SUMMARY T cells undergo metabolic reprogramming with major changes in cellular energy metabolism during activation. In patients with mitochondrial disease, clinical data were marked by frequent infections and immunodeficiency, prompting us to explore the consequences of oxidative phosphorylation dysfunction in T cells. Since cytochrome c oxidase (COX) is a critical regulator of OXPHOS, we created a mouse model with isolated dysfunction in T cells by targeting a gene, COX10, that produces mitochondrial disease in humans. COX dysfunction resulted in increased apoptosis following activation in vitro and immunodeficiency in vivo. Select T cell effector subsets were particularly affected; this could be traced to their bioenergetic requirements. In summary, the findings presented herein emphasize the role of COX particularly in T cells as a metabolic checkpoint for cell fate decisions following T cell activation, with heterogeneous effects in T cell subsets. In addition, our studies highlight the utility of translational models that recapitulate human mitochondrial disease for understanding immunometabolism.

CD8 + cytotoxic T lymphocytes (CTLs) eliminate virally infected cells through directed secretion of specialized lytic granules. Because a single CTL can kill multiple targets, degranulation must be tightly regulated. However, how CTLs regulate the termination of granule secretion remains unclear. Previous work demonstrated that centralized actin reduction at the immune synapse precedes degranulation. Using a combination of live confocal, total internal reflection fluorescence, and superresolution microscopy, we now show that, after granule fusion, actin recovers at the synapse and no further secretion is observed. Depolymerization of actin led to resumed granule secretion, suggesting that recovered actin acts as a barrier preventing sustained degranulation. Furthermore, RAB27a-deficient CTLs, which do not secrete cytotoxic granules, failed to recover actin at the synapse, suggesting that RAB27a-mediated granule secretion is required for actin recovery. Finally, we show that both actin clearance and recovery correlated with synaptic phosphatidylinositol 4,5-bisphosphate (PIP 2 ) and that alterations in PIP 2 at the immunological synapse regulate cortical actin in CTLs, providing a potential mechanism through which CTLs control cortical actin density. Our work provides insight into actin-related mechanisms regulating CTL secretion that may facilitate serial killing during immune responses.cytotoxic T lymphocytes | actin | lytic granule secretion | PIP 2 | RAB27a

X-linked lymphoproliferative disease (XLP-1) is an often-fatal primary immunodeficiency associated with the exuberant expansion of activated CD8+ T cells following Epstein-Barr virus (EBV) infection. XLP-1 is caused by defects in SAP, an adaptor protein that modulates T cell receptor (TCR)-induced signaling. SAP-deficient T cells exhibit impaired TCR restimulation-induced cell death (RICD) and diminished TCR-induced inhibition of diacylglycerol kinase alpha (DGKα), leading to increased diacylglycerol metabolism and decreased signaling through Ras and PKCθ. Here, we show that down-regulation of DGKα activity in SAP-deficient T cells restores diacylglycerol signaling at the immune synapse and rescues RICD via induction of the pro-apoptotic proteins NUR77 and NOR1. Importantly, pharmacological inhibition of DGKα prevents the excessive CD8+ T cell expansion and IFNγ production that occur in Sap-deficient mice following Lymphocytic Choriomeningitis Virus infection without impairing lytic activity. Collectively, these data highlight DGKα as a viable therapeutic target to reverse the life-threatening EBV-associated immunopathology that occurs in XLP-1 patients.

Key Points• DDR2 regulates the directional migration of neutrophils in 3D collagen matrices, but not on 2D surfaces. • DDR2 regulates directionality through increased metalloproteinase secretion and generation of collagen-derived chemotactic peptide gradients.Neutrophils express a variety of collagen receptors at their surface, yet their functional significance remains unclear. Although integrins are essential for neutrophil adhesion and migration on 2-dimensional (2D) surfaces, neutrophils can compensate for the absence of integrins in 3-dimensional (3D) lattices. In contrast, we demonstrate that the inhibition of the tyrosine-kinase collagen receptor discoidin domain receptor 2 (DDR2) has no impact on human primary neutrophil migration on 2D surfaces but is an important regulator of neutrophil chemotaxis in 3D collagen matrices. In this context, we show that DDR2 activation specifically regulates the directional migration of neutrophils in chemoattractant gradients. We further demonstrate that DDR2 regulates directionality through its ability to increase secretion of metalloproteinases and local generation of collagen-derived chemotactic peptide gradients. Our findings highlight the importance of collagen-derived extracellular signaling during neutrophil chemotaxis in 3D matrices. (Blood. 2013;121(9):1644-1650) IntroductionNeutrophils exhibit the ability to maintain robust migration under a wide array of distinct environmental conditions. For example, by increasing the rate of actin polymerization, neutrophils lacking integrins are able to retain normal migration speeds in 3D environments. 1,2 We set out to determine the role of another collagen receptor family, the discoidin domain receptors (DDRs), during neutrophil chemotaxis. The DDR family is composed of 2 members, DDR1 and DDR2. DDRs are homodimeric receptor tyrosine kinases that bind to triple-helical collagen fibers through a domain similar to discoidin 1 of the social amoeba Dictyostelium discoideum. 3,4 On binding to collagen, DDRs become activated and serve as docking sites for many signaling pathways. 5 A common outcome after DDR activation is the increase in metalloproteinase (MMP) secretion and collagen rearrangement. [6][7][8][9] As a consequence, DDR1 and DDR2 have both been associated with metastasis during tumor progression. 6 Similarly, DDR1 overexpression has been shown to enhance lymphocyte migration. 10,11 However, the mechanism underlying the enhanced leukocyte migration remains unknown.We show that circulating human primary neutrophils solely express DDR2 and establish that DDR2 regulates the ability of neutrophils to migrate directionally toward chemoattractants in a collagen I matrix. DDR2 activation induces increased MMP secretion, leading to the generation of collagen-derived chemotactic peptides that are poised to form local gradients and enhance neutrophil persistence. Methods Isolation of human blood neutrophilsBlood was collected from anonymous healthy donors enrolled in the National Institutes of Health Blood Bank research prog...

Activated phosphoinositide 3-kinase delta syndrome (APDS), also known as p110 delta-activating mutation causing senescent T cells, lymphadenopathy and immunodeficiency (PASLI), is an autosomal dominant primary human immunodeficiency (PID) caused by heterozygous gain-of-function mutations in PIK3CD, which encodes the p110δ catalytic subunit of PI3K. This recently described PID is characterized by diverse and heterogeneous clinical manifestations that include recurrent respiratory infections, lymphoproliferation, progressive lymphopenia, and defective antibody responses. A major clinical manifestation observed in the NIH cohort of patients with PIK3CD mutations is chronic Epstein–Barr virus (EBV) and/or cytomegalovirus viremia. Despite uncontrolled EBV infection, many APDS/PASLI patients had normal or higher frequencies of EBV-specific CD8+ T cells. In this review, we discuss data pertaining to CD8+ T cell function in APDS/PASLI, including increased cell death, expression of exhaustion markers, and altered killing of autologous EBV-infected B cells, and how these and other data on PI3K provide insight into potential cellular defects that prevent clearance of chronic infections.

To find an alternative endpoint for the efficacy of antismallpox treatments, bioluminescence was measured in live BALB/c mice following lethal challenge with a recombinant WR vaccinia virus expressing luciferase. Intravenous vaccinia immunoglobulin treatments were used to confer protection on a proportion of animals. Using known lethality outcomes in 200 animals and total fluxes recorded daily in live animals, we performed univariate receiver operating characteristic (ROC) curve analysis to assess whether lethality can be predicted based on bioluminescence. Total fluxes in the spleens on day 3 and in the livers on day 5 generated accurate predictive models; the area under the ROC curve (AUC) was 0.91. Multiple logistic regression analysis utilizing a linear combination of six measurements: total flux in the liver on days 2, 3, and 5; in the spleen on days 1 and 3; and in the nasal cavity on day 4 generated the most accurate predictions (AUC ‫؍‬ 0.96). This model predicted lethality in 90% of animals with only 10% of nonsurviving animals incorrectly predicted to survive. Compared with bioluminescence, ROC analysis with 25% and 30% weight loss as thresholds accurately predicted survival on day 5, but lethality predictions were low until day 9. Collectively, our data support the use of bioimaging for lethality prediction following vaccinia virus challenge and for gaining insight into protective mechanisms conferred by vaccines and therapeutics.

Immunometabolism aims to define the role of intermediary metabolism in immune cell function, with bioenergetics and the mitochondria recently taking center stage. To date, the medical literature on mitochondria and immune function extols the virtues of mouse models in exploring this biologic intersection. While the laboratory mouse has become a standard for studying mammalian biology, this model comprises part of a comprehensive approach. Humans, with their broad array of inherited phenotypes, serve as a starting point for studying immunometabolism; specifically, patients with mitochondrial disease. Using this top-down approach, the mouse as a model organism facilitates further exploration of the consequences of mutations involved in mitochondrial maintenance and function. In this review, we will discuss the emerging phenotype of immune dysfunction in mitochondrial disease as a model for understanding the role of the mitochondria in immune function in available mouse models.

Patients with mutations in ITK are susceptible to viral infections, particularly Epstein Barr Virus, suggesting that these patients have defective function of CD8+ cytolytic T lymphocytes (CTLs). Here, we evaluated the effects of ITK-deficiency on cytolysis in murine CTLs deficient in ITK, and both human and murine cells treated with an ITK inhibitor. We find that ITK-deficiency leads to a global defect in the cytolysis of multiple targets. The absence of ITK both affected CTL expansion and delayed the expression of cytolytic effectors during activation. Furthermore, absence of ITK led to a previously unappreciated intrinsic defect in degranulation. Nonetheless, these defects could be overcome by early or prolonged exposure to IL-2, or by addition of IL-12 to cultures, revealing that cytokine signaling could restore the acquisition of effector function in ITK-deficient CD8+ T cells. Our results provide new insight into the effect of ITK and suboptimal TCR signaling on CD8+ T cell function, and how these may contribute to phenotypes associated with ITK-deficiency.