Natural Killer (NK) cells play critical roles in immune defense and reproduction, yet remain the most poorly understood major lymphocyte population. Because their activation is controlled by a variety of combinatorially expressed activating and inhibitory receptors, NK cell diversity and function are closely linked. To provide an unprecedented understanding of NK cell repertoire diversity, we used mass cytometry to simultaneously analyze 35 parameters, including 28 NK cell receptors, on peripheral blood NK cells from five sets of monozygotic twins and twelve unrelated donors of defined HLA and killer cell immunoglobulin-like receptor (KIR) genotype. This analysis revealed a remarkable degree of NK cell diversity, with an estimated 6,000-30,000 phenotypic populations within an individual and >100,000 phenotypes in this population. Genetics largely determined inhibitory receptor expression, whereas activation receptor expression was heavily environmentally influenced. Therefore, NK cells may maintain self-tolerance through strictly regulated expression of inhibitory receptors, while using adaptable expression patterns of activating and costimulatory receptors to respond to pathogens and tumors. These findings further suggest the possibility that discrete NK cell subpopulations could be harnessed for immunotherapeutic strategies in the settings of infection, reproduction, and transplantation.

Innate natural killer (NK) cells are diverse at the single-cell level because of variegated expressions of activating and inhibitory receptors, yet the developmental roots and functional consequences of this diversity remain unknown. Because NK cells are critical for antiviral and antitumor responses, a better understanding of their diversity could lead to an improved ability to harness them therapeutically. We found that NK diversity is lower at birth than in adults. During an antiviral response to either HIV-1 or West Nile virus, NK diversity increases, resulting in terminal differentiation and cytokine production at the cost of cell division and degranulation. In African women matched for HIV-1 exposure risk, high NK diversity is associated with increased risk of HIV-1 acquisition. Existing diversity may therefore decrease the flexibility of the antiviral response. Collectively, the data reveal that human NK diversity is a previously undefined metric of immune history and function that may be clinically useful in forecasting the outcomes of infection and malignancy.

We describe seven Turkish children with DOCK8 deficiency who have not been previously reported. Three patients presented with typical features of recurrent or severe cutaneous viral infections, atopic dermatitis, and recurrent respiratory or gastrointestinal tract infections. However, four patients presented with other features. Patient 1-1 featured sclerosing cholangitis and colitis; patient 2-1, granulomatous soft tissue lesion and central nervous system involvement, with primary central nervous system lymphoma found on follow-up; patient 3-1, a fatal metastatic leiomyosarcoma; and patient 4-2 showed no other symptoms initially besides atopic dermatitis. Similar to other previously reported Turkish patients, but in contrast to patients of non-Turkish ethnicity, the patients’ lymphopenia was primarily restricted to CD4+ T cells. Patients had homozygous mutations in DOCK8 that altered splicing, introduced premature terminations, destabilized protein, or involved large deletions within the gene. Genotyping of remaining family members showed that DOCK8 deficiency is a fully penetrant, autosomal recessive disease. In our patients, bone marrow transplantation resulted in rapid improvement followed by disappearance of viral skin lesions, including lesions resembling epidermodysplasia verruciformis, atopic dermatitis, and recurrent infections. Particularly for patients who feature unusual clinical manifestations, immunological testing, in conjunction with genetic testing, can prove invaluable in diagnosing DOCK8 deficiency and providing potentially curative treatment.

Zhang et al. show that DOCK8-deficient T and NK cells develop cell and nuclear shape abnormalities that do not impair chemotaxis but contribute to a form of cell death they term cytothripsis. Cytothripsis of DOCK8-deficient cells prevents the generation of long-lived skin-resident memory CD8 T cells resulting in impaired immune response to skin infection.

Natural killer (NK) cells are responsible for recognizing and killing transformed, stressed, and infected cells. They recognize a set of non-antigen-specific features termed “altered self” through combinatorial signals from activating and inhibitory receptors. These natural killer cell receptors (NKR) are also expressed on CD4+ and CD8+ T cells, B cells, and monocytes, though a comprehensive inventory of NKR expression patterns across leukocyte lineages has never been performed. Using mass cytometry, we found that NKR expression patterns distinguish cell lineages in human peripheral blood. In individuals with high levels of CD57, indicative of a mature immune repertoire, NKR are more likely to be expressed on non-NK cells, especially CD8+ T cells. Mature NK and CD8+ T cell populations show increased diversity of NKR surface expression patterns, but with distinct determinants: mature NK cells acquire primarily inhibitory receptors, while CD8+ T cells attain a specific subset of both activating and inhibitory receptors, potentially imbuing them with a distinct functional role. Concurrently, monocytes show decreased expression of the generalized inhibitory receptor LILRB1, consistent with an increased activation threshold. Therefore, NKR expression is coordinately regulated as the immune system matures, resulting in the transfer of “altered self” recognition potential among leukocyte lineages. This likely reduces antigen specificity in the mature human immune system, and implies that vaccines and therapeutics that engage both its innate and adaptive branches may be more effective in the settings of aging and chronic infection.

DOCK8 mutations result in an inherited combined immunodeficiency characterized by increased susceptibility to skin and other infections. We show that when DOCK8-deficient T and NK cells migrate through confined spaces, they develop cell shape and nuclear deformation abnormalities that do not impair chemotaxis but contribute to a distinct form of catastrophic cell death we term cytothripsis. Such defects arise during lymphocyte migration in collagen-dense tissues when DOCK8, through CDC42 and p21-activated kinase (PAK), is unavailable to coordinate cytoskeletal structures. Cytothripsis of DOCK8-deficient cells prevents the generation of long-lived skin-resident memory CD8 T cells, which in turn impairs control of herpesvirus skin infections. Our results establish that DOCK8-regulated shape integrity of lymphocytes prevents cytothripsis and promotes antiviral immunity in the skin.

Epstein–Barr virus (EBV) is a human γ-herpesvirus that establishes latency and lifelong infection in host B cells while achieving a balance with the host immune response. When the immune system is perturbed through immunosuppression or immunodeficiency, however, these latently infected B cells can give rise to aggressive B cell lymphomas. Natural killer (NK) cells are regarded as critical in the early immune response to viral infection, but their role in controlling expansion of infected B cells is not understood. Here, we report that NK cells from healthy human donors display increased killing of autologous B lymphoblastoid cell lines (LCLs) harboring latent EBV compared to primary B cells. Coculture of NK cells with autologous EBV+ LCL identifies an NK cell population that produces IFNγ and mobilizes the cytotoxic granule protein CD107a. Multi-parameter flow cytometry and Boolean analysis reveal that these functional cells are enriched for expression of the NK cell receptor NKG2A. Further, NKG2A+ NK cells more efficiently lyse autologous LCL than do NKG2A− NK cells. More specifically, NKG2A+2B4+CD16−CD57−NKG2C−NKG2D+ cells constitute the predominant NK cell population that responds to latently infected autologous EBV+ B cells. Thus, a subset of NK cells is enhanced for the ability to recognize and eliminate autologous, EBV-infected transformed cells, laying the groundwork for harnessing this subset for therapeutic use in EBV+ malignancies.

Background Natural killer (NK) cells have antiviral and antitumor activity that could be harnessed for the treatment of infections and malignancies. To maintain cell viability and enhance antiviral and antitumor effects, NK cells are frequently treated with cytokines. Here we performed an extensive assessment of the effects of cytokines on the phenotype and function of human NK cells. Methods We used cytometry by time-of-flight (CyTOF) to evaluate NK cell repertoire changes after stimulation with interleukin (IL)-2, IL-15 or a combination of IL-12/IL-15/IL-18. To analyze the high dimensional CyTOF data, we used several statistical and visualization tools, including viSNE (Visualization of t-Distributed Stochastic Neighbor Embedding), Citrus (Cluster identification, characterization, and regression), correspondence analysis, and the Friedman-Rafsky test. Results All three treatments (IL-2, IL-15, and IL-12/IL-15/IL-18) increase expression of CD56 and CD69. The effects of treatment with IL-2 and IL-15 are nearly indistinguishable and characterized principally by increased expression of surface markers including CD56, NKp30, NKp44, and increased expression of functional markers, such as perforin, granzyme B, and MIP-1β. The combination of IL-12/IL-15/IL-18 induces a profound shift in the repertoire structure, decreasing expression of CD16, CD57, CD8, NKp30, NKp46, and NKG2D, and dramatically increasing expression of IFN-γ. Conclusions CyTOF provides insights into the effects of cytokines on the phenotype and function of NK cells, which could inform future research efforts and approaches to NK cell immunotherapy. There are several analytical approaches to CyTOF data, and the appropriate method should be carefully selected based on which aspect of the dataset is being explored.