IMST-54. GLIOBLASTOMA EXTRACELLULAR VESICLES INDUCE IMMUNOSUPPRESSION VIA PD-L1

Abstract

Glioblastoma multiforme (GBM) induces potent immunosuppression both within the tumor and systemically. The mechanisms underlying this have not been fully elucidated. GBM shed large numbers of extracellular vesicles (EV’s). Here, we test the hypothesis that GBM-EV’s contribute to immunosuppression. EV’s were isolated from cultured human GBM cell lines, GBM patient serum, and healthy donor serum by serial centrifugation. Nanosight nanoparticle tracking was used to determine EV frequency and size distribution. Western blot and ELISA were used to determine tumor-associated antigen and PD-L1 expression. GBM EV’s from wild type and PD-L1-overexpressing cells were cultured with healthy donor CD14+ monocytes or CD3+ T cells. Binding was determined by confocal microscopy. Induction of CD14+/HLA-DR- monocytic myeloid-derived suppressor cells (MDSC’s) and CD4+ and CD8+ regulatory T cells (Treg) were determined by flow cytometry. T cell proliferation was assessed by flow cytometry and CFSE staining. GBM’s shed large amounts of EV’s including both exosomes (<100nm) and microvesicles (100-500nm) in vitro. EV’s with similar size distribution are found in GBM patients’ serum at increased frequency compared with healthy donors. Tumor-associated antigens (ErbB2, MAGEA3) can be detected in GBM patients’ serum EV’s. Some GBM-derived EV’s express the immunosuppressive protein PD-L1 and bind both T cells and monocytes through PD-1. GBM EV’s induce MDSC differentiation in normal monocytes in vitro(27.5%+/-5.62%) compared to media (5.24%+/-0.77; P=0.0078) and this was further increased by PD-L1 overexpressing EV’s (54.28%+/-5.48%; P=0.0143). GBM EV-induced MDSC’s inhibited T cell proliferation in response to anti-CD3 (65.98%+/-5.64% vs. 40.40%+/-7.99%; P=0.0399). GBM EV’s also showed trends towards inducing both CD4 and CD8 Treg’s in a PD-L1-dependent fashion. GBM EV’s contain both immunosuppressive molecules and tumor-associated antigens. Their presence in GBM patients’ serum suggests potential to mediate systemic immunosuppression. In keeping with this, GBM EV’s induce immunosuppressive changes in leukocytes in a PD-L1-dependent fashion.