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Multiple studies identified metabolic changes within the tumor and its microenvironment during carcinogenesis. Yet, the mechanisms by which tumors affect the host metabolism are unclear. We find that systemic inflammation induced by the cancer leads to liver infiltration of myeloid cells at early extrahepatic carcinogenesis. The infiltrating immune cells via IL-6-pSTAT3 immune-hepatocyte crosstalk cause the depletion of a master metabolic regulator, HNF4a, consequently leading to systemic metabolic changes that promote breast and pancreatic cancer proliferation and a worse outcome. Preserving HNF4 levels maintains liver metabolism and restricts carcinogenesis. Standard liver biochemical tests can identify early metabolic changes and predict patients' outcomes and weight loss. Thus, the tumor induces early metabolic changes in its macro-environment with diagnostic and potentially therapeutic implications for the host.

Major histocompatibility complex class II (MHC-II) antigen presentation underlies a wide range of immune responses in health and disease. However, how MHC-II antigen presentation is regulated by the peptide-loading catalyst HLA-DM (DM), its associated modulator, HLA-DO (DO), is incompletely understood. This is due largely to technical limitations: model antigen presenting cell (APC) systems that express these MHC-II peptidome regulators at physiologically variable levels have not been described. Likewise, computational prediction tools that account for DO and DM activities are not presently available. To address these gaps, we created a panel of single MHC-II allele, HLA-DR4-expressing APC lines that cover a wide range of DO:DM ratio states. Using a combined immunopeptidomic and proteomic discovery strategy, we measured the effects DO:DM ratios have on peptide presentation by surveying over 10,000 unique DR4-presented peptides. The resulting data provide insight into peptide characteristics that influence their presentation with increasing DO:DM ratios. These include DM-sensitivity, peptide abundance, binding affinity and motif, peptide length and register positioning on the source protein. These findings have implications for designing improved HLA-II prediction algorithms and research strategies for dissecting the variety of functions that different APCs serve in the body.IN BRIEFPeptides presented by MHC-II are critical to adaptive immune function. The non-canonical MHC molecules HLA-DM and HLA-DO cooperatively regulate MHC-II function, but how varied DO-to-DM ratios across different APCs and cellular contexts might influence their immunopeptide repertoires is unclear. We address this by measuring cell lines expressing these two proteins spanning a range of relative abundances. We found that peptides could be categorized according to how robustly they were presented at different DO:DM ratios. Importantly, this presentation was only partially linked to predicted affinity to the MHC-II molecule.HIGHLIGHTSDescribe MHC-class II peptide repertoires from a unique HLA-DR4 cell line panel with increasing DO:DM ratios.Demonstrate striking and divergent changes in MHC-II immunopeptidomes that result from the tuning function of DO:DM.These findings bridge gap in understanding and predicting MHC-II antigen presentation.GRAPHICAL ABSTRACT

Individuals with narcolepsy suffer from abnormal sleep patterns due to loss of neurons that uniquely supply hypocretin (HCRT). Previous studies found associations of narcolepsy with the human leukocyte antigen (HLA)-DQ6 allele and T-cell receptor α (TRA) J24 gene segment and also suggested that in vitro-stimulated T cells can target HCRT. Here, we present evidence of in vivo expansion of DQ6-HCRT tetramer

Individuals with narcolepsy suffer from abnormal sleep patterns due to loss of neurons that uniquely supply hypocretin (HCRT). Previous studies found associations of narcolepsy with the human leukocyte antigen (HLA)-DQ6 allele and T-cell receptor α (TRA) J24 gene segment and also suggested that in vitro-stimulated T cells can target HCRT. Here, we present evidence of in vivo expansion of DQ6-HCRT tetramer+/TRAJ24+/CD4+ T cells in DQ6+ individuals with and without narcolepsy. We identify related TRAJ24+ TCRαβ clonotypes encoded by identical α/β gene regions from two patients and two controls. TRAJ24-G allele+ clonotypes only expand in the two patients, whereas a TRAJ24-C allele+ clonotype expands in a control. A representative tetramer+/G-allele+ TCR shows signaling reactivity to the epitope HCRT87–97. Clonally expanded G-allele+ T cells exhibit an unconventional effector phenotype. Our analysis of in vivo expansion of HCRT-reactive TRAJ24+ cells opens an avenue for further investigation of the autoimmune contribution to narcolepsy development., T cells from narcolepsy patients were recently reported to recognize hypocretin, a wakefulness-promoting neurohormone, suggesting autoimmune origin of the disease. Here the authors show that hypocretin-specific T cells expand both in healthy controls and in narcolepsy patients, and identify preliminary features that may distinguish them.

B cell receptors and surface-displayed peptide/MHCII complexes constitute two key components of the B-cell machinery to sense signals and communicate with other cell types during antigen-triggered activation. However, critical pathways synergizing antigen-BCR interaction and antigenic peptide-MHCII presentation remain elusive. Here, we report the discovery of factors involved in establishing such synergy. We applied a single-cell measure coupled with super-resolution microscopy to investigate the integrated function of two lysosomal regulators for peptide loading, HLA-DM and HLA-DO. In model cell lines and human tonsillar B cells, we found that tunable DM/DO stoichiometry governs DMfree activity for exchange of placeholder CLIP peptides with high affinity MHCII ligands. Compared to their naïve counterparts, memory B cells with less DMfree concentrate a higher proportion of CLIP/MHCII in lysosomal compartments. Upon activation mediated by high affinity BCR, DO tuning is synchronized with antigen internalization and rapidly potentiates DMfree activity to optimize antigen presentation for T-cell recruitment.

Mature B lymphocytes (B cells) recognize antigens using their B cell receptor (BCR) and are activated to become antibody-producing cells. In addition, and integral to the development of a high-affinity antibodies, B cells utilize the specialized major histocompatibility complex class II (MHCII) antigen presentation pathway to process BCR-bound and internalized protein antigens and present selected peptides in complex with MHCII to CD4+ T cells. This interaction influences the fate of both types of lymphocytes and shapes immune outcomes. Specific, effective, and optimally timed antigen presentation by B cells requires well-controlled intracellular machinery, often regulated by the combined effects of several molecular events. Here, we delineate and summarize these events in four steps along the antigen presentation pathway: (1) antigen capture and uptake by B cells; (2) intersection of internalized antigen/BCRs complexes with MHCII in peptide-loading compartments; (3) generation and regulation of MHCII/peptide complexes; and (4) exocytic transport for presentation of MHCII/peptide complexes at the surface of B cells. Finally, we discuss modulation of the MHCII presentation pathway across B cell development and maturation to effector cells, with an emphasis on the shaping of the MHCII/peptide repertoire by two key antigen presentation regulators in B cells: HLA-DM and HLA-DO.

HLA-DO, a non-classical class II major histocompatibility complex (MHCII), competitively inhibits HLA-DM in B cells. B cells lower DO levels during antigen-mediated activation in germinal centers, implying a regulatory function of DO in resting B cells. We transfected T2 (T/B hybrid) DR4.DM cells with DO and used index FACS to measure single cell variation in DO:DM ratios. High DO:DM ratios allowed groove occupancy by class II-associated Ii peptides (CLIP), and low DO:DM ratios enabled CLIP removal. 3D structured illumination microscopy revealed DMDO concentrated in LAMP1+ endosomes. Both DM+DO+ and DM−DO− cells express CLIP/DR complexes, but a larger fraction of CLIP/DR localizes to LAMP1+ endosomes in DM+DO+ cells. Using memory and naïve human tonsillar B cells, we saw significant downregulation of DO after cell activation. CLIP/MHCII density relative to free DM (estimated by 1-DO/DM) was generally higher in resting vs. activated B cells, especially in memory cells. This memory B cell phenotype primarily reflected higher intracellular CLIP/MHCII; upon BCR-mediated stimulation, total CLIP/MHCII fell to levels in activated naïve cells. We hypothesized that higher CLIP/MHCII and DO in resting memory B cells sets a threshold for the activation needed to efficiently recruit Tfh and selects for cells with higher affinity BCR. Using antigen stimulation of 2 clonal B cell lines expressing BCRs with different affinities for GAD65, we confirmed that, in this system, BCR affinity correlates with the amount of reduction in total CLIP/MHCII after activation. A positive correlation between BCR affinity and the extent of peptide exchange on MHCII is likely mediated by the effects of BCR affinity on signaling and antigen delivery.

l-Ascorbate, commonly known as vitamin C, serves as an antioxidant and cofactor essential for many biological processes. Distinct ascorbate biosynthetic pathways have been established for animals and plants, but little is known about the presence or synthesis of this molecule in invertebrate species. We have investigated ascorbate metabolism in the nematode Caenorhabditis elegans, where this molecule would be expected to play roles in oxidative stress resistance and as cofactor in collagen and neurotransmitter synthesis. Using high-performance liquid chromatography and gas-chromatography mass spectrometry, we determined that ascorbate is present at low amounts in the egg stage, L1 larvae, and mixed animal populations, with the egg stage containing the highest concentrations. Incubating C. elegans with precursor molecules necessary for ascorbate synthesis in plants and animals did not significantly alter ascorbate levels. Furthermore, bioinformatic analyses did not support the presence in C. elegans of either the plant or the animal biosynthetic pathway. However, we observed the complete (13)C-labeling of ascorbate when C. elegans was grown with (13)C-labeled Escherichia coli as a food source. These results support the hypothesis that ascorbate biosynthesis in invertebrates may proceed by a novel pathway and lay the foundation for a broader understanding of its biological role.

Monocytes play a critical role in the innate and adaptive immune systems, performing phagocytosis, presenting antigen, and producing cytokines. They are a heterogeneous population that has been divided in humans into classical, intermediate, and non-classical subsets, but the roles of these subsets are incompletely understood. In this study, we investigated the expression patterns of MHC class II (MHCII) and associated molecules and find that the intermediate monocytes express the highest levels of the MHC molecules, HLA-DR (tested in n = 30 samples), HLA-DP (n = 30), and HLA-DQ (n = 10). HLA-DM (n = 30), which catalyzes the peptide exchange on the MHC molecules, is also expressed at the highest levels in intermediate monocytes. To measure HLA-DM function, we measured levels of MHCII-bound CLIP (class II invariant chain peptide, n = 23), which is exchanged for other peptides by HLA-DM. We calculated CLIP:MHCII ratios to normalize CLIP levels to MHCII levels, and found that intermediate monocytes have the lowest CLIP:MHCII ratio. We isolated the different monocyte subsets (in a total of 7 samples) and analyzed their responses to selected cytokines as model of monocyte activation: two M1-polarizing cytokines (IFNγ, GM-CSF), an M2-polarizing cytokine (IL-4) and IL-10. Classical monocytes exhibit the largest increases in class II pathway expression in response to stimulatory cytokines (IFNγ, GM-CSF, IL-4). All three subsets decrease HLA-DR levels after IL-10 exposure. Our findings argue that intermediate monocytes are the most efficient constitutive antigen presenting subset, that classical monocytes are recruited into an antigen presentation role during inflammatory responses and that IL-10 negatively regulates this function across all subsets.

We surveyed the iron nutrition-responsive transcriptome of Chlamydomonas reinhardtii using RNA-Seq methodology. Presumed primary targets were identified in comparisons between visually asymptomatic iron-deficient versus iron-replete cells. This includes the known components of high-affinity iron uptake as well as candidates for distributive iron transport in C. reinhardtii. Comparison of growth-inhibited iron-limited versus iron-replete cells revealed changes in the expression of genes in chloroplastic oxidative stress response pathways, among hundreds of other genes. The output from the transcriptome was validated at multiple levels: by quantitative RT-PCR for assessing the data analysis pipeline, by quantitative proteomics for assessing the impact of changes in RNA abundance on the proteome, and by cross-species comparison for identifying conserved or universal response pathways. In addition, we assessed the functional importance of three target genes, VITAMIN C 2 (VTC2), MONODEHYDROASCORBATE REDUCTASE 1 (MDAR1), and CONSERVED IN THE GREEN LINEAGE AND DIATOMS 27 (CGLD27), by biochemistry or reverse genetics. VTC2 and MDAR1, which are key enzymes in de novo ascorbate synthesis and ascorbate recycling, respectively, are likely responsible for the 10-fold increase in ascorbate content of iron-limited cells. CGLD27/At5g67370 is a highly conserved, presumed chloroplast-localized pioneer protein and is important for growth of Arabidopsis thaliana in low iron.

The L-galactose (Smirnoff-Wheeler) pathway represents the major route to L-ascorbic acid (vitamin C) biosynthesis in higher plants. Arabidopsis thaliana VTC2 and its paralogue VTC5 function as GDP-L-galactose phosphorylases converting GDP-L-galactose to L-galactose-1-P, thus catalyzing the first committed step in the biosynthesis of L-ascorbate. Here we report that the L-galactose pathway of ascorbate biosynthesis described in higher plants is conserved in green algae. The Chlamydomonas reinhardtii genome encodes all the enzymes required for vitaminC biosynthesis via the L-galactose pathway. We have characterized recombinant C. reinhardtii VTC2 as an active GDP-L-galactose phosphorylase. C. reinhardtii cells exposed to oxidative stressshow increased VTC2 mRNA and L-ascorbate levels. Genes encoding enzymatic components of the ascorbate-glutathione system (e.g. ascorbate peroxidase, manganese superoxide dismutase, and dehydroascorbate reductase) are also up-regulated in response to increased oxidative stress.Theseresults indicate that C. reinhardtii VTC2, like its plant homologs, is a highly regulated enzyme in ascorbate biosynthesis in green algae and that, together with the ascorbate recycling system, the L-galactose pathway represents the major route for providing protective levels of ascorbate in oxidatively stressed algal cells. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

The plant VTC2 gene encodes GDP-l-galactose phosphorylase, a rate-limiting enzyme in plant vitamin C biosynthesis. Genes encoding apparent orthologs of VTC2 exist in both mammals, which produce vitamin C by a distinct metabolic pathway, and in the nematode worm Caenorhabditis elegans where vitamin C biosynthesis has not been demonstrated. We have now expressed cDNAs of the human and worm VTC2 homolog genes (C15orf58 and C10F3.4, respectively) and found that the purified proteins also display GDP-hexose phosphorylase activity. However, as opposed to the plant enzyme, the major reaction catalyzed by these enzymes is the phosphorolysis of GDP-d-glucose to GDP and d-glucose 1-phosphate. We detected activities with similar substrate specificity in worm and mouse tissue extracts. The highest expression of GDP-d-glucose phosphorylase was found in the nervous and male reproductive systems. A C. elegans C10F3.4 deletion strain was found to totally lack GDP-d-glucose phosphorylase activity; this activity was also found to be decreased in human HEK293T cells transfected with siRNAs against the human C15orf58 gene. These observations confirm the identification of the worm C10F3.4 and the human C15orf58 gene expression products as the GDP-d-glucose phosphorylases of these organisms. Significantly, we found an accumulation of GDP-d-glucose in the C10F3.4 mutant worms, suggesting that the GDP-d-glucose phosphorylase may function to remove GDP-d-glucose formed by GDP-d-mannose pyrophosphorylase, an enzyme that has previously been shown to lack specificity for its physiological d-mannose 1-phosphate substrate. We propose that such removal may prevent the misincorporation of glucosyl residues for mannosyl residues into the glycoconjugates of worms and mammals.

The Arabidopsis thaliana VTC2 gene encodes an enzyme that catalyzes the conversion of GDP-l-galactose to l-galactose 1-phosphate in the first committed step of the Smirnoff-Wheeler pathway to plant vitamin C synthesis. Mutations in VTC2 had previously been found to lead to only partial vitamin C deficiency. Here we show that the Arabidopsis gene At5g55120 encodes an enzyme with high sequence identity to VTC2. Designated VTC5, this enzyme displays substrate specificity and enzymatic properties that are remarkably similar to those of VTC2, suggesting that it may be responsible for residual vitamin C synthesis in vtc2 mutants. The exact nature of the reaction catalyzed by VTC2/VTC5 is controversial because of reports that kiwifruit and Arabidopsis VTC2 utilize hexose 1-phosphates as phosphorolytic acceptor substrates. Using liquid chromatography-mass spectroscopy and a VTC2-H238N mutant, we provide evidence that the reaction proceeds through a covalent guanylylated histidine residue within the histidine triad motif. Moreover, we show that both the Arabidopsis VTC2 and VTC5 enzymes catalyze simple phosphorolysis of the guanylylated enzyme, forming GDP and l-galactose 1-phosphate from GDP-l-galactose and phosphate, with poor reactivity of hexose 1-phosphates as phosphorolytic acceptors. Indeed, the endogenous activities from Japanese mustard spinach, lemon, and spinach have the same substrate requirements. These results show that Arabidopsis VTC2 and VTC5 proteins and their homologs in other plants are enzymes that guanylylate a conserved active site His residue with GDP-l-galactose, forming l-galactose 1-phosphate for vitamin C synthesis, and regenerate the enzyme with phosphate to form GDP.

The first committed step in the biosynthesis of l-ascorbate from d-glucose in plants requires conversion of GDP-l-galactose to l-galactose 1-phosphate by a previously unidentified enzyme. Here we show that the protein encoded by VTC2, a gene mutated in vitamin C-deficient Arabidopsis thaliana strains, is a member of the GalT/Apa1 branch of the histidine triad protein superfamily that catalyzes the conversion of GDP-l-galactose to l-galactose 1-phosphate in a reaction that consumes inorganic phosphate and produces GDP. In characterizing recombinant VTC2 from Arabidopsis thaliana as a specific GDP-l-galactose/GDP-d-glucose phosphorylase, we conclude that enzymes catalyzing each of the ten steps of the Smirnoff-Wheeler pathway from glucose to ascorbate have been identified. Finally, we identify VTC2 homologs in plants, invertebrates, and vertebrates, suggesting that a similar reaction is used widely in nature.