Abstract C102: Investigation of the mechanism of action of anti-PD-1 treatment by systematic depletion of different immune cell populations in syngeneic models
Authors: Jin Ying , Shan Yongli , An Annie Xiaoyu , Li Henry Qixiang , Ouyang Davy Xuesong 
Published: March 2020

Abstract
Background: Human cancers are heterogenic and thus response to immune checkpoint inhibitors (ICIs) such as anti-PD-1 or anti-PD-L1 antibody treatments can be vastly different, with only ~20% of treated patients responding and some initial responders developing resistance. However, the mechanism of action (MOA) underlying these differences has yet to be revealed. Immunophenotyping and/or immunogenomic profiles of baseline (without treatment) and/or pharmacodynamics (changes due to treatment) play important roles in understanding these MOAs, but so far remain elusive due to the intrinsic complexity of the tumor immunity. For instance, while it is well-known that anti-PD-1 antibodies work through activating tumor infiltrating CD8+ cytotoxic T cells (CTLs), the contributions of other immune cells, particular those among tumor infiltrating leukocytes (TILs), have yet to be defined. In this study we treated syngeneic tumor-bearing mice with anti-PD-1 antibody in combination with targeted immune cell depletion in order to understand how different lineages of immune cells impacted anti-PD-1 efficacy. Method: Selected immune cell subpopulations were depleted in four different subcutaneous murine syngeneic models (MC38, CT26.WT, EMT6 and Hepa 1-6) followed by anti-PD-1 treatment (10mg/kg). The depletion of CD8+ T cells, Treg, NK cells and macrophages were accomplished with anti-CD8 (2.43), anti-CD25 (PC-61.5.3), anti-NK1.1 (PK136) antibodies and clodronate liposomes, respectively. The effectiveness of each depletion was assessed by flow cytometry analysis of tumor, blood and spleen at the end of each study. Results: As expected, depletion of CD8+ T cells completely abolished the antitumor effects of anti-PD-1 treatment in MC38, EMT6 and CT26.WT, confirming the crucial roles of CD8+ T cells in tumor killing; Interestingly, anti-PD-1 efficacy in Hepa1-6 was only modestly attenuated by eliminating CD8+ T cells, indicating a vital role of non-CD8+ effector cells in mediating anti-PD-1 efficacy in this particular line. Depletion of NK showed a minor impact on the efficacy of anti-PD-1 treatment, whereas the depletion of macrophages largely promoted the efficacy of anti-PD-1 in MC38 and CT26.WT; however, weakening the effect of anti-PD-1 in the Hepa 1-6 model. Depletion of Treg demonstrated synergistic effects with anti-PD-1 treatment in controlling CT26.WT and EMT6 tumors. Our anti-PD-1 treatment data suggest that, first, CD8+ cells played a direct role in antitumor activity in most tumors; second, suppressive role of Treg in some tumors, i.e., MC38, CT26.WT and EMT6, once removed, can significantly sensitize them to anti-PD-1 treatment; third, macrophages also appeared to play a similar suppressive role as Treg in MC38, CT26.WT and EMT6, with the exception of Hepa 1-6; fourth, NK cells seem to contribute less in the anti-PD-1 MOA. Further detailed analysis of the immune phenotypes of TIL and peripheral blood by flow cytometry has been performed in order to provide further insight into the MOA by correlating different depletion types and the efficacy. Conclusion: Our studies show an unequivocal role of CD8+ T cells in anti-PD-1 induced tumor growth inhibition. However, other immune cell lineages may act differently upon PD-1 blockade release, presumably depending on specific tumor microenvironment.
Citation Format: Ying Jin, Yongli Shan, Annie Xiaoyu An, Henry Qixiang Li, Davy Xuesong Ouyang. Investigation of the mechanism of action of anti-PD-1 treatment by systematic depletion of different immune cell populations in syngeneic models [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C102. doi:10.1158/1535-7163.TARG-19-C102