Human Thymic CD10⁺ PD-1⁺ Intraepithelial Lymphocyte Precursors Acquire Interleukin-15 Responsiveness at the CD1a^- CD95⁺ CD28^- CCR7^- Developmental Stage

Human thymic CD8αα⁺ CD10⁺ PD-1⁺ αβ T cells selected through early agonist selection have been proposed as the putative thymic precursors of the human CD8αα⁺ intestinal intraepithelial lymphocytes (IELs). However, the progeny of these thymic precursor cells in human blood or tissues has not yet been characterized. Here, we studied the phenotypical and transcriptional differentiation of the thymic IEL precursor (IELp) lineage upon in vitro exposure to cytokines prominent in the peripheral tissues such as interleukin-15 (IL-15) and the inflammatory cytokines interleukin-12 (IL-12) and interleukin-18 (IL-18). We showed that only the CD1a^- fraction of the CD10⁺ PD-1⁺ IELp population was able to proliferate with IL-15, suggesting that this subset had acquired functionality. These cells downregulated PD-1 expression and completely lost CD10 expression, whereas other surface markers such as CD95 and CXCR3 remained highly expressed. RNA-seq analysis of the IL-15-cultured cells clearly showed induction of innate-like and effector genes. Induction of the cytotoxic machinery by the CD10⁺ PD-1⁺ population was acquired in the presence of IL-15 and was further augmented by inflammatory cytokines. Our data suggest that only the CD1a^- CD10⁺ PD-1⁺ population exits the thymus and survives in the periphery. Furthermore, PD-1 and CD10 expression is not an intrinsic property of this lineage, but rather characterizes a transient stage in differentiation. CD95 and CXCR3 expression combined with the absence of CD28, CCR7, and CD6 expression might be more powerful markers to define this lineage in the periphery.