TGF-β and IL-15 Synergize through MAPK Pathways to Drive the Conversion of Human NK Cells to an Innate Lymphoid Cell 1–like Phenotype

Abstract

Circulating NK cells are known to convert to a type 1 innate lymphoid cell (ILC1)-like phenotype in response to TGF-b exposure. However, the precise cellular changes defining this process as well as the downstream signaling pathways guiding it remain poorly defined, particularly in humans. We used mass cytometry by time-of-flight (CyTOF) to model this phenotypic shift in vitro and identify a synergistic activity of TGF-b and IL-15 in this cellular conversion. CyTOF profiling identified substantial heterogeneity in the propensity of NK cells to adopt an ILC1-like phenotype in culture, characterized by the step-wise acquisition of various markers, including CD69, CD9, CD103, and CD49a. Activating and inhibitory receptors, including NKG2A, NKG2D, KIR2DL1, KIR3DL1, NKp30, NKp44, and NKp46, were all found to be upregulated exclusively on the cellular subsets that converted most readily in response to TGF-b. An assessment of downstream TGF-b signaling identified TAK1-mediated activation of p38 MAPK as the critical pathway driving conversion. IL-15 enhanced TGF-b-mediated conversion through Ras:RAC1 signaling as well as via the activation of MEK/ERK. Interestingly, the adoption of an ILC1-like phenotype was independent of the effect of IL-15 or TGF-b on mTOR, as the culture of NK cells in the presence of mTOR inhibitors, such as rapamycin or torin1, had minimal impact on the degree of conversion. In conclusion, we have used in vitro human culture systems and CyTOF to define the conversion of circulating NK cells to an ILC1-like phenotype and have clarified the pathways responsible for this process.

The Production of Pro-angiogenic VEGF-A Isoforms by Hypoxic Human NK Cells Is Independent of Their TGF-β-Mediated Conversion to an ILC1-Like Phenotype

“…Chronic exposure of cultured circulating NK cells to TGF-β induced their conversion to an ILC1-like phenotype, as indicated by the progressive acquisition of the tissue residency markers CD9 and CD103 ( Figure 1A). As reported previously, NK cells exhibited significant heterogeneity in their propensity to convert in response to TGF-β exposure (7). NK cells cultured with TGF-β segregated into distinct single positive (CD9 + CD103 − or CD9 − CD103 + ), double positive (CD9 + CD103 + ), or doublenegative (CD9 − CD103 − ) sub-populations, with the proportion cells expressing one or both markers increasing progressively from 3 to 7 days in culture ( Figure 1B).…”

“…While the effects of TGF-β and hypoxia on NK cells are independent, it is still probable that both factors are required in vivo to illicit NK-mediated changes in vascular structure. Previous works have shown that TGF-β, in combination with other factors like IL-15, induce the expression of vital integrins, like CD9 and CD103, not only on NK cells, but also on other lymphocyte subsets like resident memory T cells (7,18,19). Such changes are essential to the migration and retention of these lymphocyte subsets into tissues (19).…”

“…dNKs also express a variety of surface markers that are shared by tissue resident NK cells from multiple organs (5), as well as innate lymphoid cell (ILC) subsets like ILC1s (6). Although dNKs, tissue resident NK cells and ILC1s were originally believed to be developmentally distinct from cytotoxic NK cells in the circulation, recent studies have identified significant plasticity in circulating NK cells, whereby they can adopt an ILC1-like phenotype in response to trophoblast or tumor-derived factors, such as transforming growth factor-β (TGF-β) (3,(7)(8)(9). This ILC1-like conversion, which is marked by reduced cytotoxicity and acquisition of tissue residency markers, including CD9, CD103, and CD49a, is not equivalent across all NK cells in the circulation, with a distinct subset of this population exhibiting a greater propensity to adopt an ILC1-like phenotype in response to chronic TGF-β exposure (7,9).…”

Galunisertib Drives Treg Fragility and Promotes Dendritic Cell-Mediated Immunity against Experimental Lymphoma

“…TGF-β and IL-15 are engaged in opposing extrinsic signals to control and contract the clonal expansion of CD8 + T cells ( Lucas et al., 2006 ; Sanjabi et al., 2009 ). In contrast, TGF-β and IL-15 play a synergistic role in promoting phenotype switch of NK cells to innate lymphoid cell suggesting considerable plasticity between them ( Hawke et al., 2020 ). We have investigated the combined therapy of galunisertib and recombinant interleukin (rIL)-15-activated DCs against a highly metastatic and aggressive murine lymphoma called Dalton lymphoma (DL) ( Hira et al., 2014 ; Klein and Klein, 1954 ).…”

Mechanisms of NK cell dysfunction in the tumor microenvironment and current clinical approaches to harness NK cell potential for immunotherapy

“…A deeper understanding of the mechanisms involved in this conversion could open perspectives for targeted immunomodulation. It was recently reported that TGF‐β and IL‐15 can synergistically induce the conversion of NK cells into ILC‐1, though the noncanonical TAK‐1/p38 MAPK and Ras/Raf/MEK/ERK pathways, respectively 75 . Interestingly, NK cell plasticity in the interaction between immune cells, tumor cells, and the TME has striking conceptual similarity with the conversion of peripheral NK cells to decidual NK cells (dNK) during pregnancy.…”