Unexpected positive control of NFκB and miR-155 by DGKα and ζ ensures effector and memory CD8+ T cell differentiation.

Signals from the T-cell receptor (TCR) and γ-chain cytokine receptors play crucial roles in initiating activation and effector/memory differentiation of CD8 T-cells. We report here that simultaneous deletion of both diacylglycerol kinase (DGK) α and ζ (DKO) severely impaired expansion of CD8 effector T cells and formation of memory CD8 T-cells after Listeria monocytogenes infection. Moreover, ablation of both DGKα and ζ in preformed memory CD8 T-cells triggered death and impaired homeostatic proliferation of these cells. DKO CD8 T-cells were impaired in priming due to decreased expression of chemokine receptors and migration to the draining lymph nodes. Moreover, DKO CD8 T-cells were unexpectedly defective in NFκB-mediated miR-155 transcript, leading to excessive SOCS1 expression and impaired γ-chain cytokine signaling. Our data identified a DGK-NFκB-miR-155-SOCS1 axis that bridges TCR and γ-chain cytokine signaling for robust CD8 T-cell primary and memory responses to bacterial infection.

Role of tumor suppressor TSC1 in regulating antigen-specific primary and memory CD8 T cell responses to bacterial infection.

The serine/threonine kinase mammalian/mechanistic target of rapamycin (mTOR) integrates various environmental cues such as the presence of antigen, inflammation, and nutrients to regulate T cell growth, metabolism, and function. The tuberous sclerosis 1 (TSC1)/TSC2 complex negatively regulates the activity of an mTOR-containing multiprotein complex called mTOR complex 1. Recent studies have revealed an essential cell-intrinsic role for TSC1 in T cell survival, quiescence, and mitochondrial homeostasis. Given the emerging role of mTOR activity in the regulation of the quantity and quality of CD8 T cell responses, in this study, we examine the role of its suppressor, TSC1, in the regulation of antigen-specific primary and memory CD8 T cell responses to bacterial infection. Using an established model system of transgenic CD8 cell adoptive transfer and challenge with Listeria monocytogenes expressing a cognate antigen, we found that TSC1 deficiency impairs antigen-specific CD8 T cell responses, resulting in weak expansion, exaggerated contraction, and poor memory generation. Poor expansion of TSC1-deficient cells was associated with defects in survival and proliferation in vivo, while enhanced contraction was correlated with an increased ratio of short-lived effectors to memory precursors in the effector cell population. This perturbation of effector-memory differentiation was concomitant with decreased expression of eomesodermin among activated TSC1 knockout cells. Upon competitive adoptive transfer with wild-type counterparts and antigen rechallenge, TSC1-deficient memory cells showed moderate defects in expansion but not cytokine production. Taken together, these findings provide direct evidence of a CD8 T cell-intrinsic role for TSC1 in the regulation of antigen-specific primary and memory responses.

Regulation of Lipid Signaling by Diacylglycerol Kinases during T Cell Development and Function.

Diacylglycerol (DAG) and phosphatidic acid (PA) are bioactive lipids synthesized when the T cell receptor binds to a cognate peptide-MHC complex. DAG triggers signaling by recruiting Ras guanyl-releasing protein 1, PKCθ, and other effectors, whereas PA binds to effector molecules that include mechanistic target of rapamycin, Src homology region 2 domain-containing phosphatase 1, and Raf1. While DAG-mediated pathways have been shown to play vital roles in T cell development and function, the importance of PA-mediated signals remains less clear. The diacylglycerol kinase (DGK) family of enzymes phosphorylates DAG to produce PA, serving as a molecular switch that regulates the relative levels of these critical second messengers. Two DGK isoforms, α and ζ, are predominantly expressed in T lineage cells and play an important role in conventional αß T cell development. In mature T cells, the activity of these DGK isoforms aids in the maintenance of self-tolerance by preventing T cell hyper-activation and promoting T cell anergy. In this review, we discuss the roles of DAG-mediated pathways, PA-effectors, and DGKs in T cell development and function. We also highlight recent work that has uncovered previously unappreciated roles for DGK activity, for instance in invariant NKT cell development, anti-tumor and anti-viral CD8 responses, and the directional secretion of soluble effectors.

Role of diacylglycerol kinases in T cell development and function.

Diacylglycerol (DAG), a second messenger generated by phospholipase Cγ1 activity upon engagement of a T-cell receptor, triggers several signaling cascades that play important roles in T cell development and function. A family of enzymes called DAG kinases (DGKs) catalyzes the phosphorylation of DAG to phosphatidic acid, acting as a braking mechanism that terminates DAG-mediated signals. Two DGK isoforms, α and ζ, are expressed predominantly in T cells and synergistically regulate the development of both conventional αß T cells and invariant natural killer T cells in the thymus. In mature T cells, the activity of these DGK isoforms aids in the maintenance of self-tolerance by preventing T-cell hyperactivation upon T cell receptor stimulation and by promoting T-cell anergy. In CD8 cells, reduced DGK activity is associated with enhanced primary responses against viruses and tumors. Recent work also has established an important role for DGK activity at the immune synapse and identified partners that modulate DGK function. In addition, emerging evidence points to previously unappreciated roles for DGK function in directional secretion and T-cell adhesion. This review describes the multitude of roles played by DGKs in T cell development and function and emphasizes recent advances in the field.

Mnk1 and 2 are dispensable for T cell development and activation but important for the pathogenesis of experimental autoimmune encephalomyelitis.

T cell development and activation are usually accompanied by expansion and production of numerous proteins that require active translation. The eukaryotic translation initiation factor 4E (eIF4E) binds to the 5' cap structure of mRNA and is critical for cap-dependent translational initiation. It has been hypothesized that MAPK-interacting kinase 1 and 2 (Mnk1/2) promote cap-dependent translation by phosphorylating eIF4E at serine 209 (S209). Pharmacologic studies using inhibitors have suggested that Mnk1/2 have important roles in T cells. However, genetic evidence supporting such conclusions is lacking. Moreover, the signaling pathways that regulate Mnk1/2 in T cells remain unclear. We demonstrate that TCR engagement activates Mnk1/2 in primary T cells. Such activation is dependent on Ras-Erk1/2 signaling and is inhibited by diacylglycerol kinases α and ζ. Mnk1/2 double deficiency in mice abolishes TCR-induced eIF4E S209 phosphorylation, indicating their absolute requirement for eIF4E S209 phosphorylation. However, Mnk1/2 double deficiency does not affect the development of conventional αß T cells, regulatory T cells, or NKT cells. Furthermore, T cell activation, in vivo primary and memory CD8 T cell responses to microbial infection, and NKT cell cytokine production were not obviously altered by Mnk1/2 deficiency. Although Mnk1/2 deficiency causes decreased IL-17 and IFN-γ production by CD4 T cells following immunization of mice with myelin oligodendrocyte glycoprotein peptide in complete Freund's adjuvant, correlating with milder experimental autoimmune encephalomyelitis scores, it does not affect Th cell differentiation in vitro. Together, these data suggest that Mnk1/2 has a minimal role in T cell development and activation but may regulate non-T cell lineages to control Th1 and Th17 differentiation in vivo.

Chronic activation of the kinase IKKß impairs T cell function and survival.

Activation of the transcription factor NF-κB is critical for cytokine production and T cell survival after TCR engagement. The effects of persistent NF-κB activity on T cell function and survival are poorly understood. In this study, using a murine model that expresses a constitutively active form of inhibitor of NF-κB kinase ß (caIKKß) in a T cell-specific manner, we demonstrate that chronic inhibitor of NF-κB kinase ß signaling promotes T cell apoptosis, attenuates responsiveness to TCR-mediated stimulation in vitro, and impairs T cell responses to bacterial infection in vivo. caIKKß T cells showed increased Fas ligand expression and caspase-8 activation, and blocking Fas/Fas ligand interactions enhanced cell survival. T cell unresponsiveness was associated with defects in TCR proximal signaling and elevated levels of B lymphocyte-induced maturation protein 1, a transcriptional repressor that promotes T cell exhaustion. caIKKß T cells also showed a defect in IL-2 production, and addition of exogenous IL-2 enhanced their survival and proliferation. Conditional deletion of B lymphocyte-induced maturation protein 1 partially rescued the sensitivity of caIKKß T cells to TCR triggering. Furthermore, adoptively transferred caIKKß T cells showed diminished expansion and increased contraction in response to infection with Listeria monocytogenes expressing a cognate Ag. Despite their functional defects, caIKKß T cells readily produced proinflammatory cytokines, and mice developed autoimmunity. In contrast to NF-κB's critical role in T cell activation and survival, our study demonstrates that persistent IKK-NF-κB signaling is sufficient to impair both T cell function and survival.