Natural killer (NK) cell suppression of T cells is a key determinant of viral pathogenesis and vaccine efficacy. This process involves perforin-dependent elimination of activated CD4 T cells during the first three days of infection. Although this mechanism requires cell-cell contact, NK cells and T cells typically reside in different compartments of lymphoid tissues at steady state. Here, we show that NK-cell suppression of T cells is associated with a transient accumulation of NK cells within T cell-rich sites of the spleen during lymphocytic choriomeningitis virus infection. The chemokine receptor CXCR3 is required for relocation to T-cell zones and suppression of antiviral T cells. Accordingly, this NK-cell migration is mediated by type I interferon (IFN)-dependent promotion of CXCR3 ligand expression. In contrast, adenoviral vectors that weakly induce type I IFN and do not stimulate NK-cell inhibition of T cells also do not promote measurable redistribution of NK cells to T-cell zones. Provision of supplemental IFN could rescue NK-cell migration during adenoviral vector immunization. Thus, type I IFN and CXCR3 are critical for properly positioning NK cells to constrain antiviral T-cell responses. Development of strategies to curtail migration of NK cells between lymphoid compartments may enhance vaccine-elicited immune responses.

Abstract Natural killer (NK) cells dictate the pathogenic outcomes of infection via both direct killing of virus-infected cells and indirect immunoregulatory killing of antiviral T cells. The latter involves a perforin-dependent mechanism targeting activated CD4 T cells during the first three days of infection. Given that perforin-dependent killing involves cell-cell contact, we speculated that NK cells must re-locate proximal to recently activated T cells within the white pulp (WP) follicles of secondary lymphoid tissue during this critical temporal window of immunoregulatory activity. As expected, virus infection prompted a gradual accumulation of NK cells within T cell rich regions of splenic WP that peaked by the third day of infection. NK cells deficient in the chemokine receptor CXCR3 exhibited impaired localization to the WP and a markedly diminished capacity to suppress antiviral T-cell and germinal center B-cell responses. Our results reveal a critical role for CXCR3 in properly positioning NK cells to prune developing antiviral T cell responses, which potentially explains loss of tolerance and enhanced immune-mediated organ damage in absence of CXCR3. Strategies to curtail localization of NK cells in the WP during immunization may be an effective means to enhance vaccine-elicited immune responses.

Natural killer (NK) cells are critical for both direct control of virus replication and regulation of potentially pathogenic antiviral responses. Yet, whether NK cells also contribute to immune control of bacterial superinfections during viral infection, a significant clinical cause of morbidity and mortality, remains unknown. We find that NK cells are vital to prevent a near complete loss of B cells and macrophages associated with the marginal zone (MZ) during chronic virus infection in mice. Establishment of chronic infection in the absence of NK cells results in hyper-susceptibility to Listeria monocytogenes infection that cannot be recapitulated by NK-cell depletion prior to bacterial challenge of chronically infected mice. Therefore, we posit that preservation of MZ leukocytes by NK cells, and not direct NK cell activity, curtails post-viral susceptibility toward bacterial superinfection. Mechanistically, we show that NK cells begin expressing the crucial MZ survival factor, B-cell activating factor (BAFF), during chronic virus infection, gradually becoming a prominent source of BAFF in the spleen. In vivo blockade of BAFF in NK-sufficient mice recapitulated the infection-associated loss of MZ cells seen in NK-cell deficient mice. These results reveal a new mechanism whereby NK cells promote bacterial resistance during chronic infection by maintaining BAFF-dependent immune structures. Disruption of this activity of NK cells during certain virus infections in humans may provoke post-viral pneumonia or invasive pneumococcal disease.

Natural killer (NK) cells suppress antiviral immunity by killing a fraction of virus-specific CD4 T cells. This occurs during the first 3 days of infection, is perforin-dependent, and coincides with NK-cell localization in the splenic white pulp. While the receptors governing this NK-cell activity are ill-defined, it is clear that negative feedback mechanisms must exist to prevent excessive pruning of T-cell responses. Comparative transcriptome analysis of red pulp and white pulp localized NK-cells derived from late in the window of immunoregulatory activity (day 3) reveals increased expression of Lag3 in white pulp NK-cells. LAG-3 is an inhibitory receptor that binds peptide-bound major histocompatibility complex II (pMHC-II). We find a progressive increase in LAG-3 protein expression on NK-cells with time post-infection and localization to the white pulp. Consistent with its putative inhibitory role, antibody-mediated neutralization of LAG-3 enhances NK-cell killing capacity in vitro. Preliminary analysis reveals reduced virus-specific T cells in mice lacking Lag3 in NK-cells (Ncr1Cre+Lag3fl/fl) relative to control mice. We hypothesize that LAG-3 upregulation on NK-cells confers inhibitory feedback signals from surrounding MHC-II+ cells to terminate NK-cell suppression of T cells. Uncovering the role of LAG-3 in NK cell immunoregulatory function will provide a novel target to constrain this suppression and enhance vaccine-elicited T cell responses.

Immunoglobulin A (IgA) is important for the maintenance of homeostasis between the host and the intestinal microbiome. Antimicrobial IgA is secreted by IgA+ plasma cells that arise from germinal center (GC) reactions within gut-associated lymphoid tissues (GALT). Dysregulation of IgA production, a common condition with an unknown etiology, can lead to the improper clearance of commensal microbes, allowing for pathogenic strains to flourish and cause disease. Thus, a better understanding of the mechanisms involved in IgA production is critical. Our lab has previously shown that natural killer (NK) cells, a type of innate lymphoid cell (ILC), suppress GC responses and long-lived humoral immunity following systemic viral infection of mice. Here we show that depletion of NK1.1-expressing ILCs (e.g. NK, ILC1, ILC3) in specific-pathogen free mice resulted in enhanced T follicular helper cell and GC B cell numbers within GALT. The numbers of lamina propria IgA+ plasma cells were increased and the expression levels of fecal IgA were elevated for weeks after depletion of NK1.1-expressing ILCs. In addition, NK1.1-depletion was associated with alterations to the intestinal microbiome. In mice with defects in cytolytic granule-mediated killing, including perforin-knockout and beige mice, depletion of NK1.1-expressing ILCs did not enhance GALT GC responses or IgA production. This suggests that a cytolytic mechanism is involved in this regulatory process, although the precise target of this killing remains undefined. Thus, innate lymphoid cells within GALT play an important role in determining the balance between host immunity and microbial commensalism, and may represent new therapeutic targets to enhance efficacy of oral vaccines.

Natural killer (NK) cells are cytotoxic innate lymphocytes that provide immune defense against pathogens and tumors, and act as potent regulators of adaptive immunity. Notably, NK cells inhibit humoral immunity by restricting the magnitude of follicular helper T-cell (TFH) and germinal center (GC) B-cell responses in a perforin-dependent manner. This suppression occurs at an early stage of infection that coincides with an anatomic redistribution of NK cells within secondary lymphoid organs. Specifically, infection with lymphocytic choriomeningitis virus (LCMV) triggers transient NK-cell redistribution from the vascularized red pulp into the white pulp of the spleen. Many of these NK cells localize at the border between T- and B-cell zones where cognate interactions between TFH and B cells facilitates development of GCs. Some NK cells also penetrate deep into the B-cell follicle. We have investigated this phenomenon using a flow cytometry-based intravascular staining method and through confocal microscopy. We have observed a novel subset of NK cells expressing CXCR5 that coincidentally arise with NK-cell redistribution to spleen regions (i.e. B-cell zone) rich in CXCL13. We hypothesize that this subset of NK cells localize in a CXCR5 dependent manner near cells participating in the early steps of GC formation, including TFH, which allows for NK-cell contact and perforin-dependent killing of GC-inducing cells that ultimately dampens the development of immunity to infection. These regulatory NK cells are an innovative target for clinical strategies aimed at subverting or enhancing humoral immune responses.

Marginal zone (MZ) B cells are an anatomically segregated population well-positioned to respond to blood borne pathogens and implicated in defense against encapsulated bacteria. MZ B cells are also an important source of the anti-inflammatory cytokine, IL-10. As such, these cells are poised to prevent secondary bacterial infection or immune pathology during chronic virus infection. Surprisingly, CD169+ MZ macrophages and CD21+CD23neg B cells were lost in mice during persistent clone 13 lymphocytic choriomeningitis virus (LCMV) infection in the absence of natural killer (NK) cells. Confocal staining revealed a near complete absence of the MZ by day 10 of infection in NKdeficient mice, which we previously found contain enhanced populations of antiviral T cells. Intriguingly, depletion of either CD4 or CD8 T cells did not restore the MZ compartment in NKdeficient mice, but combined depletion of both subsets of T cells prevented the loss. This phenomenon was independent of NKT cells. We hypothesize that NK cells play an important role in curtailing harmful inflammation and preventing pathogenic bacterial diseases during chronic viral infection by sustaining the MZ. This discovery has important implications for health, immune function, and viral clearance during persistent infection.

Natural killer (NK) cells are important in immune defense against virus infections. This is predominantly considered a function of rapid, innate NK-cell killing of virus-infected cells. However, NK cells also prime other immune cells through the release of interferon gamma (IFN-γ) and other cytokines. Additionally, NK cells share features with long-lived adaptive immune cells and can impact disease pathogenesis through the inhibition of adaptive immune responses by virus-specific T and B cells. The relative contributions of these diverse and conflicting functions of NK cells in humans are poorly defined and likely context-dependent, thereby complicating the development of therapeutic interventions. Here we focus on the contributions of NK cells to disease in diverse virus infections germane to human health.

Natural killer (NK) cells are innate lymphocytes that classically function to restrict microbial infection and tumor development. NK cells are also important regulators of adaptive immunity. Specifically, we found that NK cells inhibited antiviral humoral immunity by restricting the germinal center (GC) response via a perforin-dependent cytotoxic mechanism during the initial few days of lymphocytic choriomeningitis virus (LCMV) infection. We hypothesize that this NK cell suppression of antiviral B cells requires redistribution of activated NK cells to regions of secondary lymphoid tissues where T-B cell interactions shape the GC reaction. NK cells were predominately localized within the red pulp of the spleens of uninfected mice, but infection with LCMV triggered a redistribution of NK cells to the white pulp. At day 3 p.i, many NK cells were present in T cell rich areas, most notably at the T-B cell interface where follicular helper T cells (TFH) typically reside. In addition, a small population of NK cells penetrated deep into the B cell follicles. The redistribution of NK cells was transient, with most NK cells returning to the red pulp by day 6 p.i. We speculate that white pulp localization is stimulated by altered expression of chemokine receptors by NK cells, and that this proximity to the GC facilitates direct interactions between NK cells and TFH or GC B cells, thereby contributing to diminished GC responses in the presence of NK cells.

The goal of most vaccines is the induction of long-lived memory T and B cells capable of protecting the host from infection by cytotoxic mechanisms, cytokines and high-affinity antibodies. However, efforts to develop vaccines against major human pathogens such as HIV and HCV have not been successful, thereby highlighting the need for novel approaches to circumvent immunoregulatory mechanisms that limit the induction of protective immunity. Here, we show that mouse natural killer (NK) cells inhibit generation of long-lived virus-specific memory T- and B cells as well as virus-specific antibody production after acute infection. Mechanistically, NK cells suppressed CD4 T cells and follicular helper T cells (T-FH) in a perforin-dependent manner during the first few days of infection, resulting in a weaker germinal centre (GC) response and diminished immune memory. We anticipate that innovative strategies to relieve NK cell-mediated suppression of immunity should facilitate development of efficacious new vaccines targeting difficult-to-prevent infections.