SUMMARY The mechanisms underlying human natural killer (NK) cell phenotypic and functional heterogeneity are unknown. Here, we describe the emergence of diverse subsets of human NK cells selectively lacking expression of signaling proteins after human cytomegalovirus (HCMV) infection. The absence of B and myeloid cell-related signaling protein expression in these NK cell subsets correlated with promoter DNA hyperme-thylation. Genome-wide DNA methylation patterns were strikingly similar between HCMV-associated adaptive NK cells and cytotoxic effector T cells but differed from those of canonical NK cells. Functional interrogation demonstrated altered cytokine responsiveness in adaptive NK cells that was linked to reduced expression of the transcription factor PLZF. Furthermore, subsets of adaptive NK cells demonstrated significantly reduced functional responses to activated autologous T cells. The present results uncover a spectrum of epigenetically unique adaptive NK cell subsets that diversify in response to viral infection and have distinct functional capabilities compared to canonical NK cell subsets.

Innate lymphoid cells (ILCs) are increasingly appreciated as important participants in homeostasis and inflammation. Substantial plasticity and heterogeneity among ILC populations have been reported. Here we have delineated the heterogeneity of human ILCs through single-cell RNA sequencing of several hundreds of individual tonsil CD127(+) ILCs and natural killer (NK) cells. Unbiased transcriptional clustering revealed four distinct populations, corresponding to ILC1 cells, ILC2 cells, ILC3 cells and NK cells, with their respective transcriptomes recapitulating known as well as unknown transcriptional profiles. The single-cell resolution additionally divulged three transcriptionally and functionally diverse subpopulations of ILC3 cells. Our systematic comparison of single-cell transcriptional variation within and between ILC populations provides new insight into ILC biology during homeostasis, with additional implications for dysregulation of the immune system.

During early pregnancy, decidual innate lymphoid cells (dILCs) interact with surrounding maternal cells and invading fetal extravillous trophoblasts (EVT). Here, using mass cytometry, we characterise five main dILC subsets: decidual NK cells (dNK)1-3, ILC3s and proliferating NK cells. Following stimulation, dNK2 and dNK3 produce more chemokines than dNK1 including XCL1 which can act on both maternal dendritic cells and fetal EVT. In contrast, dNK1 express receptors including Killer-cell Immunoglobulin-like Receptors (KIR), indicating they respond to HLA class I ligands on EVT. Decidual NK have distinctive organisation and content of granules compared with peripheral blood NK cells. Acquisition of KIR correlates with higher granzyme B levels and increased chemokine production in response to KIR activation, suggesting a link between increased granule content and dNK1 responsiveness. Our analysis shows that dILCs are unique and provide specialised functions dedicated to achieving placental development and successful reproduction.