Genetic dissection of NK cell responses

The association of Natural Killer (NK) cell deficiencies with disease susceptibility has established a central role for NK cells in host defence. In this context, genetic approaches have been pivotal in elucidating and characterizing the molecular mechanisms underlying NK cell function. To this end, homozygosity mapping and linkage analysis in humans have identified mutations that impact NK cell function and cause life-threatening diseases. However, several critical restrictions accompany genetic studies in humans. Studying NK cell pathophysiology in a mouse model has therefore proven a useful tool. The relevance of the mouse model is underscored by the similarities that exist between cell-structure-sensing receptors and the downstream signaling that leads to NK cell activation. In this review, we provide an overview of how human and mouse quantitative trait locis (QTLs) have facilitated the identification of genes that modulate NK cell development, recognition, and killing of target cells

NK Cell Receptor/H2-Dk–Dependent Host Resistance to Viral Infection Is Quantitatively Modulated by H2q Inhibitory Signals

The cytomegalovirus resistance locus Cmv3 has been linked to an epistatic interaction between two loci: a Natural Killer (NK) cell receptor gene and the major histocompatibility complex class I (MHC-I) locus. To demonstrate the interaction between Cmv3 and H2k, we generated double congenic mice between MA/My and BALB.K mice and an F2 cross between FVB/N (H-2q) and BALB.K (H2k) mice, two strains susceptible to mouse cytomegalovirus (MCMV). Only mice expressing H2k in conjunction with Cmv3MA/My or Cmv3FVB were resistant to MCMV infection. Subsequently, an F3 cross was carried out between transgenic FVB/H2-Dk and MHC-I deficient mice in which only the progeny expressing Cmv3FVB and a single H2-Dk class-I molecule completely controlled MCMV viral loads. This phenotype was shown to be NK cell–dependent and associated with subsequent NK cell proliferation. Finally, we demonstrated that a number of H2q alleles influence the expression level of H2q molecules, but not intrinsic functional properties of NK cells; viral loads, however, were quantitatively proportional to the number of H2q alleles. Our results support a model in which H-2q molecules convey Ly49-dependent inhibitory signals that interfere with the action of H2-Dk on NK cell activation against MCMV infection. Thus, the integration of activating and inhibitory signals emanating from various MHC-I/NK cell receptor interactions regulates NK cell–mediated control of viral load

The Natural Selection of Herpesviruses and Virus-Specific NK Cell Receptors

During the co-evolution of cytomegalovirus (CMV) and natural killer (NK) cells, each has evolved specific tactics in an attempt to prevail. CMV has evolved multiple immune evasion mechanisms to avoid detection by NK cells and other immune cells, leading to chronic infection. Meanwhile, the host has evolved virus-specific receptors to counter these evasion strategies. The natural selection of viral genes and host receptors allows us to observe a unique molecular example of “survival of the fittest”, as virus and immune cells try to out-maneuver one another or for the virus to achieve détente for optimal dissemination in the population

Positive regulation of plasmacytoid dendritic cell function via Ly49Q recognition of class I MHC

Plasmacytoid dendritic cells (pDCs) are an important source of type I interferon (IFN) during initial immune responses to viral infections. In mice, pDCs are uniquely characterized by high-level expression of Ly49Q, a C-type lectin-like receptor specific for class I major histocompatibility complex (MHC) molecules. Despite having a cytoplasmic immunoreceptor tyrosine-based inhibitory motif, Ly49Q was found to enhance pDC function in vitro, as pDC cytokine production in response to the Toll-like receptor (TLR) 9 agonist CpG-oligonucleotide (ODN) could be blocked using soluble monoclonal antibody (mAb) to Ly49Q or H-2Kb. Conversely, CpG-ODN–dependent IFN-α production by pDCs was greatly augmented upon receptor cross-linking using immobilized anti-Ly49Q mAb or recombinant H-2Kb ligand. Accordingly, Ly49Q-deficient pDCs displayed a severely reduced capacity to produce cytokines in response to TLR7 and TLR9 stimulation both in vitro and in vivo. Finally, TLR9-dependent antiviral responses were compromised in Ly49Q-null mice infected with mouse cytomegalovirus. Thus, class I MHC recognition by Ly49Q on pDCs is necessary for optimal activation of innate immune responses in vivo

Distinct MHC class I–dependent NK cell–activating receptors control cytomegalovirus infection in different mouse strains

MCMV-infected cells are recognized by multiple MHC class I–restricted Ly49-activating receptors in genetically distinct mouse strains

Activating receptors promote NK cell expansion for maintenance, IL-10 production, and CD8 T cell regulation during viral infection

Natural killer (NK) cells have the potential to deliver both direct antimicrobial effects and regulate adaptive immune responses, but NK cell yields have been reported to vary greatly during different viral infections. Activating receptors, including the Ly49H molecule recognizing mouse cytomegalovirus (MCMV), can stimulate NK cell expansion. To define Ly49H's role in supporting NK cell proliferation and maintenance under conditions of uncontrolled viral infection, experiments were performed in Ly49h−/−, perforin 1 (Prf1)−/−, and wild-type (wt) B6 mice. NK cell numbers were similar in uninfected mice, but relative to responses in MCMV-infected wt mice, NK cell yields declined in the absence of Ly49h and increased in the absence of Prf1, with high rates of proliferation and Ly49H expression on nearly all cells. The expansion was abolished in mice deficient for both Ly49h and Prf1 (Ly49h−/−Prf1−/−), and negative consequences for survival were revealed. The Ly49H-dependent protection mechanism delivered in the absence of Prf1 was a result of interleukin 10 production, by the sustained NK cells, to regulate the magnitude of CD8 T cell responses. Thus, the studies demonstrate a previously unappreciated critical role for activating receptors in keeping NK cells present during viral infection to regulate adaptive immune responses

Ly49P recognition of cytomegalovirus-infected cells expressing H2-Dk and CMV-encoded m04 correlates with the NK cell antiviral response

Natural killer (NK) cells are crucial in resistance to certain viral infections, but the mechanisms used to recognize infected cells remain largely unknown. Here, we show that the activating Ly49P receptor recognizes cells infected with mouse cytomegalovirus (MCMV) by a process that requires the presence of H2-Dk and the MCMV m04 protein. Using H2 chimeras between H2-Db and -Dk, we demonstrate that the H2-Dk peptide-binding platform is required for Ly49P recognition. We identified m04 as a viral component necessary for recognition using a panel of MCMV-deletion mutant viruses and complementation of m04-deletion mutant (Δm04) virus infection. MA/My mice, which express Ly49P and H2-Dk, are resistant to MCMV; however, infection with Δm04 MCMV abrogates resistance. Depletion of NK cells in MA/My mice abrogates their resistance to wild-type MCMV infection, but does not significantly affect viral titers in mice infected with Δm04 virus, implicating NK cells in host protection through m04-dependent recognition. These findings reveal a novel mechanism of major histocompatability complex class I–restricted recognition of virally infected cells by an activating NK cell receptor

The NK Cell Response to Mouse Cytomegalovirus Infection Affects the Level and Kinetics of the Early CD8+ T-Cell Response

Natural killer (NK) cells and CD8+ T cells play a prominent role in the clearance of mouse cytomegalovirus (MCMV) infection. The role of NK cells in modulating the CD8+ T-cell response to MCMV infection is still the subject of intensive research. For analyzing the impact of NK cells on mounting of a CD8+ T-cell response and the contribution of these cells to virus control during the first days postinfection (p.i.), we used C57BL/6 mice in which NK cells are specifically activated through the Ly49H receptor engaged by the MCMV-encoded ligand m157. Our results indicate that the requirement for CD8+ T cells in early MCMV control inversely correlates with the engagement of Ly49H. While depletion of CD8+ T cells has only a minor effect on the early control of wild-type MCMV, CD8+ T cells are essential in the control of Δm157 virus. The frequencies of virus epitope-specific CD8+ T cells and their activation status were higher in mice infected with Δm157 virus. In addition, these mice showed elevated levels of alpha interferon (IFN-α) and several other proinflammatory cytokines as early as 1.5 days p.i. Although the numbers of conventional dendritic cells (cDCs) were reduced later during infection, particularly in Δm157-infected mice, they were not significantly affected at the peak of the cytokine response. Altogether, we concluded that increased antigen load, preservation of early cDCs' function, and higher levels of innate cytokines collectively account for an enhanced CD8+ T-cell response in C57BL/6 mice infected with a virus unable to activate NK cells via the Ly49H–m157 interaction

Evidence for high bi-allelic expression of activating Ly49 receptors

Stochastic expression is a hallmark of the Ly49 family that encode the main MHC class-I-recognizing receptors of mouse natural killer (NK) cells. This highly polygenic and polymorphic family includes both activating and inhibitory receptor genes and is one of genome's fastest evolving loci. The inhibitory Ly49 genes are expressed in a stochastic mono-allelic manner, possibly under the control of an upstream bi-directional early promoter and show mono-allelic DNA methylation patterns. To date, no studies have directly addressed the transcriptional regulation of the activating Ly49 receptors. Our study shows differences in DNA methylation pattern between activating and inhibitory genes in C57BL/6 and F1 hybrid mouse strains. We also show a bias towards bi-allelic expression of the activating receptors based on allele-specific single-cell RT–PCR in F1 hybrid NK cells for Ly49d and Ly49H expression in Ly49h+/− mice. Furthermore, we have identified a region of high sequence identity with possible transcriptional regulatory capacity for the activating Ly49 genes. Our results also point to a likely difference between NK and T-cells in their ability to transcribe the activating Ly49 genes. These studies highlight the complex regulation of this rapidly evolving gene family of central importance in mouse NK cell function

Proinflammatory cytokine signaling required for the generation of natural killer cell memory

Although natural killer (NK) cells are classified as innate immune cells, recent studies demonstrate that NK cells can become long-lived memory cells and contribute to secondary immune responses. The precise signals that promote generation of long-lived memory NK cells are unknown. Using cytokine receptor-deficient mice, we show that interleukin-12 (IL-12) is indispensible for mouse cytomegalovirus (MCMV)-specific NK cell expansion and generation of memory NK cells. In contrast to wild-type NK cells that proliferated robustly and resided in lymphoid and nonlymphoid tissues for months after MCMV infection, IL-12 receptor–deficient NK cells failed to expand and were unable to mediate protection after MCMV challenge. We further demonstrate that a STAT4-dependent IFN-γ–independent mechanism contributes toward the generation of memory NK cells during MCMV infection. Understanding the full contribution of inflammatory cytokine signaling to the NK cell response against viral infection will be of interest for the development of vaccines and therapeutics