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.

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.

Sorting nexin 27 (SNX27) recycles PSD-95, Dlg1, ZO-1 (PDZ) domain-interacting membrane proteins and is essential to sustain adequate brain functions. Here we define a fundamental SNX27 function in T lymphocytes controlling antigen-induced transcriptional activation and metabolic reprogramming. SNX27 limits the activation of diacylglycerol (DAG)-based signals through its high affinity PDZ-interacting cargo DAG kinase ζ (DGKζ). SNX27 silencing in human T cells enhanced T cell receptor (TCR)-stimulated activator protein 1 (AP-1)- and nuclear factor κB (NF-κB)-mediated transcription. Transcription did not increase upon DGKζ silencing, suggesting that DGKζ function is dependent on SNX27. The enhanced transcriptional activation in SNX27-silenced cells contrasted with defective activation of the mammalian target of rapamycin (mTOR) pathway. The analysis of Snx27 −/− mice supported a role for SNX27 in the control of T cell growth. This study broadens our understanding of SNX27 as an integrator of lipid-based signals with the control of transcription and metabolic pathways.

BackgroundThe 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.MethodsWe 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.ResultsWe 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.ConclusionsOur 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.