Distinct KIR/HLA compound genotypes affect the kinetics of human antiviral natural killer cell responses

Genetic studies suggest a role for killer cell immunoglobulin-like receptor/HLA (KIR/HLA) compound genotypes in the outcome of viral infections, but functional data to explain these epidemiological observations have not been reported. Using an in vitro model of infection with influenza A virus (IAV), we attribute functional differences in human NK cell activity to distinct KIR/HLA genotypes. Multicolor flow cytometry revealed that the HLA-C–inhibited NK cell subset in HLA-C1 homozygous subjects was larger and responded more rapidly in IFN-γ secretion and CD107a degranulation assays than its counterpart in HLA-C2 homozygous subjects. The differential IFN-γ response was also observed at the level of bulk NK cells and was independent of KIR3DL1/HLA-Bw4 interactions. Moreover, the differential response was not caused by differences in NK cell maturation status and phenotype, nor by differences in the type I IFN response of IAV-infected accessory cells between HLA-C1 and HLA-C2 homozygous subjects. These results provide functional evidence for differential NK cell responsiveness depending on KIR/HLA genotype and may provide useful insights into differential innate immune responsiveness to viral infections such as IAV.

As part of the innate immune system, NK cells present a first line of defense against viral infections and tumors (1). NK cell effector functions, such as cytotoxicity and cytokine release, are controlled by integrated signals from a large panel of both activating and inhibitory receptors (2–4). Killer cell 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 extraordinarily diverse, which led to the hypothesis that NK cell immune responses are genetically predetermined to some extent (4). This is supported by recent epidemiological observations that KIR/HLA compound genotypes with a supposedly activating profile (i.e., presence of activating KIR or lack of inhibitory KIRs or their respective ligands) are associated with resistance to HCV (5) and HIV infection (6), slower HIV disease progression (7), and better reproductive fitness (8). On the other hand, activating profiles enhance the risk for autoimmune disease such as psoriasis/psoriatic arthritis (9–13), type I diabetes (14), and scleroderma (15).

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 α1 domain, bind to the inhibitory receptors KIR2DL2 and KIR2DL3 and probably also to the activating KIR2DS2 (16, 17). In contrast, the HLA-C group 2 (HLA-C2) alleles, encoding Asp77/Lys80, bind to KIR2DL1 and possibly to KIR2DS1 (16, 17). Homozygosity for HLA-C1 alleles and KIR2DL3 is associated with resolution of HCV infection as compared with homozygosity or heterozygosity for HLA-C2 (5). In contrast, HLA-C2 alleles are associated with protection against cervical neoplasia (18) and, to some extent, nasopharyngeal carcinoma (19). Thus, KIR/HLA-C compound genotypes contribute to susceptibility or resistance to a variety of infectious diseases and cancer (20). In particular, homozygosity for HLA-C1 and KIR2DL3 may be advantageous in viral infections, but detrimental in chronic inflammatory conditions that play a role in carcinogenesis (21).

To date, the functional mechanisms responsible for these epidemiological associations are poorly defined. It has been proposed that improved resistance to virus infections among KIR2DL3/HLA-C1–positive individuals may be the result of weaker NK cell inhibition through KIR2DL3 compared with NK cell inhibition through KIR2DL1 in KIR2DL1/HLA-C2–positive individuals (5, 8, 22). Consistent with this notion, Winter et al. showed weaker binding between a KIR2DL3-Fc fusion protein and HLA-C1 transfectants than between KIR2DL1-Fc fusion protein and HLA-C2 transfectants (23). However, direct measurements by surface plasmon resonance revealed almost similar affinity between KIR2DL3 and HLA-C1 and KIR2DL1 and HLA-C2, respectively (24–26).

Apart from their role in inhibiting NK cell function, KIR and HLA-C molecules also play an essential role during the development of NK cells, because functional maturation of NK cells requires specific interaction with MHC class I molecules. For example, NK cells that express MHC class I–specific inhibitory receptors have been found to be functionally reactive, but NK cells that lack MHC class I–specific inhibitory receptors are hyporeactive to the same stimuli and do not exhibit cytotoxicity to MHC class I–deficient target cells (27, 28). Likewise, inhibitory receptor–expressing NK cells of MHC class I–deficient patients (29–31) and NK cells of β₂m^–/– (32–34), TAP^–/– (31, 35), or MHC-deficient (34, 36) mice do not kill MHC class I–deficient normal cells.

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 an influenza A virus (IAV) infection model. NK cells are thought to play an important role early after IAV infection (37, 38), and impaired NK cell responses are associated with higher susceptibility to IAV infection in mice (39). Vaccination with inactivated or attenuated virus induces significant IFN-γ responses by NK cells in young children (40), and NK cells can be directly activated by binding of the influenza hemagglutinin to the NK cytotoxicity receptors NKp44 and NKp46 (41, 42). The analysis of NK cell responses in an IAV infection model may provide information regarding the differential effect of KIR/HLA compound genotypes on NK cell effector functions. Because IAV has been a major human pathogen for more than 2,000 years (43), with annual epidemics killing between 30,000 and 50,000 people in the United States alone (44), our results may also provide useful insight on differential innate immune responsiveness to IAV infection in humans (45).

To compare the impact of KIR2DL3/HLA-C1 and KIR2DL1/HLA-C2 interactions on NK cell responsiveness, we selected 22 healthy individuals who were homozygous for the common KIR haplotype A (KIR3DL3-KIR2DL3-KIR2DL1-KIR2DL4-KIR3DL1-KIR2DS4-KIR3DL2) and homozygous for either HLA-C1 or HLA-C2 (n = 12 and 10, respectively; Table 1).

Table 1

KIR/HLA compound genotype of subjects enrolled in this study

Identification, quantitation, and characterization of HLA-C–inhibited NK cells. Because of the variegated expression of KIRs within an NK cell population, only a subset of the NK cells of any given subject is inhibited by HLA-C allotypes. The HLA-C–inhibited NK cell subset consists of KIR2DL3⁺ NK cells in HLA-C1 homozygous subjects and of KIR2DL1⁺ NK cells in HLA-C2 homozygous subjects. These HLA-C–inhibited NK cell subsets were identified in peripheral blood mononuclear cells by multicolor flow cytometry (Figure 1A). After gating on single cells (forward scatter–height versus forward scatter–area), lymphocytes (forward scatter versus side scatter), and CD56⁺ NK cells, and excluding CD3⁺ T cells, CD14⁺ monocytes, CD19⁺ B cells, and ethidium monoazide–positive (EMA⁺) dead cells, the percentage of KIR2DL3⁺ and KIR2DL1⁺ NK cells was analyzed in HLA-C1/C1 and HLA-C2/2 subjects, respectively. KIR2DL3⁺KIR2DL1⁺ double-positive NK cells were included in the KIR2DL3⁺ NK cell population in HLA-C1 homozygous subjects and in the KIR2DL1⁺ NK cell population in HLA-C2 homozygous subjects. This was justified because KIR2DL3⁺KIR2DL1⁺ NK cells were inhibited by HLA-C1 in HLA-C1 homozygous subjects and by HLA-C2 in HLA-C2 homozygous subjects, as HLA-C1/C2 heterozygous subjects were excluded from our study.

Figure 1

Identification and quantitation of HLA-C–inhibited NK cells by flow cytometry. (A) Gating strategy used to identify HLA-C–inhibited NK cells by flow cytometry. After gating on single cells (forward scatter–height versus forward scatter–area) and lymphocytes (forward scatter versus side scatter), NK cells were identified by gating on CD56⁺ cells and by excluding CD3⁺ T cells, CD14⁺ monocytes, CD19⁺ B cells, and EMA⁺ dead cells. Samples from HLA-C1/C1 subjects were stained with antibodies to KIR2DL3, and samples from HLA-C2/C2 subjects were stained with antibodies to KIR2DL1. All samples were stained with antibodies to KIR3DL1 to allow further subset analysis as shown in Figure 5. Numbers indicate the percentage of events in each quadrant. (B) Frequency of HLA-C–inhibited NK cells in PBMCs. The HLA-C–inhibited NK cell subset consists of KIR2DL3⁺ NK cells in HLA-C1 homozygous subjects and KIR2DL1⁺ NK cells in HLA-C2 homozygous subjects. Horizontal lines indicate the mean.

As shown in Figure 1A for a single subject from each group and in Figure 1B for all subjects, the frequency of HLA-C–inhibited NK cells was significantly higher in HLA-C1 homozygous than in HLA-C2 homozygous subjects, that is, KIR2DL3 was expressed on a greater percentage of NK cells in HLA-C1 homozygous subjects than was KIR2DL1 in HLA-C2 homozygous subjects (P = 0.0105; Figure 1B). Of note, there were no differences in the absolute number of NK cells between groups (HLA-C1 group, mean 253.3 NK cells/μl blood; HLA-C2 group, mean 290.9 NK cells/μl blood; P = NS) or their frequency in total PBMCs (mean, 15.5% vs. 12.9%; P = NS).

Multicolor flow cytometry demonstrated that a substantial percentage of NK cells was negative for CD25, CD117, and Nkp44 and positive for CD122, CD16, NKG2A, and NKG2D (Figure 2A), consistent with a mature phenotype (46). This phenotype extended to NK cells that were inhibited by HLA-C (Figure 2B) as well as to those NK cells that were not inhibited by HLA-C (Figure 2C) and did not differ between the HLA-C1 and the HLA-C2 homozygous groups (Figure 2, A–C).

Figure 2

Phenotype of HLA-C–inhibited NK cells in HLA-C1 and in HLA-C2 homozygous subjects. The total NK cell population (A) and the HLA-C–inhibited (B) and HLA-C–noninhibited (C) subpopulations displayed a mature phenotype and did not differ between HLA-C1 and HLA-C2 homozygous subjects. Horizontal lines indicate the mean.

To examine whether NK cells from HLA-C1 homozygous subjects displayed intrinsically different cytotoxic responses relative to HLA-C2 homozygous subjects, the level of NK cell cytotoxicity against HLA-negative K562 target cells was determined in a standard cytotoxicity assay. As shown in Supplemental Figure 1 (available online with this article; doi:10.1172/JCI32400DS1), NK cell cytotoxicity of HLA-C1 homozygous subjects did not differ from those of HLA-C2 homozygous subjects at any of the effector/target ratios or assay time points. Furthermore, IFN-γ release was studied after 5, 7, and 9 h of NK cell incubation with K562 cells and did not differ between subject groups (data not shown). Therefore, bulk NK cells of both groups did not differ in their ability to kill HLA-C–negative K562 cells (Supplemental Figure 1).

NK cells of HLA-C1 and HLA-C2 homozygous subjects display differential degranulation and cytokine responses to IAV infection. To examine NK cell responses in the context of each subject’s HLA haplotype, T cell–depleted PBMCs of HLA-C1 or HLA-C2 homozygous subjects were infected with IAV. This model had the advantage of physiological expression levels of (a) KIR on NK cells and (b) HLA molecules on autologous, IAV-infected target cells, namely monocytes. T cells were depleted from PBMCs prior to IAV infection, because T cell–derived IL-2 enhances NK cell responses (47) and because IL-2 secretion by IAV-specific memory T cells may vary among individuals based on their IAV infection history.

Because IAV-infected monocytes label poorly with ⁵¹Cr and are therefore not optimal for a traditional cytotoxicity assay, NK cells were analyzed for CD107a expression. An increase in the expression of CD107a on the cell membrane is a marker for degranulation of perforin- and granzyme-containing vesicles, corollaries for NK cell cytotoxicity (48, 49). Bulk NK cells of HLA-C1 homozygous subjects showed higher CD107a mean fluorescence intensity (MFI) than did bulk NK cells of HLA-C2 homozygous subjects during the first 3, 5, 7, 9, and 17 h of the IAV assay (Figure 3A). Differential CD107a expression was most pronounced specifically for HLA-C–inhibited NK cell populations of HLA-C1 and HLA-C2 homozygous groups (Figure 3B). In contrast, the CD107a expression level of NK cells not inhibited by HLA-C did not differ between HLA-C1 and HLA-C2 homozygous subjects (Figure 3C).

Figure 3

HLA-C–inhibited NK cells of HLA-C1 homozygous subjects show a stronger increase in CD107a expression after IAV infection than do HLA-C–inhibited NK cells of HLA-C2 homozygous subjects. Upregulation of CD107a, a surrogate marker of degranulation and cytotoxicity, is shown at the indicated times after IAV infection of CD3-depleted PBMCs for the total NK cell population (A) and the HLA-C–inhibited (B) and HLA-C–noninhibited (C) NK cell subpopulations. Values are mean ± SEM. n = 7 per group.

Next, IFN-γ production was studied as additional readout of NK cell function. To better understand the differential response kinetics of HLA-C–inhibited NK cells in the 2 subject groups, the percentage of IFN-γ–secreting HLA-C–inhibited NK cells was determined 3, 5, 7, 9, and 17 h after IAV infection using T cell–depleted PBMCs from 5 donors in each group. Using an IFN-γ secretion assay, we calculated how many HLA-C–inhibited NK cells were secreting IFN-γ at each time point after IAV infection relative to the frequency of NK cells at the end of the assay (17-h time point). The results were displayed as a percentage of the 17-h time point rather than as absolute values in order to assess the early kinetics of the NK cell response and to eliminate an effect of differences in the absolute response strength between individuals. Consistent with the results of the CD107a assay (Figure 3B), HLA-C–inhibited (i.e., KIR2DL3⁺) NK cells from HLA-C1 homozygous subjects displayed a faster response than did HLA-C–inhibited (i.e., KIR2DL1⁺) NK cells from HLA-C2 homozygous subjects, as evidenced by a greater slope of the response curve during the first 5 h of IAV infection (Figure 4A).

Figure 4

HLA-C–inhibited NK cells of HLA-C1 homozygous subjects secrete IFN-γ more rapidly after IAV infection than do HLA-C–inhibited NK cells of HLA-C2 homozygous subjects. (A and C) Kinetic analysis of the IFN-γ response of (A) HLA-C–inhibited NK cells and (C) the total NK cell population for a subgroup of HLA-C1 and HLA-C2 homozygous subjects. The respective NK cell populations were identified in IAV-infected, CD3-depleted PBMCs in a cytokine-secretion assay by flow cytometry. Mean and SEM at each time point of the infection assay are shown relative to the study end point at 17 h (expressed as ratio). n = 5 per group. (B) Frequency of IFN-γ⁺, HLA-C–inhibited NK cells at the 9-h time point of the IAV assay relative to the 17-h time point (expressed as 9-h/17-h ratio). (D) IFN-γ released by the total NK cell population during the first 9h of the IAV assay relative to IFN-γ released during the total 17h of the IAV assay (set as 100%). Experiments in B and D were performed as in A, but included a larger number of HLA-C1 (n = 9) and HLA-C2 homozygous subjects (n = 8). Horizontal lines indicate the mean.

These results were confirmed by assessing the percentage of IFN-γ–secreting HLA-C–inhibited NK cells at the 9-h and 17-h time points for a larger number of subjects in the HLA-C1 and HLA-C2 homozygous groups. As shown in Figure 4B, the 9-h/17-h ratio of IFN-γ–secreting HLA-C–inhibited NK cells was significantly higher for the HLA-C1 homozygous group than for the HLA-C2 homozygous group (P = 0.021). There was no significant difference in the percentage of IFN-γ–secreting HLA-C–inhibited NK cells in the HLA-C1 and HLA-C2 homozygous groups at the final, 17-h assay time point (data not shown). These results indicated that there may be functional differences between HLA-C1 and HLA-C2 homozygous subjects due to faster response kinetics of KIR2DL3⁺ NK cells from HLA-C1 homozygous subjects compared with KIR2DL1⁺ NK cells from HLA-C2 homozygous subjects.

To investigate whether the differential response of the HLA-C–inhibited NK cell subsets was associated with a differential overall bulk NK cell response, IFN-γ release into the supernatant was measured by enzyme immunoassay (EIA) at 3, 5, 7, 9, 12, 15, and 17 h after IAV infection. Figure 4C shows the amount of IFN-γ released into the supernatant at each time point relative to the total amount released during the entire 17 h of the assay (set as 100%). Consistent with the previous results, bulk NK cells of the HLA-C1 homozygous group released IFN-γ faster than did bulk NK cells of the HLA-C2 homozygous group. The time interval from the start of the assay to the time point at which 50% of the total IFN-γ release was achieved was significantly shorter for the HLA-C1 homozygous group than for the HLA-C2 homozygous group (P = 0.024; Figure 4C).

This differential response kinetics of the bulk NK cell population was confirmed by studying samples from a larger number of subjects in the HLA-C1 and HLA-C2 homozygous groups at the 9- and 17-h time points. As shown in Figure 4D, bulk NK cells of HLA-C1 homozygous subjects released a greater percentage of the total IFN-γ amount within the first 9 h of the 17-h infection assay than did bulk NK cells of HLA-C2 homozygous subjects (P = 0.006). As mentioned above, the total amount of IFN-γ released during the full 17 h of the assay did not significantly differ between HLA-C1 and HLA-C2 homozygous subjects (mean IFN-γ, HLA-C1 homozygous, 1,423 pg/ml; HLA-C2 homozygous 832 pg/ml; P = NS).

To evaluate whether NK cell–independent factors might contribute to this differential response, we asked whether macrophages and dendritic cells from HLA-C1 and HLA-C2 homozygous subjects responded differentially to IAV infection. Supernatants from the same IAV assays used to study NK cell functions were used to assess production of IFN-α and -β. Those cytokines were chosen because they are produced by dendritic cells and by virus-infected cells (in our IAV assay mainly by monocytes) and influence NK cell function. However, IFN-α and -β release did not differ between HLA-C1 and HLA-C2 homozygous groups (data not shown). Collectively, these data show a differential response of HLA-C1– and HLA-C2–inhibited NK cells in HLA-C1 and HLA-C2 homozygous subjects. Thus, HLA-C genotypes may influence the strength of the early NK cell response when HLA-C allotypes are present on target cells.

KIR3DL1 and HLA-Bw4 do not mediate any confounding effect. To analyze potential confounding effects, we examined a possible influence of KIR3DL1, a locus that displays significant polymorphism and is associated with HLA-Bw4–mediated inhibition of NK cells (50–52). This was important because a substantial proportion of KIR2DL1⁺ and KIR2DL3⁺ cells coexpressed KIR3DL1 (Figure 5A), because coexpression of multiple inhibitory KIRs affects the functional capacity of NK cells (53), and because the KIR3DL1 ligand, HLA-Bw4, was observed less frequently in the HLA-C1 homozygous group (6 of 12 subjects, 50.0%) than in the HLA-C2 homozygous group (8 of 10 subjects, 80.0%). Antibodies against KIR3DL1 were therefore included in the flow cytometry staining panel, and KIR3DL1⁺ cells were excluded from the analysis. As shown in Figure 5, B and C, the differences in the time kinetics described above for the total HLA-C–inhibited NK cell population of HLA-C1 and HLA-C2 homozygous subjects were completely reproducible after exclusion of KIR3DL1⁺ cells from the assays. Consistent with our above findings, the 9-h/17-h ratio of cytokine-secreting NK cells after IAV infection was significantly higher for HLA-C1 homozygous subjects than for HLA-C2 homozygous subjects (P = 0.008; Figure 5B). Moreover, detailed kinetic analysis confirmed that the response pattern of HLA-C–inhibited NK cells of both groups did not change after exclusion of KIR3DL1⁺ cells (Figure 5C). Thus, the differential response of HLA-C–inhibited NK cells of HLA-C1 and HLA-C2 homozygous subjects was independent of KIR3DL1.

Figure 5

The differential response of HLA-C–inhibited NK cell subsets in HLA-C1 and HLA-C2 homozygous subjects is not influenced by KIR3DL1 and Bw4 expression. (A) Examples of KIR2DL3⁺KIR3DL1⁺ cells in an HLA-C1 homozygous subject and KIR2DL1⁺KIR3DL1⁺ NK cells in an HLA-C2 homozygous subject. Both plots are gated on CD56⁺CD3^– NK cells. Numbers indicate the percentage of events in each quadrant. (B) Frequency of IFN-γ–secreting NK cells in the HLA-C–inhibited, KIR3DL1^– population 9 h after IAV infection relative to 17 h after IAV infection (expressed as 9-h/17-h ratio). Horizontal lines indicate the mean. (C) Frequencies of IFN-γ–producing cells in the HLA-C–inhibited cell population (reproduced from Figure 4A for reference) and the KIR3DL1- and HLA-Bw4–independent, HLA-C–inhibited NK cell population at various time points of the IAV infection assay. The frequency of the respective IFN-γ–secreting NK cell population in each subject group is shown as a ratio relative to the maximum frequency at the 17-h time point. Mean and SEM are indicated. n = 5 per group.

A growing number of epidemiological studies suggest an influence of the KIR/HLA compound genotypes on the outcome of a variety of diseases (5–9, 11, 12). However, to our knowledge, functional data to explain these genetic data have not been reported to date. Here we describe a model system to investigate human NK cell responses to IAV infection. In this model, NK cell responses were studied in the context of physiological expression levels of KIR on NK cells and HLA molecules on autologous, IAV-infected monocytes. Multicolor flow cytometry analysis of NK cell subsets allowed us to attribute differences in frequency and antiviral function of HLA-C–inhibited NK cell activity to distinct KIR/HLA-C compound genotypes.

For this study, we chose a unique cohort of well-characterized subjects who were all homozygous for the KIR haplotype A, the most common KIR haplotype worldwide. Homozygosity for this haplotype is found in 30% of white individuals and 56% of Japanese individuals (45). Moreover, nearly all KIR haplotypes worldwide contain KIR2DL1 along with either KIR2DL3 or KIR2DL2. HLA-C allotypes, the ligands for KIR2DL molecules, therefore almost always inhibit a subset of each individual’s NK cell population. In contrast, inhibition by HLA-A and -B allotypes is less common, because it has only been shown for HLA-A3/-A11 (54, 55) and HLA-Bw4 (56, 57), which bind to KIR3DL2 and KIR3DL1, respectively.

A distinction between the effect of the compound genotypes HLA-C2/KIR2DL1 and HLA-C1/KIR2DL3 on the outcome of various diseases has been previously reported and attributed to differential activation of NK cells (5, 18, 19, 21, 45). To address the functional basis for this finding, we studied the frequency, phenotype, and function of NK cells from healthy subjects who had both KIR2DL3 and KIR2DL1 (and not KIR2DL2), but were homozygous for either HLA group C1 or group C2 alleles. We found that HLA-C1 homozygous subjects displayed a significantly higher frequency of HLA-C–inhibited NK cells than did HLA-C2 homozygous subjects (26.3% vs. 16.3%; P = 0.019). This observation is consistent with recent in vitro studies showing that NK cells acquire the HLA-C1–specific KIR2DL3 at earlier time points during development from hematopoietic cells and with higher frequency than the HLA-C2–specific KIR2DL1 (58). Functional differences in the properties of the KIR2DL3 and KIR2DL1 promoters (59) may account for the higher frequency of NK cells expressing KIR2DL3 compared with KIR2DL1.

We also demonstrated that HLA-C–inhibited KIR2DL3⁺ NK cells from HLA-C1 homozygous subjects secreted more IFN-γ at earlier time points after infection and displayed greater degranulation than did HLA-C–inhibited KIR2DL1⁺ NK cells from HLA-C2 homozygous subjects. Because all subjects were KIR2DL3⁺ and KIR2DL1⁺, these results suggest that NK cell inhibition through interaction between KIR2DL3 and HLA-C1 is weaker than the inhibition conferred through KIR2DL1/HLA-C2 interaction. Given that the affinities of KIR2DL1 and KIR2DL3 for their respective ligands are very similar (24, 25, 26), the basis for weaker inhibition by KIR2DL3 could be differences in negative signaling controlled by the cytoplasmic tails (26). Thus, our data provide a functional correlate for previous epidemiological observations (5, 18, 19, 21, 45).

Our study extends the results of previous publications that used similar in vitro infection systems (38, 47). First, Draghi et al. reported that NK cells are stimulated by IAV-infected dendritic cells (38), and He et al. observed that the strength of NK cell responses in IAV-infected PBMCs differed among subjects (47). This may be explained, at least in part, by differences in IAV-specific memory T cell responses, because T cells were not depleted in the latter study and T cell–derived IL-2 was clearly shown to enhance NK cell responses. However, as we show now, KIR/HLA interaction also affects NK cell responsiveness, especially in the early phase of this viral infection. Second, Artavanis-Tsakonas et al. studied IFN-γ responses of human NK cells to Plasmodium falciparum–infected erythrocytes in vitro (60). The presence of the KIR3DL2*002 allele was associated with stronger NK cell responses in this parasite infection model. However, due to the lack of HLA expression on erythrocytes, the underlying mechanisms of KIR/ligand interactions could not be evaluated (60).

Because of the complexity of factors that regulate NK cell activation, we also considered potential confounders. Although the HLA-C1 homozygous and HLA-C2 homozygous groups were identical with regard to the genes present on their KIR haplotypes, subjects were diverse with regard to presence of Bw4, the ligand for KIR3DL1. Nevertheless, after exclusion of KIR3DL1⁺ cells, the difference between the responses of HLA-C–inhibited NK cells in HLA-C1 homozygous versus HLA-C2 homozygous groups was preserved (Figure 5, B and C). Thus, the observed difference between HLA-C1 homozygous and HLA-C2 homozygous groups appeared to be independent of Bw4/KIR3DL1 interactions. Although it is not possible to control for all genetic differences in a functional study, the fact that we still observed differences in the overall IFN-γ responses between NK cells from the HLA-C1 homozygous and HLA-C2 homozygous groups strengthens our conclusions.

An additional factor for further investigation is the possibility that HLA-C–bound viral or host peptides contribute to the differential inhibitory effect of HLA-C1 and HLA-C2 molecules. Such peptides may be produced in response to infection and introduce allosteric changes in HLA-C molecules or enhance the binding of HLA-C molecules by their cognate KIR receptor. Indeed, KIR preference for certain peptides sequences has been previously demonstrated in studies examining NK cell recognition of HLA-B27–expressing target cells (61, 62) and in KIR2DL recognition of HLA-C molecules (63, 64). However, we consider it unlikely that differential peptide recognition explains the better NK cell response conferred by the KIR2DL3/HLA-C1 compound genotype compared with the KIR2DL1/HLA-C2 compound genotype for the following reasons. First, each HLA-C group is composed of multiple HLA-C alleles, which do not contain a common peptide binding motif (65). Therefore, they all present different peptides, even though they are recognized by the same KIR. Second, the HLA molecules of a given cell display a large number of different peptides, so that only a small percentage (<1%) of all HLA molecules is occupied by the same self or viral peptide.

We also considered that NK cells from HLA-C1 homozygous and HLA-C2 homozygous groups may have intrinsically different responsiveness. This is an interesting topic in light of recent studies implicating a role of inhibitory receptors for MHC class I in the education and eventual functional capacity of NK cells (27, 28, 66–69). According to these prior studies, NK cells that can be inhibited by MHC class I are functionally reactive because they express the corresponding inhibitory receptors. In contrast, NK cells that cannot be inhibited by MHC class I are hyporeactive because they lack the corresponding MHC class I–specific inhibitory receptors. However, the HLA-C1 and HLA-C2 homozygous patients in our study did not differ in their cytotoxic response to HLA-negative K562 cells. Functional differences only became evident in a more physiologic model of IAV infection in the context of HLA-C–expressing autologous cells.

While the previous studies compared NK cells that expressed MHC-specific inhibitory receptors with those that did not (27, 28, 66–69), our study focused on NK cell subsets that all expressed MHC-specific inhibitory receptors, but were inhibited by different groups of HLA-C molecules. The observation that HLA-C1– and HLA-C2–inhibited NK cells displayed differential functional responsiveness to IAV infection is an important extension of a previous observation Johansson et al. made in transgenic mice (70). By generating mice expressing different single MHC class I molecules, the authors show that individual MHC class I molecules have a differential impact on NK cell function (70). Thus, differential inhibition of NK cell function by recognition of distinct class I allotypes does not only occur in transgenic mice, but also in humans with different HLA-C ligands.

To our knowledge, there is presently no in vivo infection model to demonstrate that differential response kinetics in the first 9 h of NK cell activation result in earlier or better viral control. However, it should be noted that NK cells are abundant in inflamed and noninflamed human lymph nodes (71, 72) and thus may exert a rapid antiviral effect. Fast-replicating viruses such as IAV reach significant viral titers within 24 h of infection (73); human NK cells respond within 24 h to IAV (47); and IFN-γ levels in nasopharyngeal lavage fluid correlate significantly to IAV titers, clinical symptoms, oral temperature, and nasal discharge (74). In addition to its antiviral effect, NK cell responses promote early CD8 T cell responses against viruses (75), and depletion of NK cells from mice has been shown to result in inefficient priming of virus-specific T cells (76).

In summary, this study presents an in vitro model to study the functional impact of KIR/HLA interactions in an autologous system of HLA-C–expressing, IAV-infected monocytes and KIR-expressing NK cells. The data provide what we believe to be the first functional evidence in support of previous genetic studies that describe associations between distinct KIR/HLA compound genotypes and susceptibility to infectious diseases (5).

View Supplemental data

We thank Jon W. Yewdell and Jack R. Bennink (NIAID, NIH) for the IAV; Ruy Ribeiro and Alan Perelson (Los Alamos National Laboratory, Los Alamos, New Mexico, USA) for help with statistical analysis; and Thomas A. Fleisher and Margaret Brown (NIH Clinical Center) for complete blood count, white blood cell differential analysis, and immune status analysis. We are grateful to Stephen K. Anderson (SAIC-Frederick Inc. and National Cancer Institute–Frederick); Eric O. Long and Sumati Rajagopalan (NIAID, NIH); and Eui-Cheol Shin, Sukanya Raghuraman, and Joo Chun Yoon (NIDDK, NIH) for helpful discussions and critical review of the manuscript. G. Ahlenstiel is the recipient of Deutsche Forschungsgemeinschaft grant AH173/1-1. This project has been funded in part with federal funds from the NIDDK, NIH, intramural research program. This research was supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. This project has been funded in part with federal funds from the National Cancer Institute, NIH, under contract N01-CO-12400. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. government.

Address correspondence to: Barbara Rehermann, Immunology Section, Liver Diseases Branch, NIDDK, NIH, DHHS, 10 Center Drive, Building 10, Room 9B16C, Bethesda, Maryland 20892-1800, USA. Phone: (301) 402-7144; Fax: (301) 402-0491; E-mail: rehermann@nih.gov.

Nonstandard abbreviations used: EIA, enzyme immunoassay; EMA, ethidium monoazide; IAV, influenza A virus; KIR, killer cell immunoglobulin-like receptor.

Conflict of interest: The authors have declared that no conflict of interest exists.

Reference information: J. Clin. Invest.118:1017–1026 (2008). doi:10.1172/JCI32400

1. Hamerman, J.A., Ogasawara, K., Lanier, L.L. 2005. NK cells in innate immunity. Curr. Opin. Immunol. 17:29-35.
   View this article via: CrossRef PubMed Google Scholar
2. Ljunggren, H.G., Karre, K. 1990. In search of the ‘missing self’: MHC molecules and NK cell recognition. Immunol. Today. 11:237-244.
   View this article via: CrossRef PubMed Google Scholar
3. Moretta, A., et al. 1990. A novel surface antigen expressed by a subset of human CD3- CD16+ natural killer cells. Role in cell activation and regulation of cytolytic function. J. Exp. Med. 171:695-714.
   View this article via: CrossRef PubMed Google Scholar
4. Parham, P. 2005. MHC class I molecules and KIRs in human history, health and survival. Nat. Rev. Immunol. 5:201-214.
   View this article via: CrossRef PubMed Google Scholar
5. Khakoo, S.I., et al. 2004. HLA and NK cell inhibitory receptor genes in resolving hepatitis C virus infection. Science. 305:872-874.
   View this article via: CrossRef PubMed Google Scholar
6. Jennes, W., et al. 2006. Cutting edge: resistance to HIV-1 infection among african female sex workers is associated with inhibitory KIR in the absence of their HLA ligands. J. Immunol. 177:6588-6592.
   View this article via: PubMed Google Scholar
7. Martin, M.P., et al. 2002. Epistatic interaction between KIR3DS1 and HLA-B delays the progression to AIDS. Nat. Genet. 31:429-434.
   View this article via: PubMed Google Scholar
8. Hiby, S.E., et al. 2004. Combinations of maternal KIR and fetal HLA-C genes influence the risk of preeclampsia and reproductive success. J. Exp. Med. 200:957-965.
   View this article via: CrossRef PubMed Google Scholar
9. Holm, S.J., et al. 2005. Distinct HLA-C/KIR genotype profile associates with guttate psoriasis. J. Invest. Dermatol. 125:721-730.
   View this article via: CrossRef PubMed Google Scholar
10. Luszczek, W., et al. 2004. Gene for the activating natural killer cell receptor, KIR2DS1, is associated with susceptibility to psoriasis vulgaris. Hum. Immunol. 65:758-766.
    View this article via: CrossRef PubMed Google Scholar
11. Nelson, G.W., et al. 2004. Cutting edge: heterozygote advantage in autoimmune disease: hierarchy of protection/susceptibility conferred by HLA and killer Ig-like receptor combinations in psoriatic arthritis. J. Immunol. 173:4273-4276.
    View this article via: PubMed Google Scholar
12. Martin, M.P., et al. 2002. Cutting edge: susceptibility to psoriatic arthritis: influence of activating killer Ig-like receptor genes in the absence of specific HLA-C alleles. J. Immunol. 169:2818-2822.
    View this article via: PubMed Google Scholar
13. Williams, F., et al. 2005. Activating killer cell immunoglobulin-like receptor gene KIR2DS1 is associated with psoriatic arthritis. Hum. Immunol. 66:836-841.
    View this article via: CrossRef PubMed Google Scholar
14. Nikitina-Zake, L., Rajalingham, R., Rumba, I., Sanjeevi, C.B. 2004. Killer cell immunoglobulin-like receptor genes in Latvian patients with type 1 diabetes mellitus and healthy controls. Ann. N. Y. Acad. Sci. 1037:161-169.
    View this article via: CrossRef PubMed Google Scholar
15. Momot, T., et al. 2004. Association of killer cell immunoglobulin-like receptors with scleroderma. Arthritis Rheum. 50:1561-1565.
    View this article via: CrossRef PubMed Google Scholar
16. Colonna, M., Borsellino, G., Falco, M., Ferrara, G.B., Strominger, J.L. 1993. HLA-C is the inhibitory ligand that determines dominant resistance to lysis by NK1- and NK2-specific natural killer cells. Proc. Natl. Acad. Sci. U. S. A. 90:12000-12004.
    View this article via: CrossRef PubMed Google Scholar
17. Wagtmann, N., Rajagopalan, S., Winter, C.C., Peruzzi, M., Long, E.O. 1995. Killer cell inhibitory receptors specific for HLA-C and HLA-B identified by direct binding and by functional transfer. Immunity. 3:801-809.
    View this article via: CrossRef PubMed Google Scholar
18. Carrington, M., et al. 2005. Hierarchy of resistance to cervical neoplasia mediated by combinations of killer immunoglobulin-like receptor and human leukocyte antigen loci. J. Exp. Med. 201:1069-1075.
    View this article via: CrossRef PubMed Google Scholar
19. Butsch Kovacic, M., et al. 2005. Variation of the killer cell immunoglobulin-like receptors and HLA-C genes in nasopharyngeal carcinoma. Cancer Epidemiol. Biomarkers Prev. 14:2673-2677.
    View this article via: CrossRef PubMed Google Scholar
20. Carrington, M., Martin, M.P. 2006. The impact of variation at the KIR gene cluster on human disease. Curr. Top. Microbiol. Immunol. 298:225-257.
    View this article via: PubMed Google Scholar
21. Hawes, S.E., Kiviat, N.B. 2002. Are genital infections and inflammation cofactors in the pathogenesis of invasive cervical cancer? J. Natl. Cancer Inst. 94:1592-1593.
    View this article via: PubMed Google Scholar
22. Parham, P. 2004. Immunology. NK cells lose their inhibition. Science. 305:786-787.
    View this article via: CrossRef PubMed Google Scholar
23. Winter, C.C., Gumperz, J.E., Parham, P., Long, E.O., Wagtmann, N. 1998. Direct binding and functional transfer of NK cell inhibitory receptors reveal novel patterns of HLA-C allotype recognition. J. Immunol. 161:571-577.
    View this article via: PubMed Google Scholar
24. Vales-Gomez, M., Reyburn, H.T., Mandelboim, M., Strominger, J.L. 1998. Kinetics of interaction of HLA-C ligands with natural killer cell inhibitory receptors. Immunity. 9:337-344.
    View this article via: CrossRef PubMed Google Scholar
25. Maenaka, K., Juji, T., Stuart, D.I., Jones, E.Y. 1999. Crystal structure of the human p58 killer cell inhibitory receptor (KIR2DL3) specific for HLA-Cw3-related MHC class I. Structure. 7:391-398.
    View this article via: CrossRef PubMed Google Scholar
26. Rajagopalan, S., Long, E.O. 2005. Understanding how combinations of HLA and KIR genes influence disease. J. Exp. Med. 201:1025-1029.
    View this article via: CrossRef PubMed Google Scholar
27. Anfossi, N., et al. 2006. Human NK cell education by inhibitory receptors for MHC class I. Immunity. 25:331-342.
    View this article via: CrossRef PubMed Google Scholar
28. Fernandez, N.C., et al. 2005. A subset of natural killer cells achieves self-tolerance without expressing inhibitory receptors specific for self-MHC molecules. Blood. 105:4416-4423.
    View this article via: CrossRef PubMed Google Scholar
29. Vitale, M., et al. 2002. Analysis of natural killer cells in TAP2-deficient patients: expression of functional triggering receptors and evidence for the existence of inhibitory receptor(s) that prevent lysis of normal autologous cells. Blood. 99:1723-1729.
    View this article via: CrossRef PubMed Google Scholar
30. Furukawa, H., et al. 1999. Tolerance of NK and LAK activity for HLA class I-deficient targets in a TAP1-deficient patient (bare lymphocyte syndrome type I). Hum. Immunol. 60:32-40.
    View this article via: CrossRef PubMed Google Scholar
31. Zimmer, J., et al. 1998. Activity and phenotype of natural killer cells in peptide transporter (TAP)-deficient patients (type I bare lymphocyte syndrome). J. Exp. Med. 187:117-122.
    View this article via: CrossRef PubMed Google Scholar
32. Bix, M., et al. 1991. Rejection of class I MHC-deficient haemopoietic cells by irradiated MHC-matched mice. Nature. 349:329-331.
    View this article via: CrossRef PubMed Google Scholar
33. Liao, N.S., Bix, M., Zijlstra, M., Jaenisch, R., Raulet, D. 1991. MHC class I deficiency: susceptibility to natural killer (NK) cells and impaired NK activity. Science. 253:199-202.
    View this article via: CrossRef PubMed Google Scholar
34. Hoglund, P., et al. 1991. Recognition of beta 2-microglobulin-negative (beta 2m-) T-cell blasts by natural killer cells from normal but not from beta 2m- mice: nonresponsiveness controlled by beta 2m- bone marrow in chimeric mice. Proc. Natl. Acad. Sci. U. S. A. 88:10332-10336.
    View this article via: CrossRef PubMed Google Scholar
35. Ljunggren, H.G., Van Kaer, L., Ploegh, H.L., Tonegawa, S. 1994. Altered natural killer cell repertoire in Tap-1 mutant mice. Proc. Natl. Acad. Sci. U. S. A. 91:6520-6524.
    View this article via: PubMed Google Scholar
36. Grigoriadou, K., Menard, C., Perarnau, B., Lemonnier, F.A. 1999. MHC class Ia molecules alone control NK-mediated bone marrow graft rejection. Eur. J. Immunol. 29:3683-3690.
    View this article via: CrossRef PubMed Google Scholar
37. Liu, B., et al. 2004. Interleukin-18 improves the early defence system against influenza virus infection by augmenting natural killer cell-mediated cytotoxicity. J. Gen. Virol. 85:423-428.
    View this article via: CrossRef PubMed Google Scholar
38. Draghi, M., et al. 2007. NKp46 and NKG2D recognition of infected dendritic cells is necessary for NK cell activation in the human response to influenza infection. J. Immunol. 178:2688-2698.
    View this article via: PubMed Google Scholar
39. Dong, L., Mori, I., Hossain, M.J., Kimura, Y. 2000. The senescence-accelerated mouse shows aging-related defects in cellular but not humoral immunity against influenza virus infection. J. Infect. Dis. 182:391-396.
    View this article via: CrossRef PubMed Google Scholar
40. He, X.S., et al. 2006. Cellular immune responses in children and adults receiving inactivated or live attenuated influenza vaccines. J. Virol. 80:11756-11766.
    View this article via: CrossRef PubMed Google Scholar
41. Mandelboim, O., et al. 2001. Recognition of haemagglutinins on virus-infected cells by NKp46 activates lysis by human NK cells. Nature. 409:1055-1060.
    View this article via: CrossRef PubMed Google Scholar
42. Arnon, T.I., et al. 2001. Recognition of viral hemagglutinins by NKp44 but not by NKp30. Eur. J. Immunol. 31:2680-2689.
    View this article via: CrossRef PubMed Google Scholar
43. Langmuir, A.D., Worthen, T.D., Solomon, J., Ray, C.G., Petersen, E. 1985. The Thucydides syndrome. A new hypothesis for the cause of the plague of Athens. N. Engl. J. Med. 313:1027-1030.
    View this article via: PubMed Google Scholar
44. Osterholm, M.T. 2005. Preparing for the next pandemic. N. Engl. J. Med. 352:1839-1842.
    View this article via: CrossRef PubMed Google Scholar
45. Khakoo, S.I., Carrington, M. 2006. KIR and disease: a model system or system of models? Immunol. Rev. 214:186-201.
    View this article via: CrossRef PubMed Google Scholar
46. Freud, A.G., Caligiuri, M.A. 2006. Human natural killer cell development. Immunol. Rev. 214:56-72.
    View this article via: CrossRef PubMed Google Scholar
47. He, X.S., et al. 2004. T cell-dependent production of IFN-gamma by NK cells in response to influenza A virus. J. Clin. Invest. 114:1812-1819.
    View this article via: JCI PubMed Google Scholar
48. Betts, M.R., et al. 2003. Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation. J. Immunol. Methods. 281:65-78.
    View this article via: CrossRef PubMed Google Scholar
49. Alter, G., Malenfant, J.M., Altfeld, M. 2004. CD107a as a functional marker for the identification of natural killer cell activity. J. Immunol. Methods. 294:15-22.
    View this article via: CrossRef PubMed Google Scholar
50. Gardiner, C.M., et al. 2001. Different NK cell surface phenotypes defined by the DX9 antibody are due to KIR3DL1 gene polymorphism. J. Immunol. 166:2992-3001.
    View this article via: PubMed Google Scholar
51. Carr, W.H., Pando, M.J., Parham, P. 2005. KIR3DL1 polymorphisms that affect NK cell inhibition by HLA-Bw4 ligand. J. Immunol. 175:5222-5229.
    View this article via: PubMed Google Scholar
52. Yawata, M., et al. 2006. Roles for HLA and KIR polymorphisms in natural killer cell repertoire selection and modulation of effector function. J. Exp. Med. 203:633-645.
    View this article via: CrossRef PubMed Google Scholar
53. Yu, J., et al. 2007. Hierarchy of the human natural killer cell response is determined by class and quantity of inhibitory receptors for self-HLA-B and HLA-C ligands. J. Immunol. 179:5977-5989.
    View this article via: PubMed Google Scholar
54. Dohring, C., Scheidegger, D., Samaridis, J., Cella, M., Colonna, M. 1996. A human killer inhibitory receptor specific for HLA-A1,2. J. Immunol. 156:3098-3101.
    View this article via: PubMed Google Scholar
55. Pende, D., et al. 1996. The natural killer cell receptor specific for HLA-A allotypes: a novel member of the p58/p70 family of inhibitory receptors that is characterized by three immunoglobulin-like domains and is expressed as a 140-kD disulphide-linked dimer. J. Exp. Med. 184:505-518.
    View this article via: CrossRef PubMed Google Scholar
56. Cella, M., Longo, A., Ferrara, G.B., Strominger, J.L., Colonna, M. 1994. NK3-specific natural killer cells are selectively inhibited by Bw4-positive HLA alleles with isoleucine 80. J. Exp. Med. 180:1235-1242.
    View this article via: CrossRef PubMed Google Scholar
57. Gumperz, J.E., Litwin, V., Phillips, J.H., Lanier, L.L., Parham, P. 1995. The Bw4 public epitope of HLA-B molecules confers reactivity with natural killer cell clones that express NKB1, a putative HLA receptor. J. Exp. Med. 181:1133-1144.
    View this article via: CrossRef PubMed Google Scholar
58. Fischer, J.C., et al. 2007. Relevance of C1 and C2 epitopes for hemopoietic stem cell transplantation: role for sequential acquisition of HLA-C-specific inhibitory killer Ig-like receptor. J. Immunol. 178:3918-3923.
    View this article via: PubMed Google Scholar
59. Davies, G.E., et al. 2007. Identification of bidirectional promoters in the human KIR genes. Genes Immun. 8:245-253.
    View this article via: CrossRef PubMed Google Scholar
60. Artavanis-Tsakonas, K., et al. 2003. Activation of a subset of human NK cells upon contact with Plasmodium falciparum-infected erythrocytes. J. Immunol. 171:5396-5405.
    View this article via: PubMed Google Scholar
61. Peruzzi, M., Wagtmann, N., Long, E.O. 1996. A p70 killer cell inhibitory receptor specific for several HLA-B allotypes discriminates among peptides bound to HLA-B*2705. J. Exp. Med. 184:1585-1590.
    View this article via: CrossRef PubMed Google Scholar
62. Peruzzi, M., Parker, K.C., Long, E.O., Malnati, M.S. 1996. Peptide sequence requirements for the recognition of HLA-B*2705 by specific natural killer cells. J. Immunol. 157:3350-3356.
    View this article via: PubMed Google Scholar
63. Zappacosta, F., Borrego, F., Brooks, A.G., Parker, K.C., Coligan, J.E. 1997. Peptides isolated from HLA-Cw*0304 confer different degrees of protection from natural killer cell-mediated lysis. Proc. Natl. Acad. Sci. U. S. A. 94:6313-6318.
    View this article via: CrossRef PubMed Google Scholar
64. Rajagopalan, S., Long, E.O. 1997. The direct binding of a p58 killer cell inhibitory receptor to human histocompatibility leukocyte antigen (HLA)-Cw4 exhibits peptide selectivity. J. Exp. Med. 185:1523-1528.
    View this article via: CrossRef PubMed Google Scholar
65. Rammensee, H., Bachmann, J., Emmerich, N.P., Bachor, O.A., Stevanovic, S. 1999. SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics. 50:213-219.
    View this article via: CrossRef PubMed Google Scholar
66. Valiante, N.M., et al. 1997. Functionally and structurally distinct NK cell receptor repertoires in the peripheral blood of two human donors. Immunity. 7:739-751.
    View this article via: CrossRef PubMed Google Scholar
67. Kim, S., et al. 2005. Licensing of natural killer cells by host major histocompatibility complex class I molecules. Nature. 436:709-713.
    View this article via: CrossRef PubMed Google Scholar
68. Raulet, D.H., Vance, R.E. 2006. Self-tolerance of natural killer cells. Nat. Rev. Immunol. 6:520-531.
    View this article via: CrossRef PubMed Google Scholar
69. Yokoyama, W.M., Kim, S. 2006. How do natural killer cells find self to achieve tolerance? Immunity. 24:249-257.
    View this article via: CrossRef PubMed Google Scholar
70. Johansson, S., et al. 2005. Natural killer cell education in mice with single or multiple major histocompatibility complex class I molecules. J. Exp. Med. 201:1145-1155.
    View this article via: CrossRef PubMed Google Scholar
71. Ferlazzo, G., et al. 2004. The abundant NK cells in human secondary lymphoid tissues require activation to express killer cell Ig-like receptors and become cytolytic. J. Immunol. 172:1455-1462.
    View this article via: PubMed Google Scholar
72. Fehniger, T.A., et al. 2003. CD56bright natural killer cells are present in human lymph nodes and are activated by T cell-derived IL-2: a potential new link between adaptive and innate immunity. Blood. 101:3052-3057.
    View this article via: CrossRef PubMed Google Scholar
73. Fritz, R.S., et al. 1999. Nasal cytokine and chemokine responses in experimental influenza A virus infection: results of a placebo-controlled trial of intravenous zanamivir treatment. J. Infect. Dis. 180:586-593.
    View this article via: CrossRef PubMed Google Scholar
74. Kaiser, L., Fritz, R.S., Straus, S.E., Gubareva, L., Hayden, F.G. 2001. Symptom pathogenesis during acute influenza: interleukin-6 and other cytokine responses. J. Med. Virol. 64:262-268.
    View this article via: CrossRef PubMed Google Scholar
75. Robbins, E., Marcus, P.I. 1964. Mitotically synchronized mammalian cells: a simple method for obtaining large populations. Science. 144:1152-1153.
    View this article via: CrossRef PubMed Google Scholar
76. Liu, Z.X., Govindarajan, S., Okamoto, S., Dennert, G. 2000. NK cells cause liver injury and facilitate the induction of T cell-mediated immunity to a viral liver infection. J. Immunol. 164:6480-6486.
    View this article via: PubMed Google Scholar
77. Brosterhus, H., et al. 1999. Enrichment and detection of live antigen-specific CD4(+) and CD8(+) T cells based on cytokine secretion. Eur. J. Immunol. 29:4053-4059.
    View this article via: CrossRef PubMed Google Scholar
78. Nain, M., et al. 1990. Tumor necrosis factor-alpha production of influenza A virus-infected macrophages and potentiating effect of lipopolysaccharides. J. Immunol. 145:1921-1928.
    View this article via: PubMed Google Scholar
79. Gong, J.H., et al. 1991. Influenza A virus infection of macrophages. Enhanced tumor necrosis factor-alpha (TNF-alpha) gene expression and lipopolysaccharide-triggered TNF-alpha release. J. Immunol. 147:3507-3513.
    View this article via: PubMed Google Scholar
80. Achdout, H., et al. 2003. Enhanced recognition of human NK receptors after influenza virus infection. J. Immunol. 171:915-923.
    View this article via: PubMed Google Scholar
81. Rehermann, B., et al. 1995. The cytotoxic T lymphocyte response to multiple hepatitis B virus polymerase epitopes during and after acute viral hepatitis. J. Exp. Med. 181:1047-1058.