Dara M. Strauss-Albee

Genetic and Environmental Determinants of Human NK Cell Diversity Revealed by Mass Cytometry

Both genetics and environment contribute to human NK cell diversity. NK Cell Nature Versus Nurture Natural killer (NK) cells were first discovered because of their ability to kill tumor cells without any previous exposure. However, this population is actually quite heterogeneous: Different subgroups of NK cells express different combinations of activating and inhibiting receptors that govern their specificity. Now, Horowitz et al. use mass cytometry to examine NK cell diversity in humans. The authors examined 35 parameters simultaneously in 5 sets of monozygotic twins as well as 12 unrelated donors. They found up to 30,000 phenotypic NK cell populations in a given individual. What’s more, by comparing the twins versus unrelated donors, they determined that although genetics primarily determined inhibitory receptor expression, activating receptors were controlled by the environment. These data suggest that inhibitory receptors may contribute more to NK cell self-tolerance, whereas activating receptors may guide response to pathogens and tumors. 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 37 parameters, including 28 NK cell receptors, on peripheral blood NK cells from 5 sets of monozygotic twins and 12 unrelated donors of defined human leukocyte antigen (HLA) and killer cell immunoglobulin-like receptor (KIR) genotype. This analysis revealed a remarkable degree of NK cell diversity, with an estimated 6000 to 30,000 phenotypic populations within an individual and >100,000 phenotypes in the donor panel. 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.

Human NK cell repertoire diversity reflects immune experience and correlates with viral susceptibility

Human natural killer cell diversity is a metric of immune function associated with less effective antiviral response. The downside of diversity The adaptive immune system exemplifies the benefits of diversity, allowing for individual responses to specific pathogens. Natural killer (NK) cells are diverse at the single-cell level, but the contribution of this diversity to NK cell–mediated immunity has been unclear. Strauss-Albee et al. found that contrary to adaptive immune cells, human NK cell diversity is lower at birth than in adults. Moreover, diversification as a result of antiviral response decreases the flexibility of future antiviral responses. Indeed, high NK cell diversity was associated with increased risk of HIV-1 acquisition in African women. These data suggest that preexisting NK cell diversity should be considered in the context of viral infections. 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.

Coordinated Regulation of NK Receptor Expression in the Maturing Human Immune System

NK cells are responsible for recognizing and killing transformed, stressed, and infected cells. They recognize a set of non–Ag-specific features termed “altered self” through combinatorial signals from activating and inhibitory receptors. These NKRs are also expressed on CD4+ and CD8+ T cells, B cells, and monocytes, although 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, NKRs 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, whereas 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 leukocyte Ig–like receptor subfamily b member 1, 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 Ag 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.

Mass Cytometry Analytical Approaches Reveal Cytokine-Induced Changes in Natural Killer Cells.

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 they performed an extensive assessment of the effects of cytokines on the phenotype and function of human NK cells.

Application of Mass Cytometry (CyTOF) for Functional and Phenotypic Analysis of Natural Killer Cells.

Mass cytometry is a novel platform for high-dimensional phenotypic and functional analysis of single cells. This system uses elemental metal isotopes conjugated to monoclonal antibodies to evaluate up to 42 parameters simultaneously on individual cells with minimal overlap between channels. The platform can be customized for analysis of both phenotypic and functional markers. Here, we will describe methods to stain, collect, and analyze intracellular functional markers and surface phenotypic markers on natural killer cells.

NKG2A-Expressing Natural Killer Cells Dominate the Response to Autologous Lymphoblastoid Cells Infected with Epstein-Barr Virus.

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.

Human NK Cell Diversity in Viral Infection: Ramifications of Ramification

Natural killer (NK) cells are a unique lymphocyte lineage with remarkable agility in the rapid destruction of virus-infected cells. They are also the most poorly understood class of lymphocyte. A spectrum of activating and inhibitory receptors at the NK cell surface leads to an unusual and difficult-to-study mechanism of cellular recognition, as well as a very high capacity for diversity at the single-cell level. Here, we review the evidence for the role of NK cells in the earliest stage of human viral infection, and in its prevention. We argue that single-cell diversity is a logical evolutionary adaptation for their position in the immune response and contributes to their ability to kill virus-infected cells. Finally, we look to the future, where emerging single-cell technologies will enable a new generation of rigorous and clinically relevant studies on NK cells accounting for all of their unique and diverse characteristics.

The newborn human NK cell repertoire is phenotypically formed but functionally reduced.

Infection is a leading cause of death worldwide in babies under 1 month of age. Better vaccines and therapeutics are desperately needed for this vulnerable population.

The natural killer cell response to West Nile virus in young and old individuals with or without a prior history of infection

West Nile virus (WNV) typically leads to asymptomatic infection but can cause severe neuroinvasive disease or death, particularly in the elderly. Innate NK cells play a critical role in antiviral defenses, yet their role in human WNV infection is poorly defined. Here we demonstrate that NK cells mount a robust, polyfunctional response to WNV characterized by cytolytic activity, cytokine and chemokine secretion. This is associated with downregulation of activating NK cell receptors and upregulation of NK cell activating ligands for NKG2D. The NK cell response did not differ between young and old WNV-naïve subjects, but a history of symptomatic infection is associated with more IFN-γ producing NK cell subsets and a significant decline in a specific NK cell subset. This NK repertoire skewing could either contribute to or follow heightened immune pathogenesis from WNV infection, and suggests that NK cells could play an important role in WNV infection in humans.

Association between Latent Proviral Characteristics and Immune Activation in Antiretrovirus-Treated Human Immunodeficiency Virus Type 1-Infected Adults

ABSTRACT Generalized immune activation during HIV infection is associated with an increased risk of cardiovascular disease, neurocognitive disease, osteoporosis, metabolic disorders, and physical frailty. The mechanisms driving this immune activation are poorly understood, particularly for individuals effectively treated with antiretroviral medications. We hypothesized that viral characteristics such as sequence diversity may play a role in driving HIV-associated immune activation. We therefore sequenced proviral DNA isolated from peripheral blood mononuclear cells from HIV-infected individuals on fully suppressive antiretroviral therapy. We performed phylogenetic analyses, calculated viral diversity and divergence in the env and pol genes, and determined coreceptor tropism and the frequency of drug resistance mutations. Comprehensive immune profiling included quantification of immune cell subsets, plasma cytokine levels, and intracellular signaling responses in T cells, B cells, and monocytes. These antiretroviral therapy-treated HIV-infected individuals exhibited a wide range of diversity and divergence in both env and pol genes. However, proviral diversity and divergence in env and pol, coreceptor tropism, and the level of drug resistance did not significantly correlate with markers of immune activation. A clinical history of virologic failure was also not significantly associated with levels of immune activation, indicating that a history of virologic failure does not inexorably lead to increased immune activation as long as suppressive antiretroviral medications are provided. Overall, this study demonstrates that latent viral diversity is unlikely to be a major driver of persistent HIV-associated immune activation. IMPORTANCE Chronic immune activation, which is associated with cardiovascular disease, neurologic disease, and early aging, is likely to be a major driver of morbidity and mortality in HIV-infected individuals. Although treatment of HIV with antiretroviral medications decreases the level of immune activation, levels do not return to normal. The factors driving this persistent immune activation, particularly during effective treatment, are poorly understood. In this study, we investigated whether characteristics of the latent, integrated HIV provirus that persists during treatment are associated with immune activation. We found no relationship between latent viral characteristics and immune activation in treated individuals, indicating that qualities of the provirus are unlikely to be a major driver of persistent inflammation. We also found that individuals who had previously failed treatment but were currently effectively treated did not have significantly increased levels of immune activation, providing hope that past treatment failures do not have a lifelong “legacy” impact.