Cord blood-derived V62+and V62-T cells acquire differential cell state compositions upon in vitro expansion

Human cord blood-derived & gamma;6 T cells (CB & gamma;6) display a highly diverse TCR & gamma;6 repertoire and have a unique subtype composition different from fetal or adult peripheral blood counterparts. We expanded CB & gamma;6 in vitro using an irradiated Epstein-Barr virus-transformed feeder cell-based modified rapid expansion protocol (REP). Singlecell RNA sequencing tracked progressive differentiation of naive CB & gamma;6 into cells expressing neoantigen-reactive tumor-infiltrating lymphocyte as well as tissue-resident memory precursor-like and antigen-presenting cell-like gene signatures. TCR & gamma;6 clonal tracing revealed a bias toward cytotoxic effector differentiation in a much larger proportion of V62- clones compared to V62+ 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 & gamma;6 T cells already in operation at early postnatal stage and highlighted key areas of consideration in optimizing cell manufacturing processes.