CD8 T Cells Mediate Host Death Following Lcmv Clone 13 Infection in Dgkα Deficiency

Abstract

Diacylglycerol is a potent element of intracellular secondary signaling cascades whose production is enhanced by cell surface receptor agonism and function is regulated by enzymatic degradation by diacylglycerol kinases. In T cells, stringent regulation of the activity of this second messenger maintains an appropriate balance between effector function and anergy. In our study, we demonstrate that the DGKα is an indispensable regulator of T cell receptor-mediated activation of CD8 T cells in LCMV Clone 13 viral infection. In the absence of DGKα in mice, LCMV Clone 13 viral inoculation, a noncytopathic infection, resulted in a pathologic, proinflammatory state and host mortality. This was first observed in a systemic loss of function model, where significant mortality observed in DGKα-/- mice following LCMV Clone 13 when compared to wildtype (73% vs 6%, respectively, p < 0.0001). A similar trend towards increased post-infection mortality was observed in mice with CD8 T cell specific DGKα deficiency. The model was generated by chimeric bone marrow transplant of CD8-/- and DGKα-/- bone marrow, so that reconstituted marrow contained a DGKα deficiency specific to CD8 T cells. When compared to mice transplanted with DGKα sufficient bone marrow, increased mortality was observed in mice with CD8 T cell specific absence of DGKα (20% vs 80%, respectively, p=0.07). In contrast, when systemic DGKα-/- mice were treated with a CD8 depleting antibody prior to LCMV Clone 13 infection, none of the CD8 depleted mice suffered mortality, demonstrating DGKα-deficient CD8 T cells are necessary for the observed mortality. Next, an immunophenotype of the systemic knockout model during LCMV Clone 13 infection was established to determine the mechanism behind the increased mortality. This revealed a significant increase in the frequency of antigen (glycoprotein 33-41) specific CD8 T cells, and a significant decrease in serum viral load when compared to a similarly infected wildtype mouse. No appreciable differences in frequency of Treg or Th1 CD4 T cells were observed, but there was a discordant increase in Tfh CD4 T cells and a decrease in germinal center B cells. Myeloid subsets of macrophages, monocytes, and granulocytes were similar between infected DGKα-/- mice and wildtype controls. Following LCMV Clone 13 infection, transcriptomic analysis of RNA sequencing data gathered from sorted virus-specific, DGKα-deficient CD8 T cells revealed an enrichment of multiple cell cycling gene sets, and transcriptional upregulation of genes associated with glycolysis, effector function, and TCR signaling as compared to DGKα-sufficient, virus-specific CD8 T cells. These transcriptomic differences were confirmed phenotypically in vivo following competitive adoptive transfer of virus-specific, wildtype and DGKα-/- CD8 T cells into the same host. Following LCMV Clone 13 infection, a significant expansion of DGKα-/- virus-specific CD8 T cells occurred as compared to DGKα replete virus-specific CD8 T cells. Further, those deficient in DGKα displayed enhanced effector function, evidenced by significantly increased per-cell production of granzyme B and interferon gamma. In total, our data demonstrate that CD8 T cell expression of DGKα is necessary to prevent immunopathologic host death in the setting of LCMV Clone 13 infection. In its absence in CD8 T cells, a noncytopathic viral infection can propagate an overwhelming, mortal proinflammatory phenotype.