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.

SUMMARY Here we describe a multiplexed immunohistochemical platform, with computational image processing workflows including image cytometry, enabling simultaneous evaluation of 12 biomarkers in one formalin-fixed paraffin-embedded tissue section. To validate this platform, we used tissue microarrays containing 38 archival head and neck squamous cell carcinomas, and revealed differential immune profiles based on lymphoid and myeloid cell densities, correlating with human papilloma virus status and prognosis. Based on these results, we investigated 24 pancreatic ductal adenocarcinomas from patients who received neoadjuvant GVAX vaccination, and revealed that response to therapy correlated with degree of mono-myelocytic cell density, and percentages of CD8+ T cells expressing T cell exhaustion markers. These data highlight the utility of in situ immune monitoring for patient stratification, and provide digital image processing pipelines (https://github.com/multiplexIHC/cppipe) to the community for examining immune complexity in precious tissue sections, where phenotype and tissue architecture are preserved to thus improve biomarker discovery and assessment.

Pancreas ductal adenocarcinoma (PDAC) has one of the worst five-year survival rates of all solid tumors, and thus new treatment strategies are urgently needed. Here we report that targeting Bruton’s Tyrosine Kinase (BTK), a key B cell and macrophage kinase, restores T cell-dependent anti-tumor immune responses, thereby inhibiting PDAC growth and improving responsiveness to standard-of-care chemotherapy (CTX). We report that PDAC tumor growth depends on crosstalk between B cells and FcRγ+ tumor-associated macrophages, resulting in TH2-type macrophage programming via BTK activation in a phosphatidylinositide 3-kinase (PI3K)γ-dependent manner. Treatment of PDAC-bearing mice with the BTK inhibitor PCI32765 (ibrutinib) or by PI3Kγ inhibition reprogrammed macrophages toward a TH1 phenotype that fostered CD8+ T cell cytotoxicity, and suppressed PDAC growth, indicating that BTK signaling mediates PDAC immunosuppression. These data indicate that pharmacological inhibition of BTK in PDAC can reactivate adaptive immune responses, presenting a new therapeutic modality for this devastating tumor type.

Mechanisms of immune suppression in peripheral tissues counteract protective immunity to prevent immunopathology and are coopted by tumors for immune evasion. While lymphatic vessels facilitate T cell priming, they also exert immune suppressive effects in lymph nodes at steady-state. Therefore, we hypothesized that peripheral lymphatic vessels acquire suppressive mechanisms to limit local effector CD8+ T cell accumulation in murine skin. We demonstrate that nonhematopoietic PD-L1 is largely expressed by lymphatic and blood endothelial cells and limits CD8+ T cell accumulation in tumor microenvironments. IFNγ produced by tissue-infiltrating, antigen-specific CD8+ T cells, which are in close proximity to tumor-associated lymphatic vessels, is sufficient to induce lymphatic vessel PD-L1 expression. Disruption of IFNγ-dependent crosstalk through lymphatic-specific loss of IFNγR boosts T cell accumulation in infected and malignant skin leading to increased viral pathology and tumor control, respectively. Consequently, we identify IFNγR as an immunological switch in lymphatic vessels that balances protective immunity and immunopathology leading to adaptive immune resistance in melanoma.