Anti-Pd-1 Reduces The Immuno-Suppressive Phenotype Of Myeloid Subsets In The Murine Hepa 1-6 Tumor

Abstract Hepatocellular carcinoma (HCC) is the third leading cause of cancer related deaths and is refractory to standard chemo and radiation therapies. Clinical trials are tackling unresectable HCC through combination approaches using immunotherapies and have shown evidence of improved survival but additional treatment options are needed. Hepa 1-6 is a murine model for HCC characterized by suppressive M2 tumor-associated macrophages (TAM) and reduced costimulatory molecule expression in dendritic cells (DC), which impairs T cell activity. To expose new options for therapeutic intervention, we performed immunophenotypic analysis in Hepa 1-6 tumor-bearing mice treated with checkpoint blockade (anti-PD-1) or isotype control antibodies. Flow cytometry and FluoroSpot were used to measure tumor infiltrates and provide a deep dive into the biology of the immune response. Our results demonstrated Hepa 1-6 tumors were responsive to anti-PD-1 treatment with an increased time to progression of 60%. The response coincided with several functional changes in the lymphocyte compartment consistent with enhanced anti-tumor activity. Anti-PD-1 increased tumor infiltrating CD8 T cells, upregulated ICOS expression, and increased the frequency of polyfunctional T cells producing IFNγ, TNFα, and IL-2. Granzyme B and IFNγ responses were also increased in tumor-derived NK and NKT cells. FluoroSpot revealed that white blood cells from anti-PD-1 treated mice were more responsive to stimulation with Hepa 1-6 cells suggesting anti-PD-1 increased systemic anti-tumor specific activity. Examination of the myeloid subsets indicated anti-PD-1 reduced immunosuppressive pressure in the tumor as the number of tumor-infiltrating granulocytic myeloid-derived suppressor cells (G-MDSC) decreased in treated mice. We also measured Arg1 and iNOS expression. In TAMs, iNOS activity is associated with enhanced anti-tumor efficacy. Although the increase in M1 TAMs triggered by anti-PD-1 did not reach significance (p>0.05), iNOS expression increased significantly by 34% in total TAMs. In tumor-derived MDSCs, Arg1 and iNOS increased with treatment. Notably, Arg1 and iNOS mediate MDSC immunosuppressive function via catabolism of L-arginine, suggesting some pro-tumor activity can also be triggered by checkpoint blockade. Finally, DCs play a key role in T cell activation. Analysis revealed the dominant B7 co-stimulatory molecule CD86 was upregulated in the DC1 subset following anti-PD-1 treatment. Together, these data demonstrate that anti-PD-1 enhances lymphocyte activity and inhibits Hepa 1-6 tumor growth. This can potentially be attributed to reduced G-MDSC recruitment, reprograming of TAMs from an M2 to an M1 phenotype, and enhanced DC-mediated T cell activation. Further investigation into combination therapies to enhance the response to checkpoint inhibition in the hepa 1-6 model can inform new strategies to treat HCC. Citation Format: David W. Draper, Philip Lapinski, Brogan Yarzabek, Hillary Evens, Scott Wise, Maryland Franklin. Anti-PD-1 reduces the immuno-suppressive phenotype of myeloid subsets in the murine Hepa 1-6 tumor [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 5537.