Sorting Nexin 27-dependent regulation of Lck and CD4 tunes the initial stages of T-cell activation.

Sorting nexin (SNX) 27 is a unique member of the SNX family of proteins that mediates the endosome-to-plasma membrane trafficking of cargos bearing a PSD95/Dlg1/ZO-1 (PDZ)-binding motif. In brain, SNX27 regulates synaptic plasticity, and its dysregulation contributes to cognitive impairment and neuronal degeneration. In T lymphocytes, SNX27 partners with diacylglycerol (DAG) kinase ζ (DGKζ) to facilitate polarized traffic and signaling at the immune synapse (IS). By silencing SNX27 expression in a human T cell line, we demonstrate that SNX27 is a key regulator of the early T cell tyrosine-based signaling cascade. SNX27 transcriptionally controls CD4 abundance in resting conditions, and that of its associated molecule, Lck. This guarantees the adequate recruitment of Lck at the IS that is indispensable for subsequent activation of tyrosine phosphorylation regulated events. In contrast, reduced SNX27 expression enhances NFκB-dependent induction of CXCR4 and triggers production of lytic enzymes and pro-inflammatory cytokines. These results provide mechanistic explanation to previously described SNX27 function in the control of immune synapse organization and indicate that impaired SNX27 expression contributes to CD4 T cell dysfunction.

Partial loss of Sorting Nexin 27 resembles age- and Down syndrome-associated T cell dysfunctions.

BACKGROUND: Sorting Nexin 27 (SNX27)-retromer complex facilitates cargo recycling from endosomes to the plasma membrane. SNX27 downregulation in neurons, as the result of Trisomy 21 (T21), has been linked with cognitive deficits due to impairment of AMPA and NMDA receptor recycling. Studies in human T cell lines likewise demonstrated that SNX27 regulates the correct delivery of cargoes to the immune synapse limiting the activation of pro-inflammatory pathways. Nevertheless, the physiological consequences of partial SNX27 loss in T cell homeostasis are still unclear. RESULTS: In this study, we have explored the consequences of T cell specific partial SNX27 downregulation in mice. T cells with partial SNX27 deficiency show a marked deficit in the CD4+ T cell pool, a hallmark of aging in mice and humans, and a well-characterized comorbidity of individuals with Down syndrome (DS). When analyzed ex vivo, CD4+ T cells with partial SNX27 deletion demonstrate enhanced proliferation but diminished IL-2 production. In contrast, the CD8+ population show enhanced expression of pro-inflammatory cytokines and lytic enzymes. CONCLUSIONS: This mouse model supports the relevance of SNX27 in the organization of the immune synapse, previously described in cell lines, as well as in the control of T cell homeostasis. Individuals with DS experiment an acceleration of the aging process, which particularly affects the immune and central nervous systems. Thus, we hypothesize that reduced SNX27 expression in DS could contribute to the dysregulation of these systems and further research in SNX27 will shed light on the molecular factors underlying the phenotypes observed in people with DS and its contribution to aging.

Diacylglycerol kinases: A look into the future of immunotherapy.

Cancer still represents the second leading cause of death right after cardiovascular diseases. According to the World Health Organization (WHO), cancer provoked around 10 million deaths in 2020, with lung and colon tumors accounting for the deadliest forms of cancer. As tumor cells become resistant to traditional therapeutic approaches, immunotherapy has emerged as a novel strategy for tumor control. T lymphocytes are key players in immune responses against tumors. Immunosurveillance allows identification, targeting and later killing of cancerous cells. Nevertheless, tumors evolve through different strategies to evade the immune response and spread in a process called metastasis. The ineffectiveness of traditional strategies to control tumor growth and expansion has led to novel approaches considering modulation of T cell activation and effector functions. Program death receptor 1 (PD-1) and cytotoxic T-lymphocyte antigen 4 (CTLA-4) showed promising results in the early 90s and nowadays are still being exploited together with other drugs for several cancer types. Other negative regulators of T cell activation are diacylglycerol kinases (DGKs) a family of enzymes that catalyze the conversion of diacylglycerol (DAG) into phosphatidic acid (PA). In T cells, DGKα and DGKζ limit the PLCγ/Ras/ERK axis thus attenuating DAG mediated signaling and T cell effector functions. Upregulation of either of both isoforms results in impaired Ras activation and anergy induction, whereas germline knockdown mice showed enhanced antitumor properties and more effective immune responses against pathogens. Here we review the mechanisms used by DGKs to ameliorate T cell activation and how inhibition could be used to reinvigorate T cell functions in cancer context. A better knowledge of the molecular mechanisms involved upon T cell activation will help to improve current therapies with DAG promoting agents.

Identification of Host PDZ-Based Interactions with the SARS-CoV-2 E Protein in Human Monocytes.

Proteins containing PDZ (post-synaptic density, PSD-95/disc large, Dlg/zonula occludens, ZO-1) domains assemble signaling complexes that orchestrate cell responses. Viral pathogens target host PDZ proteins by coding proteins containing a PDZ-binding motif (PBM). The presence of a PBM in the SARS-CoV-2 E protein contributes to the virus's pathogenicity. SARS-CoV-2 infects epithelia, but also cells from the innate immune response, including monocytes and alveolar macrophages. This process is critical for alterations of the immune response that are related to the deaths caused by SARS-CoV-2. Identification of E-protein targets in immune cells might offer clues to understanding how SARS-CoV-2 alters the immune response. We analyzed the interactome of the SARS-CoV-2 E protein in human monocytes. The E protein was expressed fused to a GFP tag at the amino terminal in THP-1 monocytes, and associated proteins were identified using a proteomic approach. The E-protein interactome provided 372 partners; only 8 of these harbored PDZ domains, including the cell polarity protein ZO-2, the chemoattractant IL-16, and syntenin. We addressed the expression and localization of the identified PDZ proteins along the differentiation of primary and THP-1 monocytes towards macrophages and dendritic cells. Our data highlight the importance of identifying the functions of PDZ proteins in the maintenance of immune fitness and the viral alteration of inflammatory response.

SARS-CoV-2 Spike Protein and Its Receptor Binding Domain Promote a Proinflammatory Activation Profile on Human Dendritic Cells.

Dendritic cells (DCs) are the most potent antigen-presenting cells, and their function is essential to configure adaptative immunity and avoid excessive inflammation. DCs are predicted to play a crucial role in the clinical evolution of the infection by the severe acute respiratory syndrome (SARS) coronavirus (CoV)-2. DCs interaction with the SARS-CoV-2 Spike protein, which mediates cell receptor binding and subsequent fusion of the viral particle with host cell, is a key step to induce effective immunity against this virus and in the S protein-based vaccination protocols. Here we evaluated human DCs in response to SARS-CoV-2 S protein, or to a fragment encompassing the receptor binding domain (RBD) challenge. Both proteins increased the expression of maturation markers, including MHC molecules and costimulatory receptors. DCs interaction with the SARS-CoV-2 S protein promotes activation of key signaling molecules involved in inflammation, including MAPK, AKT, STAT1, and NFκB, which correlates with the expression and secretion of distinctive proinflammatory cytokines. Differences in the expression of ACE2 along the differentiation of human monocytes to mature DCs and inter-donor were found. Our results show that SARS-CoV-2 S protein promotes inflammatory response and provides molecular links between individual variations and the degree of response against this virus.

Diacylglycerol kinase α inhibition cooperates with PD-1-targeted therapies to restore the T cell activation program.

BACKGROUND: Antibody-based therapies blocking the programmed cell death-1/ligand-1 (PD-1/PD-L1) axis have provided unprecedent clinical success in cancer treatment. Acquired resistance, however, frequently occurs, commonly associated with the upregulation of additional inhibitory molecules. Diacylglycerol kinase (DGK) α limits the extent of Ras activation in response to antigen recognition, and its upregulation facilitates hypofunctional, exhausted T cell states. Pharmacological DGKα targeting restores cytotoxic function of chimeric antigen receptor and CD8+ T cells isolated from solid tumors, suggesting a mechanism to reverse T cell exhausted phenotypes. Nevertheless, the contribution of DGKα downstream of the PD-1/PD-L1 inhibitory axis in human T cells and the consequences of combining DGKα and anti-PD-1/PD-L1 inhibitors are still unresolved relevant issues. MATERIALS AND METHODS: We used a human triple parameter reporter cell line to investigate DGKα contribution to the PD-1/PD-L1 inhibitory pathway. We also addressed the impact of deleting DGKα expression in the growth dynamics and systemic tumor-derived effects of a PD-1-related tumor model, the MC38 colon adenocarcinoma. RESULTS: We identify DGKα as a contributor to the PD-1/PD-L1 axis that strongly limits the Ras/ERK/AP-1 pathway. DGKα function reinforces exhausted T cell phenotypes ultimately promoting tumor growth and generalized immunosuppression. Pharmacological DGKα inhibition selectively enhances AP-1 transcription and, importantly, cooperates with antibodies blocking the PD-1/PD-L1 interrelation. CONCLUSIONS: Our results indicate that DGKα inhibition could provide an important mechanism to revert exhausted T lymphocyte phenotypes and thus favor proper anti-tumor T cell responses. The cooperative effect observed after PD-1/PD-L1 and DGKα blockade offers a promising strategy to improve the efficacy of immunotherapy in the treatment of cancer.

Sorting Nexin 27 Enables MTOC and Secretory Machinery Translocation to the Immune Synapse.

Sorting nexin 27 (SNX27) association to the retromer complex mediates intracellular trafficking of cargoes containing PSD95/Dlg1/ZO-1 (PDZ)-binding C-terminal sequences from endosomes to the cell surface, preventing their lysosomal degradation. Antigen recognition by T lymphocyte leads to the formation of a highly organized structure named the immune synapse (IS), which ensures cell-cell communication and sustained T cell activation. At the neuronal synapse, SNX27 recycles PDZ-binding receptors and its defective expression is associated with synaptic dysfunction and cognitive impairment. In T lymphocytes, SNX27 was found localized at recycling endosomal compartments that polarized to the IS, suggesting a function in polarized traffic to this structure. Proteomic analysis of PDZ-SNX27 interactors during IS formation identify proteins with known functions in cytoskeletal reorganization and lipid regulation, such as diacylglycerol (DAG) kinase (DGK) ζ, as well as components of the retromer and WASH complex. In this study, we investigated the consequences of SNX27 deficiency in cytoskeletal reorganization during IS formation. Our analyses demonstrate that SNX27 controls the polarization towards the cell-cell interface of the PDZ-interacting cargoes DGKζ and the retromer subunit vacuolar protein sorting protein 26, among others. SNX27 silencing abolishes the formation of a DAG gradient at the IS and prevents re-localization of the dynactin complex component dynactin-1/p150Glued, two events that correlate with impaired microtubule organizing center translocation (MTOC). SNX27 silenced cells show marked alteration in cytoskeleton organization including a failure in the organization of the microtubule network and defects in actin clearance at the IS. Reduced SNX27 expression was also found to hinder the arrangement of signaling microclusters at the IS, as well as the polarization of the secretory machinery towards the antigen presenting cells. Our results broaden the knowledge of SNX27 function in T lymphocytes by showing a function in modulating IS organization through regulated trafficking of cargoes.

Diacylglycerol kinase ζ limits IL-2-dependent control of PD-1 expression in tumor-infiltrating T lymphocytes.

BACKGROUND: The inhibitory functions triggered by the programmed cell death-1 (PD-1) receptor following binding to its ligand (PD-L1) protect healthy organs from cytotoxic T cells, and neutralize antitumor T cell attack. Antibody-based therapies to block PD-1/PD-L1 interaction have yielded notable results, but most patients eventually develop resistance. This failure is attributed to CD8+ T cells achieving hyporesponsive states from which recovery is hardly feasible. Dysfunctional T cell phenotypes are favored by a sustained imbalance in the diacylglycerol (DAG)- and Ca2+-regulated transcriptional programs. In mice, DAG kinase ζ (DGKζ) facilitates DAG consumption, limiting T cell activation and cytotoxic T cell responses. DGKζ deficiency facilitates tumor rejection in mice without apparent adverse autoimmune effects. Despite its therapeutic potential, little is known about DGKζ function in human T cells, and no known inhibitors target this isoform. METHODS: We used a human triple parameter reporter cell line to examine the consequences of DGKζ depletion on the transcriptional restriction imposed by PD-1 ligation. We studied the effect of DGKζ deficiency on PD-1 expression dynamics, as well as the impact of DGKζ absence on the in vivo growth of MC38 adenocarcinoma cells. RESULTS: We demonstrate that DGKζ depletion enhances DAG-regulated transcriptional programs, promoting interleukin-2 production and partially counteracting PD-1 inhibitory functions. DGKζ loss results in limited PD-1 expression and enhanced expansion of cytotoxic CD8+ T cell populations. This is observed even in immunosuppressive milieus, and correlates with the reduced ability of MC38 adenocarcinoma cells to form tumors in DGKζ-deficient mice. CONCLUSIONS: Our results, which define a role for DGKζ in the control of PD-1 expression, confirm DGKζ potential as a therapeutic target as well as a biomarker of CD8+ T cell dysfunctional states.

Interplay Between SNX27 and DAG Metabolism in the Control of Trafficking and Signaling at the IS.

Recognition of antigens displayed on the surface of an antigen-presenting cell (APC) by T-cell receptors (TCR) of a T lymphocyte leads to the formation of a specialized contact between both cells named the immune synapse (IS). This highly organized structure ensures cell-cell communication and sustained T-cell activation. An essential lipid regulating T-cell activation is diacylglycerol (DAG), which accumulates at the cell-cell interface and mediates recruitment and activation of proteins involved in signaling and polarization. Formation of the IS requires rearrangement of the cytoskeleton, translocation of the microtubule-organizing center (MTOC) and vesicular compartments, and reorganization of signaling and adhesion molecules within the cell-cell junction. Among the multiple players involved in this polarized intracellular trafficking, we find sorting nexin 27 (SNX27). This protein translocates to the T cell-APC interface upon TCR activation, and it is suggested to facilitate the transport of cargoes toward this structure. Furthermore, its interaction with diacylglycerol kinase ζ (DGKζ), a negative regulator of DAG, sustains the precise modulation of this lipid and, thus, facilitates IS organization and signaling. Here, we review the role of SNX27, DAG metabolism, and their interplay in the control of T-cell activation and establishment of the IS.

Diacylglycerol kinase ζ promotes actin cytoskeleton remodeling and mechanical forces at the B cell immune synapse.

Diacylglycerol kinases (DGKs) limit antigen receptor signaling in immune cells by consuming the second messenger diacylglycerol (DAG) to generate phosphatidic acid (PA). Here, we showed that DGKζ promotes lymphocyte function-associated antigen 1 (LFA-1)-mediated adhesion and F-actin generation at the immune synapse of B cells with antigen-presenting cells (APCs), mostly in a PA-dependent manner. Measurement of single-cell mechanical force generation indicated that DGKζ-deficient B cells exerted lower forces at the immune synapse than did wild-type B cells. Nonmuscle myosin activation and translocation of the microtubule-organizing center (MTOC) to the immune synapse were also impaired in DGKζ-deficient B cells. These functional defects correlated with the decreased ability of B cells to present antigen and activate T cells in vitro. The in vivo germinal center response of DGKζ-deficient B cells was also reduced compared with that of wild-type B cells, indicating that loss of DGKζ in B cells impaired T cell help. Together, our data suggest that DGKζ shapes B cell responses by regulating actin remodeling, force generation, and antigen uptake-related events at the immune synapse. Hence, an appropriate balance in the amounts of DAG and PA is required for optimal B cell function.

Biological regulation of diacylglycerol kinases in normal and neoplastic tissues: New opportunities for cancer immunotherapy.

In the recent years, the arsenal of anti-cancer therapies has evolved to target T lymphocytes and restore their capacity to destroy tumor cells. However, the clinical success is limited, with a large number of patients that never responds and others that ultimately develop resistances. Overcoming the hypofunctional state imposed by solid tumors to T cells has revealed critical but challenging due to the complex strategies that tumors employ to evade the immune system. The Diacylglycerol kinases (DGK) limit DAG-dependent functions in T lymphocytes and their upregulation in tumor-infiltrating T lymphocytes contribute to limit T cell cytotoxic potential. DGK blockade could reinstate T cell attack on tumors, limiting at the same time tumor cell growth, thanks to the DGK positive input into several oncogenic pathways. In this review we summarize the latest findings regarding the regulation of specific DGK isoforms in healthy and anergic T lymphocytes, as well as their contribution to oncogenic phenotypes. We will also revise the latest advances in the search for pharmacological inhibitors and their potential as anti-cancer agents, either alone or in combination with immunomodulatory agents.

Diacylglycerol Kinase Malfunction in Human Disease and the Search for Specific Inhibitors.

The diacylglycerol kinases (DGKs) are master regulator kinases that control the switch from diacylglycerol (DAG) to phosphatidic acid (PA), two lipids with important structural and signaling properties. Mammalian DGKs distribute into five subfamilies that regulate local availability of DAG and PA pools in a tissue- and subcellular-restricted manner. Pharmacological manipulation of DGK activity holds great promise, given the critical contribution of specific DGK subtypes to the control of membrane structure, signaling complexes, and cell-cell communication. The latest advances in the DGK field have unveiled the differential contribution of selected isoforms to human disease. Defects in the expression/activity of individual DGK isoforms contribute substantially to cognitive impairment, mental disorders, insulin resistance, and vascular pathologies. Abnormal DGK overexpression, on the other hand, confers the acquisition of malignant traits including invasion, chemotherapy resistance, and inhibition of immune attack on tumors. Translation of these findings into therapeutic approaches will require development of methods to pharmacologically modulate DGK functions. In particular, inhibitors that target the DGKα isoform hold particular promise in the fight against cancer, on their own or in combination with immune-targeting therapies.

Diacylglycerol kinase control of protein kinase C.

The diacylglycerol kinases (DGK) are lipid kinases that transform diacylglycerol (DAG) into phosphatidic acid (PA) in a reaction that terminates DAG-based signals. DGK provide negative regulation to conventional and novel protein kinase C (PKC) enzymes, limiting local DAG availability in a tissue- and subcellular-restricted manner. Defects in the expression/activity of certain DGK isoforms contribute substantially to cognitive impairment and mental disorders. Abnormal DGK overexpression in tumors facilitates invasion and resistance to chemotherapy preventing tumor immune destruction by tumor-infiltrating lymphocytes. Effective translation of these findings into therapeutic approaches demands a better knowledge of the physical and functional interactions between the DGK and PKC families. DGKζ is abundantly expressed in the nervous and immune system, where physically and functionally interacts with PKCα. The latest discoveries suggest that PDZ-mediated interaction facilitates spatial restriction of PKCα by DGKζ at the cell-cell contact sites in a mechanism where the two enzymes regulate each other. In T lymphocytes, DGKζ interaction with Sorting Nexin 27 (SNX27) guarantees the basal control of PKCα activation. SNX27 is a trafficking component required for normal brain function whose deficit has been linked to Alzheimer's disease (AD) pathogenesis. The enhanced PKCα activation as the result of SNX27 silencing in T lymphocytes aligns with the recent correlation found between gain-of-function PKCα mutations and AD and suggests that disruption of the mechanisms that provides a correct spatial organization of DGKζ and PKCα may lie at the basis of immune and neuronal synapse impairment.

Transcriptional Activity of FOXO Transcription Factors Measured by Luciferase Assays.

The Forkhead box O (FOXO) family of transcription factors translates environmental cues into gene expression. FOXO factors are crucial for the maintenance of cell homeostasis, with important roles in cell fate decisions and differentiation. Identification of FOXO target genes requires strict validation by several methods. Luciferase-based reporters are a valuable starting point for determining the transcription-promoting capacity of potential FOXO-binding sites in candidate genes. Luciferase, an enzyme found in bioluminescent organisms catalyzes oxidation of luciferin to produce oxyluciferin together with light, which can be easily detected and measured with a luminometer. Due to their many advantages, transcriptional assays based on luciferase activity are widely used; they are easy, highly reproducible, and very sensitive. Continued improvements in luciferase-based vectors and measurement reagents confer considerable versatility. Luciferase-based reporters are also a reliable approach in the search for unknown components in the signaling pathways that control FOXO factor activity.We previously reported that FOXO transcription factors control expression of the enzyme diacylglycerol kinase α (DGKα) in T cells. DGKα consumes diacylglycerol, a lipid that activates several mitogenic pathways. Here, we describe the use of a luciferase-based promoter bearing the FOXO-binding sites of the DGKα gene to explore the relationship between the expression of this enzyme and stress conditions in NIH3T3 mouse fibroblasts. Our data support a role for FOXO factors in promoting high DGKα levels in conditions of growth factor deprivation. DGKα regulation by FOXO factors correlates with the reported alterations in DGKα expression during cell transformation and cancer progression.

Diacylglycerol kinase α inactivation is an integral component of the costimulatory pathway that amplifies TCR signals.

The arsenal of cancer therapies has evolved to target T lymphocytes and restore their capacity to destroy tumor cells. T cells rely on diacylglycerol (DAG) to carry out their functions. DAG availability and signaling are regulated by the enzymes diacylglycerol kinase (DGK) α and ζ, whose excess function drives T cells into hyporesponsive states. Targeting DGKα is a promising strategy for coping with cancer; its blockade could reinstate T-cell attack on tumors while limiting tumor growth, due to positive DGKα functions in several oncogenic pathways. Here, we made a side-by-side comparison of the effects of commercial pharmacological DGK inhibitors on T-cell responses with those promoted by DGKα and DGKζ genetic deletion or silencing. We show the specificity for DGKα of DGK inhibitors I and II and the structurally similar compound ritanserin. Inhibitor treatment promoted Ras/ERK (extracellular signal-regulated kinase) signaling and AP-1 (Activator protein-1) transcription, facilitated DGKα membrane localization, reduced the requirement for costimulation, and cooperated with enhanced activation following DGKζ silencing/deletion. DGKiII and ritanserin had similar effects on TCR proximal signaling, but ritanserin counteracted long-term T-cell activation, an effect that was potentiated in DGKα-/- cells. In contrast with enhanced activation triggered by pharmacological inhibition, DGKα silencing/genetic deletion led to impaired Lck (lymphocyte-specific protein tyrosine kinase) activation and limited costimulation responses. Our results demonstrate that pharmacological inhibition of DGKα downstream of the TCR provides a gain-of-function effect that amplifies the DAG-dependent signaling cascade, an ability that could be exploited therapeutically to reinvigorate T cells to attack tumors.

Sorting nexin 27 interactome in T-lymphocytes identifies zona occludens-2 dynamic redistribution at the immune synapse.

T Lymphocyte recognition of antigens leads to the formation of a highly organized structure termed immune synapse (IS) by analogy with the neuronals synapse. Sorting nexin 27 (SNX27) controls the endosomal traffic of PSD95, Dlg1, ZO-1 (PDZ) domain-interacting proteins, and its alteration is associated with impaired synaptic function and neurological diseases. In T-lymphocytes, SNX27-positive vesicles polarize to the IS, the identity of SNX27 interactors in these conditions nonetheless remains unknown. Here we used proteomics to analyze the SNX27 interactome purified from IS-forming T cells, and confirmed the conserved nature of the SNX27/WASH/retromer association in hematopoietic cells. Furthermore, our comparative interactome analysis of SNX27 wild-type and a mutant-deficient for PDZ cargo recognition identified the epithelial cell-cell junction protein zona occludens-2 (ZO-2) as an IS component. Biochemistry and microscopy approaches in T cells confirmed SNX27/ZO-2 PDZ-dependent interaction, and demonstrated its role controlling the dynamic localization of ZO-2 at the IS. This study broadens our knowledge of SNX27 function in T lymphocytes, and suggests that pathways that delimit polarized structures in nervous and epithelial systems also participate in IS regulation.

Diacylglycerol Kinase ζ Limits Cytokine-dependent Expansion of CD8+ T Cells with Broad Antitumor Capacity.

Interleukin-2 and -15 drive expansion/differentiation of cytotoxic CD8+ T cells that eliminate targets via antigen-independent killing. This property is clinically relevant for the improvement of T cell-based antitumor therapies. Diacylglycerol kinase α and ζ (DGKα/ζ) metabolize the diacylglycerol generated following antigen recognition by T lymphocytes. Enhanced expression of these two lipid kinases in tumor-infiltrating CD8+ T cells promotes a hyporesponsive state that contributes to tumor immune escape. Inhibition of these two enzymes might thus be of interest for potentiating conventional antigen-directed tumor elimination. In this study, we sought to characterize the contribution of DGKα and ζ to antigen-independent cytotoxic functions of CD8+ T cells. Analysis of DGKζ-deficient mice showed an increase in bystander memory-like CD8+ T cell populations not observed in DGKα-deficient mice. We demonstrate that DGKζ limits cytokine responses in an antigen-independent manner. Cytokine-specific expansion of DGKζ-deficient CD8+ T cells promoted enhanced differentiation of innate-like cytotoxic cells in vitro, and correlated with the more potent in vivo anti-tumor responses of DGKζ-deficient mice engrafted with the murine A20 lymphoma. Our studies reveal a isoform-specific function for DGKζ downstream of IL-2/IL-15-mediated expansion of innate-like cytotoxic T cells, Pharmacological manipulation of DGKζ activity is of therapeutic interest for cytokine-directed anti-tumor treatments.