Cord blood–derived V(δ)2(+) and V(δ)2(−) 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(δ)2(−) clones compared to V(δ)2(+) 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.