The tumor microenvironment is a highly complex ecosystem of diverse cell types, which shape cancer biology and impact the responsiveness to therapy. Here, we analyze the microenvironment of esophageal squamous cell carcinoma (ESCC) using single-cell transcriptome sequencing in 62,161 cells from blood, adjacent nonmalignant and matched tumor samples from 11 ESCC patients. We uncover heterogeneity in most cell types of the ESCC stroma, particularly in the fibroblast and immune cell compartments. We identify a tumor-specific subset of CST1+ myofibroblasts with prognostic values and potential biological significance. CST1+ myofibroblasts are also highly tumor-specific in other cancer types. Additionally, a subset of antigen-presenting fibroblasts is revealed and validated. Analyses of myeloid and T lymphoid lineages highlight the immunosuppressive nature of the ESCC microenvironment, and identify cancer-specific expression of immune checkpoint inhibitors. This work establishes a rich resource of stromal cell types of the ESCC microenvironment for further understanding of ESCC biology.

The human fallopian tube harbors the cell of origin for the majority of high-grade serous ''ovarian'' cancers (HGSCs), but its cellular composition, particularly the epithelial component, is poorly characterized. We perform single-cell transcriptomic profiling of around 53,000 individual cells from 12 primary fallopian specimens to map their major cell types. We identify 10 epithelial subpopulations with diverse transcriptional programs. Based on transcriptional signatures, we reconstruct a trajectory whereby secretory cells differentiate into ciliated cells via a RUNX3 high intermediate. Computational deconvolution of advanced HGSCs identifies the ''early secretory'' population as a likely precursor state for the majority of HGSCs. Its signature comprises both epithelial and mesenchymal features and is enriched in mesenchymal-type HGSCs (p = 6.7 3 10 À27 ), a group known to have particularly poor prognoses. This cellular and molecular compendium of the human fallopian tube in cancer-free women is expected to advance our understanding of the earliest stages of fallopian epithelial neoplasia.

Metastatic cancer involving spread to the peritoneal cavity is referred to as peritoneal carcinomatosis and has a very poor prognosis. Activating the anti-tumor immune response in the characteristically immune-suppressive peritoneal environment presents a potential strategy to treat this disease. In this study, we show that a toll-like receptor (TLR) and C-type lectin receptor (CLR) agonist pairing of monophosphoryl lipid A (MPL) and trehalose-6,6'-dicorynomycolate (TDCM) effectively inhibits tumor growth and ascites development in a mouse model of aggressive mammary cancer-induced peritoneal carcinomatosis. MPL/TDCM treatment similarly inhibited peritoneal EL4 tumor growth and ascites development. These effects were not observed in mice lacking B cells or mice lacking CD19, which are deficient in B-1a cells, an innate-like B cell population enriched in the peritoneal cavity. Remarkably, adoptive transfer of B-1a cells, but not splenic B cells from WT mice restored MPL/TDCM-induced protection in mice with B cell defects. Treatment induced B-1 cells to rapidly produce high levels of natural IgM reactive against tumor-associated carbohydrate antigens. Consistent with this, we found significant deposition of IgM and C3 on peritoneal tumor cells as early as 5 days post-treatment. Mice unable to secrete IgM or complement component C4 were not protected by MPL/TDCM treatment, indicating tumor killing was mediated by activation of the classical complement pathway. Collectively, our findings reveal an unsuspected role for B-1 cell-produced natural IgM in providing protection against tumor growth in the peritoneal cavity, thereby highlighting potential opportunities to develop novel therapeutic strategies for the prevention and treatment of peritoneal metastases.

B-1 cells produce natural antibodies which provide an integral first line of defense against pathogens while also performing important homeostatic housekeeping functions. In this study, we demonstrate programmed cell death 1 ligand 2 (PD-L2) regulates the production of natural antibodies against phosphorylcholine (PC). Naïve PD-L2-deficient (PD-L2−/−) mice produced significantly more PC-reactive IgM and IgA. This afforded PD-L2−/− mice with selectively enhanced protection against PC-expressing non-typeable Haemophilus influenzae (NTHi), but not PC-negative NTHi, relative to wild type mice. PD-L2−/− mice had significantly increased PC-specific CD138+ splenic plasmablasts bearing a B-1a phenotype, and produced PC-reactive Abs largely of the T15 idiotype. Importantly, PC-reactive B-1 cells expressed PD-L2 and irradiated chimeras demonstrated B cell-intrinsic PD-L2 expression regulated PC-specific Ab production. In addition to increased PC-specific IgM, naïve PD-L2−/− mice and irradiated chimeras reconstituted with PD-L2−/− B cells had significantly higher levels of IL-5 – a potent stimulator of B-1 cell Ab production. PDL2 mAb blockade of wild type B-1 cells in culture significantly increased CD138 and Blimp1 expression and PC-specific IgM, but did not affect proliferation. PDL2 mAb blockade significantly increased IL-5+ T cells in culture. Both IL-5 neutralization and STAT5 inhibition blunted the effects of PDL2 mAb blockade on B-1 cells. Thus, B-1 cell-intrinsic PD-L2 expression inhibits IL-5 production by T cells and thereby limits natural Ab production by B-1 cells. These findings have broad implications for the development of therapeutic strategies aimed at altering natural Ab levels critical for protection against infectious disease, autoimmunity, allergy, cancer, and atherosclerosis.

Tn is a carbohydrate antigen uniquely exposed on tumor mucins and thus, an ideal target for immunotherapy. However, it has been difficult to elicit protective antibody responses against Tn antigen and other tumor associated carbohydrate antigens. Our study demonstrates this can be attributed to PD-1 immuno-inhibition. Our data show a major role for PD-1 in suppressing mucin- and Tn-specific B-cell activation, expansion, and antibody production important for protection against Tn-bearing tumor cells. These Tn/mucin-specific B cells belong to the innate-like B-1b cell subset typically responsible for T cell–independent antibody responses. Interestingly, PD-1–mediated regulation is B cell–intrinsic and CD4+ cells play a key role in supporting Tn/mucin-specific B cell antibody production in the context of PD-1 deficiency. Mucin-reactive antibodies produced in the absence of PD-1 inhibition largely belong to the IgM subclass and elicit potent antitumor effects via a complement-dependent mechanism. The identification of this role for PD-1 in regulating B cell–dependent antitumor immunity to Tn antigen highlights an opportunity to develop new therapeutic strategies targeting tumor associated carbohydrate antigens.