Single-cell transcriptional profiling identifies a spectrum of unconventional intraepithelial T lineage cells in human cord blood [bulk RNA-Seq]

Conventional CD4 and CD8 single positive T cell lineages constitute the main differentiation pathway in the thymus. In human thymus, a minor TCRαβ differentiation pathway diverges from the early double positive stage, consisting of CD10+ PD-1+ cells. These cells are phenotypically and functionally similar to murine agonist-selected intraepithelial T lymphocyte precursors (IELps) which home to the small intestine. Here, the progeny of the human agonist-selected IEL lineage was identified in antigen-inexperienced cord blood (CB) with a polyclonal T cell receptor (TCR) repertoire exhibiting a bias towards early TCR alpha chain rearrangements and elevated autoreactive indices. Single-cell RNA sequencing allowed further delineation of this unconventional lineage in CB. Trajectory analysis, along with TCR repertoire analysis and transcriptomics, suggests a precursor-progeny relationship with the thymic IELps. The distinct, heterogeneous CB population can now be defined as CD3+ TCRαβ+ CD4- CCR7- CD26-. Besides recent thymic emigrants, this population also consists of newly identified effector clusters and previously described populations: the suppressive NK receptor expressing CD8+ Treg population, the KIR/NKG2A+ EOMES+  virtual memory population and the CD8αα+ T cell populations. The population shows a discriminating stable Helios expression and is exclusively able to downregulate CD8β expression, resulting in double negative T cells. The functional properties of this population suggest that the cells expand on inflammatory cues and exert cytotoxic and proinflammatory activity. To identify additional marker genes in cord blood for unconventional intraepithelial T cells, 3 T cell populations were sorted from cord blood: I. TCRγδ+, II. CD3+/low PD-1+ (unconventional intraepithelial T cells) and III. CD3+ PD-1- (conventional T cells). For each sort 3 replicates from 3 different donors wereused in a differential expression analysis with DeSeq2 to identify additional markers. The same populations were sorted from post natal thymus (also 3 replicates from 3 different donors per cell population) to use as a comparison.