Background: PD-1 blockade achieves objective responses in 30-55% of melanoma patients, but there remain 45-70% of patients where it is ineffective, either as a single agent or in combination with anti-CTLA-4. We lack salvage strategies for this population and a mechanistic understanding of checkpoint inhibitor refractory melanoma. Previously, we reported that patients with liver metastasis have worse response rate and survival than those without liver metastasis despite treatment with checkpoint immunotherapy and cutaneous biopsy samples from patients with liver metastasis have significantly less activated tumor-infiltrating lymphocytes (TILs) than those with metastasis elsewhere. However, the mechanism of how liver metastasis influences the systemic antitumor immune response in this context is unclear. We hypothesize that tumor antigen-specific tolerance can be induced when metastatic cells invade the liver, resulting in reduced systemic antitumor immunity and immunotherapy resistance.

Results: In this study, we developed a syngeneic two-site tumor animal model in which tumor cells are injected simultaneously into a subcutaneous (SQ) site and into the liver or other organ sites of wild-type C57BL/6 mice to investigate the unique effects of the intrahepatic tumor on antitumor immunity at a distant site. We found that the presence of tumor or tumor antigen within the liver, as opposed to other organs, reduced systemic antitumor immunity and effector T cell function that is evident at the distant SQ site. Tetramer-based flow cytometry and single cell-RNAseq analysis were done on the TILs within the SQ site and the results suggest that tumor-specific CD8 T cells were preferentially dysfunctional but not sequestered by the liver or clonally deleted, with reduced expression of activation markers and effector cytokines (ICOS, CTLA-4, IFNγ, and TNFα). Collectively, the data suggest a liver and antigen-specific immunoregulatory process mediated by the coordinated activation of Foxp3+ regulatory T cells (with increased expression of CTLA-4 and ICOS) and significant changes in the non-T cell immune composition at the distant SQ site in the liver-tumor bearing mice. Genetic studies using a mouse strain expressing the diphtheria toxin receptor under the control of Foxp3 (Foxp3-DTR) with or without the systemic depletion of Tregs revealed that in the absence of Tregs, there was no difference in the distal immunity against the SQ tumor between mice with and without experimental liver metastasis. Finally, we found that this form of systemic antitumor effector T cell dysfunction was not reversed by anti-PD-1 monotherapy but rather by combination therapy with the deletion (anti-CTLA-4) or destabilization (EZH2 inhibitor) of Tregs, suggesting a distinct regulatory mechanism contributing to anti-PD-1 resistance in the setting of liver metastasis that could be overcome to achieve complete tumor regression at both sites.

Conclusion: Our results reveal a novel mechanism of checkpoint inhibitor resistance and suggest a strategy to improve the outcome of immunotherapy treatments for stage IV cancer patients with liver metastasis.

Citation Format: James C. Lee, Sadaf Mehdizadeh, Arabella Young, Jennifer Bridge, Ilgiz A. Mufazalov, Adil Daud, Jeffrey A. Bluestone. Liver metastasis mediated control of distant tumor-specific immunity and response to checkpoint immunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1031.