Introduction: Natural Killer (NK) cells are part of the innate lymphoid cell (ILC) family with seemingly opposing roles in contributing to inflammatory immune responses or inhibiting T and B cell activity, thereby serving as rheostats of adaptive immunity. NK cell function is influenced by cytokines and other microenvironment factors, which can alter NK cell responses and result in remarkably diverse phenotypes and functions. Recently, we and others identified distinct regulatory NK cell-like ILCs that directly suppress T cell responses in various malignancies and in chronic viral infections. Unlike conventional cytotoxic NK cells, ‘regulatory’ NK cell-like ILCs exhibit the ability to suppress T cell proliferation and function. The ontogeny of regulatory NK cell-like ILCs remains in question and signals that promote the development of NK cell-like regulatory ILCs have not been defined.

Methods: Using single-cell RNA-sequencing (scRNAseq) of peripheral blood samples from allogeneic hematopoietic stem cell transplant (HSCT) recipients, we identified a unique circulating regulatory NK cell-like ILC population in individuals protected from the development of graft-versus-host disease (GVHD). We further define cytokines that induce conventional CD56^bright NK cells to develop a regulatory NK cell-like ILC phenotype and demonstrate these ‘induced’ regulatory NK cell-like ILCs directly suppress T cell responses.

Results: In scRNA-seq studies of HSCT recipients, a distinct TGFb1 high CD56^bright NK cell population was observed in HSCT recipients protected from GVHD. We further define IL-2 and TGF-β1 as being key cytokines capable of inducing circulating CD56^bright NK cells to acquire a phenotype consistent with regulatory NK cell-like ILCs. This includes low IFN-γ and TNF-α expression, loss of cytotoxic potential, and acquiring the ability to directly suppress T cells. While conventional NK cell-associated cytokines were inhibited, we noted these ‘induced’ regulatory NK cell-like ILCs cells acquired the capacity to produce TGF-β1, similar to NK-like regulatory ILC observed in allogeneic HSCT recipients protected from GVHD. These induced regulatory NK cell-like ILCs maintained expression of canonical NK cell receptor genes and did not acquire a transcriptional profile consistent with other ILC family members. Instead, a unique transcriptional program was observed, with an increased expression of IKZF2 (HELIOS), ZNF683 (HOBIT) and ITGAE (CD103). While IL-2 and TGF-β1 could synergize to promote immunoregulatory functions in NK cells, this phenotype was not stable as induced regulatory NK-like ILCs lost the ability to secrete TGF-β1 and upregulated IFN-γ and TNF-α upon exposure to inflammatory cytokines.

Conclusions: These findings define a unique population of CD56^bright NK cells in the context of allogeneic HSCT in recipients who do not develop GVHD, and identify key roles for IL-2 and TGF-β1 inducing immunosuppressive functions in circulating NK cells.