Dynamic reprogramming of metabolism is essential for T cell effector function and memory formation. However, the regulation of metabolism in exhausted CD8+ T (Tex) cells is poorly understood. We found that during the first week of chronic lymphocytic choriomeningitis virus (LCMV) infection, before severe dysfunction develops, virus-specific CD8+ T cells were already unable to match the bioenergetics of effector T cells generated during acute infection. Suppression of T cell bioenergetics involved restricted glucose uptake and use, despite persisting mechanistic target of rapamycin (mTOR) signaling and up-regulation of many anabolic pathways. PD-1 regulated early glycolytic and mitochondrial alterations and repressed transcriptional coactivator PGC-1α. Improving bioenergetics by overexpression of PGC-1α enhanced function in developing Tex cells. Therapeutic reinvigoration by anti-PD-L1 reprogrammed metabolism in a subset of Tex cells. These data highlight a key metabolic control event early in exhaustion and suggest that manipulating glycolytic and mitochondrial metabolism may enhance checkpoint blockade outcomes.

Mutation of predicted 3′‐‐>5′ exonuclease active site residues of Saccharomyces cerevisiae POL3 DNA polymerase (delta) or deletion of the PMS1 mismatch repair gene lead to relative (to wild type) spontaneous mutation rates of approximately 130 and 41, respectively, measured at a URA3 reporter gene inserted near to a defined replication origin. The POL3 exonuclease‐deficient mutant pol3‐01 generated most classes of single base mutation in URA3, indicating a broad specificity that generally corresponds to that of the PMS1 system. pol3‐01 pms1 haploid cells ceased growth after a few divisions with no unique terminal cell morphology. A pol3‐01/pol3‐01 pms1/pms1 diploid was viable and displayed an estimated URA3 relative mutation rate of 2 × 10(4), which we calculate to be catastrophically high in a haploid. The relationship between the relative mutation rates of pol3‐01 and pms1 was multiplicative, indicating action in series. The PMS1 transcript showed the same cell cycle periodicity as those of a set of DNA replication genes that includes POL3, suggesting PMS1 is co‐regulated with these genes. We propose that the POL3 3′‐‐>5′ exonuclease and the PMS1 mismatch repair system act on a common pathway analogous to the dnaQ‐‐>mutHLS pathway of DNA replication error correction in Escherichia coli.

To illuminate genes and mechanisms for humoral immunity we performed a mouse genetic screen for mutations that do not affect the first wave of antibody to immunization but disrupt response maturation and persistence. The first two mutants identified were loss-of-function mutations in DOCK8. DOCK8 mutant B cells were unable to form marginal zone B cells or to persist in germinal centers and undergo affinity maturation. DOCK8 mutation disrupted the concentration of ICAM-1 in the B cell immune synapse but did not alter other aspects of B cell antigen receptor signaling. Humoral immunodeficiency due to DOCK8 mutation provides evidence that organization of an immune synapse is critical for signaling the survival of B cell subsets required for long-lasting immunity.

We have isolated a multicopy suppressor of the temperature-sensitive growth phenotype of organisms carrying mutations of DBF4, a gene that is required for the initiation of chromosomal DNA replication in Saccharomyces cerevisiae and that interacts with the CDC7 protein kinase. periodically during the cell cycle, peaking at the G2/M boundary. CDCS on a multicopy plasmid also suppresses temperature-sensitive cdc15, cdc2O, and dbf2 mutations which affect mitosis during the cell cycle.Protein phosphorylation by protein kinases plays an important role in regulating both the mitotic cell cycle and meiosis in eukaryotes (28, 49). In the yeast Saccharomyces cerevisiae, genes encoding more than 30 protein kinases have been identified (16). Among these, at least four protein kinase genes, CDC28, CDC7, DBF2, and HRR25, are associated with DNA metabolism in the mitotic cell cycle. CDC28 is essential for cell growth and is required both for entry into the S phase and for the G2/M transition (35). Although the cellular abundance of the Cdc28 protein remains constant throughout the cell cycle (30), its protein kinase is periodically active and is regulated by a physical interaction with G, and G2 cyclins (8, 37, 45). CDC7 is also essential for cell growth and is required for the GJIS transition. It has recently been shown that the Cdc7 polypeptide has an associated protein kinase activity and is phosphorylated in vivo (18). Its execution point is just before the initiation of DNA replication (12). After the CDC7 execution point, no protein synthesis is required for the initiation of DNA replication (15). The abundance of the CDC7 transcript remains constant throughout the cell cycle (42), and this is likely to be true for the Cdc7 polypeptide also. However, no direct relationship between the Cdc28 and Cdc7 protein kinases has been elucidated to date.Deletion of DBF2 is not lethal because a homolog ofDBF2 (DBF20) is able to substitute for DBF2 function (48). However, in dbf2 mutants DNA synthesis is transiently delayed and the cell cycle is blocked in late nuclear division at the restrictive temperature, suggesting that the Dbf2 protein is required for completion of the S and M phases (20). The DBF2 transcript accumulates periodically during the cell * Corresponding author.cycle (20), and it is likely that this is also true of the Dbf2 protein. HRR25 is not essential for cell growth, but deletion of HRR25 results in a delay at the G2/M boundary (17), suggesting an important role in the cell cycle. Mutant cells carrying hrr25 exhibit sensitivity to methyl methanesulfonate and X rays (17), suggesting that the gene is also required for DNA repair.In an attempt to identify the various proteins that might interact either with the Cdc7 protein kinase or with both the Cdc28 and Cdc7 protein kinases, we isolated a multicopy suppressor of the temperature-sensitive growth phenotype of organisms carrying cdc7 mutations (24). The suppressor was identified as the DBF4 gene, whose execution point is just before initiation of DNA replication ...

T-cell receptor signaling in CD4+CD8+ double positive thymocytes determines cell survival and lineage commitment, but the genetic and molecular basis of this process is poorly defined. To address this issue, we used ethylnitrosourea mutagenesis to identify a novel T-lineage-specific gene, Themis, which is critical for the completion of positive selection. Themis contains a tandem repeat of a unique globular domain (CABIT), which includes a cysteine motif that defines a family of 5 uncharacterized vertebrate proteins with orthologs in most animal species. Themis-deficient thymocytes showed no major impairment in early T-cell receptor signaling, but exhibited altered expression of cell cycle and survival genes before and during positive selection. These data suggest a unique role for Themis in sustaining positive selection.

Deletion of the Saccharomyces cerevisiae gene YOL008W, here referred to as COQ10, elicits a respiratory defect as a result of the inability of the mutant to oxidize NADH and succinate. Both activities are restored by exogenous coenzyme Q 2 . Respiration is also partially rescued by COQ2, COQ7, or COQ8/ABC1, when these genes are present in high copy. Unlike other coq mutants, all of which lack Q 6 , the coq10 mutant has near normal amounts of Q 6 in mitochondria. Coq10p is widely distributed in bacteria and eukaryotes and is homologous to proteins of the "aromatic-rich protein family" Pfam03654 and to members of the START domain superfamily that have a hydrophobic tunnel implicated in binding lipophilic molecules such as cholesterol and polyketides. Analysis of coenzyme Q in polyhistidine-tagged Coq10p purified from mitochondria indicates the presence 0.032-0.034 mol of Q 6 /mol of protein. We propose that Coq10p is a Q 6 -binding protein and that in the coq10 mutant Q 6 it is not able to act as an electron carrier, possibly because of improper localization.Coenzyme Q (ubiquinone) is an essential electron carrier of the mitochondrial respiratory chain. Its main function is to accept electrons from the NADH-and succinate-coenzyme Q reductases and to donate them to the bc 1 complex (1). Biosynthesis of coenzyme Q in eukaryotes occurs in mitochondria. The benzoquinone ring of coenzyme Q 6 (Q 6 ) of Saccharomyces cerevisiae has a polyprenyl side chain with six isoprenoid units (2). At least nine yeast nuclear genes (COQ1-9) defined by nine complementation groups of a pet 3 mutant collection have been inferred to participate in the synthesis of Q 6 based on the biochemical properties of the mutant mitochondria (3, 4). Mutations in each of the nine genes block the NADH and succinate-cytochrome c reductase activities of mitochondria, which can be restored by addition of coenzyme Q 2 (5). Additionally, coq mutants lack Q 6 and coq3, coq4, coq5, coq6, coq7, coq8/abc1, and coq9 mutants accumulate 3-hexaprenyl-4-hydroxybenzoic acid, an early intermediate in Q 6 biosynthesis (Refs. 4 and 6 -13, see Ref. 11 for details of the pathway in yeast). Except for demethoxy-Q 6 , which accumulates in certain coq7 point mutants (7,14), other intermediates of the pathway are not detected in coq mutants (6 -12). These observations indicate that the pathway is stringently regulated and/or that most of the intermediates are degraded when biosynthesis of Q 6 is arrested. COQ gene products are located in, or are peripherally associated with the inner membrane of mitochondria (9 -15) where they constitute a pathway that is similar to but diverges from the one in bacteria in at least two steps (16,17).In the present study we report a novel phenotype displayed by mutants with a deletion of reading frame YOL008W, which we have named COQ10. The coq10 mutant is similar to other coq mutants in which optimal oxidation of NADH and succinate by isolated mitochondria depends on addition of coenzyme Q 2 . Unlike the coq mutants, which lack Q 6 , the coq...

Deficiency in the guanine nucleotide exchange factor DOCK8 causes a human immunodeficiency syndrome associated with recurrent sinopulmonary and viral infections. We have recently identified a DOCK8-deficient mouse strain, carrying an ethylnitrosourea-induced splice-site mutation that shows a failure to mature a humoral immune response due to the loss of germinal centre B cells. In this study we turned to T-cell immunity to investigate further the human immunodeficiency syndrome and its association with decreased peripheral CD4+ and CD8+ T cells. Characterisation of the DOCK8-deficient mouse revealed T-cell lymphopenia, with increased T-cell turnover and decreased survival. Egress of mature CD4+ thymocytes was reduced with increased migration of these cells to the chemokine CXCL12. However, despite the two-fold reduction in peripheral naïve T cells, the DOCK8-deficient mice generated a normal primary CD8+ immune response and were able to survive acute influenza virus infection. The limiting effect of DOCK8 was in the normal survival of CD8+ memory T cells after infection. These findings help to explain why DOCK8-deficient patients are susceptible to recurrent infections and provide new insights into how T-cell memory is sustained.

Two temperature‐sensitive DNA polymerase II mutants (pol2‐9 and pol2‐18) of the yeast Saccharomyces cerevisiae were isolated by the plasmid shuffling method. DNA polymerase II activity partially purified from both mutants was thermolabile, while DNA polymerase I and III activities remained thermotolerant. At the restrictive temperature, the pol2 mutants were defective in chromosomal DNA replication and exhibited the dumbbell terminal morphology typical of DNA replication mutants. The POL2 transcript accumulated periodically during the cell cycle, peaking at the G1/S boundary in the same manner as the transcripts of more than 10 other DNA replication genes. These results indicate that DNA polymerase II participates in nuclear DNA replication. The similarities in structure and activities between the DNA polymerases of yeast and mammals make it likely that mammalian DNA polymerase epsilon too is required for chromosomal DNA replication.