Background Malignant pleural effusions and peritoneal carcinomatosis are associated with poor outcomes in patients with cancer.1–3 Macrophages in these serous body cavities express the phosphatidylserine receptor Tim-4.4–8 Prior reports demonstrated that Tim-4 abrogation is associated with improved anti-tumor activity.9–11 Whether macrophages expressing Tim-4 contribute to immunosuppression in the serous body cavities has not been previously investigated.
Methods We retrospectively annotated sites of metastases in 500 patients with lung cancer and assessed for clinical outcomes. Utilizing a combination of flow cytometry, immunohistochemistry, and antibody biodistribution assays, we surveyed for Tim-4 expression across various tissues and cell types. We performed flow cytometry on 55 consecutive pleural and peritoneal effusions from patients with lung cancer. We utilized murine models of peritoneal carcinomatosis to determine whether Tim-4 abrogation could enhance the anti-tumor efficacy of anti-PD-1 therapy. We characterized CD8+ T cells with high levels of phosphatidylserine (PShigh) with flow cytometry, cytotoxicity assays, and paired single cell RNA and TCR sequencing. Confocal microscopy was utilized to visualize interactions between Tim-4+ macrophages and PShigh CD8+ T cells.
Results Metastatic disease involvement of the pleural or peritoneal cavity was associated with reduced response rate and progression-free and overall survival. We demonstrate that Tim-4 is highly expressed on pleural and peritoneal macrophages and other select resident macrophages, but not on monocytes, tumor-associated macrophages, or tumor cells in mice and humans. High levels of Tim-4 on macrophages from fluid biospecimens is associated with reduced levels CD39+ CD8+ T cells, which comprise the tumor-reactive portion of CD8+ T lymphocytes. In order to further elucidate the mechanism of Tim-4+ macrophage-mediated immunosuppression, we established a murine model of peritoneal carcinomatosis with MC38 and CT26 colon carcinoma. Genetic or pharmacologic abrogation of Tim-4 improved the efficacy of anti-PD-1 therapy and was associated with enhanced CD39+ CD8+ T cell numbers. In parallel, we observed in mice and humans that CD8+ T cell activation results in PS upregulation despite not undergoing cell death. PShigh CD8+ T cells expressed genes associated with cytotoxicity, activation/exhaustion, and proliferation, and mediated greater cytotoxicity. Mechanistic studies revealed that Tim-4 mediates sequestration of PShigh CD8+ T cells by macrophages which subsequently impedes CD8+ T cell cytotoxicity of tumor cells.
Conclusions We demonstrate that Tim-4+ resident macrophages impair anti-tumor CD8+ T cell immunity in the serous body cavities and Tim-4 blockade represents on a novel therapeutic strategy to overcome resistance to immune checkpoint blockade (figure 1).
Ethics Approval The retrospective clinical analysis was approved by Memorial Sloan Kettering Cancer Center IRB #16-1566. The human biospecimen analyses were approved by Memorial Sloan Kettering Cancer Center IRB #06-107 and 14-091.
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