Preclinical mouse models suggest that the gut microbiome modulates tumor response to checkpoint blockade immunotherapy; however, this has not been well-characterized in human cancer patients. Here we examined the oral and gut microbiome of melanoma patients undergoing anti–programmed cell death 1 protein (PD-1) immunotherapy (n = 112). Significant differences were observed in the diversity and composition of the patient gut microbiome of responders versus nonresponders. Analysis of patient fecal microbiome samples (n = 43, 30 responders, 13 nonresponders) showed significantly higher alpha diversity (P < 0.01) and relative abundance of bacteria of the Ruminococcaceae family (P < 0.01) in responding patients. Metagenomic studies revealed functional differences in gut bacteria in responders, including enrichment of anabolic pathways. Immune profiling suggested enhanced systemic and antitumor immunity in responding patients with a favorable gut microbiome as well as in germ-free mice receiving fecal transplants from responding patients. Together, these data have important implications for the treatment of melanoma patients with immune checkpoint inhibitors.

A collection of 130 new plant cell wall glycan-directed monoclonal antibodies (mAbs) was generated with the aim of facilitating in-depth analysis of cell wall glycans. An enzyme-linked immunosorbent assay-based screen against a diverse panel of 54 plant polysaccharides was used to characterize the binding patterns of these new mAbs, together with 50 other previously generated mAbs, against plant cell wall glycans. Hierarchical clustering analysis was used to group these mAbs based on the polysaccharide recognition patterns observed. The mAb groupings in the resulting cladogram were further verified by immunolocalization studies in Arabidopsis (Arabidopsis thaliana) stems. The mAbs could be resolved into 19 clades of antibodies that recognize distinct epitopes present on all major classes of plant cell wall glycans, including arabinogalactans (both protein-and polysaccharide-linked), pectins (homogalacturonan, rhamnogalacturonan I), xyloglucans, xylans, mannans, and glucans. In most cases, multiple subclades of antibodies were observed to bind to each glycan class, suggesting that the mAbs in these subgroups recognize distinct epitopes present on the cell wall glycans. The epitopes recognized by many of the mAbs in the toolkit, particularly those recognizing arabinose-and/or galactose-containing structures, are present on more than one glycan class, consistent with the known structural diversity and complexity of plant cell wall glycans. Thus, these cell wall glycan-directed mAbs should be viewed and utilized as epitope-specific, rather than polymer-specific, probes. The current world-wide toolkit of approximately 180 glycan-directed antibodies from various laboratories provides a large and diverse set of probes for studies of plant cell wall structure, function, dynamics, and biosynthesis.

BackgroundDNA methylation is an epigenetic mechanism central to development and maintenance of complex mammalian tissues, but our understanding of its role in intestinal development is limited.ResultsWe use whole genome bisulfite sequencing, and find that differentiation of mouse colonic intestinal stem cells to intestinal epithelium is not associated with major changes in DNA methylation. However, we detect extensive dynamic epigenetic changes in intestinal stem cells and their progeny during the suckling period, suggesting postnatal epigenetic development in this stem cell population. We find that postnatal DNA methylation increases at 3′ CpG islands (CGIs) correlate with transcriptional activation of glycosylation genes responsible for intestinal maturation. To directly test whether 3′ CGI methylation regulates transcription, we conditionally disrupted two major DNA methyltransferases, Dnmt1 or Dnmt3a, in fetal and adult intestine. Deficiency of Dnmt1 causes severe intestinal abnormalities in neonates and disrupts crypt homeostasis in adults, whereas Dnmt3a loss was compatible with intestinal development. These studies reveal that 3′ CGI methylation is functionally involved in the regulation of transcriptional activation in vivo, and that Dnmt1 is a critical regulator of postnatal epigenetic changes in intestinal stem cells. Finally, we show that postnatal 3′ CGI methylation and associated gene activation in intestinal epithelial cells are significantly altered by germ-free conditions.ConclusionsOur results demonstrate that the suckling period is critical for epigenetic development of intestinal stem cells, with potential important implications for lifelong gut health, and that the gut microbiome guides and/or facilitates these postnatal epigenetic processes.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0763-5) contains supplementary material, which is available to authorized users.

CD25 knock-out (CD25KO) mice spontaneously develop Sjögren Syndrome (SS)-like inflammation. We investigated the role of commensal bacteria by comparing CD25KO mice housed in conventional or germ-free conditions. Germ-free CD25KO mice have greater corneal barrier dysfunction, lower goblet cell density, increased total lymphocytic infiltration score, increased expression of IFN-γ, IL-12 and higher a frequency of CD4IFN-γ cells than conventional mice. CD4 T cells isolated from female germ-free CD25KO mice adoptively transferred to naive immunodeficient RAG1KO recipients caused more severe Sjögren-like disease than CD4 T cells transferred from conventional CD25KO mice. Fecal transplant in germ-free CD25KO mice reversed the spontaneous dry eye phenotype and decreased the generation of pathogenic CD4IFN-γ cells. Our studies indicate that lack of commensal bacteria accelerates the onset and severity of dacryoadenitis and generates autoreactive CD4T cells with greater pathogenicity in the CD25KO model, suggesting that the commensal bacteria or their metabolites products have immunoregulatory properties that protect exocrine glands in the CD25KO SS model.

Clostridioides difficile infections occur upon ecological / metabolic disruptions to the normal colonic microbiota, commonly due to broad-spectrum antibiotic use. Metabolism of bile acids through a 7α-dehydroxylation pathway found in select members of the healthy microbiota is regarded to be the protective mechanism by which C. difficile is excluded. These 7α-dehydroxylated secondary bile acids are highly toxic to C. difficile vegetative growth, and antibiotic treatment abolishes the bacteria that perform this metabolism. However, the data that supports the hypothesis that secondary bile acids protect against C. difficile infection is supported only by in vitro data and correlative studies. Here we show that bacteria that 7α-dehydroxylate primary bile acids protect against C. difficile infection in a bile acid-independent manner. We monoassociated germ-free, wildtype or Cyp8b1-/- (cholic acid-deficient) mutant mice and infected them with C. difficile spores. We show that 7α-dehydroxylation (i.e., secondary bile acid generation) is dispensable for protection against C. difficile infection and provide evidence that Stickland metabolism by these organisms consumes nutrients essential for C. difficile growth. Our findings indicate secondary bile acid production by the microbiome is a useful biomarker for a C. difficile-resistant environment but the microbiome protects against C. difficile infection in bile acid-independent mechanisms.