Natural killer (NK) cells form a first line of defense against virus-infected cells by recognizing alterations in human leukocyte antigen (HLA) expression. NK cells recognize HLA via a panel of killercell immunoglobulin-like receptors (KIRs). Upon binding to HLA, KIRs will transmit inhibitory or activatory signals, with inhibition dominating over activation in a healthy state. As part of the innate immune system, natural killer (NK) cells present a first line of defense against viral infections and tumors. NK cell effector functions, such as cytotoxicity and cytokine release, are controlled by integrated signals from a large panel of both activating and inhibitory receptor. Killercell immunoglobulin-like receptors (KIRs) on NK cells and their ligands, HLA class I molecules, play an essential part in this tight regulation. Both, the KIR gene cluster and the HLA class I loci are known to be extraordinarily diverse, which led to the hypothesis that NK cell immune responses are genetically predetermined to some extent. This is supported by recent epidemiological observations that KIR/HLA compound genotypes with a supposedly activating profile (presence of activating KIR or lack of inhibitory KIRs or their respective ligands) are associated with resistance to HCV (Khakoo et al., Science 2004;305:872-874) and human immunodeficiency virus (Martin et al., Nat Genet 2002;31:429-434). Several of these studies take into account HLA-C, the gene locus encoding ligands for KIR2DL receptor, where a functional dimorphism determines KIR specificity. HLA-C group 1 (HLA-C1) alleles, encoding ser77/asp80 of the HLA-Cw a1 domain, bind to the inhibitory receptors KIR2DL2 and KIR2DL3, and probably also to the activating KIR2DS2. In contrast, the HLA-C group 2 (HLA-C2) alleles, encoding asp77/lys80, bind to KIR2DL1 and possibly to KIR2DS1. Homozygosity for HLA-C1 alleles and KIR2DL3 is associated with resolution of HCV infection as compared to homozygosity or heterozygosity for HLA-C2 and KIR2DL1 (Khakoo et al., Science 2004;305:872-874). To date, the functional mechanisms responsible for these epidemiological associations are poorly defined. To examine the effect of KIR/HLA compound genotypes on NK cells at the functional level we developed an in vitro influenza virus infection model and attributed functional differences in human NK cell activity to distinct KIR/HLA genotypes. In order to characterize NK cell responsiveness in the context of different HLA alleles, we studied the kinetics of NK cell responses in a unique and well-characterized cohort of subjects with distinct KIR/HLA compound genotypes in response to influenza A virus (IAV) infection. Multicolor flow cytometry revealed that the HLA-C-inhibited NK cell subset in HLA-C1 homozygous subjects was larger, and that it responded more rapidly in IFN-g secretion and CD107a degranulation assays than its counterpart in HLA-C2 homozygous subjects. The differential IFN-g response was also observed at the level of bulk NK cells. It was independent of KIR3DL1/HLA-Bw4 interactions. Neither was it due to differences in NK cell maturation status or phenotype nor to differences in the type I IFN response of influenza A virus-infected accessory cells between HLA-C1 and HLA-C2 homozygous subjects. These results provide the first functional evidence for differential NK cell responsiveness, depending on KIR/HLA genotypes, against viral infection. Based on these results we have initiated studies to analyze the NK cell response in response to HCV. As a first approach we examined whether the virus itself affects NK cells directly after direct exposure. Based on two previous reports that recombinant, truncated HCV has been proposed that HCV has the capacity to impair NK cell function upon direct contact (J Exp Med 2002;195:35-41;J Exp Med 2002;195:43-49). However, the relevance of these findings has not been verified with HCV E2 expressed as part of intact virions. To answer this question we employed a new cell culture system generating infectious HCV particles with genotype 1a and 2a structural proteins, and analyzed direct and indirect effects of HCV on human NK cells. Antibody-mediated crosslinking of CD16 stimulated and antibody-mediated crosslinking of CD81 inhibited NK cell activation and IFN-g production. However, infectious HCV itself had no effect even at titers that far exceeded HCV RNA and protein concentrations in the blood of infected patients. Consistent with these results, anti-CD81 but not HCV inhibited NK cell cytotoxicity. These results were independent of the presence or absence of HCV-binding antibodies and independent of the presence or absence of other peripheral blood mononuclear cell populations. Conclusion: HCV 1a or 2a envelope proteins do not modulate NK cell function when expressed as a part of infectious HCV particles. Without direct inhibition by HCV, NK cells may become activated by cytokines in acute HCV infection and contribute to infection outcome and disease pathogenesis. In a third project we used the same in vitro culture system to study HCVs effect on another population of innate immune cells. Previous studies suggested a functional impairment of dendritic cells (DCs) in patients with chronic hepatitis C. To investigate whether this effect was mediated by a direct interaction of HCV with DCs we studied the effects of cell culture-derived, infectious HCV on PBMC, ex vivo isolated plasmacytoid, and myeloid DCs and in vitro generated monocyte-derived DCs of healthy blood donors. We found that HCV inhibited TLR9 (CpG and herpes simples virus)-mediated IFN-alpha production by PBMC and plasmacytoid DC. This inhibitory effect was also observed in response to UV-inactivated, non-infectious HCV, and it was not abrogated by neutralizing antibodies, thus did not appear to require DC infection. Influenza A virus restored maturation and TLR9-mediated IFN-alpha production. In contrast to its effect on plasmacytoid DCs, HCV did not inhibit TLR3- and TLR4-mediated maturation and IL-12, IL-6, IL-10, IFN-gamma and TNF-alpha production by myeloid DCs and monocyte-derived DCs. Likewise, HCV did neither alter the capacity of myeloid DCs nor monocyte-derived DCs to induce CD4 T cell proliferation. While phagocytosis of apoptotic hepatoma cells by monocyte-derived DCs resulted in DC maturation, this effect was independent of whether the phagocytosed Huh7.5.1 cells were infected with HCVcc or not. In contrast to HCV, vaccinia virus inhibited maturation and TNF-alpha expression of myeloid DC as well as maturation and IL-6 and IL-10 production of monocyte-derived DC. Thus, HCV inhibited plasmacytoid DCs, but not myeloid and monocytoid-derived DCs via a direct interaction that did not require infection and the response of plasmacytoid DCs to other viruses such as influenza A virus was not impaired.
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