Abstract 2046: Potential suppression of B effector cells by exosomal PD-L1 on anti-cancer stem cell immunity

Abstract

Abstract We have reported that cancer stem cell (CSC) vaccination induces CSC-specific T and B cell responses associated with therapeutic efficacy which can be enhanced by anti-PD-L1 administration. Tumor cells have been recognized to suppress T cell function via secretion of exosomes that express PD-L1. Whether a similar suppression by CSCs on B cells exists is unknown. In this study, 4T1 (breast cancer) and CT26 (colon cancer) tumor-bearing mice were administered with anti-CD20 mAb to deplete B cells prior to anti-PD-L1 mAb administration. Depletion of B cells as compared to the controls resulted in more aggressive tumor growth, and the anti-tumor efficacy of anti-PD-L1 was significantly reduced for both 4T1 and CT26, indicating the involvement of host B cells in the anti-tumor effect of anti-PD-L1. Administration of anti-PD-L1 partially recovered the humoral immune response, confirming the PD-L1/PD-1 pathway involvement in B cell suppression. We detected elevated expression of PD-1 on activated B cells, and higher PD-L1 expression on ALDHhigh CSCs than on ALDHlow non-CSCs. We co-cultured purified B cells with ALDHhigh CSCs with or without the addition of anti-PD-L1 mAb and found that the reduction of IgG secretion of B cells suppressed by CSCs could be rescued by anti-PD-L1 in a dose-dependent manner. These experiments indicate that CSCs suppress the IgG production by B cells via the PD-L1/PD-1 axis. In a different model, we isolated exosomes from D5 (melanoma) tumor cell culture media by differential ultracentrifugation and verified the isolated exosomes by transmission electron microscopy and nanoparticle tracking analysis as well as by exosome markers: CD63, HRS, ALIX, and TSG101. We generated Rab27ako D5 and F10 (melanoma) cells to delete exosomes by the CRISPR/Cas9 gene editing system. Rab27a knockout significantly decreased the exosome production from D5 and F10 tumor cells. We also generated PD-L1ko D5 and F10 cells. While PD-L1 was expressed in both wild type tumor cells and tumor-derived exosomes, it was absent in the PD-L1ko D5 and F10 cells and the exosomes isolated from the PD-L1ko cells as evident by western blot and flow cytometry. Furthermore, IFNγ failed to augment the PD-L1 expression in D5 and F10 cells treated with PD-L1-targeted CRISPR/Cas9, confirming the knockout of the PD-L1 gene in these tumor cells. Deletion of Rab27a or PD-L1 did not affect the proliferation of the tumor cells. Knockout of PD-L1 did not influence exosome secretion, nor did the knockout of Rab27a influence cell-surface PD-L1 expression. Based on these findings, the use of Rab27ako and PD-L1ko D5 and F10 CSCs will allow us to further characterize the potential suppression of exosomal PD-L1 on host B cells as well as T cells. Citation Format: You Qin, Kexing Lyu, Sifei Yu, Ming Lin, Max Wicha, Alfred E. Chang, Qiao Li. Potential suppression of B effector cells by exosomal PD-L1 on anti-cancer stem cell immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2046.