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.

Key Points Clonal-like expansion of NK cells in response to CMV infection causes stable imprints in the human KIR repertoire. Education by inhibitory KIRs promotes the expansion of NK cells, causing repertoire skewing and a bias for self-specific inhibitory KIRs.

Understanding innate immune responses in COVID-19 is important to decipher mechanisms of host responses and interpret disease pathogenesis. Natural killer (NK) cells are innate effector lymphocytes that respond to acute viral infections but might also contribute to immunopathology. Using 28-color flow cytometry, we here reveal strong NK cell activation across distinct subsets in peripheral blood of COVID-19 patients. This pattern was mirrored in scRNA-seq signatures of NK cells in bronchoalveolar lavage from COVID-19 patients. Unsupervised high-dimensional analysis of peripheral blood NK cells furthermore identified distinct NK cell immunotypes that were linked to disease severity. Hallmarks of these immunotypes were high expression of perforin, NKG2C, and Ksp37, reflecting increased presence of adaptive NK cells in circulation of patients with severe disease. Finally, arming of CD56bright NK cells was observed across COVID-19 disease states, driven by a defined protein-protein interaction network of inflammatory soluble factors. This study provides a detailed map of the NK cell activation landscape in COVID-19 disease.

SummaryInfection by human cytomegalovirus (HCMV) leads to NKG2C-driven expansion of adaptive natural killer (NK) cells, contributing to host defense. However, approximately 4% of all humans carry a homozygous deletion of the gene that encodes NKG2C (NKG2C−/−). Assessment of NK cell repertoires in 60 NKG2C−/− donors revealed a broad range of NK cell populations displaying characteristic footprints of adaptive NK cells, including a terminally differentiated phenotype, functional reprogramming, and epigenetic remodeling of the interferon (IFN)-γ promoter. We found that both NKG2C− and NKG2C+ adaptive NK cells expressed high levels of CD2, which synergistically enhanced ERK and S6RP phosphorylation following CD16 ligation. Notably, CD2 co-stimulation was critical for the ability of adaptive NK cells to respond to antibody-coated target cells. These results reveal an unexpected redundancy in the human NK cell response to HCMV and suggest that CD2 provides “signal 2” in antibody-driven adaptive NK cell responses.

No abstract

Experimental design: Sixteen patients received fludarabine/cyclophosphamide conditioning 68 combined with total lymphoid irradiation followed by adoptive immunotherapy with IL-2-69 activated haploidentical NK cells. 70

Stem cell transplantation across HLA barriers may trigger NK cell-mediated graft-vs-leukemia effects leading to improved survival for patients with hematological malignancies. However, the genetic algorithm based on killer cell Ig-like receptor (KIR) and HLA genes used to predict NK cell alloreactivity have yielded discrepant results. Accordingly, it has been difficult to define transplantation settings that favor NK cell alloreactivity. In this study, we have used multiparameter flow cytometry to simultaneously analyze the cell surface expression of all four major inhibitory KIR and CD94/NKG2A to determine the size of the alloreactive NK cell repertoires in 31 individuals homozygous for the group A KIR haplotype. We observed a vast variability in the frequencies of cells with an alloreactive potential, ranging from 0 to 62% of the total NK cell population depending on which, and how many, KIR ligands were missing in theoretical recipients. This analysis required a functional examination of KIR3DL2-single positive NK cells, showing that this subset was hyporesponsive in individuals harboring the cognate ligands HLA-A3/A11. The results provide new insights into the variability of the functional alloreactive NK cell repertoire and have implications for donor selection in hematopoietic stem cell transplantation and adoptive NK cell-based immunotherapy.

The functional capacity of NK cells is dynamically tuned by integrated signals from inhibitory and activating cell surface receptors in a process termed NK cell education. However, the understanding of the cellular and molecular mechanisms behind this functional tuning is limited. In this study, we show that the expression of the adhesion molecule and activation receptor DNAX accessory molecule 1 (DNAM-1) correlates with the quantity and quality of the inhibitory input by HLA class I–specific killer cell Ig-like receptors and CD94/NKG2A as well as with the magnitude of functional responses. Upon target cell recognition, the conformational state of LFA-1 changed in educated NK cells, associated with rapid colocalization of both active LFA-1 and DNAM-1 at the immune synapse. Thus, the coordinated expression of LFA-1 and DNAM-1 is a central component of NK cell education and provides a potential mechanism for controlling cytotoxicity by functionally mature NK cells.