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524 Tim-4+ resident macrophages impair anti-tumor immunity in the serous body cavities By sequestering viable and cytotoxic CD8+ T cells expressing high levels of phosphatidylserine
  1. Andrew Chow1,
  2. Sara Schad1,
  3. Michael Green2,
  4. Matthew Hellmann1,
  5. Nicholas Ceglia1,
  6. Viola Allaj1,
  7. Giulia Zago1,
  8. Nisarg Shah1,
  9. Sai Sharma1,
  10. Marissa Mattar1,
  11. Joseph Chan1,
  12. Hira Rizvi1,
  13. Hong Zhong1,
  14. Cailian Liu1,
  15. Yonina Bykov1,
  16. Dmitriy Zamarin1,
  17. Hongyu Shi1,
  18. Sadna Budhu1,
  19. Corrin Wohlhieter1,
  20. Fathema Uddin1,
  21. Aditi Gupta1,
  22. Inna Khodos1,
  23. Jessica Waninger2,
  24. Angel Qin2,
  25. Vinod Balachandran1,
  26. Weiping Zou2,
  27. Sohrab Shah1,
  28. Andrew McPherson1,
  29. Katherine Panageas1,
  30. Jason Lewis1,
  31. Justin Perry1,
  32. Elisa de Stanchina1,
  33. Triparna Sen1,
  34. John Poirier3,
  35. Jedd Wolchok1,
  36. Charles Rudin1 and
  37. Taha Merghoub1
  1. 1Memorial Sloan Kettering Cancer Center, New York, NY, USA
  2. 2University of Michigan, Ann Arbor, MI, USA
  3. 3NYU Langone Medical Center, New York, NY, USA


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.

Abstract 524 Figure 1

After activation by antigen-presenting cells in the lymph nodes, viable CD8+ T cells express high levels of phosphatidylserine, which coincides with a highly proliferative and cytotoxic state. As they migrate towards tumors cells in the serous body cavities, they are sequestered by Tim-4+ resident macrophages which impede their anti-tumor cytotoxicity. Tim-4 abrogation can alleviate this sequestration and enhance anti-tumor immunity

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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