Protein kinase C-θ (PKC-θ) translocates to the center of the immunological synapse, but the underlying mechanism and its importance in T cell activation are unknown. We found that the PKC-θ V3 domain is necessary and sufficient for IS localization mediated by Lck-dependent association with CD28. We identified a conserved proline-rich motif in V3 required for CD28 association and IS localization. CD28 association was essential for PKC-θ-mediated downstream signaling and TH2 and TH17, but not TH1, differentiation. Ectopic V3 expression sequestered PKC-θ from the IS and interfered with its functions. These results identify a unique mode of CD28 signaling, establish a molecular basis for the IS localization of PKC-θ, and implicate V3-based “decoys” as therapeutic modalities for T cell-mediated inflammatory diseases.

Regulatory T cells (Treg cells), which maintain immune homeostasis and self-tolerance, form an immunological synapse (IS) with antigen-presenting cells (APCs). However, signaling events at the Treg IS remain unknown. Here we show that protein kinase C-η (PKC-η) associated with CTLA-4 and was recruited to the Treg IS. PKC-η-deficient Treg cells displayed defective suppressive activity, including suppression of tumor immunity but not autoimmune colitis. Phosphoproteomic analysis revealed an association between CTLA-4-PKC-η and the GIT-PIX-PAK complex, an IS-localized focal adhesion complex. Defective activation of this complex in PKC-η-deficient Treg cells was associated with reduced CD86 depletion from APCs by Treg cells. These results reveal a novel CTLA-4-PKC-η signaling axis required for contact-dependent suppression, implicating this pathway as a potential cancer immunotherapy target.

Inducible costimulator (ICOS) signaling fuels the stepwise development of T follicular helper (TFH) cells. However, a signaling pathway unique to ICOS has not been identified. We show that TANK-binding kinase 1 (TBK1) associates with ICOS via a conserved motif, IProx, which shares homology with tumor necrosis factor receptor (TNFR)-associated factors, TRAF2 and TRAF3. Disruption of this motif abolishes the association with TBK1, thus identifying a TBK1-binding consensus. Mutation of this motif in ICOS, or depletion of TBK1 in T cells severely impaired the differentiation of germinal center (GC) TFH, B cell and antibody responses, but was dispensable for early TFH differentiation. These results reveal a novel ICOS-TBK1 signaling pathway that specifies GC TFH cell commitment.

SWAP-70-like adapter of T cells (SLAT; also known as Def6) is a novel guanine nucleotide exchange factor for Rho GTPases that has been previously shown to play a role in CD4+ T cell activation and Th1/Th2 differentiation. However, the role of SLAT/Def6 in autoimmunity and its associated Th1- and Th17-specific responses has not yet been clearly elucidated. We used a prototypical and pathologically relevant Th1/Th17-mediated autoimmune model, that is, experimental autoimmune encephalomyelitis, to assess the role of SLAT/Def6 in autoantigen-specific T cell response. We found that T cell-expressed SLAT/Def6 was critical for experimental autoimmune encephalomyelitis development and pathogenesis, as evidenced by the resistance of Def6-deficient (Def6−/−) mice to clinical signs of the disease associated with a lack of CNS inflammation and demyelination in myelin oligodendrocyte glycoprotein-immunized Def6−/− mice. Moreover, Def6 deficiency resulted in a severely diminished myelin oligodendrocyte glycoprotein-specific CD4+ T cell proliferation as well as a defect in IFN-γ and IL-17 production in secondary lymphoid organs and the CNS. Lastly, Def6−/− CD4+ T cells were grossly deficient in their ability to differentiate into Th17 cells both in vitro and in vivo in a T cell-intrinsic manner. Therefore, our study establishes T cell-expressed SLAT/Def6 as a pivotal positive regulator of Th17 inflammatory responses and, thus, essential in controlling autoimmune and inflammatory diseases.

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SLAT (also known as DEF6) promotes T cell activation and differentiation by regulating NFAT-Ca 2+ signaling. However, its role in TCR-mediated inside-out signaling, which induces integrin activation and T cell adhesion, a central process in T cell immunity and inflammation, has not been explored. Here, we show that SLAT is crucial for TCR-induced adhesion to ICAM-1 and affinity maturation of LFA-1 in CD4 + T cells. Mechanistic studies revealed that SLAT interacts, through its PH domain, with a key component of inside-out signaling, namely the active form of the small GTPase Rap1 (which has two isoforms, Rap1A and Rap1B). This interaction has been further shown to facilitate the interdependent recruitment of Rap1 and SLAT to the T cell immunological synapse upon TCR engagement. Furthermore, a SLAT mutant lacking its PH domain drastically inhibited LFA-1 activation and CD4 + T cell adhesion. Finally, we established that a constitutively active form of Rap1, which is present at the plasma membrane, rescues the defective LFA-1 activation and ICAM-1 adhesion in SLAT-deficient (Def6 −/− ) T cells. These findings ascribe a new function to SLAT, and identify Rap1 as a target of SLAT function in TCR-mediated inside-out signaling.

Protein kinase C-θ (PKCθ) is an important component of proximal T cell receptor (TCR) signaling. We previously identified the amino-terminal C2 domain of PKCθ as a phosphotyrosine (pTyr)–binding domain. Using a mutant form of PKCθ that cannot bind pTyr (PKCθHR2A), we showed that pTyr binding by PKCθ was required for TCR-induced T cell activation, proliferation, and TH2 cell differentiation but not for T cell development. Using tandem mass spectrometry and coimmunoprecipitation, we identified the kinase ζ-associated protein kinase of 70 kDa (Zap70) as a binding partner of the PKCθ pTyr-binding pocket. Tyr126 of Zap70 directly bound to PKCθ, and the interdomain B residues Tyr315 and Tyr319 were indirectly required for binding to PKCθ, reflecting their role in promoting the open conformation of Zap70. PKCθHR2A-expressing CD4+ T cells displayed defects not only in known PKCθ-dependent signaling events, such as nuclear factor κB (NF-κB) activation and TH2 cell differentiation, but also in full activation of Zap70 itself and in the activating phosphorylation of linker of activation of T cells (LAT) and phospholipase C-γ1 (PLCγ1), signaling proteins that are traditionally considered to be activated independently of PKC. These findings demonstrate that PKCθ plays an important role in a positive feedback regulatory loop that modulates TCR-proximal signaling and, moreover, provide a mechanistic explanation for earlier reports that documented an important role for PKCθ in T cell Ca2+ signaling. This PKCθ-Zap70 interaction could potentially serve as a promising and highly selective immunosuppressive drug target in autoimmunity and organ transplantation.

We reported that protein kinase C-h (PKCh) forms a novel (to our knowledge) signaling complex with the checkpoint inhibitory protein CTLA-4 in regulatory T cells (Tregs). This complex is required for the contact-dependent suppressive activity of Tregs, including suppression of antitumor immunity. However, the importance of PKCh in protective immunity mediated by T effector cells remains unclear. We used mice with germline or conditional Treg-specific deletion of Prkch, the PKCh-encoding gene, to explore CD8 + T cell-dependent antiviral immunity using the lymphocytic choriomeningitis virus Armstrong strain acute infection model as well as the in vitro activation of murine or human CD8 + T cells. Five days following infection, germline Prkch 2/2 mice displayed enhanced viral clearance compared with control mice. Similarly, Prkch Treg-specific conditional knockout mice also showed improved viral clearance and displayed enhanced expression of granzyme B and IFN-g by both virus-specific and total CD8 + T cells, demonstrating that enhanced viral clearance in germline Prkch 2/2 mice is caused by PKCh deficiency in Tregs and the resulting functional defect of Prkch 2/2 Tregs. In addition, purified Prkch 2/2 mouse CD8 + T cells as well as PRKCH knockdown human CD8 + T cells displayed intact, or even enhanced, T cell activation in vitro as measured by proliferation and expression of granzyme B and IFN-g. Thus, global PKCh deletion does not impair overall CD8 + T cell-mediated immunity, including antiviral immunity, implying that selective pharmacological PKCh inhibition could be safely used in vivo to inhibit undesired contact-dependent suppression by Tregs and, thus, enhance tumor-specific and, likely, virus-specific immunity.