Cutting Edge: Resistance to HIV-1 Infection among African Female Sex Workers Is Associated with Inhibitory KIR in the Absence of Their HLA Ligands¹

The NK cells play an important role in the innate immune system by providing the first line of defense against viral infections and tumors (1). NK cell activity is partially controlled by distinct inhibitory and activating killer Ig-like receptors (KIR)⁴ that recognize specific ligands on potential target cells (2). NK effector functions occur only when inhibitory signals are overcome by activating signals. KIR are encoded by 15 polymorphic genes located on chromosome 19 and contain two or three external Ig-like domains (2D, 3D) with either long (2DL, 3DL) or short (2DS, 3DS) cytoplasmic tails, corresponding to their function as inhibitory or activating receptors, respectively (3). Several inhibitory KIR have well-defined HLA class I ligands. KIR2DL2 and KIR2DL3 bind to HLA-Cw group 1 (HLA-C1), which have asparagine at position 80, whereas KIR2DL1 binds the mutually exclusive HLA-Cw group 2 (HLA-C2) with a lysine at this position (4). KIR3DL1 binds HLA-B molecules with the serologically defined Bw4 epitope, specified by five variable amino acids spanning positions 77–83 (5). The alternative serotype, Bw6, is not known to bind any KIR. Despite high sequence similarity with inhibitory receptors, activating 2DS and 3DS KIR show either weak or undetectable binding to HLA class I (6, 7, 8).

The extreme levels of population diversity and rapid evolution of both KIR and HLA genes suggest that they are under pathogen-mediated selection (9). KIR and HLA loci map to separate chromosomes resulting in variation in the number and kind of KIR/HLA ligand pairs, potentially influencing disease outcome at the individual level. Indeed, specific KIR/HLA genotypes favoring NK cell activation were found to protect against disease progression after HIV-1 or hepatitis C virus infection (10, 11) and were associated with increased susceptibility to autoimmune disorders like psoriatic arthritis (12) and type I diabetes (13).

Rare individuals remain HIV-seronegative despite frequent unprotected exposure to the virus and several mechanisms of resistance have been proposed (HIV-exposed seronegative (ESN), reviewed in ref. 14). HLA polymorphism has been shown to play a role in protection against HIV infection (15, 16), but its mode of action remains incompletely resolved. In this study, we analyzed the genes encoding KIR and their HLA ligands in ESN female sex workers (FSWs), HIV-1-seropositive (SP) FSWs, and HIV-seronegative female blood donors (FBDs) from Abidjan, Côte d’Ivoire.

Twenty-one ESN and 20 SP FSWs were enrolled at a confidential FSW clinic in Abidjan, Côte d’Ivoire, between June 1998 and May 2000. The women were followed as part of a clinical trial testing the efficacy of a nonoxynol-9 microbicide gel (17). Twenty-five HIV-seronegative FBDs were enrolled at the blood transfusion center in Abidjan, Côte d’Ivoire. The study was approved by ethical committees of the Ministry of Health, Côte d’Ivoire, and the Institute of Tropical Medicine, Antwerp, Belgium, and by the Institutional Review Board of the Centers for Disease Control and Prevention, Atlanta, GA. All subjects gave written informed consent before enrollment.

Whole blood was drawn in EDTA tubes (BD Biosciences). Plasma was separated from whole blood by centrifugation and tested for HIV by ELISA and Western blot, and confirmed by HIV RT-PCR. PBMC were separated from whole blood by gradient centrifugation and stored in liquid nitrogen.

Genomic DNA was extracted from PBMC using a QIAamp DNA blood mini kit (Qiagen). KIR typing was performed with the PCR sequence-specific primer technique as previously reported (18). In the assessment of KIR genotypes, group A haplotypes were defined by the presence of KIR2DL1, KIR2DL3, KIR3DL1, and KIR2DS4. Group B haplotypes were defined by lack of KIR3DL1 and KIR2DS4 in the presence of inhibitory KIR2DL2 and KIR2DL5 and one or more of the activating KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS5, and KIR3DS1 (19). HLA-B and -C typing at the allele group level was performed by PCR sequence-specific oligonucleotides methodology (Tepnel Lifecodes).

Differences in continuous variables were analyzed with Mann-Whitney U tests. Fisher’s exact tests were used to calculate statistical significances and exact 95% confidence intervals (CIs) of odds ratios of allele frequency differences. Occurrence of trend was analyzed with the exact non-parametric Cochran-Armitage test. Level of statistical significance for all analyses was set at p < 0.05. Statistical analyses were performed with SAS version 9.1 (The SAS Institute).

Twenty-one ESN FSWs, 20 SP FSWs, and 25 FBDs were included in the study (Table I). First, we analyzed the frequencies of individual KIR genes and KIR genotypes. ESN FSWs showed significantly higher frequencies of inhibitory KIR2DL2 and KIR2DL5 than SP FSWs (Fig. 1,A). ESN FSWs also showed higher frequencies of activating KIR2DS2 and KIR2DS3, but the differences were not statistically significant (Fig. 1,B). All study subjects displayed KIR3DL1 and KIR2DS4 genes, therefore only AA and AB genotypes were present. ESN FSWs more frequently displayed AB genotypes, whereas SP FSWs more frequently displayed AA genotypes (Fig. 1,C). In general, FBDs showed frequencies of individual KIR genes and KIR genotypes in between those from ESN and SP FSWs (Fig. 1).

Next, we analyzed the presence of inhibitory KIR in association with their known HLA ligand genes (Tables II and III). First, we analyzed gene frequencies of the inhibitory receptors KIR2DL2 and KIR2DL3, which segregate as alleles of the same gene locus, together with their HLA-C1 ligand (Table II). There were significantly more KIR2DL2/KIR2DL3 heterozygotes among ESN FSWs, whereas SP FSWs showed a markedly higher proportion of KIR2DL3/KIR2DL3 homozygotes. There was a trend toward a higher frequency of HLA-C1/C2 heterozygotes among SP FSWs, potentially resulting in more inhibitory NK signals via KIR2DL receptors. More specifically, we found that the increased frequency of homozygous KIR2DL3 among SP FSWs was significant only if both HLA-C1 and -C2 were present. ESN FSWs retained an increased frequency of heterozygous KIR2DL2/KIR2DL3 only in the absence of their HLA-C1 ligand gene (i.e., C2/C2 homozygous). The trends for the KIR/HLA comparisons between ESN FSWs and FBDs were similar to those between ESN and SP FSWs, although they did not always reach statistical significance probably because of the low sample size.

Next, we analyzed frequencies of inhibitory KIR3DL1 and activating KIR3DS1, also alleles of the same gene locus, together with the HLA-Bw4 ligand KIR3DL1 (Table III). KIR3DL1 and KIR3DS1 frequencies were similar among ESN and SP FSWs. SP FSWs tended to be more frequently homozygous for HLA-Bw4, on its own and in combination with KIR3DL1, potentially resulting in more NK inhibition via KIR3DL1. A significantly higher proportion of ESN FSWs who were homozygous for KIR3DL1 lacked HLA-Bw4 (i.e., Bw6 homozygous), resulting in the absence of NK inhibition via KIR3DL1. Here, the trends for the KIR/HLA comparisons between ESN FSWs and FBDs did not confirm those between ESN and SP FSWs: similar proportions of ESN FSWs and FBDs were HLA-Bw4 and KIR3DL1/HLA-Bw6 homozygous. None of the subjects included in the study were homozygous for KIR3DS1 (Table III). Slow progression toward AIDS was previously associated with KIR3DS1 in the presence of HLA-Bw4 alleles with isoleucine at position 80 (Bw4–80Ile) (10). In our study, frequencies of Bw4–80Ile were similar among ESN, SP FSWs, and FBDs. Compared with ESN FSWs, SP FSWs displayed a higher proportion of the alternative HLA-Bw4 allele with a threonine at position 80 (p = 0.028). No statistically significant interactions were observed between KIR3DS1 and HLA-B alleles, possibly as the result of the low frequency of KIR3DS1 in this population (12% compared with 42% among healthy Caucasians; see Ref. 18). No correlations were found between the observed KIR2DL/HLA-C and KIR3DL1/HLA-B interactions (data not shown).

At the time of sample collection, HIV-1 seroprevalence was 32% among FSWs (20), and 14% among their clients (21). HIV-1 seroincidence among FSWs participating in the nonoxynol-9 trial was 4% (17). Using self-reported data, ESN FSWs in Abidjan were estimated to have on average 52 unprotected exposures to HIV-1 per year (22). None of the FSWs tested in Abidjan showed the CCR5 32-bp deletion (23), which is in agreement with other populations in Africa (24). Together, this suggests that ESN FSWs in Abidjan are at high risk for acquiring HIV-1 infection. ESN FSWs enrolled in this study reported a median duration of commercial sex work of 3.5 years, ranging from as short as 2 mo to as long as 20 years (Table I). Putative HIV-1 resistance factors may be selected for in ESN subjects with a longer history of high-risk behavior. To test this, we correlated the observed KIR/HLA associations with the duration of commercial sex work of the ESN FSWs. For the KIR and KIR/HLA combinations that reached statistical significance in Tables II and III, we observed a statistically significant trend over SP and ESN FSWs who had done more and <3 years of commercial sex work (Fig. 2).

In this study, the ESN status of FSWs was associated with the occurrence of certain inhibitory KIR genes in the absence of their cognate HLA genes: KIR2DL2/KIR2DL3 heterozygosity in the absence of HLA-C1, and independent from this, KIR3DL1 homozygosity in the absence of HLA-Bw4. Together, 38% of ESN FSWs showed at least one such KIR/HLA mismatch compared with 0% of SP FSWs (p = 0.006). Absence of HLA ligands for inhibitory KIR may lower the threshold for NK cell activation via activating KIR, resulting in NK cytotoxic activity and early elimination of HIV-infected cells. In agreement with this, a higher frequency of ESN FSWs possessed AB KIR genotypes, which contain a higher number of activating KIR. In that respect, our data corroborate a recent study showing increased NK cell-mediated cytotoxicity and cytokine and β-chemokine secretion among ESN intravascular drug users (25). In contrast with this, SP FSWs were characterized by corresponding inhibitory KIR/HLA ligand pairs that can directly inhibit NK cell effector functions: KIR2DL3 homozygosity in the presence of heterozygous HLA-C1/C2, and independent from this, a trend toward KIR3DL1/HLA-Bw4 homozygosity. Together, 61% of SP FSWs showed at least one such inhibitory KIR/HLA pair compared with 19% of ESN FSWs (p = 0.009). In addition, SP FSWs more frequently showed AA KIR genotypes that contain lower numbers of activating KIR.

Although SP FSWs showed a wide range of CD4 counts (median, 437 cells/μl; range, 144-1712 cells/μl; Table I), it is unclear to what extent rapid and slow progressor patients were equally represented; this cannot be known in a cross-sectional study. Thus, it cannot be ruled out that KIR/HLA combinations related to differential disease progression in the SP group have influenced our results. However, although less pronounced, the KIR and HLA frequency differences among ESN and SP FSWs were generally confirmed by comparisons between ESN FSWs and FBDs. This supports the conclusion that the observed associations are related to HIV resistance rather than differential HIV disease progression.

Despite large gene frequency differences, the small sample size resulted in KIR/HLA interactions with relatively weak statistical significance. Because of the exploratory nature of the study, the testing of distinct hypotheses rather than a comprehensive screening for genes and associations, and restriction of HLA-B and -C allele analysis to four distinct KIR binding groups, correction for multiple testing was not applied. Exact testing for small sample sizes was performed for all analyses. Nevertheless, confirmation of our findings in larger populations of healthy vs HIV-infected subjects, and in functional studies, is needed.

Our data are the first to show that KIR/HLA interactions may influence susceptibility to virus transmission. Moreover, the KIR/HLA gene combinations favoring NK activation over inhibition by lack of HLA ligands for inhibitory KIR may be more straightforward than those previously described for protection against viral disease progression. Indeed, delayed onset of AIDS was associated with the activating KIR3DS1 receptor in combination with its only presumptive HLA-B Bw4–80Ile ligand (10). Resolution of hepatitis C virus infection was found to be associated with corresponding KIR2DL3/HLA-C1 gene combinations, only putatively resulting in the weakest inhibitory signals (11).

The non-classical HLA class I molecules HLA-E and -G are ligands for the inhibitory NK cell receptors CD94/NKG2A and KIR2DL4, respectively. A recent study found genetic variants of HLA-E and HLA-G with a potentially lower affinity for their inhibitory NK cell receptors to be associated with a decreased risk of HIV transmission (26). These findings further support a role for NK cells in protection against virus transmission and suggest that parallel inhibitory NK receptor/HLA ligand mechanisms may be at play.

We thank the community of female sex workers in Abidjan for their cooperation, and Joris Menten and Odette Tossou for help with data analysis.

The authors have no financial conflict of interest.