Progenitor effects and unique transcriptomic signatures linked to differentiation phenotype in clonally expanded antigen-specific CD8 T memory stem cells

Memory stem CD8+ T cells (TSCM) are a long-lived T-cell subset with stem cell-like properties, playing a key role in antiviral immunity. Despite their importance, comprehensive single-cell transcriptomic profiling of antigen-specific TSCM has not been previously conducted. In this study, an in vitro single-cell colony expansion protocol was used to investigate human CD8+ T-cell clones specific for cytomegalovirus (CMV) epitopes. Clonal lineages from selected donors were generated by single-cell sorting of dextramer-positive CD8+ T cells with varied effector and memory phenotypes, all specific for one of 2 HLA-restricted CMV epitopes. Phenotypic and functional characterization of clonal lineages derived from antigen-specific TSCM revealed differentiated memory and effector subsets (TCM, TEM, TEMRA) as well as TSCM, with the latter subset featuring multi-potentiality and multi-cytokine production. Studying TSCM-derived progeny of these varied differentiation phenotypes in single-cell transcriptomic analysis revealed strong progenitor-to-progeny effects, whereby daughter cells from a shared progenitor clustered closely regardless of progeny differentiation phenotype, suggesting a dominant impact associated with heritable genes. TSCM-derived progeny that retained a TSCM phenotype expressed canonical early-memory genes such as IL7R, CCR7, SELL, and CD27, along with novel transcripts consistently shared across donors and epitopes. The transcriptional features of clonal lineages are strongly dependent on the progenitor cell, but TSCM have an additional transcriptomic signature, likely underpinning their unique differentiation and functional characteristics. These findings have important implications for identification of long-lived and multipotent CD8 T cells for immunotherapy and immunization against viral infections.