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

Abstract

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.

In vitro OP9-DL1 co-culture and subsequent maturation in the presence of IL-21 generates tumor antigen-specific T cells with a favorable less-differentiated phenotype and enhanced functionality

“…Human unconventional agonist selected T cells are characterized by PD-1 expression in thymus, which is gradually lost in the corresponding population in CB, and also after IL-15-induced proliferation. 34,42 PD-1 expression on our CB-derived in vitro generated T cells was, however, low (when IL-21 alone or in combination with IL-15 was added) or absent (with the other cytokine mixes). On the other hand, the previously described unconventional T cells express only low levels of CD62L that are even more downregulated after IL-15-induced proliferation, 34,42 which we also observed after feeder expansion when IL-15 was added, but not with IL-21.…”

“…34,42 PD-1 expression on our CB-derived in vitro generated T cells was, however, low (when IL-21 alone or in combination with IL-15 was added) or absent (with the other cytokine mixes). On the other hand, the previously described unconventional T cells express only low levels of CD62L that are even more downregulated after IL-15-induced proliferation, 34,42 which we also observed after feeder expansion when IL-15 was added, but not with IL-21. These distinct phenotypes mark differences between in vitro agonist selected T cells and unconventional agonist selected T cells isolated directly from human thymus or CB.…”

“…Recently, our group described an in vivo agonist selected population which acquires cytotoxic functionality and NK-like properties when cultured in the presence of IL-15. 34 In 18,35 The synergistic effects of both cytokines could be attributed to activation of distinct downstream mediators, amplifying cytokine production and cytotoxicity. IL-21 signals predominantly through Jak3, STAT1 and STAT3, whereas downstream mediators of IL-15 are STAT5 and ERK1/2.…”

Single-cell profiling identifies a spectrum of human unconventional intraepithelial T lineage cells

“…Similar to the PNT PD-1 + population, the CB UTCs extensively proliferated in the presence of interleukin-15 (IL-15)(fig. S5A-B) 36 . CD26 expression was upregulated by the UTCs in culture, while Helios proved to be a stable distinguishing feature between the CTCs and UTCs, even after proliferation (fig.…”

“…Allophycocyanin (APC)/AF647-conjugated: CD4 (Miltenyi, 130-113-250), CD158b CD4/CD14/CD19/CD235-depleted CB MNCs were sorted into CD3 + TCRγδ -CD4 -CD8α + PD-1 -(PD-1population) and CD3 +/low TCRγδ -CD4 -CD8α + PD-1 + (PD-1 + population) for the transcriptome, TCR and proteome analyses. The PNT populations were sorted as previously described 36 . For the single cell assay, CD4/CD14/CD19/CD235-depleted CB MNCs were sorted into CD3 +/low TCRγδ -CD4 -CD8α + PD-1 + (sort 1), CD3 +/low TCRγδ -CD4 -CD8α + CCR7 -EVI2B + (sort 2), CD3 + TCRγδ -CD4 -PD-1and CD3 +/low TCRγδ -CD4 -PD-1 + (sort 3), CD3 + TCRγδ -CD4 -CCR7 + EVI2Band CD3 +/low TCRγδ -CD4 -CCR7 -EVI2B + (sort 4).…”