Knowledge of immune cell phenotypes in the tumor microenvironment is essential for understanding mechanisms of cancer progression and immunotherapy response. We profiled 45,000 immune cells from eight breast carcinomas, as well as matched normal breast tissue, blood, and lymph nodes, using single-cell RNA-seq. We developed a preprocessing pipeline, SEQC, and a Bayesian clustering and normalization method, Biscuit, to address computational challenges inherent to single-cell data. Despite significant similarity between normal and tumor tissue-resident immune cells, we observed continuous phenotypic expansions specific to the tumor microenvironment. Analysis of paired single-cell RNA and T cell receptor (TCR) sequencing data from 27,000 additional T cells revealed the combinatorial impact of TCR utilization on phenotypic diversity. Our results support a model of continuous activation in T cells and do not comport with the macrophage polarization model in cancer. Our results have important implications for characterizing tumor-infiltrating immune cells.

Summary Regulatory T (Treg) cells reside in lymphoid organs and barrier tissues where they control different types of inflammatory responses. Treg cells are also found in human cancers, and studies in animal models suggest that they contribute to cancer progression. However, properties of human intratumoral Treg cells and those present in corresponding normal tissue remain largely unknown. Here, we analyzed features of Treg cells in untreated human breast carcinomas, normal mammary gland, and peripheral blood. Tumor-resident Treg cells were potently suppressive and their gene expression pattern resembled that of normal breast tissue, but not of activated peripheral blood Treg cells. Nevertheless, a number of cytokine and chemokine receptor genes, most notably CCR8, were upregulated in tumor-resident Treg cells in comparison to normal tissue resident ones. Our studies suggest that targeting CCR8 for the depletion of tumor-resident Treg cells may represent a promising immunotherapeutic approach for the treatment of breast cancer.

Infection with antibiotic-resistant bacteria, such as vancomycin-resistant Enterococcus (VRE), is a dangerous and costly complication of broad-spectrum antibiotic therapy 1,2 . How antibioticmediated elimination of commensal bacteria promotes infection by antibiotic-resistant bacteria is a fertile area for speculation with few defined mechanisms. Here we demonstrate that antibiotic treatment of mice notably downregulates intestinal expression of RegIIIγ (also known as Reg3g), a secreted C-type lectin that kills Gram-positive bacteria, including VRE. Downregulation of RegIIIγ markedly decreases in vivo killing of VRE in the intestine of antibiotic-treated mice. Stimulation of intestinal Toll-like receptor 4 by oral administration of lipopolysaccharide re-induces RegIIIγ, thereby boosting innate immune resistance of antibiotic-treated mice against VRE. Compromised mucosal innate immune defence, as induced by broad-spectrum antibiotic therapy, can be corrected by selectively stimulating mucosal epithelial Toll-like receptors, providing a potential therapeutic approach to reduce colonization and infection by antibiotic-resistant microbes.Infections caused by highly antibiotic-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA) and VRE, are an increasing menace in hospitalized patients 3,4 . Treatment of serious VRE infections is limited by the paucity of effective antibiotics 5 . VRE colonizes the gastrointestinal tract and it is likely that systemic bloodstream infections are the result of dissemination from the intestine 6 . It has been widely assumed that antibiotic treatment, by eliminating commensal flora, opens intestinal niches and provides increased access to nutrients, thereby enhancing VRE survival and proliferation. Recent studies, however, have demonstrated that commensal microbes in the intestine induce expression of proteins that restrict bacterial survival and growth 7,8 . Thus, whereas commensal microbes may directly restrict VRE proliferation, an alternative hypothesis is that commensal microbes inhibit VRE indirectly by activating mucosal innate immune defenses.Correspondence and requests for materials should be addressed to E.G.P. (Fig. 1b). RegIIIγ is a secreted lectin with potent bactericidal activity against Gram-positive bacteria that is expressed by intestinal epithelial and Paneth cells 7 . Expression of RegIIIγ is dependent on TLR-MyD88-mediated signals in intestinal epithelial cells and is induced by commensal microbes 7,9 . To determine whether RegIIIγ mediates in vivo killing of VRE in the intestine, we injected a blocking polyclonal antiserum 10 against RegIIIγ into ileal loops of wild-type mice before inoculation of VRE. The number of surviving VRE bacteria was increased by over 400% in intestines treated with RegIIIγ-specific antiserum (Fig. 1c), indicating that RegIIIγ mediates in vivo VRE killing.Administration of the broad-spectrum antibiotic combination metronidazole, neomycin and vancomycin (MNV), to which VRE is resistant, markedly increases ...

Accurate identification of tumor-derived somatic variants in plasma circulating cell-free DNA (cfDNA) requires understanding the various biologic compartments contributing to the cfDNA pool. We sought to define the technical feasibility of a high-intensity sequencing assay of cfDNA and matched white-blood cell (WBC) DNA covering a large genomic region (508 genes, 2Mb, >60,000X raw-depth) in a prospective study of 124 metastatic cancer patients, with contemporaneous matched tumor tissue biopsies, and 47 non-cancer controls. The assay displayed a high sensitivity and specificity, allowing for de novo detection of tumor-derived mutations and inference of tumor mutational burden, microsatellite instability, mutational signatures and sources of somatic mutations identified in cfDNA. The vast majority of cfDNA mutations (81.6% in controls and 53.2% in cancer patients) had features consistent with clonal hematopoiesis (CH). This cfDNA sequencing approach revealed that CH constitutes a pervasive biological phenomenon emphasizing the importance of matched cfDNA-WBC sequencing for accurate variant interpretation.

Listeria monocytogenes is a food-borne bacterial pathogen that causes systemic infection by traversing the intestinal mucosa. Although MyD88-mediated signals are essential for defense against systemic L. monocytogenes infection, the role of Toll-like receptor and MyD88 signaling in intestinal immunity against this pathogen has not been defined. We show that clearance of L. monocytogenes from the lumen of the distal small intestine is impaired in MyD88−/− mice. The distal ileum of wild-type (wt) mice expresses high levels of RegIIIγ, which is a bactericidal lectin that is secreted into the bowel lumen, whereas RegIIIγ expression in MyD88−/− mice is nearly undetectable. In vivo depletion of RegIIIγ from the small intestine of wt mice diminishes killing of luminal L. monocytogenes, whereas reconstitution of MyD88-deficient mice with recombinant RegIIIγ enhances intestinal bacterial clearance. Experiments with bone marrow chimeric mice reveal that MyD88-mediated signals in nonhematopoietic cells induce RegIIIγ expression in the small intestine, thereby enhancing bacterial killing. Our findings support a model of MyD88-mediated epithelial conditioning that protects the intestinal mucosa against bacterial invasion by inducing RegIIIγ.

Transient ablation of regulatory T cells in a murine model of breast carcinogenesis inhibits primary tumor and lung metastatic growth and enhances the therapeutic effect of radiotherapy, but not immune checkpoint blockade.

The liver is believed to promote tolerance, which may be beneficial due to its constant exposure to foreign Ags from the portal circulation. Although dendritic cells (DCs) are critical mediators of immune responses, little is known about human liver DCs. We compared freshly purified liver DCs from surgical specimens with autologous blood DCs. Liver and blood DCs were equally immature, but had distinct subset compositions. BDCA-1+ DCs represented the most prevalent liver DC subset, whereas the majority of peripheral blood DCs were CD16+. Upon TLR4 ligation, blood DCs secreted multiple proinflammatory cytokines, whereas liver DCs produced substantial amounts of IL-10. Liver DCs induced less proliferation of allogeneic T cells both in a primary MLR and after restimulation. Similarly, Ag-specific CD4+ T cells were less responsive to restimulation when initially stimulated by autologous liver DCs rather than blood DCs. In addition, liver DCs generated more suppressive CD4+ CD25+FoxP3+ T regulatory cells and IL-4-producing Th2 cells via an IL-10-dependent mechanism. Our findings are critical to understanding hepatic immunity and demonstrate that human liver DCs promote immunologic hyporesponsiveness that may contribute to hepatic tolerance.

TLRs are recognized as promoters of tissue damage, even in the absence of pathogens. TLR binding to damage-associated molecular patterns (DAMPs) released by injured host cells unleashes an inflammatory cascade that amplifies tissue destruction. However, whether TLRs possess the reciprocal ability to curtail the extent of sterile inflammation is uncertain. Here, we investigated this possibility in mice by studying the role of conventional DCs (cDCs) in liver ischemia/reperfusion (I/R) injury, a model of sterile inflammation. Targeted depletion of mouse cDCs increased liver injury after I/R, as assessed by serum alanine aminotransferase and histologic analysis. In vitro, we identified hepatocyte DNA as an endogenous ligand to TLR9 that promoted cDCs to secrete IL-10. In vivo, cDC production of IL-10 required TLR9 and reduced liver injury. In addition, we found that inflammatory monocytes recruited to the liver via chemokine receptor 2 were downstream targets of cDC IL-10. IL-10 from cDCs reduced production of TNF, IL-6, and ROS by inflammatory monocytes. Our results implicate inflammatory monocytes as mediators of liver I/R injury and reveal that cDCs respond to DAMPS during sterile inflammation, providing the host with protection from progressive tissue damage.