Blinatumomab-driven T-cell activation in αβ and γδ T-cell subsets: Insights from in vitro assays

Blinatumomab (BLN) is a bispecific T-cell engager that has revolutionized the treatment of B-cell precursor acute lymphoblastic leukemia (BCP-ALL), significantly improving outcomes in both adults and children. By simultaneously binding to CD19 on B cells and CD3 on T cells, BLN triggers target cell-dependent T-cell activation, resulting in the cytolysis of CD19+ BCP-ALL cells. Despite the remarkable clinical advancements achieved with BLN, the immunological mechanisms underlying treatment response or failure remain poorly characterized. {gamma}{delta} T cells are attractive candidates for adoptive T-cell therapy due to potent cytotoxicity, capacity to present antigens, broad lysis of different tumor entities, and low alloreactivity. Because {gamma}{delta} T cells can also be redirected by BLN, we systematically studied BLN-driven effector functions of conventional {beta} and unconventional {gamma}{delta} T cells. We evaluated cytotoxicity and cytokine/effector release in freshly isolated and in vitro-expanded {beta} and {gamma}{delta} T cells from healthy adults against CD19 BCP-ALL lines (NALM-6, HAL-01), and profiled dynamic phenotypic alterations by multiparametric flow cytometry. CD19 targets were consistently reduced in the presence of BLN. Freshly isolated {beta}, especially CD8, displayed superior BLN-mediated cytotoxicity as compared to {gamma}{delta} T cells, with donor-dependent variability in {gamma}{delta} killing. Notably, zoledronate-expanded V{gamma}9V{delta}2 {gamma}{delta} T-cell lines achieved cytotoxicity comparable to PHA-expanded {beta} cells. However, {gamma}{delta} T-cell-killing benefited from higher BLN concentration when challenged with high tumor load. BLN induced CD3 down-modulation in {beta} T cells but not in {gamma}{delta} T cells, alongside higher soluble Fas ligand in {beta} cultures, consistent with stronger early activation, preceding activation-induced cell death. {gamma}{delta} T cells showed no such changes, suggesting reduced susceptibility to activation-induced cell death. Single-cell RNA and flow analyses corroborated these findings, showing robust activation/exhaustion programs in {beta} T cells and a stable effector-memory state with low checkpoint expression in {gamma}{delta} T cells. Together, these data reveal subset-specific BLN responses and support expanded V{gamma}9V{delta}2 {gamma}{delta} T cells as a rational adoptive partner to BLN -- particularly in settings of favorable antigen density/low tumor burden -- providing complementary cytotoxicity with potentially reduced inflammatory liability. These findings provide a framework for combining {gamma}{delta} T-cell-based therapies in BLN-treated patients for improving BLN efficacy in BCP-ALL patients.