Cord blood-derived V_δ2⁺ and V_δ2^- T cells acquire differential cell state compositions upon in vitro expansion

Human cord blood-derived γδ T cells (CB_γδ) display a highly diverse TCR_γδ repertoire and have a unique subtype composition different from fetal or adult peripheral blood counterparts. We expanded CB_γδ in vitro using an irradiated Epstein-Barr virus-transformed feeder cell-based modified rapid expansion protocol (REP). Single-cell RNA sequencing tracked progressive differentiation of naïve CB_γδ into cells expressing neoantigen-reactive tumor-infiltrating lymphocyte as well as tissue-resident memory precursor-like and antigen-presenting cell-like gene signatures. TCR_γδ clonal tracing revealed a bias toward cytotoxic effector differentiation in a much larger proportion of V_δ2^- clones compared to V_δ2⁺ clones, resulting in the former being more cytotoxic at the population level. These clonotype-specific differentiation dynamics were not restricted to REP and were recapitulated upon secondary nonviral antigen stimulations. Thus, our data showed intrinsic cellular differences between major subtypes of human γδ T cells already in operation at early postnatal stage and highlighted key areas of consideration in optimizing cell manufacturing processes.