IMMU-32. The intra-tumoral heterogeneity of T cell antigens in glioblastoma

Abstract The limited effectiveness of current glioblastoma treatments can be attributed, at least in part, to the intricate intra- and inter-tumoral heterogeneity. To entangle this complexity, we employed a multi-omics approach, focusing on the immunological landscape in three distinct tumor regions: the hypoxic/necrotic center (CORE), the contrast-enhancing rim (CER) and the peritumoral infiltration zone (INF). As this region is likely to be left behind after surgery and contributes to tumor recurrence, we mainly focus on the peritumoral infiltration zone, Our study offers a comprehensive regional map of T cell antigens derived from a multi-omics methodology encompassing HLA ligandomics, transcriptomics, and whole-exome sequencing. We provide novel knowledge on the intra-tumoral heterogeneity of both HLA-I and -II naturally presented tumor-specific peptides in glioblastoma. A total of 31,227 unique HLA-I and 22,340 unique HLA-II peptides from all three zones were identified in 12/15 patients. Overlap analysis of glioblastoma-derived peptides and a benign tissue immunopeptidome database (https://hla-ligand-atlas.org) identified 44% of HLA-I and 44% of HLA-II peptides as tumor-exclusive. Among these exclusive HLA class I ligands, 16%, 17%, and 15% and of the HLA-II tumor-associated antigens (TAAs) 16%, 19%, and 42%, were exclusively presented in the INF, CER, or CORE zones, respectively. 6% of HLA-I and 1% of HLA-II peptides were presented with high frequency among all three zones. Top-ranking targets underwent further functional characterization through in vitro immunogenicity assays with autologous regional tumor infiltrating lymphocytes (TIL) and peripheral blood mononuclear cells (PBMC), revealing peptide-specific T cell memory responses for both types of immune cells compartments. This extensive exploration sheds light on the intra- and inter-tumoral heterogeneity of naturally presented T cell antigens as well as their immune recognition within the tumor and in the periphery.