The Peptidyl-Prolyl cis-trans isomerase, Pin1, associates with Protein Kinase C θ via a critical Phospho-Thr-Pro motif in the V3 regulatory domain.

Protein kinase C-θ (PKCθ) is a member of the novel PKC subfamily known for its selective and predominant expression in T lymphocytes where it regulates essential functions required for T cell activation and proliferation. Our previous studies provided a mechanistic explanation for the recruitment of PKCθ to the center of the immunological synapse (IS) by demonstrating that a proline-rich (PR) motif within the V3 region in the regulatory domain of PKCθ is necessary and sufficient for PKCθ IS localization and function. Herein, we highlight the importance of Thr335-Pro residue in the PR motif, the phosphorylation of which is key in the activation of PKCθ and its subsequent IS localization. We demonstrate that the phospho-Thr335-Pro motif serves as a putative binding site for the peptidyl-prolyl cis-trans isomerase (PPIase), Pin1, an enzyme that specifically recognizes peptide bonds at phospho-Ser/Thr-Pro motifs. Binding assays revealed that mutagenesis of PKCθ-Thr335-to-Ala abolished the ability of PKCθ to interact with Pin1, while Thr335 replacement by a Glu phosphomimetic, restored PKCθ binding to Pin1, suggesting that Pin1-PKCθ association is contingent upon the phosphorylation of the PKCθ-Thr335-Pro motif. Similarly, the Pin1 mutant, R17A, failed to associate with PKCθ, suggesting that the integrity of the Pin1 N-terminal WW domain is a requisite for Pin1-PKCθ interaction. In silico docking studies underpinned the role of critical residues in the Pin1-WW domain and the PKCθ phospho-Thr335-Pro motif, to form a stable interaction between Pin1 and PKCθ. Furthermore, TCR crosslinking in human Jurkat T cells and C57BL/6J mouse-derived splenic T cells promoted a rapid and transient formation of Pin1-PKCθ complexes, which followed a T cell activation-dependent temporal kinetic, suggesting a role for Pin1 in PKCθ-dependent early activation events in TCR-triggered T cells. PPIases that belong to other subfamilies, i.e., cyclophilin A or FK506-binding protein, failed to associate with PKCθ, indicating the specificity of the Pin1-PKCθ association. Fluorescent cell staining and imaging analyses demonstrated that TCR/CD3 triggering promotes the colocalization of PKCθ and Pin1 at the cell membrane. Furthermore, interaction of influenza hemagglutinin peptide (HA307-319)-specific T cells with antigen-fed antigen presenting cells (APCs) led to colocalization of PKCθ and Pin1 at the center of the IS. Together, we point to an uncovered function for the Thr335-Pro motif within the PKCθ-V3 regulatory domain to serve as a priming site for its activation upon phosphorylation and highlight its tenability to serve as a regulatory site for the Pin1 cis-trans isomerase.

Experimental Melanoma Immunotherapy Model Using Tumor Vaccination with a Hematopoietic Cytokine.

Fms-like tyrosine kinase 3 ligand (Flt3L) is a hematopoietic cytokine that promotes the survival and differentiation of dendritic cells (DCs). It has been used in tumor vaccines to activate innate immunity and enhance antitumor responses. This protocol demonstrates a therapeutic model using cell-based tumor vaccine consisting of Flt3L-expressing B16-F10 melanoma cells along with phenotypic and functional analysis of immune cells in the tumor microenvironment (TME). Procedures for cultured tumor cell preparation, tumor implantation, cell irradiation, tumor size measurement, intratumoral immune cell isolation, and flow cytometry analysis are described. The overall goal of this protocol is to provide a preclinical solid tumor immunotherapy model, and a research platform to study the relationship between tumor cells and infiltrating immune cells. The immunotherapy protocol described here can be combined with other therapeutic modalities, such as immune checkpoint blockade (anti-CTLA-4, anti-PD-1, anti-PD-L1 antibodies) or chemotherapy in order to improve the cancer therapeutic effect of melanoma.

Leveraging the Treg-intrinsic CTLA4-PKCη signaling pathway for cancer immunotherapy.

BACKGROUND: Our previous studies revealed a critical role of a novel CTLA4-protein kinase C-eta (PKCη) signaling axis in mediating the suppressive activity of regulatory T cells (Tregs) in antitumor immunity. These studies have employed adoptive transfer of germline PKCη-deficient (Prkch-/-) Tregs into Prkch+/+ mice prior to tumor implantation. Here, we extended these findings into a biologically and clinically more relevant context. METHODS: We have analyzed the role of PKCη in antitumor immunity and the tumor microenvironment (TME) in intact tumor-bearing mice with Treg-specific or CD8+ T cell-specific Prkch deletion, including in a therapeutic model of combinatorial treatment. In addition to measuring tumor growth, we analyzed the phenotype and functional attributes of tumor-infiltrating immune cells, particularly Tregs and dendritic cells (DCs). RESULTS: Using two models of mouse transplantable cancer and a genetically engineered autochthonous hepatocellular carcinoma (HCC) model, we found, first, that mice with Treg-specific Prkch deletion displayed a significantly reduced growth of B16-F10 melanoma and TRAMP-C1 adenocarcinoma tumors. Tumor growth reduction was associated with a less immunosuppressive TME, indicated by increased numbers and function of tumor-infiltrating CD8+ effector T cells and elevated expression of the costimulatory ligand CD86 on intratumoral DCs. In contrast, CD8+ T cell-specific Prkch deletion had no effect on tumor growth or the abundance and functionality of CD8+ effector T cells, consistent with findings that Prkch-/- CD8+ T cells proliferated normally in response to in vitro polyclonal or specific antigen stimulation. Similar beneficial antitumor effects were found in mice with germline or Treg-specific Prkch deletion that were induced to develop an autochthonous HCC. Lastly, using a therapeutic model, we found that monotherapies consisting of Treg-specific Prkch deletion or vaccination with irradiated Fms-like tyrosine kinase 3 ligand (Flt3L)-expressing B16-F10 tumor cells post-tumor implantation significantly delayed tumor growth. This effect was more pronounced in mice receiving a combination of the two immunotherapies. CONCLUSION: These findings demonstrate the potential utility of PKCη inhibition as a viable clinical approach to treat patients with cancer, especially when combined with adjuvant therapies.

Protein Kinase C-η Deficiency Does Not Impair Antiviral Immunity and CD8+ T Cell Activation.

We reported that protein kinase C-η (PKCη) 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 PKCη in protective immunity mediated by T effector cells remains unclear. We used mice with germline or conditional Treg-specific deletion of Prkch, the PKCη-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 -/- 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-γ by both virus-specific and total CD8+ T cells, demonstrating that enhanced viral clearance in germline Prkch -/- mice is caused by PKCη deficiency in Tregs and the resulting functional defect of Prkch -/- Tregs. In addition, purified Prkch -/- 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-γ. Thus, global PKCη deletion does not impair overall CD8+ T cell-mediated immunity, including antiviral immunity, implying that selective pharmacological PKCη 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.

Human DEF6 deficiency underlies an immunodeficiency syndrome with systemic autoimmunity and aberrant CTLA-4 homeostasis.

Immune responses need to be controlled tightly to prevent autoimmune diseases, yet underlying molecular mechanisms remain partially understood. Here, we identify biallelic mutations in three patients from two unrelated families in differentially expressed in FDCP6 homolog (DEF6) as the molecular cause of an inborn error of immunity with systemic autoimmunity. Patient T cells exhibit impaired regulation of CTLA-4 surface trafficking associated with reduced functional CTLA-4 availability, which is replicated in DEF6-knockout Jurkat cells. Mechanistically, we identify the small GTPase RAB11 as an interactor of the guanine nucleotide exchange factor DEF6, and find disrupted binding of mutant DEF6 to RAB11 as well as reduced RAB11+CTLA-4+ vesicles in DEF6-mutated cells. One of the patients has been treated with CTLA-4-Ig and achieved sustained remission. Collectively, we uncover DEF6 as player in immune homeostasis ensuring availability of the checkpoint protein CTLA-4 at T-cell surface, identifying a potential target for autoimmune and/or cancer therapy.

Role of TRAFs in Signaling Pathways Controlling T Follicular Helper Cell Differentiation and T Cell-Dependent Antibody Responses.

Follicular helper T (TFH) cells represent a highly specialized CD4+ T cell subpopulation that supports the generation of germinal centers (GC) and provides B cells with critical signals promoting antibody class switching, generation of high affinity antibodies, and memory formation. TFH cells are characterized by the expression of the chemokine receptor CXCR5, the transcription factor Bcl-6, costimulatory molecules ICOS, and PD-1, and the production of cytokine IL-21. The acquisition of a TFH phenotype is a complex and multistep process that involves signals received through engagement of the TCR along with a multitude of costimulatory molecules and cytokines receptors. Members of the Tumor necrosis factor Receptor Associated Factors (TRAF) represent one of the major classes of signaling mediators involved in the differentiation and functions of TFH cells. TRAF molecules are the canonical adaptor molecules that physically interact with members of the Tumor Necrosis Factor Receptor Superfamily (TNFRSF) and actively modulate their downstream signaling cascades through their adaptor function and/or E3 ubiquitin ligase activity. OX-40, GITR, and 4-1BB are the TRAF-dependent TNFRSF members that have been implicated in the differentiation and functions of TFH cells. On the other hand, emerging data demonstrate that TRAF proteins also participate in signaling from the TCR and CD28, which deliver critical signals leading to the differentiation of TFH cells. More intriguingly, we recently showed that the cytoplasmic tail of ICOS contains a conserved TANK-binding kinase 1 (TBK1)-binding motif that is shared with TBK1-binding TRAF proteins. The presence of this TRAF-mimicking signaling module downstream of ICOS is required to mediate the maturation step during TFH differentiation. In addition, JAK-STAT pathways emanating from IL-2, IL-6, IL-21, and IL-27 cytokine receptors affect TFH development, and crosstalk between TRAF-mediated pathways and the JAK-STAT pathways can contribute to generate integrated signals required to drive and sustain TFH differentiation. In this review, we will introduce the molecular interactions and the major signaling pathways controlling the differentiation of TFH cells. In each case, we will highlight the contributions of TRAF proteins to these signaling pathways. Finally, we will discuss the role of individual TRAF proteins in the regulation of T cell-dependent humoral responses.

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

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.

Phosphoproteomics Reveals Regulatory T Cell-Mediated DEF6 Dephosphorylation That Affects Cytokine Expression in Human Conventional T Cells.

Regulatory T cells (Tregs) control key events of immune tolerance, primarily by suppression of effector T cells. We previously revealed that Tregs rapidly suppress T cell receptor (TCR)-induced calcium store depletion in conventional CD4+CD25- T cells (Tcons) independently of IP3 levels, consequently inhibiting NFAT signaling and effector cytokine expression. Here, we study Treg suppression mechanisms through unbiased phosphoproteomics of primary human Tcons upon TCR stimulation and Treg-mediated suppression, respectively. Tregs induced a state of overall decreased phosphorylation as opposed to TCR stimulation. We discovered novel phosphosites (T595_S597) in the DEF6 (SLAT) protein that were phosphorylated upon TCR stimulation and conversely dephosphorylated upon coculture with Tregs. Mutation of these DEF6 phosphosites abrogated interaction of DEF6 with the IP3 receptor and affected NFAT activation and cytokine transcription in primary Tcons. This novel mechanism and phosphoproteomics data resource may aid in modifying sensitivity of Tcons to Treg-mediated suppression in autoimmune disease or cancer.

NFκB-Pim-1-Eomesodermin axis is critical for maintaining CD8 T-cell memory quality.

T-cell memory is critical for long-term immunity. However, the factors involved in maintaining the persistence, function, and phenotype of the memory pool are undefined. Eomesodermin (Eomes) is required for the establishment of the memory pool. Here, we show that in T cells transitioning to memory, the expression of high levels of Eomes is not constitutive but rather requires a continuum of cell-intrinsic NFκB signaling. Failure to maintain NFκB signals after the peak of the response led to impaired Eomes expression and a defect in the maintenance of CD8 T-cell memory. Strikingly, we found that antigen receptor [T-cell receptor (TCR)] signaling regulates this process through expression of the NFκB-dependent kinase proviral integration site for Moloney murine leukemia virus-1 (PIM-1), which in turn regulates NFκB and Eomes. T cells defective in TCR-dependent NFκB signaling were impaired in late expression of Pim-1, Eomes, and CD8 memory. These defects were rescued when TCR-dependent NFκB signaling was restored. We also found that NFκB-Pim-1 signals were required at memory to maintain memory CD8 T-cell longevity, effector function, and Eomes expression. Hence, an NFκB-Pim-1-Eomes axis regulates Eomes levels to maintain memory fitness.

Chemical proteomic map of dimethyl fumarate-sensitive cysteines in primary human T cells.

Dimethyl fumarate (DMF) is an electrophilic drug that is used to treat autoimmune conditions, including multiple sclerosis and psoriasis. The mechanism of action of DMF is unclear but may involve the covalent modification of proteins or DMF serving as a prodrug that is converted to monomethyl fumarate (MMF). We found that DMF, but not MMF, blocked the activation of primary human and mouse T cells. Using a quantitative, site-specific chemical proteomic platform, we determined the DMF sensitivity of >2400 cysteine residues in human T cells. Cysteines sensitive to DMF, but not MMF, were identified in several proteins with established biochemical or genetic links to T cell function, including protein kinase Cθ (PKCθ). DMF blocked the association of PKCθ with the costimulatory receptor CD28 by perturbing a CXXC motif in the C2 domain of this kinase. Mutation of these DMF-sensitive cysteines also impaired PKCθ-CD28 interactions and T cell activation, designating the C2 domain of PKCθ as a key functional, electrophile-sensing module important for T cell biology.

Protein Kinase C Enzymes in the Hematopoietic and Immune Systems.

The protein kinase C (PKC) family, discovered in the late 1970s, is composed of at least 10 serine/threonine kinases, divided into three groups based on their molecular architecture and cofactor requirements. PKC enzymes have been conserved throughout evolution and are expressed in virtually all cell types; they represent critical signal transducers regulating cell activation, differentiation, proliferation, death, and effector functions. PKC family members play important roles in a diverse array of hematopoietic and immune responses. This review covers the discovery and history of this enzyme family, discusses the roles of PKC enzymes in the development and effector functions of major hematopoietic and immune cell types, and points out gaps in our knowledge, which should ignite interest and further exploration, ultimately leading to better understanding of this enzyme family and, above all, its role in the many facets of the immune system.

A TRAF-like motif of the inducible costimulator ICOS controls development of germinal center TFH cells via the kinase TBK1.

Signaling via the inducible costimulator ICOS fuels the stepwise development of follicular helper T cells (TFH cells). However, a signaling pathway unique to ICOS has not been identified. We found here that the kinase TBK1 associated with ICOS via a conserved motif, IProx, that shares homology with the tumor-necrosis-factor receptor (TNFR)-associated factors TRAF2 and TRAF3. Disruption of this motif abolished the association of TBK1 with ICOS, TRAF2 and TRAF3, which identified a TBK1-binding consensus. Alteration of this motif in ICOS or depletion of TBK1 in T cells severely impaired the differentiation of germinal center (GC) TFH cells and the development of GCs, interfered with B cell differentiation and disrupted the development of antibody responses, but the IProx motif and TBK1 were dispensable for the early differentiation of TFH cells. These results reveal a previously unknown ICOS-TBK1 signaling pathway that specifies the commitment of GC TFH cells.

Protein kinase C-η controls CTLA-4-mediated regulatory T cell function.

Regulatory T (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 cell IS remain unknown. Here we show that the kinase PKC-η associated with CTLA-4 and was recruited to the Treg cell IS. PKC-η-deficient Treg cells displayed defective suppressive activity, including suppression of tumor immunity but not of autoimmune colitis. Phosphoproteomic and biochemical analysis revealed an association between CTLA-4-PKC-η and the GIT2-αPIX-PAK complex, an IS-localized focal adhesion complex. Defective activation of this complex in PKC-η-deficient Treg cells was associated with reduced depletion of CD86 from APCs by Treg cells. These results reveal a CTLA-4-PKC-η signaling axis required for contact-dependent suppression and implicate this pathway as a potential cancer immunotherapy target.

The yin and yang of protein kinase C-theta (PKCθ): a novel drug target for selective immunosuppression.

Protein kinase C-theta (PKCθ) is a protein kinase C (PKC) family member expressed predominantly in T lymphocytes, and extensive studies addressing its function have been conducted. PKCθ is the only T cell-expressed PKC that localizes selectively to the center of the immunological synapse (IS) following conventional T cell antigen stimulation, and this unique localization is essential for PKCθ-mediated downstream signaling. While playing a minor role in T cell development, early in vitro studies relying, among others, on the use of PKCθ-deficient (Prkcq(-/-)) T cells revealed that PKCθ is required for the activation and proliferation of mature T cells, reflecting its importance in activating the transcription factors nuclear factor kappa B, activator protein-1, and nuclear factor of activated T cells, as well as for the survival of activated T cells. Upon subsequent analysis of in vivo immune responses in Prkcq(-/-) mice, it became clear that PKCθ has a selective role in the immune system: it is required for experimental Th2- and Th17-mediated allergic and autoimmune diseases, respectively, and for alloimmune responses, but is dispensable for protective responses against pathogens and for graft-versus-leukemia responses. Surprisingly, PKCθ was recently found to be excluded from the IS of regulatory T cells and to negatively regulate their suppressive function. These attributes of PKCθ make it an attractive target for catalytic or allosteric inhibitors that are expected to selectively suppress harmful inflammatory and alloimmune responses without interfering with beneficial immunity to infections. Early progress in developing such drugs is being made, but additional studies on the role of PKCθ in the human immune system are urgently needed.

SLAT regulates CD8+ T cell clonal expansion in a Cdc42- and NFAT1-dependent manner.

After antigenic stimulation, CD8(+) T cells undergo clonal expansion and differentiation into CTLs that can mount a strong defense against intracellular pathogens and tumors. SWAP-70-like adapter of T cells (SLAT), also known as Def6, is a novel guanine nucleotide exchange factor for the Cdc42 GTPase and plays a role in CD4(+) T cell activation and Th cell differentiation by controlling Ca(2+)/NFAT signaling, but its requirement in CD8(+) T cell response has not been explored. Using a range of transgenic and knockout in vivo systems, we show that SLAT is required for efficient expansion of CD8(+) T cells during the primary response but is not necessary for CTL differentiation. The reduced clonal expansion observed in the absence of SLAT resulted from a CD8(+) T cell-intrinsic proliferation defect and a reduced IL-2-dependent cell survival. On a molecular level, we show that Def6 deficiency resulted in defective TCR/CD28-induced NFAT translocation to the nucleus in CD8(+) T cells. Constitutively active Cdc42 or NFAT1 mutants fully restored the impaired expansion of Def6(-/-) CD8(+) T cells. Taken together, these data describe a new and pivotal role of SLAT-mediated NFAT activation in CD8(+) T cells, providing new insight into the signaling pathways involved in CD8(+) T cell proliferation.

PKC-theta-mediated signal delivery from the TCR/CD28 surface receptors.

Protein kinase C-theta (PKCθ) is a key enzyme in T lymphocytes, where it plays an important role in signal transduction downstream of the activated T cell antigen receptor (TCR) and the CD28 costimulatory receptor. Interest in PKCθ as a potential drug target has increased following recent findings that PKCθ is essential for harmful inflammatory responses mediated by Th2 (allergies) and Th17 (autoimmunity) cells as well as for graft-versus-host disease (GvHD) and allograft rejection, but is dispensable for beneficial responses such as antiviral immunity and graft-versus-leukemia (GvL) response. TCR/CD28 engagement triggers the translocation of the cytosolic PKCθ to the plasma membrane (PM), where it localizes at the center of the immunological synapse (IS), which forms at the contact site between an antigen-specific T cell and antigen-presenting cells (APC). However, the molecular basis for this unique localization, and whether it is required for its proper function have remained unresolved issues until recently. Our recent study resolved these questions by demonstrating that the unique V3 (hinge) domain of PKCθ and, more specifically, a proline-rich motif within this domain, is essential and sufficient for its localization at the IS, where it is anchored to the cytoplasmic tail of CD28 via an indirect mechanism involving Lck protein tyrosine kinase (PTK) as an intermediate. Importantly, the association of PKCθ with CD28 is essential not only for IS localization, but also for PKCθ-mediated activation of downstream signaling pathways, including the transcription factors NF-κB and NF-AT, which are essential for productive T cell activation. Hence, interference with formation of the PKCθ-Lck-CD28 complex provides a promising basis for the design of novel, clinically useful allosteric PKCθ inhibitors. An additional recent study demonstrated that TCR triggering activates the germinal center kinase (GSK)-like kinase (GLK) and induces its association with the SLP-76 adaptor at the IS, where GLK phosphorylates the activation loop of PKCθ, converting it into an active enzyme. This recent progress, coupled with the need to study the biology of PKCθ in human T cells, is likely to facilitate the development of PKCθ-based therapeutic modalities for T cell-mediated diseases.

Protein kinase Cθ C2 domain is a phosphotyrosine binding module that plays a key role in its activation.

Protein kinase Cθ (PKCθ) is a novel PKC that plays a key role in T lymphocyte activation. To understand how PKCθ is regulated in T cells, we investigated the properties of its N-terminal C2 domain that functions as an autoinhibitory domain. Our measurements show that a Tyr(P)-containing peptide derived from CDCP1 binds the C2 domain of PKCθ with high affinity and activates the enzyme activity of the intact protein. The Tyr(P) peptide also binds the C2 domain of PKCδ tightly, but no enzyme activation was observed with PKCδ. Mutations of PKCθ-C2 residues involved in Tyr(P) binding abrogated the enzyme activation and association of PKCθ with Tyr-phosphorylated full-length CDCP1 and severely inhibited the T cell receptor/CD28-mediated activation of a PKCθ-dependent reporter gene in T cells. Collectively, these studies establish the C2 domain of PKCθ as a Tyr(P)-binding domain and suggest that the domain may play a major role in PKCθ activation via its Tyr(P) binding.

Antigen-independent signalosome of CARMA1, PKCθ, and TNF receptor-associated factor 2 (TRAF2) determines NF-κB signaling in T cells.

NF-κB activation is essential for T-cell responses, and costimulatory molecules in the TNF receptor (TNFR) superfamily are viewed as a major source of this signal. Although the TNFR family recruits TNFR-associated factor (TRAF) molecules leading to IKKα/ß/γ activation, it is not clear whether simple binding of TRAFs explains why they are such strong activators of NF-κB and so important for T-cell immunity. We now show that one TNFR family member, OX40 (CD134), after ligation by OX40L, assembles a unique complex that not only contains TRAF2, RIP, and IKKα/ß/γ but also CARMA1, MALT1, BCL10, and PKC, molecules previously shown to regulate NF-κB activation through the T-cell receptor (TCR). The OX40 signalosome is formed in membrane microdomains irrespective of TCR engagement, and strongly promotes NF-κB activation only if CARMA1 and PKC are recruited. This NF-κB signal allows effector/memory T cells to survive when antigen is no longer available. Thus, by recruiting TCR-related intracellular molecules into the TRAF2 complex, OX40 provides the T cell with a high level of NF-κB activity needed for longevity.

Protein kinase C θ deficiency increases resistance of C57BL/6J mice to Plasmodium berghei infection-induced cerebral malaria.

Protein kinase C θ (PKCθ) functions as a core component of the immunological synapse and serves as a key protein in the integrated T-cell antigen receptor (TCR)/CD28-induced signaling cascade leading to T-cell activation. However, the involvement of PKCθ in host-mediated immune responses to pathogens has not been thoroughly investigated. We tested the consequences of PKCθ ablation on the host response to infection by Plasmodium berghei ANKA (PbA). We found that both PKCθ(+/+) and PKCθ(-/-) C57BL/6J mice are susceptible to infection with PbA. However, despite a similar parasite burden, PKCθ(+/+) mice had an earlier onset of neurological signs, characteristics of experimental cerebral malaria (ECM), resulting in an earlier death. These mice suffered from an early and pronounced splenomegaly with a concomitant increase in the total number of CD4(+) splenic T cells. In contrast, a large proportion of PbA-infected PKCθ(-/-) mice overcame the acute phase characterized by neurological symptoms and survived longer than PKCθ(+/+) mice. The partial resistance of PKCθ(-/-) mice to ECM was associated with an impaired production of Th1-type cytokines, including gamma interferon and tumor necrosis factor alpha/lymphotoxin-α, which are known to exacerbate symptoms leading to ECM. In addition, PbA infection-induced LFA-1 expression in CD8(+) T cells was suppressed in PKCθ-deficient T cells, suggesting a diminished ability to adhere to endothelial cells and sequester in brain microvasculature, which may explain the decrease in neurological symptoms. These data implicate PKCθ in CD4(+) Th1(+) and CD8(+) T-cell-mediated immune responses during PbA infection that contribute to the development of ECM.