No abstract

Caspase activity is critical for both T-cell survival and death. However, little is known regarding what determines caspase activity in cycling T cells. Interleukin (IL)-2 and IL-15 confer very different susceptibilities to T-cell death. We therefore considered that IL-2 and IL-15 differentially regulate caspase activity to influence T-cell survival. We observed that IL-2-cultured primary murine effector T cells manifested elevated levels of caspase-3 activity compared with IL-15-cultured T cells. T cell receptor (TCR) restimulation further increased caspase activity and induced considerable cell death in IL-2-cultured T cells, but provoked only a minimal increase of caspase activity and cell death in IL-15-cultured T cells. IL-2 sensitization to cell death was caspase-3 mediated. Interestingly, increased active caspase-3 levels with IL-2 were independent of active initiator caspase-8 and caspase-9 that were similar with IL-2 and IL-15. Rather, caspase-3 activity was inhibited by posttranslational S-nitrosylation in IL-15-cultured T cells, but not in the presence of IL-2. This paralleled increased reactive nitrogen and oxygen species with IL-15 and reduced glycolysis. Taken together, these data suggest that the metabolic state conferred by IL-15 inhibits T-cell apoptosis in part by maintaining low levels of active caspase-3 via S-nitrosylation.

Resting T cells undergo a rapid metabolic shift to glycolysis upon activation in the presence of interleukin (IL)-2, in contrast to oxidative mitochondrial respiration with IL-15. Paralleling these different metabolic states are striking differences in susceptibility to restimulation-induced cell death (RICD); glycolytic effector T cells are highly sensitive to RICD, whereas non-glycolytic T cells are resistant. It is unclear whether the metabolic state of a T cell is linked to its susceptibility to RICD. Our findings reveal that IL-2-driven glycolysis promotes caspase-3 activity and increases sensitivity to RICD. Neither caspase-7, caspase-8, nor caspase-9 activity is affected by these metabolic differences. Inhibition of glycolysis with 2-deoxyglucose reduces caspase-3 activity as well as sensitivity to RICD. By contrast, IL-15-driven oxidative phosphorylation actively inhibits caspase-3 activity through its glutathionylation. We further observe active caspase-3 in the lipid rafts of glycolytic but not non-glycolytic T cells, suggesting a proximity-induced model of self-activation. Finally, we observe that effector T cells during influenza infection manifest higher levels of active caspase-3 than naive T cells. Collectively, our findings demonstrate that glycolysis drives caspase-3 activity and susceptibility to cell death in effector T cells independently of upstream caspases. Linking metabolism, caspase-3 activity, and cell death provides an intrinsic mechanism for T cells to limit the duration of effector function.

An effective adaptive immune response requires rapid T cell proliferation, followed by equally robust cell death. These two processes are coordinately regulated to allow sufficient magnitude of response followed by its rapid resolution, while also providing the maintenance of T cell memory. Both aspects of this T cell response are characterized by profound changes in metabolism; glycolysis drives proliferation whereas oxidative phosphorylation supports the survival of memory T cells. While much is known about the separate aspects of T cell expansion and contraction, considerably less is understood regarding how these processes might be connected. We report a link between the induction of glycolysis in CD8+ T cells and upregulation of the inhibitor of complex I and oxidative phosphorylation, methylation-controlled J protein (MCJ). MCJ acts synergistically with glycolysis to promote caspase-3 activity. Effector CD8+ T cells from MCJ-deficient mice manifest reduced glycolysis and considerably less active caspase-3 compared to wild-type cells. Consistent with these observations, in non-glycolytic CD8+ T cells cultured in the presence of IL-15, MCJ expression is repressed by methylation, which parallels their reduced active caspase-3 and increased survival compared to glycolytic IL-2-cultured T cells. Elevated levels of MCJ are also observed in vivo in the highly proliferative and glycolytic subset of CD4-CD8- T cells in Fas-deficient lpr mice. This subset also manifests elevated levels of activated caspase-3 and rapid cell death. Collectively, these data demonstrate tight linkage of glycolysis, MCJ expression, and active caspase-3 that serves to prevent the accumulation and promote the timely death of highly proliferative CD8+ T cells.

We recently reported that the Thermotogales acquired the ability to synthesize vitamin B 12 by acquisition of genes from two distantly related lineages, Archaea and Firmicutes (K. S. Swithers et al., Genome Biol. Evol. 4:730 -739, 2012). Ancestral state reconstruction suggested that the cobinamide salvage gene cluster was present in the Thermotogales' most recent common ancestor. We also predicted that Thermotoga lettingae could not synthesize B 12 de novo but could use the cobinamide salvage pathway to synthesize B 12 . In this study, these hypotheses were tested, and we found that Tt. lettingae did not synthesize B 12 de novo but salvaged cobinamide. The growth rate of Tt. lettingae increased with the addition of B 12 or cobinamide to its medium. It synthesized B 12 when the medium was supplemented with cobinamide, and no B 12 was detected in cells grown on cobinamide-deficient medium. Upstream of the cobinamide salvage genes is a putative B 12 riboswitch. In other organisms, B 12 riboswitches allow for higher transcriptional activity in the absence of B 12 . When Tt. lettingae was grown with no B 12 , the salvage genes were upregulated compared to cells grown with B 12 or cobinamide. Another gene cluster with a putative B 12 riboswitch upstream is the btuFCD ABC transporter, and it showed a transcription pattern similar to that of the cobinamide salvage genes. The BtuF proteins from species that can and cannot salvage cobinamides were shown in vitro to bind both B 12 and cobinamide. These results suggest that Thermotogales species can use the BtuFCD transporter to import both B 12 and cobinamide, even if they cannot salvage cobinamide. The Thermotogales order is one of the deepest bacterial lineages in the "ribosomal tree of life" (1-3). Thermotogales genomes are subjected to frequent gene transfers (4), with the largest number of transfers from archaea and firmicutes (5). Our recent study provided strong evidence that this order has acquired two different gene clusters, corrinoid synthesis from the firmicutes and cobinamide salvage gene cluster from various archaeal and bacterial organisms. These gene clusters allow for de novo synthesis of vitamin B 12 , also termed cobalamin (6), and the synthesis of B 12 from partial B 12 molecules, called cobinamides. The B 12 cofactor is required by all domains of life, and de novo synthesis requires over 30 enzymes to produce an active form of B 12 (7). B 12 is required as a growth factor for many bacteria and archaea that do not have genes for its de novo synthesis. Only 50% of sequenced bacterial genomes that indicate a need for B 12 appear to encode the ability to synthesize B 12 (8). Some of these bacteria salvage incomplete corrinoid rings called cobinamides and use these as precursors to synthesize an active form of B 12 (9). Other microorganisms import B 12 from the environment using a B 12 /cobinamide BtuFCD ABC transporter (10).We recently explored the evolutionary origins of B 12 -related genes in the Thermotogales and showed that some members of the order, l...