Our studies approached this problem using integrative omics
Our studies approached this problem using integrative -omics analysis of human NK-ieILC1s from distinct microenvironments and functional subsets, including helper and adaptive phenotypes. At both the transcriptional and epigenetic levels, we find that the adaptive subset, CD57+ cytotoxic NK cells, segregated away from a spectrum of more closely related subsets (CD57–, CD56bright, tonsillar NK, and ieILC1s). Beyond this, a small set of signature genes distinguished the five subsets from one another, with a substantial mixing and matching of transcriptional modules to create patchwork expression programs. For example, RUNX2, an osteoblast-restricted gene, was selectively expressed in CD56bright NK Adrucil (Komori, 2010). KLF3, originally thought to be an erythrocyte TF (Crossley et al., 1996, Vu et al., 2011), was selectively expressed in circulating NK cells, but not those found in mucosal tissue. Unexpectedly, circulating CD56dim NK cells shared a small set of genes with ieILC1s residing in a mucosal microenvironment, further underscoring the patchwork nature of NK-ieILC1 expression programs. Although additional studies are required, the CD56dim-ieILC1 shared module may derive from a unique combination of TF expression that includes ZNF683 (HOBIT, ieILC1), PRDM1 (BLIMP1, CD56dim), and IKZF3 (AIOLOS, both). A major finding to emerge from this study is a conserved regulatory scheme deployed by both innate and adaptive lymphocytes, which is coordinated by a cohort of TFs. The scheme functionally compartmentalizes effector cells that circulate between blood and peripheral tissues from self-renewing precursors that traffic to lymph nodes. The latter regulatory scheme utilizes the transcriptional activators TCF1-LEF1 and MYC in NK (CD56bright), T memory, and T naive compartments. Moreover, human B, T, and NK cells suppress effector pathways via expression of BACH2, which may be under the control of AHR signaling (Richer et al., 2016). The BACH2-mediated brake appears to be released in all of these cells upon expression of BLIMP1, which feeds back to repress the TCF1-LEF1 and MYC module, while effector programs are activated by MAF. In support of this unified model, TCF1 promotes renewal and maintenance of hematopoietic stem cells (Wu et al., 2012), TSCM, which exhibit a heightened capacity to undergo IL-7- and IL-15-driven homeostatic proliferation (Gattinoni et al., 2011), CD4+ T follicular helper cells (Choi et al., 2015), and Ly49H+ MCMV-specific naive and memory NK cells (Lau et al., 2018). With regard to translational implications, TCF1 also is expressed in a subset of CXCR5+CD8+ T cells that proliferate in response to anti-PD1 treatments for chronic infection and cancer (Im et al., 2016). In contrast, TCF7-LEF1 were both poorly expressed in the adaptive CD56dimCD57+ NK subset, which exhibits a more terminally differentiated state reminiscent of CD8+ TEM and CD4+TH1 cells (Choi et al., 2015, Herndler-Brandstetter et al., 2018). Our analyses identified conserved enhancers in TCF7 and LEF1 loci that are targeted by BLIMP1 in differentiated mouse cells, likely repressing their expression. A similar switch is governed by these TFs in mouse counterparts of human NK subsets, as well as in macaques, a non-human primate. The parallels between regulatory programs in NK and T cells may extend to additional TFs. Specifically, we observed a reciprocity between ZEB1 and ZEB2 expression in proliferative precursors (CD56bright, TSCM, TCM) and effector cells (CD56dim, TEM), respectively (Guan et al., 2018). Our integrative and single cell analyses revealed potential developmental relationships between circulating human NK cells. Transcriptome, epigenome and protein expression data displayed gradients of activity from CD56bright to CD57− transitional cells to the CD57+ NK subset. This trajectory is consistent with some reports indicating that CD56bright cells can serve as precursors to the CD56dim subset (Freud et al., 2017, Yu et al., 2010). Thus, the TCF7-LEF1-MYC module in CD56bright NKs may function to preserve progenitor populations, which can continually replenish CD57+ adaptive cells following induction of BLIMP1. However, we do not exclude alternative cause-effect relationships between transcriptional modules, especially since fate mapping studies of NK cells are not possible in humans. Distinct expression programs may reflect homing potential or localization of NK subsets, rather than a discrete developmental pathway. In support of this scenario, all cells sharing the TCF1-LEF1-MYC module, including naive CD8 T cells, traffic between the blood and secondary lymphoid organs, as indicated by expression of CD62L and CCR7. Indeed, we found that expression programs of tonsillar NK cells were nearly indistinguishable from the CD56bright subset. In contrast, CD56dim NK and TEM cells, which share the BLIMP1-MAF module, traffic between blood and non-lymphoid organs. Consistent with recent studies, a third regulatory module driven by the TF HOBIT (ZNF683) appears to drive retention of ieILC1 and resident memory T cells (TRM) in a variety of tissues (Jameson and Masopust, 2018).