Not so cold after all: tumor infiltrating CD8+ T cells in EBV-positive Burkitt lymphoma are quiescent, not exhausted

Abstract / SummarySurvival outcomes for pediatric Burkitt lymphoma (BL) substantially vary depending on geography (50-90%), which also serves as a proxy for the prevalence of Epstein-Barr virus (EBV) within the tumors. Although BL is considered an immunologically "cold" tumor with few tumor-infiltrating lymphocytes (TILs), their functional status has not been fully evaluated, especially for EBV-positive disease. Here, we characterize the exhaustion and activation profiles of T cells in the tumor microenvironment (TME) of EBV-positive BL using orthogonal methods, single-cell gene expression analysis, spectral flow cytometry, and immuno-histochemistry staining (IHC). We found that CD8+ TILs displayed a mosaic of immune inhibitory gene expression encoding, PD1, TIGIT, LAG3 and HAVCR2/TIM3. IHC validated the expression of PD1 and TIGIT on CD8+ TILs, as well as their respective ligands, PDL-1, PVR, and Nectin-2 on malignant B cells. Despite exhaustion-associated signatures, CD8+ TILs retain cytotoxic potential, expressing granules (i.e. Granzyme A, Perforin) and cytokines (i.e. IFN{gamma}) and demonstrate an increased uptake of metabolites such as glucose, arginine, and methionine. In peripheral blood, pediatric BL patients exhibited a significantly higher abundance of PD1+TIGIT+ CD8+ T cells compared to healthy children. Notably, these circulating T cells from BL patients express significantly lower levels of TOX, suggesting they are not irreversibly dysfunctional. Together, our results indicate that CD8+ T cells both in the TME and in circulation of children with BL are not terminally exhausted but remain poised for functional re-invigoration. These findings support the potential integration of immune checkpoint inhibitors into combination chemotherapeutic regimens to improve outcomes for these children. SignificanceEBV-positive BL tumors contain functional, metabolically active CD8+ T cells. Circulating PD1+TIGIT+CD8+ T cells found in BL patients blood are a biomarker for those in the tumor microenvironment.