During activation, T cells undergo metabolic reprogramming, which imprints distinct functional fates. We determined that on PD-1 ligation, activated T cells are unable to engage in glycolysis or amino acid metabolism but have an increased rate of fatty acid β-oxidation (FAO). PD-1 promotes FAO of endogenous lipids by increasing expression of CPT1A, and inducing lipolysis as indicated by elevation of the lipase ATGL, the lipolysis marker glycerol and release of fatty acids. Conversely, CTLA-4 inhibits glycolysis without augmenting FAO, suggesting that CTLA-4 sustains the metabolic profile of non-activated cells. Because T cells utilize glycolysis during differentiation to effectors, our findings reveal a metabolic mechanism responsible for PD-1-mediated blockade of T-effector cell differentiation. The enhancement of FAO provides a mechanistic explanation for the longevity of T cells receiving PD-1 signals in patients with chronic infections and cancer, and for their capacity to be reinvigorated by PD-1 blockade.

S. canis Cas9 is a natural CRISPR enzyme that uses two motif insertions to enable flexible targeting of DNA sequences.

No abstract

Maintenance of peripheral tolerance is essential for homeostasis of the immune system. While central tolerance mechanisms result in deletion of the majority of self-reactive T cells, T lymphocytes specific for self-antigens also escape this process and circulate in the periphery. To control the development of autoimmunity, multiple mechanisms of peripheral tolerance have evolved, including T cell anergy, deletion and suppression by regulatory T cells (Treg). The pathway consisting of the PD-1 receptor (CD279) and its ligands PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC; CD273) plays a vital role in the induction and maintenance of peripheral tolerance. This pathway also regulates the balance between stimulatory and inhibitory signals needed for effective immunity and maintenance of T cell homeostasis. In contrast to this important beneficial role in maintaining T cell homeostasis, PD-1 mediates potent inhibitory signals that prevent the expansion and function of T effector cells and have detrimental effects on anti-viral and anti-tumor immunity. In spite of the compelling studies on the significant functional role of PD-1 in mediating inhibition of activated T cells, little is known about how PD-1 blocks T cell activation. Here, we will provide a brief overview of the signaling events that are regulated by PD-1 triggering and we will discuss their implications on cell intrinsic and extrinsic mechanisms that determine the fate and function of T effector cells.

An enhancer within intron 1 of the amyloid precursor protein gene (APPb) of zebrafish is identified functionally using a novel approach. Bacterial artificial chromosomes (BACs) were retrofitted with enhancer traps, and expressed as transgenes in zebrafish. Expression from both transient assays and stable lines were used for analysis. Although the enhancer was active in specific nonneural cells of the notochord when placed with APPb gene promoter proximal elements its function was restricted to, and absolutely required for, specific expression in neurons when juxtaposed with additional far-upstream promoter elements of the gene. We demonstrate that expression of green fluorescent protein fluorescence resembling the tissue distribution of APPb mRNA requires both the intron 1 enhancer and ∼28 kb of DNA upstream of the gene. The results indicate that tissue-specificity of an isolated enhancer may be quite different from that in the context of its own gene. Using this enhancer and upstream sequence, polymorphic variants of APPb can now more closely recapitulate the endogenous pattern and regulation of APPb expression in animal models for Alzheimer's disease. The methodology should help functionally map multiple noncontiguous regulatory elements in BACs with or without gene-coding sequences.

A loxP-transposon retrofitting strategy for generating large nested deletions from one end of the insert DNA in bacterial artificial chromosomes and P1 artificial chromosomes was described recently [Chatterjee, P. K. & Coren, J. S. (1997) Nucleic Acids Res. 25, 2205-2212]. In this report, we combine this procedure with direct sequencing of nested-deletion templates by using primers located in the transposon end to illustrate its value for position-specific single-nucleotide polymorphism (SNP) discovery from chosen regions of large insert clones. A simple ampicillin sensitivity screen was developed to facilitate identification and recovery of deletion clones free of transduced transposon plasmid. This directed approach requires minimal DNA sequencing, and no in vitro subclone library generation; positionally oriented SNPs are a consequence of the method. The procedure is used to discover new SNPs as well as physically map those identified from random subcloned libraries or sequence databases. The deletion templates, positioned SNPs, and markers are also used to orient large insert clones into a contig. The deletion clone can serve as a ready resource for future functional genomic studies because each carries a mammalian cell-specific antibiotic resistance gene from the transposon. Furthermore, the technique should be especially applicable to the analysis of genomes for which a full genome sequence or radiation hybrid cell lines are unavailable. I dentifying polymorphic sites in the genome is a basic aspect of molecular genetics and genomics. The process is needed for a variety of purposes, ranging from the development of polymorphic marker sets useful as a tool for genetic analysis of a chromosomal region or full genome scan, to the initial identification of variants or mutations in a newly discovered gene (1, 2). In most cases, the identity of base differences and their location relative to a gene or other polymorphic sites is either useful or required. Recent estimates of the number of singlenucleotide polymorphisms (SNPs) needed for whole genome association studies in humans vary from several thousand to several hundred thousand (1, 3); thus, efficient and cost-effective methods for identifying a large number of SNPs with the required characteristics of dense yet even spacing, and of known order over large uncharacterized regions of the genome, is of interest. A comparison of two methods to develop a densely ordered map of SNPs covering a 4-Mb region of the human genome was recently reported (4). In one approach, large-insert bacterial clones, bacterial artificial chromosomes (BACs) (5) and P1 artificial chromosomes (PACs) (6), spanning this region were fragmented and reconstructed in 2-kb plasmid libraries, which were then sequenced. This shotgun procedure is efficient in identifying SNPs; however, to approach a map of 30-kb average SNP spacing, bidirectional sequencing of approximately 500 randomly chosen subclones per 100 kb of genomic sequence was required. Multiple BAC and PAC clones mapping to the region...

The proteins associated with parental, adenoviral DNA in productively‐infected HeLa cells have been examined both directly and indirectly. HeLa cells infected with 32P‐labelled Ad2 were irradiated with u.v. light at various points in the infectious cycle. Following degradation of the DNA, nuclear proteins carrying cross‐linked nucleotides, or oligonucleotides, were distinguished from virion phosphoproteins by the resistance of their 32P radioactivity to 1 M NaOH. The major core protein of the virion, protein VII, was found to be associated with viral DNA throughout infection, even when cells were infected at a multiplicity of 0.14. Micrococcal nuclease digestion of intranuclear viral DNA 4 h after infection liberated two nucleoprotein particles containing viral DNA, neither of which co‐migrated with HeLa cell mononucleosomes. These results indicate that core protein VII remains associated with parental adenoviral DNA during productive infections. The observation that protein VII can be cross‐linked to DNA in cells infected at very low multiplicity, together with the results of a comparison of proteins cross‐linkable to viral DNA in cells infected by wild‐type virus and a non‐infectious mutant containing the precursor to protein VII, suggest that nucleoproteins comprising viral DNA and protein VII must be the templates for expression of pre‐early and early viral genes.

CRISPR-associated (Cas) DNA-endonucleases are remarkably effective tools for genome engineering, but have limited target ranges due to their protospacer adjacent motif (PAM) requirements. We demonstrate a critical expansion of the targetable sequence space for a type II-A CRISPR-associated enzyme through identification of the natural 5 0-NAAN-3 0 PAM preference of Streptococcus macacae Cas9 (SmacCas9). To achieve efficient editing activity, we graft the PAM-interacting domain of SmacCas9 to its well-established ortholog from Streptococcus pyogenes (SpyCas9), and further engineer an increased efficiency variant (iSpyMac) for robust genome editing activity. We establish that our hybrids can target all adenine dinucleotide PAM sequences and possess robust and accurate editing capabilities in human cells.