The serine/threonine protein kinase C (PKC) theta has been firmly implicated in T cell-mediated immunity. Because its role in macrophages has remained undefined, we employed PKCtheta-deficient (PKCtheta −/−) mice in order to investigate if PKCtheta plays a role in macrophage-mediated immune responses during bacterial infections. Our results demonstrate that PKCtheta plays an important role in host defense against the Gram-negative, intracellular bacterium Salmonella typhimurium, as reflected both by markedly decreased survival and a significantly enhanced number of bacteria in spleen and liver of PKCtheta −/− mice, when compared to wild-type mice. Of note, albeit macrophages do not express detectable PKCtheta, PKCtheta mRNA expression was found to be profoundly upregulated during the first hours of lipopolysaccharide (LPS)/interferon-gamma (IFNgamma)-, but not IL-4-mediated cell polarization conditions in vitro. Mechanistically, despite expressing normal levels of classically activated macrophage (CAM) markers, PKCtheta-deficient CAMs expressed significantly higher levels of the anti-inflammatory cytokine IL-10 in vivo and in vitro when challenged with S. typhimurium or LPS/IFNgamma. Neutralization of IL-10 recovered immune control to S. typhimurium infection in PKCtheta-deficient macrophages. Taken together, our data provide genetic evidence that PKCtheta promotes a potent pro-inflammatory CAM phenotype that is instrumental to mounting protective anti-bacterial immunity. Mechanistically, PKCtheta exerts a host-protective role against S. typhimurium infection, and acts as an essential link between TLR4/IFNgammaR signaling and selective suppression of the anti-inflammatory cytokine IL-10 at the onset of CAM differentiation in the course of a bacterial infection.

NEDDylation is a posttranslational modification whereby the ubiquitin-like molecule NEDD8 is attached to protein substrates in a process dependent on NEDD8-activating enzyme regulatory subunit (NAE1). NEDDylation is emerging as a regulator of cancer biology, but its precise role in antitumor immunity has not been thoroughly characterized. In this study, we examine the impact of NEDDylation in CD8+ T cell–mediated antitumor responses. Analysis of publicly available single-cell RNA sequencing databases revealed that CD8+ tumor-infiltrating lymphocytes showed increased expression of NEDD8 during their differentiation into effector memory cells. In vitro activation of mouse and human CD8+ T cells drove the upregulation of the NEDDylation enzymatic pathway, resulting in an enrichment of NEDDylated proteins. In vivo tumor challenge assays demonstrated that CD8+ T cells lacking NAE1 exhibited reduced antitumor capability and a less activated phenotype with compromised differentiation into effector cells. Upregulating NEDDylation by knocking out deNEDDylase sentrin-specific protease 8 increased the in vitro cytotoxic capability of CD8+ CAR T cells. In addition, LC MS/MS proteomic analyses of NAE1-deficient CD8+ T cells and CD8+ T cells treated with the NEDDylation inhibitor MLN4924 showed a pronounced impairment in metabolic pathways, including glycolysis and oxidative phosphorylation. In this context, we validated lactate dehydrogenase A, α-enolase, and hexokinase 1, which are relevant glycolytic enzymes, as NEDD8 targets. In line with this, NEDDylation-deficient CD8+ T cells demonstrated reduced transcription, protein expression, and enzymatic activity of lactate dehydrogenase. In summary, we uncover NEDDylation as a critical regulator of CD8+ T cell–mediated antitumor immunity.

Background: The Protein kinase D3 (PKD3) has been implicated in signal transduction downstream of the T cell receptor (TCR). However, its role for the activation of primary T lymphocytes has not been elucidated so far. Methods: Expression of PKD isoforms in primary murine T cells was determined by RT-PCR and SDS-Page. A germline PKD3-knockout mouse line was analyzed for its immune response to OVA/alum intraperitoneal immunization. Phenotyping of the T cell compartment ex vivo as well as upon stimulation in vitro was performed by flow cytometry. Additionally, cytokine expression was assessed by flow cytometry, RT-PCR and Luminex technology. Results: PKD expression in T cells is modulated by TCR stimulation, leading to a rapid down-regulation on mRNA and on protein level. PKD3-deficient mice respond to immunization with enhanced T follicular helper cell generation. Furthermore, peripheral PKD3-deficient CD4⁺ T cells express more interleukin-2 than wild type CD4⁺ T cells upon TCR stimulation ex vivo. However, purified naïve CD4⁺ T cells do not differ in their phenotype upon differentiation in vitro from wild type T cells. Moreover, we observed a shift towards an effector/memory phenotype of splenic T cells at steady state, which might explain the contradictory results obtained with pan-T cells ex vivo and naïve-sorted T cells. Conclusion: While PKD3-deficiency in vivo in mice leads to a skewing of the T cell compartment towards a more activated phenotype, this kinase seems to be dispensable for naïve CD4⁺ T cell differentiation in vitro.

Cerebral malaria (CM) is the severe progression of an infection with Plasmodium falciparum, causing detrimental damage to brain tissue and is the most frequent cause of Plasmodium falciparum mortality. The critical role of brain-infiltrating CD8⁺ T cells in the pathophysiology of CM having been revealed, our investigation focuses on the role of NR2F6, an established immune checkpoint, as a candidate driver of CM pathology. We employed an experimental mouse model of CM based on Plasmodium berghei ANKA (PbA) infection to compare the relative susceptibility of Nr2f6-knock-out and wild-type C57BL6/N mice. As a remarkable result, Nr2f6 deficiency confers a significant survival benefit. In terms of mechanism, we detected less severe endotheliopathy and, hence, less damage to the blood–brain barrier (BBB), accompanied by decreased sequestered parasites and less cytotoxic T-lymphocytes within the brain, manifesting in a better disease outcome. We present evidence that NR2F6 deficiency renders mice more resistant to experimental cerebral malaria (ECM), confirming a causal and non-redundant role for NR2F6 in the progression of ECM disease. Consequently, pharmacological inhibitors of the NR2F6 pathway could be of use to bolster BBB integrity and protect against CM.

Members of the Protein kinases D (PKD) family are described as regulators of T cell responses. From the two T cell-expressed isoforms PKD2 and PKD3, so far mainly the former was thoroughly investigated and is well understood. Recently, we have investigated also PKD3 using conventional as well as conditional T cell-specific knockout models. These studies suggested PKD3 to be a T cell-extrinsic regulator of the cells' fate. However, these former model systems did not take into account possible redundancies with the highly homologous PKD2. To overcome this issue and thus properly unravel PKD3's T cell-intrinsic functions, here we additionally used a mouse model overexpressing a constitutively active isoform of PKD3 specifically in the T cell compartment. These transgenic mice showed a slightly higher proportion of central memory T cells in secondary lymphoid organs and blood. This effect could not be explained via differences upon polyclonal stimulation in vitro, however, may be connected to the observed developmental aberrances in the CD8 single positive compartment during thymic development. Lastly, the observed alterations in the CD8(+) T cell compartment did not impact proper immune response upon immunization with ovalbumin or in a subcutaneous tumour model suggesting only a small to absent biological relevance. Taking together the knowledge of all our published studies on PKD3 in the T cell compartment, we now conclude that T cell-intrinsic PKD3 is a fine-tuner of central memory T cell as well as CD8 single positive thymocyte development.

Background: Cerebral malaria (CM) is a severe and often fatal complication of Plasmodium falciparum infection that causes devastating brain injury largely through immune-mediated mechanisms. Pathogenic brain-infiltrating CD8⁺ T cells are key drivers of CM pathology, yet the intracellular signals enabling their harmful autoimmune-like activity remain poorly defined. Here, we identify protein kinase C θ (PKCθ), a central antigen receptor-signalling mediator, as a critical contributor to experimental cerebral malaria (ECM). Methods/Results: Using a PKCθ null allele mouse strain on a C57BL/6N background, we demonstrate that PKCθ deficiency significantly improves survival in Plasmodium berghei ANKA (PbA)-infected mice without altering parasite burdens in the blood or brain. Mechanistically, loss of PKCθ skews T cell differentiation towards central memory (Tcm) rather than effector memory (Tem) phenotypes, thereby reducing effector differentiation and sequestration of CD8⁺ T cells in the cerebral microvasculature. This prevents extensive neurovascular damage, preserves neural tissue integrity, and alleviates neurological signs and symptoms. Our findings provide genetic evidence that PKCθ drives CD8⁺ T cell-mediated brain injury in ECM. Conclusions: These results underscore the potential for repurposing clinically PKCθ inhibitors as host-targeted interventions to protect against cerebral injury and improve outcomes in patients with CM.