GZMK+ T cells in glioblastoma and an activated, non-exhausted phenotype.

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e14071 Background: Glioblastoma (GBM) bears a survival estimate below 10% at 5 years, despite surgery and standard chemoradiation. Immunotherapeutic strategies have not improved disease outcomes, primarily due to the brain’s uniquely immunosuppressive tumor microenvironment (TME). Our group formerly described a GZMK + effector T cells subpopulation, enriched in GBM, which has been so far poorly characterized in its function and spatial localization. Methods: By using 27-parameters flow cytometry, we profiled 162.466 tumor infiltrating lymphocytes (TILs) from 8 newly diagnosed GBM patients (pts) surgically treated at our Institution and matched peripheral blood mononuclear cells (PBMC). Formalin-fixed paraffin embedded (FFPE) GBM slides were stained by hematoxylin-eosin (H&E) and imaging mass cytometry (IMC). Tumor regions of interest (7-13 per case) were selected on H&E and mapped to IMC slides (Hyperion Imaging System). A CCR5/CXCR3-based flow cytometry gating strategy was established to enrich GZMK+ CD8+ T cells from PBMC by excluding naïve (CCR7+CD95-), NK(CD56+) and MAIT(CD161 hi ) cells. CD8+ T cells from PBMC were stimulated in vitro with cytokines present in the GBM TME (IL-1β, IL-2, IL-6, IL-12, IL-15 and TNF). Cells were cultured for 7 days and phenotyped by flow cytometry; cell fate and division were tracked using CellTraceViolet labelling. Results: Flow cytometry analyses confirmed the high presence of GZMK+ and GZMK+/GZMB+ double positive CD8+ T cell populations within GBM tissue. In particular, we identified a GZMK+ subpopulation (~40% of CD8+ T cells) displaying a memory, activated, non-exhausted phenotype (CCR5+, CD69+, CD27+, PD1+, TIGIT-, CXCR3+, CCR4+, HLA-DR+). IMC revealed that CD8⁺ T cells were spatially localized in close proximity to activated myeloid cells (IBA1⁺, CD16⁺, CD68⁺, CD163⁺, CD14⁺, HLA-DR⁺), consistent with either microglia or macrophages. GZMK+ CD8+ T cells isolation from PBMCs based on CCR5/CXCR3 co-expression, led to an enrichment of up to 95% of GZMK+ CD8+ T cells. Compared to other CD8+ memory T cell populations, bona fide GZMK+ CD8+ T cells sorted from PBMC exhibited enhanced degranulation capacity (CD107a), higher INFg production and increased polyfunctionality upon stimulation with PMA-Ionomycin or aCD3/aCD28+IL-2. In vitro stimulation with IL-12+IL-15 induced GZMB expression in GZMK+ CD8+ T cells from PBMCs, recapitulating the GZMK+GZMB+ CD8+ TILs phenotype observed in GBM and establishing a robust in vitro model to study GBM-infiltrating T cells. Conclusions: GBM infiltrating GZMK+ CD8+ T cells display a memory, activated, non-exhausted phenotype. In vitro IL-12+IL-15 stimulation induces GZMB expression in GZMK⁺ CD8⁺ T cells, partially recapitulating the phenotype of GBM-infiltrating T cells. Collectively, these findings identify the predominant activated GZMK⁺ CD8⁺ T-cell population as a promising therapeutic target in GBM.

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