Certain individuals remain seronegative despite repeated exposures to HIV. The study of these exposed uninfected (EU) individuals may help identify features of natural resistance to this virus. A recent study of Vietnamese injection drug users (IDU) demonstrated that NK cells are more activated in EU than in seroconverters and low-risk controls . This suggests that NK cells may be involved in conferring protection from HIV infection seen in EU.
NK cells play an important role in innate immunity by providing protection against viruses and tumor cells . Their function is regulated by the integration of activating and inhibitory signals transmitted through cell surface receptors [3,4]. Killer Immunoglobulin-like receptors (KIR) are a family of activating and inhibitory receptors that participate in the activation of NK cells. Although the gene content of a KIR genome is highly variable, a few framework loci, such as KIR3DS/L1, are found on nearly all haplotypes . KIR3DS1 is an activating KIR gene with an unknown ligand and is allelic with KIR3DL1, an inhibitory receptor that binds to HLA Bw4 .
In HIV-infected individuals, co-expression of KIR3DS1 with a subset of HLA Bw4 alleles having an isoleucine at position 80 (HLA Bw4-I80) is correlated with slower disease progression . Given that NK cells may be involved in the resistance demonstrated by EU, we sought to determine whether either KIR3DS1 or HLA Bw4 or a combination of these alleles were also associated with protection from infection. Previous work examining NK receptor expression in EU from an African population and a Vietnamese IDU group found patterns reflecting increased NK activation potential compared with controls, but no increase in KIR3DS1 or link between KIR3DS1 and HLA Bw4-I80 [8,9]. The small sample size of the populations studied together with the frequency of KIR3DS1 expression in these populations, however, may have precluded detecting an association between KIR3DS1 expression with or without co-expression of HLA-Bw4 alleles and protection from infection [8,9]. Here we compared the distribution of KIR3DS/L1 and HLA B genotypes of a larger cohort of North American EU and subjects in HIV primary infection (PI) and report a significantly increased proportion of KIR3DS1 homozygosity in these EU versus the HIV-susceptible PI population.
Eighty EU and 304 individuals in PI were included in this study. Participants who were in PI were members of the Montreal PI cohort, which recruits individuals who are within 1 year of infection as determined using the criteria established by the Acute HIV Infection Early Disease Research Program sponsored by the National Institutes of Health and follows them over 2 years . EU were recruited from a prospective cohort of active HIV-negative IDU at high risk for HIV acquisition , and among HIV-negative partners of serodiscordant couples followed in medical clinics in Montreal . Subjects were followed longitudinally every 6 months. Follow-up included assessment of the frequency of high-risk behavior for HIV acquisition, blood draws and monitoring of HIV serostatus. All EU subjects maintained a negative HIV enzyme immunoassay (HIV EIA) test despite at least five documented exposures. Parenteral exposure was defined as sharing needles with known HIV-infected partners and mucosal exposure was defined as unprotected sex with a known HIV-infected partner. None of the EU subjects were CCR5Δ32 homozygotes. Informed consent was obtained from all study subjects, and the research conformed to ethical guidelines of all the authors' institutions.
Genomic DNA was extracted from peripheral blood mononuclear cells (PBMC) or Epstein–Barr virus (EBV)-transformed cells using a QIAamp DNA blood kit (QIAGEN, Inc., Mississauga, Ontario, Canada). KIR3DS/L1 genotyping was performed by polymerase chain reaction (PCR) with sequence-specific primers based on previously published studies [7,13]. Two pairs of specific primers for each locus were used for amplification of KIR3DL1 and KIR3DS1. Primers for NKG2A were also included as a positive control. All primer sequences and amplification conditions are available upon request. Subjects were typed for MHC class I expression using the line probe assay (Innogenetics Inc, Alpharetta, Georgia, USA) and by sequencing (Atria Genetics, South San Francisco, California, USA) if high resolution typing was needed to resolve the assignment of B alleles to the Bw4 or Bw6 public specificities based on amino acids at positions 77 to 83.
Statistical analysis and graphical presentation were performed using GraphPad InStat 3.05 and GraphPad Prism 4.01. Fisher's exact test was used to compare proportions of selected genotypes between EU and PI. A two-tailed chi-squared (χ2) test was used to examine differences in the distribution of genotypes between EU and PI subjects. Hardy–Weinberg equilibrium was evaluated using a χ2 test with frequencies of each allele derived from the combined population (KIR3DS1 = 0.22). A P-value of less than 0.05 was considered significant.
The EU and PI populations were mainly composed of Caucasians (93%) living in the same geographic region (Montreal) and there were no significant between-group differences in racial composition (data not shown). All individuals were genotyped for KIR3DS/L1 and MHC class IB locus alleles. Whereas the distribution of the three KIR3DS/L1 genotypes: KIR3DS1 homozygote (3DS1hmz), KIR3DS1/KIR3DL1 heterozygote (3DS/L1htz) and KIR3DL1 homozygotes (3DL1hmz) at this locus in the PI population did not deviate statistically from the Hardy–Weinberg (H–W) equilibrium (χ2 = 2.17, P = 0.1407), the same distribution in the EU population diverged from H–W equilibrium (χ2 = 13.20, P < 0.001). The deviation from H–W equilibrium of EU but not of PI was reflected in a statistically significant difference in the distribution of the 3 KIR3DS/L1 genotypes between the two groups (χ2 test, P = 0.0283, degree of freedom = 2, Table 1). When each of the frequencies for 3DS1hmz, 3DS/L1htz and 3DL1hmz were compared in EU versus PI subjects, we found that there was a significant increase of 3DS1hmz individuals in EU [odds ratio (OR) = 2.87, 95% confidence interval (CI) = 1.28–6.46, P = 0.0132] as shown in Table 1; however, the proportions of 3DS/L1htz and 3DL1hmz were similar. Thus these results suggest an increase in the proportion of individuals with the activating genotype KIR3DS1hmz in our EU population.
Although the known ligand for KIR3DL1 is HLA Bw4, a molecular interaction between KIR3DS1 and either HLA-Bw4 or HLA Bw4-I80 has yet to be demonstrated experimentally despite an association of the KIR3DS1 and HLA Bw4-I80 genes with slower HIV disease progression [7,14–16]. The possible interaction of the HLA Bw4 supertype or of the subset of alleles expressing an isoleucine at position 80 (HLA Bw4-I80) with KIR3DS/L1 prompted us to assess the distribution of these HLA alleles in EU and PI subjects. No significant differences or trends in the between group distribution of these alleles were found (Table 1).
We next examined whether there were between group differences in the distribution of KIR3DS/L1 genotypes in association with their known or putative HLA ligand (HLA Bw4 or HLA Bw4-I80). Figure 1a shows that the proportion of individuals having at least one copy of KIR3DS1 in the presence of HLA Bw4-I80 was similar in EU and PI subjects.
A recent study found an increased proportion of 3DL1hmz EU that were HLA Bw6 homozygotes (i.e. lacked the HLA Bw4 receptor for 3DL1) . As shown in Fig. 1b, however, we could not confirm this observation in our cohort. For all analyses testing associations of KIR3DS/L1 and HLA Bw4 or HLA Bw4-I80, no significant differences were found between EU and PI except those reflecting the increased frequency of KIR3DS1hmz in EU described above (data not shown). This suggests that HLA genotypes, alone or in combination with KIR3DS1, implicated in modulating the rate of HIV disease progression in infected subjects do not appear to be a factor in preventing HIV infection of the EU population studied here.
We have studied the distribution of KIR3DS/L1 genotypes and of their known or possible ligands in HIV-resistant EU and HIV-PI subjects. Subjects in PI were selected as a control population for these studies because they are susceptible to HIV infection and eliminate any effect that disease progression could have on skewing KIR or HLA genotypes. We have shown that KIR3DS/L1 genotypes were not in H–W equilibrium in EU due to an increased proportion of 3DS1hmz in these subjects. The presence of this activating KIR gene (or the absence of the inhibitory KIR3DL1) could contribute to the increased level of NK cell activation in EU described in previous studies [1,9,17]. Whether the KIR3DS1hmz genotype translates into more activated NK cell function remains to be demonstrated. It is unclear whether it is the absence of an inhibitory KIR3DL1 gene or the presence of two copies of KIR3DS1 that is more important for resistance to HIV infection. In the former case, expression levels of KIR3DL1 alleles, their inhibitory capacity or whether they are expressed at all (such as KIR3DL1*004) may modulate susceptibility to HIV in heterozygous individuals expressing one copy of KIR3DS1 [18–21]. A recent report provides support for a role for high expression KIR3DL1 allotypes in combination with HLA-Bw4 in protection from HIV disease progression . Selection of greater NK receptor inhibitory potential during the NK functional maturation process termed licensing may permit superior activation when signaling through the receptor is disrupted such as during viral infection [22,23]. Therefore an analysis of the distribution of KIR3DL1 allotypes in EU and PI subjects is warranted to determine whether these alleles also play a role in HIV resistance. It is also possible that two copies of KIR3DS1 are required for increased potency of NK cells for limiting virus spread before infection can become established. Finally, we cannot exclude that a gene in linkage disequilibrium with KIR3DS1 mediates the protective effect.
Although we cannot rule out that the size of our sample was too small to observe between group differences, we did not observe even a trend towards an effect of expression of MHC class I alleles HLA Bw4 or HLA Bw4-I80 either alone or in combination with KIR3DS/L1 genotypes when we compared our study groups. Although, interactions between KIR3DS1 and HLA-Bw4 molecules have not been experimentally demonstrated, it has recently been shown that KIR3DL1/HLA-Bw4 binding is improved by the presence of peptide [14,15,21,24]. In EU subjects, in whom a productive infection is never established, the impact of HIV peptide would have relevance limited to a time interval between transmission and control/clearance of infection. In this scenario, the interaction of KIR3DS/L1 with its ligand may be less important than possessing NK cells with a greater potential for activation in the context of all MHC class I types expressed on potential HIV infected donors. In addition, the impact of co-expression of KIR3DS1 and HLA Bw4-I80 on HIV disease progression has recently been called into question as some investigators have failed to demonstrate synergy between expression of these molecules in relation to markers of clinical HIV disease progression [25,26]. The difference between these results and those reported by Martin et al. may be explained by the differences in patient status, clinical outcome measures used as markers of disease progression or statistical methodology used to analyze results [7,25–27].
In an African EU population, female sex workers had decreased genetic pairing of an inhibitory KIR with its HLA ligand, which would reduce signaling through inhibitory receptors . For example, they found more KIR3DL1hmz/HLA Bw6hmz (i.e. Bw4 −/−) in their EU group than controls. Although this result was not confirmed in this study, the overall concept that potential for improved NK activation or reduced NK inhibition is linked with resistance to HIV infection is a consistent finding . Neither of the studies that have examined NK receptor patterns in EU found an increase in KIR3DS1hmz in the EU subjects [8,9]. The discrepancy between these findings and ours could be explained by differential frequency of KIR3DS/L1 in African, Vietnamese and North American populations but also by study population sample size.
We would like to acknowledge Dr M. Martin and Dr M. Carrington for providing the sequence for the primers used for KIR3DS/L1 genotyping, Dr G. Alter and Dr M. Altfeld for helpful discussion of the results, Mr Mario Legault and Mr Martin Rioux for coordination of the Montreal PI and IDU cohorts, respectively. We would also like to acknowledge the investigators of the Montreal PI cohort for subject recruitment and clinical follow-up.
Sponsorship: This study received support from the Canadian Institutes for Health and Research #MOP-79515, the Fonds de la Recherche en Santé du Québec (FRSQ). S.B. was supported by a PhD scholarship from the Canadian Network for Vaccines and Immunotherapeutics (CANVAC) Network Centre for Excellence. J.P.R. is a scientific scholar receiving support from FRSQ.
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