in PKC-θ -/- T cells restored ability to inhibit iTreg differentiation. Furthermore, knockdown or overexpression of the AKT downstream targets FoxO1 and FoxO3a was found to inhibit or promote iTreg differentiation in PKC-θ -/- T cells, indicating that the AKT-FoxO1/3A pathway plays an inhibitory role in the differentiation of iTreg downstream of PKC-θ. In the other hand, we show that naive PKC-θ -/- T cells were defective in Th17 differentiation in vitro, whereas in vitro differentiation of Th1 and Th2 appeared normal. PKC-θ -/- T cells had notably lower levels of Stat3, a transcription factor required for Th17 differentiation. PKC-θ-mediated activation of the Stat3 promoter was inhibited by dominant negative AP-1 and IκB kinase β mutations, but stimulated by WT AP-1 and IκB kinase β, suggesting that PKC-θ stimulates Stat3 transcription via the AP-1 and NF-κB pathways. We conclude that PKC-θ is able to control T cell-mediated immune responses by shifting the balance between the differentiation of Th17 inflammatory and regulatory T cells.