Differential Activation of V _γ4V _δ1 and V _γ9V _δ2 Cord Blood Derived Gamma Delta T Cells upon Exposure to EBV-Lcl and K562 Cells

Gamma-delta (γδ) T cells have emerged as a promising candidate for adoptive cellular immunotherapy. To harness and maximize the anti-leukemia properties of these cells, we sort to comprehensively profile the transcriptomic signatures and immune repertoire of in vitro expanded γδ T cell products. Given the reported diverse TCR _γδ repertoire and naïve nature of γδ T cells found in human cord blood (CB _γδ), we serially track the molecular and cellular changes in these cells upon activation in expansion cultures. Based on the established viral reactivities of γδ T cell as well as prior studies showing their cross reactivities against leukemia and cancer cells, we had previously shown that stimulating CB _γδ with an irradiated EBV-LCL feeder cell-based rapid expansion protocol (REP) is capable of generating cell products with potent and specific cytotoxicity against human AML cells. In the present study, using single cell RNA sequencing (scRNA-seq) coupled with single cell TCR _γδ repertoire analysis, we compared the transcription signatures between our REP expanded γδ T cell (REP _γδ) and non-manipulated γδ T cells reported in literatures, showing the progressive acquisition of an adult PB derived γδ T cell (PB _γδ)-like cell states. Time course analysis demonstrated complex T cell activation and maturation trajectories correlating with variable level of clonal induction throughout the course of in vitro expansion. At the end of expansion, the harvested REP _γδ are predominantly of the V _γ4V _δ1 subtype. Nevertheless, upon exposing REP _γδ to target leukemia cell line K562, outgrowth of other non-V _γ4V _δ1 as well as the semi-invariant V _γ9V _δ2 cells were observed. Taken together, our data shows that as CB _γδ expand and differentiate in culture, they adopt an adult PB _γδ-like program. More importantly, our data highlights the rich clonal composition of in vitro expanded CB _γδ, with different clonotypes being variably activated upon exposure to different stimuli. Such characteristics can potentially overcome the challenges of cancer heterogeneity and cell persistence, with the potential of improving outcomes in cell immunotherapy.