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. Author manuscript; available in PMC: 2016 Dec 26.
Published in final edited form as: Cell Rep. 2016 Nov 22;17(9):2210–2220. doi: 10.1016/j.celrep.2016.10.075

HIV-1 control by NK cells via reduced interaction between KIR2DL2 and HLA-C*12:02/C*14:03

Zhansong Lin 1, Kimiko Kuroki 2, Nozomi Kuse 1, Xiaoming Sun 1, Tomohiro Akahoshi 1, Ying Qi 3, Takayuki Chikata 1, Takuya Naruto 1, Madoka Koyanagi 1, Hayato Murakoshi 1, Hiroyuki Gatanaga 1,5, Shinichi Oka 1,5, Mary Carrington 3,4, Katsumi Maenaka 3, Masafumi Takiguchi 1,*
PMCID: PMC5184766  NIHMSID: NIHMS827657  PMID: 27880898

SUMMARY

Natural killer (NK) cells control viral infection in part through the interaction between killer cell immunoglobulin-like receptors (KIRs) and their HLA ligands. We investigated 504 ART-free Japanese patients chronically infected with HIV-1 and identified two KIR/HLA combinations, KIR2DL2/HLA-C*12:02 and KIR2DL2/HLA-C*14:03, that impact suppression of HIV-1 replication. KIR2DL2+ NK cells suppressed viral replication in HLA-C*14:03+ or HLA-C*12:02+ cells to a significantly greater extent than did KIR2DL2− NK cells in vitro. Functional analysis showed that the binding between HIV-1-derived peptide and HLA-C*14:03 or HLA-C*12:02 influenced KIR2DL2+NK cell activity through reduced expression of the peptide-HLA (pHLA) complex on the cell surface (i.e. reduced KIR2DL2 ligand expression), rather than through reduced binding affinity of KIR2DL2 to the respective pHLA complexes. Thus, KIR2DL2/HLA-C*12:02 and KIR2DL2/HLA-C*14:03 compound genotypes have protective effects on control of HIV-1 through a mechanism involving KIR2DL2-mediated NK cell recognition of virus-infected cells, providing additional understanding of NK cells in HIV-1 infection.

Graphical Abstract

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INTRODUCTION

NK cells play an important role in the eradication of target cells infected with viruses, such as HCV (Khakoo et al., 2004), Ebola (Williams et al., 2015) and HIV-1 (Alter et al., 2011; Barker et al., 2007). NK cell function is tuned by the engagement of several different receptors expressed on the cell surface, including the killer cell immunoglobulin-like receptor (KIR) family, which consists of both inhibitory and activating members (Kulkarni et al., 2008). The interaction between KIR and their HLA class I ligands on the target cells plays an important role in the regulation of NK cell maturation (Anfossi et al., 2006; Orr and Lanier, 2010; Yawata et al., 2008) and anti-viral functions (Altfeld and Goulder, 2007; Carrington et al., 2008; Kulkarni et al., 2008). The presence of specific KIR-HLA combinations influences the pathogenesis of viral infection, such as KIR3DS1 plus HLA-B Bw4-80I (HLA-A/B alleles belonging to the Bw4 group and containing an isoleucine at position 80), which associates with slower progression to AIDS (Martin et al., 2002). The presence of KIR3DL1 and its HLA-Bw4-80I ligand confers stronger killing of HIV-1-infected cells and consequently, better clinical outcomes in HIV-1 infection (Alter et al., 2009; Boudreau et al., 2016; Boulet et al., 2010; Song et al., 2014).

HLA-C has been thought to play a less important role relative to HLA-A/-B in defense against viral infection via T cell immunity since it is expressed at a significantly lower level on the cell surface (Apps et al., 2015). However, a recent report showed that increasing HLA-C expression associates independently with protection against multiple outcomes of HIV-1 infection (Apps et al., 2013). This and several other studies (Blais et al., 2012; Makadzange et al., 2010) support an important role of HLA-C restricted CTLs in control of HIV-1. Moreover, HLA-C alleles are the ligands for KIR2D, influencing NK cell function after viral infection. KIR2DL3+ individuals homozygous for HLA-C1 (HLA-C alleles with asparagine at position 80) had better clinical outcomes after HCV infection (Khakoo et al., 2004), suggesting a protective effect of this KIR2D/HLA-C combination against the virus. Both the surface expression of HLA ligands and the pHLA-KIR binding affinity influence NK cell activity (Altfeld and Goulder, 2007). Unlike HLA-A and HLA-B alleles, the surface expression level of HLA-C alleles is not down-regulated by Nef and therefore HLA-C may be relatively stable after HIV-1 infection (Cohen et al., 1999; Le Gall et al., 1998). However, a recent study demonstrated that some HIV strains are able to down-modulate HLA-C on the infected cell surface by Vpu (Apps et al., 2016). Thus, binding between peptide-HLA-C complex and KIR2D, which is sensitive to sequence variation of the bound peptide, might be key in regulating the activity of NK cells (Alter et al., 2011; Fadda et al., 2012; Thananchai et al., 2009).

KIR2DL2, a B haplotype-specific inhibitory receptor expressed on NK cells, is capable of recognizing HIV-1 derived peptides in the context of HLA-C molecules in virus-infected target cells (Alter et al., 2011; Fadda et al., 2012; Van Teijlingen et al., 2014). Both in vitro and in vivo screenings discovered that the presence of KIR2DL2 (Alter et al., 2011) or certain KIR2DL2/HLA-C1 (Van Teijlingen et al., 2014) compound genotypes associate with specific sequence variations in the HIV-1 genome. These viral mutations confer stronger binding of KIR2DL2 to the pHLA complex resulting in the inhibition of KIR2DL2+ NK cells, and consequent escape of HIV-1-infected target cells from NK cell-mediated killing activity in vitro(Alter et al., 2011; Van Teijlingen et al., 2014). These studies suggest that KIR2DL2+ NK cells exert immune pressure on the virus, but direct genetic evidence to support KIR2DL2 protection in HIV-1 infection is lacking.

All KIR studies in HIV-1 infection published to date have been performed using Caucasian or African samples. Previous studies in Caucasian and African cohorts reported that the presence of some specific KIR genes and/or KIR-HLA gene combinations confers better HIV-1 control (Alter et al., 2011; Fadda et al., 2012; Qi et al., 2006; Song et al., 2014; Van Teijlingen et al., 2014). Japanese have different HLA and KIR frequency distributions from Caucasians or Africans ((Bunce et al., 1997; Denis et al., 2005; Hollenbach et al., 2010; Miyashita et al., 2006; Norman et al., 2013), so the effects of various KIRs on viral infection may be distinct from that in other populations. We here recruited 504 treatment naïve Japanese patients chronically infected with HIV-1 and tested for correlations between the presence of each KIR gene or KIR/HLA combination and the clinical outcome of HIV-1 infection in this genetically distinct population. We further investigated the mechanisms for the observed genetic effects of certain KIR/HLA combinations.

RESULTS

Effect of 2DS2/2DL2 with HLA-C*12:02 or HLA-C*14:03 on HIV-1 pVL in chronically HIV-1-infected Japanese individuals

To determine the presence of each KIR gene among our cohort of 504 treatment-naïve Japanese individuals chronically infected with HIV-1, we first performed low-resolution analysis for KIR using multiplex PCR-SSP (Kulkarni et al., 2010). Three haplotype B-specific genes, KIR2DL2, 2DS2, and 2DS3, showed significantly lower frequencies in our cohort than that observed in the Caucasian and African cohorts (p<0.0001) (Figure S1). These results concur with previous reports showing that the A haplotype is more common in the Japanese population than haplotype B (Yawata et al., 2002). To assess the influence of KIR on HIV-1 infection, we tested for correlations between the presence of specific KIR haplotypes and individual genes on plasma viral load (pVL) in our cohort. The presence of haplotype B-specific genes had a weak beneficial effect on the control of HIV-1 (p=0.02884) (Figure 1A), but none of the individual haplotype B-specific KIR genes showed a significant effect on pVL on their own, though 2DS2 and 2DL2 genes trended towards protection (Table S1).

Figure 1. The combinatorial effect of KIR2DL2/S2 and HLA-C1 alleles on HIV-1 pVL.

Figure 1

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(A) The effect of haplotype B-specific genes on pVL. Haplotypes were assigned according to the following scheme: A/A are individuals without any of the haplotype B-specific genes; A/B: individuals possessing all the haplotype A-specific genes and at least one haplotype B-specific gene; B/B: individuals who lacks some haplotype A-specific genes. (B) The effect of HLA-C1 homozygotes on pVL. (C) The effect of HLA-C1 in combination with KIR2DL2/S2 on pVL. All results are given in (A-C) as median and interquartile range. P values were calculated by the Mann-Whitney U test or the Kruskal-Wallis test when comparing two or more groups, respectively.

HLA-C alleles can be divided into two groups, HLA-C1 or HLA-C2, based on differences at positions 77 and 80 (Winter and Long, 1997). Previous studies showed that KIR2DL2 binds more strongly to HLA-C1 than to HLA-C2 (Moesta et al., 2008; Winter et al., 1998). We observed that HLA-C1 homozygotes showed weakly lower pVL compared to all others (Figure 1B), which could be due to HLA-C*12:02, an HLA-C1 allele known to associate with protection in Japanese (Naruto et al., 2012). There was no significant beneficial effect on pVL in the KIR2DL2-HLA-C1 double-positive group as compared to all others (Figure 1C). Since there is strong linkage disequilibrium between KIR2DL2 and KIR2DS2 (Moesta and Parham, 2012), we investigated these two KIR genes together and term them as KIR2DL2/S2. We analyzed the effect of each HLA-C1 allele with KIR2DL2/S2 in order to determine whether the weak protection observed for HLA-C1/C1 may be due to an individual HLA-C1 allele(s) in combination with its cognate receptor, KIR2DL2/S2. HLA-C*12:02 and HLA-C*14:03 both showed effects in combination with KIR2DL2/S2 on the control of HIV-1 infection (Table 1). Individuals having KIR2DL2/S2 and HLA-C*12:02 or HLA-C*14:03 showed significantly lower pVL than those having one alone or neither (Figure 2A, B), and in both cases, formal tests for interactions indicated a synergistic effect between KIR2DL2/S2 with HLA-C*1202 and also with C*14:03 (Table 2). However, KIR2DL2/S2 did not show an effect in combination with HLA-C*14:02 (Figure 2B), even though this allele differs from HLA-C*14:03 by only one substitution at position 21 outside of the peptide binding groove. Individuals having HLA-C*12:02 or HLA-C*14:03 but not KIR2DL2/S2 showed significantly though weakly lower pVL than double-negative individuals (Figure 2A, B), supporting previous work (Naruto et al., 2012). Notably, the test for genetic interaction where the three variables, KIR2DL2/S2, HLA-C*12:02, and KIR2DL2/S2 plus HLA-C*12:02, were included in the model showed that HLA-C*12:02 has a significant effect even after accounting for its protective effect in the presence of KIR2DL2/S2 (Table 2). This suggests a dual mechanism of HLA-C*12:02 protection; one involving NK cell and the other CTL recognition. The protection conferred by HLA-C*14:03, however, is likely due only to its interaction with KIR2DL2/S2 (Table 2). Finally, we analyzed the effect of other haplotype-B specific KIRs and their HLA ligands on the pVL, but no significant effects were observed (Figure S2).

Table 1.

The effect of each C1 allotype allele and KIR2DL2/S2 on pVL.

HLA-C Allele N Double Positive Others P value
N Median pVL IQR N Median pVL IQR
01:02 139 24 4.506 3.966–4.975 479 4.544 3.959–000 0.928
03:03 101 19 4.623 3.973–4.982 484 4.538 3.955–5.000 0.976
03:04 112 18 4.498 3.895–5.368 485 4.544 3.961–5.000 0.974
07:02 129 21 4.643 4.041–4.965 482 4.531 3.953–5.000 0.901
08:01 62 11 4.079 3.898–5.556 492 4.544 3.970–4.998 0.836
12:02 136 26 3.872 3.398–4.540 477 4.544 4.000–5.000 <0.001
14:02 71 12 4.266 3.997–5.108 491 4.544 3.954–5.000 0.843
14:03 67 10 3.835 3.133–4.237 493 4.544 3.975–5.000 0.003
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P values were calculated by Mann-Whitney U test.

Figure 2. Effect of KIR2DL2/S2 and HLA-C*12:02 or HLA-C*14:03 on HIV-1 pVL.

Figure 2

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(A) The effect of HLA-C*12:02 and KIR2DL2/S2 on pVL. (B) The effect of HLA-C*14:03 or HLA-C*14:02 and KIR2DL2/S2 on pVL. All results are given in A and B as median and interquartile range. P values were calculated by the Mann-Whitney U test or the Kruskal-Wallis test when comparing two or more groups, respectively.

Table 2.

The synergistic effects of KIR/HLA combinations on pVL.

Three variables in each model N Mean logVL SE P value
KIR2DL2/S2 85 4.21 0.09 0.82
Others 418 4.24 0.10
HLA-C*12:02 136 4.11 0.09 0.02
Others 367 4.33 0.10
Double positive 26 3.99 0.18 0.03
Others 477 4.45 0.06
KIR2DL2/S2 85 4.02 0.14 0.31
Others 418 4.13 0.14
HLA-C*14:03 67 3.98 0.14 0.10
Others 436 4.17 0.14
Double positive 10 3.76 0.27 0.03
Others 493 4.40 0.07
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P values were generated by logistical regression model with interaction term.

Stronger NK cell activation through reduced recognition of KIR2DL2 for HIV-1-infected HLA-C*14:03+ target cells

The genetic analysis suggested that NK cells more effectively suppress HIV-1 replication among HLA-C*14:03+KIR2DL2/S2+ HIV-1-infected individuals than in those with HLA-C*14:02+KIR2DL2/S2+ or HLA-C*14:03+KIR2DL2/S2-. Therefore, we investigated the function of the NK cells from HLA-C*14:02+ vs. HLA-C*14:03+ subjects. We established 721.221-CD4 cells transfected with HLA-C*14:02 or HLA-C*14:03 (termed .221-C1402 and .221-C1403, respectively). .221-C1402 and .221-C1403 cells expressed HLA-C*14 at same level on the cell surface (Figure S3A left). The cell lines were then infected with HIV-1 (NL4-3 and NL4-3gagHXB2, respectively) and co-cultured with KIR2DL2/S2+ or KIR2DL2/S2− NK cells (CD3-CD56+CD158b+) isolated from healthy donors (see Methods). KIR2DL2/S2+ NK cells suppressed HIV-1 replication in .221-C1403 at a significantly higher level than did KIR2DL2/S2− NK cells, whereas these 2 NK cell subsets suppressed HIV-1 replication at the same level in .221-C1402 cells (Figure 3A left). In addition, 2DL2/S2+ NK cells suppressed HIV-1 replication in .221-C1403 at a significantly higher level than that in .221-C1402, whereas 2DL2/S2− NK cells suppressed HIV-1 replication in these 2 target cell lines at the same level (Figure 3A right). The NK cell reaction assay showed that KIR2DL2/S2+ NK cells produced significantly higher levels of reaction markers (IFN-γ and CD107a) after stimulation with HIV-1-infected .221-C1403 cells than they did after stimulation with .221-C1402 cells whereas 2DL2/S2− NK cells produced similar levels of reaction markers after stimulation with .221-C1403 or .221-C1402 cells infected with HIV-1 (Figure 3B). These results indicate that KIR2DL2/S2+ NK cells are more strongly reacted and suppressive of viral replication in the presence of HLA-C*14:03 ligand relative to other KIR/HLA combinations. Further confirmation of these results was generated using primary CD4+ T cells from HLA-C*14:02 or HLA-C*14:03 homozygous donors (Figure 3C).

Figure 3. Inhibition of viral replication in HLA-C*14:02 or 14:03 positive target cells by NK cells.

Figure 3

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(A) Percentage of viral suppression in .221-C1402 or .221-C1403 cell lines infected with NL4-3gagHXB2 by bulk NK cells sorted from three KIR2DL2/S2+ or three 2DL2/S2− individuals. Viral suppression ability of NK cells were tested in triplicate samples from each individual. The left panel shows the comparison of the viral suppression level between KIR2DL2/S2+ vs. 2DL2/S2− NK cells; the right panel compares the viral suppression level for each individual depending on HLA genotype. Viral suppression ability of KIR2DL2/S2+ and 2DL2/S2− NK cells for .221 cells infected with NL4-3gagHXB2 was 89.6 ± 4.2% and 87.1 ± 8.0%, respectively.

(B) Comparison of NK cell reactivity upon stimulation with NL4-3gagHXB2-infected .221-C1402 and .221-C1403 cells for each individual. Relative NK cell reactivity was calculated by determining the frequency of IFN-g+ and/or CD107a+ NK cells as described in materials and methods. (C) Percentage of viral suppression using NL4-3gagHXB2 virus-infected CD4 T cells separated from HLA-C*14:02 or -C*14:03 homozygous patients by bulk NK cells sorted from three KIR2DL2/S2+ or three 2DL2/S2− individuals. Viral suppression ability of NK cells were tested in triplicate from each individual. The left panel shows the comparison of the viral suppression level between KIR2DL2/S2+ vs. 2DL2/S2− NK cells; the right panel compares the viral suppression levels for each individual. (D) Reactivity of bulk NK cells sorted from KIR2DL2/S2+ individuals after stimulation with RMA-S-C1402 or -C1403 cell lines pulsed with epitope peptide Gag-LL8 (30 uM). (E) Expression of HLA molecules on RMA-S-C1402 or -C1403 cell lines pulsed with epitope peptides Gag-LL8. (F) Binding affinity of HLA-C*14:02 or C*14:03 monomers folded with Gag-LL8 to KIR2DL2 or KIR2DS2 molecules were measured by surface plasmon resonance. The KD values were determined using equilibrium binding curves and Scatchard analysis of equilibrium binding. In A-E, P values (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001) were calculated by the Mann-Whitney U test. All of the data were acquired by triplicated experiments.

We next tested the ability of NK cells to recognize cells expressing HLA-C*14:02 or HLA-C*14:03 pulsed with a HLA-C*14-restricted HIV-1-derived CTL epitope peptide, Gag-LL8 (Horton et al., 2006). HLA-C*14:02 or HLA-C*14:03 were transfected into the RMA-S cell line (RMA-S-C1402 and RMA-S-C1403, respectively). RMA-S-C1402 and RMA-S-C1403 cells expressed similar level of HLA-C*14 on the cell surface at 26 °C or at 37 °C (Figure S3A right). The cells were pulsed with Gag-LL8 and used as targets. Bulk NK cells from KIR2DL2/S2+ donors stimulated with Gag-LL8-pulsed RMA-S-C1403 showed a significantly higher reaction level than those stimulated with Gag-LL8-pulsed RMA-S-C1402 (Figure 3D), in concert with results using HIV-1-infected .221 targets (Figure 3A–C). This difference in reaction level may be due to distinct binding affinities of Gag-LL8 for HLA-C*14:02 vs. HLA-C*14:03 molecules, which in turn may result in differential levels of stability of the HLA molecules, thereby affecting KIR recognition. Indeed, HLA-C*14:02 molecules showed a significantly greater expression index in the presence of Gag-LL8 than did HLA-C*14:03 molecules (Figure 3E). The greater reaction and viral suppression conferred by C*14:03 in the presence of KIR2DL2/S2, yet higher expression index of Gag-LL8/HLA-C*14:02 complexes would suggest that the mechanism distinguishing the effects of HLA-C*14:02 vs. HLA-C*14:03 involves inhibition by KIR2DL2 rather than activation by KIR2DS2.

We tested whether the binding affinity of KIR for its ligand affects NK cell function using surface plasmon resonance (SPR) analysis. KIR2DS2 exhibited little binding to these HLA molecules (Figure 3F), indicating that the mechanism of differential viral control across HLA genotypes does not involve KIR2DS2. Further, the binding affinity of KIR2DL2 for Gag-LL8-HLA-C*14:02 was similar to that for Gag-LL8-HLA-C*14:03 (Figure 3F), excluding the possibility that different affinities of KIR2DL2 for these two HLA-C*14 molecules results in the observed differential levels of HIV control. Taken together, the results raise the possibility that lower expression of Gag-LL8-HLA-C*14:03 on the cell surface of HIV-1-infected HLA-C*14:03+ target cells may reduce the level of KIR2DL2-mediated NK cell inhibition relative to that involving HLA-C*14:02 target cells.

In order to determine whether these results were specific to the Gag-LL8 epitope, we measured the expression index of 4 additional HLA-C*14-restricted HIV-1-derived peptides and showed that all 4 peptides bind to HLA-C*14:02 with significantly higher affinity than they do to HLA-C*14:03 molecules (Figure S3B). Thus, differential expression levels of HLA-C*14:02 vs. HLA-C*14:03 in the context of HIV-1-bound peptides likely affects KIR2DL2 recognition and outcome to HIV-1 infection.

Effective function of KIR2DL2/S2+NK cells for HLA-C*12:02+ target cells infected with HIV-1

We tested whether NK cells from KIR2DL2/S2+ subjects were capable of suppressing HIV-1-infected HLA-C*12:02+ cells to a greater extent than NK cells from KIR2DL2/S2 negative subjects. No difference was observed, however, between NK cells sorted from KIR2DL2/S2+ vs. those from KIR2DL2/S2− donors in their ability to suppress the replication of NL4-3 (Figure 4A left), suggesting that the effect of KIR2DL2/S2 with HLA-C*12:02 is due to a different mechanism than that involving the two subtypes of HLA-C*14. Previous studies suggested that sequence variations within HLA binding peptides may influence NK cell activity (Fadda et al., 2012). We previously demonstrated that a Val to Ala substitution at Pol 464, which is located at the 9th position of the HLA-C*12:02-restricted epitope Pol-IY10, significantly impairs the killing activity of Pol-IY10-specific CTLs (Honda et al., 2011). In order to determine whether this mutation has an effect on the suppression of viral proliferation by KIR2DL2/S2 NK cells, we performed viral suppression assays on both WT and 9A-mutant viruses. KIR2DL2/S2+NK cells suppressed the replication of the 9A virus in HIV-1-infected .221-C*1202 cells more effectively than KIR2DL2/S2− NK cells (Figure 4A right), whereas both NK cells suppressed the replication of WT viruses to the same extent (Figure 4A left). In addition, 2DL2/S2+ NK cells suppressed the replication of the 9A virus at a significantly higher level than that of WT virus, whereas 2DL2/S2− NK cells suppressed that of both viruses at the same level (Figure 4A right). The NK cell reaction assay showed that KIR2DL2/S2+ NK cells produced significantly higher levels of reaction markers after stimulation with the 9A virus-infected .221-C1202 cells than they did after stimulation with the WT virus-infected .221-C1202 cells, whereas 2DL2/S2− NK cells produced similar levels of reaction markers after stimulation with both cells (Figure 4B). The ability of KIR2DL2/S2+ NK cells to suppress viral replication more robustly than KIR2DL2/S2− NK cells was confirmed in the experiments using primary CD4+ T cells infected in vitro from HLA-C*12:02 homozygous donors (Figure 4C).

Figure 4. Inhibition of viral replication in HLA-C*12:02 positive target cells by NK cells.

Figure 4

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(A) Percentage of viral suppression in NL4-3 (WT) or NL4-3pol 464-10-9A (9A) virus-infected .221-C1202 cells by bulk NK cells sorted from 3 KIR2DL2/S2+ or 3 2DL2/S2- individuals. Viral suppression ability of NK cells was tested in triplicate from each individual. The left panel shows the comparison of the viral suppression level between the KIR2DL2/S2+ vs. 2DL2/S2− NK cells; the right panel compares the viral suppression level between target cells infected with WT and 9A viruses for each individual. (B) The comparison of NK cell reactivity upon stimulation with WT and 9A mutant virus-infected .221-C1202 cells for each individual. Relative NK cell reactivity was calculated by determining the frequency of IFN-g+ and/or CD107a+ NK cells as described in materials and methods. (C) Percentage of viral suppression in WT- or 9A mutant virus-infected CD4 T cells isolated from an HLA-C*12:02 homozygous healthy donor by bulk NK cells sorted from three KIR2DL2/S2+ or three 2DL2/S2− individuals. Viral suppression ability of NK cells was tested in triplicate from each individual. The left panel shows the comparison of the viral suppression level between KIR2DL2/S2+ vs. 2DL2/S2− NK cells; the right panel compares the viral suppression level between target cells infected with WT and 9A viruses for each individual. (D) Reactivity of bulk NK cells sorted from KIR2DL2/S2+ individuals after stimulation with RMA-S-C1202 cell lines pulsed with Pol-IY10 (WT) or 9A mutant peptide (100 uM). (E) Expression level of HLA molecules on RMA-S-C1202 cell lines pulsed with the Pol-IY10 (WT) or 9A mutant peptides. (F) Binding affinity of HLA-C*12:02 monomers folded with WT or mutant Pol-IY10 to KIR2DL2 or KIR2DS2 molecules were measured by surface plasmon resonance. WT-1 and WT-2 represented the duplicated experiments for the WT peptide-HLA complexes; 9A-1 and 9A-2 represented the duplicated experiments for the mutant (the V9A mutation in Pol-IY10 epitope) peptide-HLA complex. The KD values were determined using equilibrium binding curves and Scatchard analysis of equilibrium binding. In A-E, P values (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001) were calculated by the Mann-Whitney U test. All of the data were acquired by triplicate experiments.

NK cell functional assays revealed that RMA-S-C1202 cells pre-pulsed with the mutant peptide stimulated KIR2DL2/S2+ NK cells at significantly higher levels than did those prepulsed with the WT peptide (Figure 4D), suggesting that the 9A mutation influenced the recognition of the target cells by NK cells via KIR2DL2/S2. To clarify how the 9A mutation influences KIR2DL2/S2+ NK cell reactivity, we measured the expression index of the 9A and WT peptides to HLA-C*12:02 molecules. The results showed that the 9A mutant peptide had a significantly weaker expression index (Figure 4E), raising the possibility that the 9A mutation reduced the expression level of HLA-C*12:02 molecules on cells infected with the 9A mutant virus. We further tested for direct binding of KIR2DL2 and KIR2DS2 to HLA-C*12:02-peptide complexes by using SPR. The binding affinity of KIR2DL2 to WT Pol-IY10-HLA-C*12:02 was similar to that observed for the mutant Pol-IY10-HLA-C*12:02 complexes (KD 21 μM vs 23 or 24 μM) whereas KIR2DS2 did not bind to these HLA molecules (Figure 4F).

These results support a model in which NK cells expressing KIR2DL2 are more readily reacted in response to the HLA-C*12:02-associated escape mutation in the Pol-IY10 epitope than they are to the WT form of this epitope due to lower expression of HLA-C*12:02 when complexed with the mutant peptide. Thus, the protective effects of both HLA-C*14:03 and -C*12:02 appear to be mediated by the lower expression of these allotypes in the presence of corresponding HIV peptides and diminished KIR2DL2 recognition, resulting in greater NK cell activation.

DISCUSSION

In the present study, we first performed genetic analyses using the same cohort of 504 treatment naïve Japanese individuals chronically infected with HIV-1 in which the effect of HLA alleles on clinical outcome was previously analyzed (Naruto et al., 2012). We identified two KIR/HLA combinations, KIR2DL2/S2/HLA-C*12:02 and KIR2DL2/S2/HLA-C*14:03 that significantly correlated with lower pVL in the Japanese population, in which HLA-C*12:02 and C*14:03 were detected at a frequency of 22.3% and 13.3%, respectively (Ikeda et al., 2015). Previous studies showed the combined effect of KIR2DL2 and two HLA-C alleles, HLA-C*01:02 (Alter et al., 2011; Fadda et al., 2012) and C*03:04 (Van Teijlingen et al., 2014), on NK cell function and HIV-1 infection in Caucasian cohorts, which have extremely low frequencies of HLA-C*12:02 and C*14:03.

The results of viral suppression and NK cell reaction assays demonstrated that the presence of KIR2DL2/S2 combined with HLA-C*14:03 confers stronger inhibition of HIV-1 replication and a higher level of IFN-γ secretion and degranulation than does the presence of either one alone. Interestingly, HLA-C*14:02 did not show such an effect with KIR2DL2/S2 in the genetic analysis, nor did it in the viral suppression or NK cell reaction assays. HLA-C*14:02 and -C*14:03 differ by only a single substitution at position 21 located outside of the peptide binding cleft. NK cell reaction assays using RMA-S-C1402 or RMA-S-C1403 pre-pulsed with the Gag-LL8 epitope peptide demonstrated that KIR2DL2/S2+ NK cells stimulated with the peptide-pulsed RMA-S-C1403 cells were reacted at a significantly higher level than those stimulated with peptide-pulsed RMA-S-C1402 cells. This result suggested the possibility of lower expression of HLA-C*14:03 on cells relative to HLA-C*14:02, perhaps due to lower peptide affinity for HLA-C*14:03 relative to that for HLA-C*14:02. Indeed, HLA stabilization assay showed that five HIV-1 peptides had higher binding affinity to HLA-C*14:02 than to HLA-C*14:03. Thus, the single amino acid difference between HLA-C*14:02 and HLA-C*14:03 outside of the binding cleft influences the binding affinity of the peptide bound to these HLA molecules and consequently modulates the recognition of HIV-1-infected target cells by NK cells through KIR2DL2.

Several studies have reported that peptide sequence variations may enhance the binding of inhibitory KIRs to the pHLA complexes and consequently down-regulate the activity of NK cells (Alter et al., 2011; Fadda et al., 2012; Holzemer et al., 2015; Thananchai et al., 2009; Van Teijlingen et al., 2014). However, we showed that the 9A mutation in Pol-IY10 did not influence the direct binding of KIR2DL2 to the peptide-HLA-C*12:02 complex, even though this mutation resulted in greater KIR2LD2+ NK cell activation and resulted in stronger inhibition of replication of the virus carrying this mutation. Rather, the HLA stabilization assay showed that the 9A mutation reduced peptide-HLA binding affinity. Taken together, our data suggest that peptide sequence variation influences the peptide-HLA binding affinity, thereby altering the expression level of pHLA on the cells surface, which consequently regulates the recognition by and activation of NK cells without changing the KIR-pHLA binding affinity. We analyzed the association between this mutation and HLA-C*12:02 in 363 chronically HIV-1 infected individuals and showed that this mutation is detected at a significantly higher frequency in HLA-C*12:02+ individuals than in those missing this allele (Figure S4A), confirming a previous smaller cohort study (Honda et al., 2011). There is no significant difference in the frequency of the 9A escape mutation between KIR2DL2/S2+ and KIR2DL2/S2− HLA-C*12:02+ individuals (Figure S4B), excluding the selection of this mutation by NK cells.

HIV-1 sequence variations have been reported to be selected by NK cell mediated immune pressure (Alter et al., 2011; Van Teijlingen et al., 2014). These KIR-associated sequence variations impair NK cell function by facilitating the binding and recognition of specific HLA alleles to related inhibitory KIRs. On the other hand, it is widely known that strong immune pressure conferred by HIV-1-specific CTLs selects escape mutations within HIV-1 epitopes. These mutations accumulate not only in individuals possessing related HLA alleles but also in populations to some extent (Chikata et al., 2014; Kawashima et al., 2009; Moore et al., 2002). The impact of these CTL selected escape mutations on KIR recognition and NK cell function is now beginning to be considered. A previous study reported that a single amino acid variant within an HLA-Cw4-restricted epitope impaired both CTL recognition and NK cell function by increasing the direct binding of the pHLA complex to KIR2DL1 molecules, implying that a CTL escape mutation can also affect NK cell recognition (Thananchai et al., 2009). We showed that the 9A escape mutation selected by the HLA-C*12:02-restricted CTLs, which accumulates in HLA-C*12:02+ individuals without reversion (Honda et al., 2011), activates KIR2DL2+ NK cells, leading to suppressed replication of the mutant virus replication. The 9A mutation was detected in approximately 40% of KIR2DL2+HLA-C*12:02+ Japanese individuals (Figure S4A). These findings suggest that the 9A mutation enhances the recognition of 2DL2+ NK cells and contribute to the protective effect of the KIR2DL2/HLA-C*12:02 compound genotype on HIV-1 control at the population level.

We describe herein a comprehensive study on the synergistic effect of KIR-HLA combinations in the Japanese population and identified two new combinations, KIR2LD2/HLA-C*12:02 and KIR2LD2/HLA-C*14:03, that have protective effects on HIV-1 control. Furthermore, the mechanism involves NK cell reaction via decreased KIR2DL2 recognition of HIV-1-infected cells. The impact of a CTL escape mutation on KIR recognition and NK cell reaction that we describe herein underscore the delicate network between the acquired and innate immune systems upon HIV-1 infection.

EXPERIMENTAL PROCEDURES

Ethics statement

The study was approved by the Ethics Committees of Kumamoto University (RINRI-540 and GENOME-210) and the National Center for Global Health and Medicine (NCGM-A-000172-00). The written informed consent was obtained from all individuals according to the Declaration of Helsinki.

Study subjects

504 treatment naïve HIV-1 chronically infected individuals and 4 HIV-1 seronegative individuals were recruited. Their plasma and PBMCs were separated from whole blood. HLA genotypes of the HLA-A, B and C allele were identified by the Luminex microbead method at the NPO HLA laboratory (Kyoto, Japan).

KIR typing

The low-resolution KIR typing which is aimed to detect the presence or absence of 16 KIR genes was performed by using multiplex PCR-SSP (Kulkarni et al., 2010). Based on the two haplotypes, individuals were classified as follow: A/A: individuals without any of the haplotype B-specific genes (or we can say individuals having only 3DL3, 2DL3, 2DP1, 2DL1, 2DL1, 3DP1, 2DL4, 3DL1, 2DS4 and 3DL2); A/B: individuals possessing all the haplotype A-specific genes (2DL3, 3DL1 and 2DS4) and at least one haplotype B-specific gene (2DS2, 2DL2, 2DL5, 2DS5 3DS1, 2DS3 and 2DS1); B/B: individuals who lacks some of the haplotype A-specific genes (especially 2DS4).

NK cell sorting and culture

Cryopreserved PBMC samples from four healthy donors (all had KIR2DL3 whereas three had KIR2DL2/S2 but one did not) were thawed, divided and immediately stained with anti-CD3 mAb, anti-CD16 mAb, anti-CD56 mA, GL183 anti-CD158b mAb (BD Pharmingen™) and 7AAD (BD Pharmingen™). CD158b is the cell surface marker for KIR2DL2, KIR2DS2 and KIR2DL3 which share similar extracellular structure. One thousands of CD3-CD16+CD56+CD158b+7AAD− cells were sorted into a 96-well plate by using a FACSAria I (BD Biosciences). Sorted NK cells from 2DL2/S2+ or 2DL2/S2− donors were stained with anti-KIR2DL3 mAbs (R&D Systems) and the frequency of KIR2DL3+ cells was measured by flow cytometry. NK cells from 2DL2/S2+ donors contained only 4.1–5.9% KIR2DL3+ cells, whereas all of NK cells from 2DL2/S2− donors expressed KIR2DL3. Sorted cells were cultured in cellgroSCGM serum-free medium (CellGenix) supplemented with 10% FBS and 400U/ml rIL-2 for 1-2 months. These cultured NK cells were tested for the expression of CD158b and all of them expressed CD158b. The reactivity of the cultured NK cells were tested against K562 cells by measuring the frequency of NK cells producing IFN-γ or expressing CD107a. They were 99.3 %–100 %.

Target cells

721.221-C1202-CD4 cells were generated in our lab as previously described (Honda et al., 2011). 721.221 -C1402 and -C1403 cell lines were generated by the transfection of HLA-C*14:02 and -C*14:03 genes into 721.221-CD4 cell lines, respectively. These cells express the same level of HLA-C14 on the cell surface. RMA-S-C1202 cells were generated in our lab as previously described (Kuse et al., 2015). RMA-S-C1402 and -C1403 cell lines were generated by the transfection of HLA-C*14:02 and -C*14:03 genes into RMA-S cell lines, respectively. RMA-S cell line is a TAP2 defected cell line derived from mice. They express high levels of empty MHC molecules (i.e. without binding peptide) on the cell surface when cultured at 26 °C and extremely low levels when cultured at 37 °C (Ljunggren et al., 1990). CD4+ T cells were separated from donors homozygous for HLA-C*12:02, -C*14:02, or -C*14:03 by using MACS separation columns (Miltenyi Biotec).

HIV-1 clones

The proviral clones NL4-3 (Akari et al., 2000) and the chimeric virus clone NL4-3gagHXB2 (Fujiwara et al., 2005) were previously described. These two clones were used as wild type (WT) virus controls. The NL4-3pol 464-10-9A (9A mutant virus) was generated by introducing the Pol463-10-9A mutation into NL-432 using site-directed mutagenesis (Invitrogen) as previously described (Honda et al., 2011).

Viral suppression assay

Target cells (CD4 T cells or 721.221 cell lines) were incubated with a given HIV-1 clone for 6 h at 37 °C after 30min centrifugation with 1000 rpm at 4 °C. After 3 times wash by R10, the infected cells were co-cultured with different sorted NK cell lines (E:T ratio=0.1:1). From day 4 to day 7 post infection, 30 ul of supernatant was collected and stored at −30°C; and the concentration of p24 Ag was measured by p24 ELISA by using HIV-1 p24 Ag ELISA kit (ZeptoMEtrix). The percentage of suppression of HIV-1 replication was calculated as follow: % suppression= (1-concentration of p24 Ag in the supernatant of HIV-1-infected target cells co-cultured with an NK cell line/concentration of p24 Ag in the supernatant of HIV-1-infected target cells without effectors) × 100.

NK cell stimulation and reaction assay

For HIV-1 infected target cells, 721.221 cell lines were infected with specific HIV-1 strains for 3 days; viral infectivity were measured by intracellular p24 staining. Target cells with similar frequency of p24 positive populations were selected and co-cultured with a specific NK cell line (E/T ratio=0.1:1) for 1 day. For peptide-pulsed RMA-S cells, RMA-S cell lines were pre-cultured at 26 °C for 18h and then incubated with different concentrations of a specific synthesized peptide at same temperature for 1h; cells were then co-cultured with a specific NK cell line (E/T ratio=0.1:1) for 1h at 37 °C; Brefeldin A (6ug/ml) and Alexa Fluor® 647 labeled anti-mouse CD107a (LAMP-1) mAb (BioLegend) were added and cells were cultured for another 5h; NK cells were then stained with PE labeled anti-CD56 mAb (BD Pharmingen™), FITC labeled anti-CD158b mAb (BD Pharmingen™) and 7AAD (BD Pharmingen™), and analyzed by FACS (FACSCanto II).

CD107a/IFN-γ double positive and single positive 7AAD-CD56+CD158b+ cells were all counted as reacted NK cell population. For HIV-1 infected target cells, relative NK cell reactivity (%) was calculated as follow: % of reacted NK cells co-cultured with HIV-1-infected target cell lines – % of reacted NK cells co-cultured with uninfected target cell lines. For peptide-pulsed RMA-S cells, NK cell reactivity index was calculated as follow: (% of reacted NK cells co-cultured with peptide-pulsed RMA-S cell lines- % of reacted NK cells without target cells) / (% of reacted NK cells co-cultured RMA-S cell lines without peptide pulsing - % of reacted NK cells without target cells).

Peptide-HLA stabilization assay

The binding of peptides to HLA class I molecules were measured as previously described (Takamiya et al., 1994). Briefly, RMA-S cell lines were pre-cultured at 26 °C for 18 h to fully express and present empty HLA class I molecules on the cell surface and then incubated with different concentrations of peptide at same temperature for 1h; cells were then incubated at 37 °C for another 3h. After incubation, cells were stained with anti-HLA class I γ3 domain mAb TP25.99 (Tanabe et al., 1992) and subsequently with FITC-conjugated sheep IgG (Jackson ImmunoResearch Laboratories). The surface expression of these HLA-C molecules was measured by flow cytometry (FACSCanto II). The HLA expression index was calculated as follow: (MFI of RMA-S cells pre-pulsed with peptide –MFI of RMA-S cells without peptide pulsing) / (MFI of RMA-S cells kept at 26°C forever – MFI of RMA-S cells without peptide pulsing). RMA-S cell lines expressing HLA-C*14:02 or HLA-C*14:03 cultured at 26° C present the same level of HLA-C14 on the cell surface.

Peptide-HLA complex and KIR binding assay

HLA-C*12:02, HLA-C*14:02 and HLA-C*14:03 monomers were made as previously described (Fan et al., 2001), respectively. HLA-C*12:02 monomers were folded with the following peptides: ILKEPVHGVY (WT Pol-IY10) and ILKEPVHGAY (9A Pol-IY10), respectively. HLA-C*14:02 and C*14:03 monomers were folded with the peptide LYNTVATL (Gag-LL8). Surface plasmon resonance studies were performed by using a BIAcore3000 (GE Healthcare) as previously described (Dong et al., 2004; Thananchai et al., 2009). Experiments were performed at 25°C. HBS-EP buffer (GE Healthcare) was used as the running buffer.

KD values and kinetic were measured either by Scatchard plots or by curve fitting of the data to the Langmuir binding isotherm. All analysis was performed by using BIAevaluation software (version 4.1.1; GE Healthcare) and graphs were made by using Origin software (version 7; Microcal Software, Northampton, Massachusetts, USA).

Statistical analysis

We performed statistical analyses by using Prism (Graphpad Software) and Origin Software. For the comparison between two groups, we used Mann-Whitney U test. For the comparison between more than three groups, we used Kruskal-Wallis test. We performed logistical regression model with interaction term to analyze the synergistic effect of KIR/HLA combinations on pVL.

Supplementary Material

1
2

Acknowledgments

The authors thank Sachiko Sakai for her secretarial assistance and Yoshiko Tamura for technical assistance.

This research was supported by a grant-in-aid for AIDS Research (H24-AIDS-007) from the Ministry of Health, Labour, and Welfare and by a grant-in-aid (26293240, 25870550) for scientific research from the Ministry of Education, Science, Sports and Culture, Japan. This project has been funded in whole or in part with federal funds from the Frederick National Laboratory for Cancer Research, under Contract No. HHSN261200800001E. 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. This Research was supported in part by the Intramural Research Program of the NIH, Frederick National Lab, Center for Cancer Research.

Footnotes

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

AUTHOR CONTRI BUTIONS

Z.L. performed experiments, analysed data, and wrote the manuscript. K.K. and K.M. performed SPR analysis. N.K., X. S., H. M., and T.A. performed experiments of NK cell purification and culture. T.C., T. N., and M. K performed experiments on KIR typing and viral sequencing. H.G. and S.O. supplied samples and clinical data from patients. M.T. Y. Q. performed statistical analysis. M.C. performed statistical analysis and wrote the manuscript. M. T. designed the study, supervised all experiments, and wrote the manuscript. All authors revised and edited the manuscript.

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Supplementary Materials

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NIHMS827657-supplement-1.pdf (345.9KB, pdf)
2
NIHMS827657-supplement-2.docx (16.5KB, docx)

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