of April 10, 2019.This information is current as

with an AIDS-Protective PolymorphismRestricted CTL Selective Pressure Associated

−with HLA-C Suggests Enhanced HLA-CHigh Frequency of HIV Mutations Associated

Rowland-JonesIan James, Mina John, Tao Dong and Sarah L.Griffin, Stephen Taylor, Keyi Xu, Huiping Yan, Hao Wu, Marie-Eve Blais, Yonghong Zhang, Tim Rostron, Harry

ol.1103472http://www.jimmunol.org/content/early/2012/04/02/jimmun

published online 2 April 2012J Immunol 

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The Journal of Immunology

High Frequency of HIV Mutations Associated with HLA-CSuggests Enhanced HLA-C–Restricted CTL SelectivePressure Associated with an AIDS-Protective Polymorphism

Marie-Eve Blais,* Yonghong Zhang,*,† Tim Rostron,‡ Harry Griffin,* Stephen Taylor,x

Keyi Xu,{ Huiping Yan,† Hao Wu,† Ian James,‖ Mina John,‖ Tao Dong,†,‡,1 and

Sarah L. Rowland-Jones*,1

Delayed HIV-1 disease progression is associated with a single nucleotide polymorphism upstream of theHLA-C gene that correlates

with differential expression of the HLA-C Ag. This polymorphism was recently shown to be a marker for a protective variant in

the 39UTR of HLA-C that disrupts a microRNA binding site, resulting in enhanced HLA-C expression at the cell surface. Whether

individuals with “high” HLA-C expression show a stronger HLA-C–restricted immune response exerting better viral control than

that of their counterparts has not been established. We hypothesized that the magnitude of the HLA-C–restricted immune

pressure on HIV would be greater in subjects with highly expressed HLA-C alleles. Using a cohort derived from a unique narrow

source epidemic in China, we identified mutations in HIV proviral DNA exclusively associated with HLA-C, which were used as

markers for the intensity of the immune pressure exerted on the virus. We found an increased frequency of mutations in

individuals with highly expressed HLA-C alleles, which also correlated with IFN-g production by HLA-C–restricted CD8+

T cells. These findings show that immune pressure on HIV is stronger in subjects with the protective genotype and highlight

the potential role of HLA-C–restricted responses in HIV control. This is, to our knowledge, the first in vivo evidence supporting

the protective role of HLA-C–restricted responses in nonwhites during HIV infection. The Journal of Immunology, 2012, 188:

000–000.

Several host genetic determinants can influence HIV repli-cation and immune responsiveness, thereby having animpact on disease progression. Three genome-wide asso-

ciation studies have confirmed the central role of the HLA locus in

HIV disease progression by identifying two single nucleotidepolymorphisms (SNPs) associated with steady-state plasma HIVRNA levels (1–3). One is in strong linkage disequilibrium (LD)with HLA-B*5701, an allele strongly associated with goodclinical outcome of HIV infection. The second SNP lies 35 kbupstream of the HLA-C locus and is associated with increasedexpression of both HLA-C mRNA and protein at the cell surfacein subjects homozygous for the protective “C” allele (1, 4). Al-though previous studies have reported that 235 SNP is in LD withHLA-B*5701, several groups have shown that 235 SNP inde-pendently associates with HIV control (1, 4, 5). The 235 SNP isalso in strong LD with a variant found in the 39UTR of the HLA-Cgene, which was recently shown to regulate binding of themicroRNA miR-148a to its target site, thereby altering HLA-Cexpression (6). This strongly suggests that HLA-C expressionlevels play a direct role in HIV-1 control, most probably throughthe mobilization of an HLA-C–restricted immune response.HLA-C is interesting in the context of HIV infection as HIV-

negative replication factor protein selectively downregulatesHLA-A and HLA-B while maintaining HLA-C and HLA-E ex-pression at the cell surface (7). Therefore, highly expressed HLA-Calleles could trigger HIV-specific responses through either in-creased Ag presentation to cytotoxic CD8 T cells (CTLs), bindingto killing inhibitory receptors (KIRs) on NK cells, or a combina-tion of these mechanisms. The fact that HLA-C–restricted CTLsexert pressure on HIV is an indication of their effectivenessin vivo, and there is a growing body of evidence for the role ofHLA-C–mediated responses in other viral infections (4, 6, 8–10).This implies that HLA-C–restricted responses play an underap-preciated role in HIV control. The protective effect of the 235SNP was clearly shown in whites, but, with the exception of onestudy that showed no protection in African Americans, there are

*Nuffield Department of Medicine, John Radcliffe Hospital, Oxford OX3 9DS,United Kingdom; †Beijing You’An Hospital, Capital Medical University, Beijing100069, People’s Republic of China; ‡Medical Research Council Human Immunol-ogy Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Ox-ford OX3 9DS, United Kingdom; xComputational Biochemistry Research Group,Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX39DS, United Kingdom; {Beijing Ditan Hospital, Capital Medical University, Beijing100015, People’s Republic of China; and ‖Centre for Clinical Immunology and Bio-medical Statistics, Institute for Immunology and Infectious Diseases, Murdoch Uni-versity and Royal Perth Hospital, Perth, Western Australia 6155, Australia

1T.D. and S.L.R.-J. contributed equally to this work.

Received for publication December 5, 2011. Accepted for publication February 29,2012.

This work was supported by Medical Research Council UK, the Li Ka Shing Founda-tion, Royal Society UK, the Beijing Natural Science Foundation (7111005), the BeijingTalents Building Projects (PYZZ091016001765), the Beijing Fengtai Health Bureauand Beijing Municipal Science & Technology Commission (D09050703590904,D09050703560903, and D09050703590901), and the China National Science & Tech-nology Key Program (2012ZX10001-006). M.-E.B. holds a postdoctoral fellowshipfrom Fonds de la Recherche en Sante du Quebec, and Y.Z. was funded by the Drs.Richard Charles and Esther Yewpick Lee Charitable Foundation.

Address correspondence and reprint requests to Dr. Sarah L. Rowland-Jones, NuffieldDepartment of Medicine, John Radcliffe Hospital, Oxford OX3 9DS, U.K. E-mailaddress: sarah.rowland-jones@ndm.ox.ac.uk

The online version of this article contains supplemental material.

Abbreviations used in this article: A-mut, HLA-A–associated mutation; ART,antiretroviral treatment; B-mut, HLA-B–associated mutation; C-mut, HLA-C–associatedmutation; ICS, intracellular cytokine staining; KIR, killing inhibitory receptor; LD,linkage disequilibrium; mVL, mean plasma viral load; SNP, single nucleotide poly-morphism.

Copyright� 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00

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no data available from other ethnic groups (11). As geneticdeterminants vary among genetic backgrounds, it is crucial toassess the impact of this variant in additional populations to un-derstand how HLA-C–restricted immune responses might con-tribute to HIV control.We studied the protective effect of 235 SNP in a unique cohort

of former plasma donors in rural China where subjects remainedfree of antiretroviral therapy (ART) in the first 9–10 y of infectionwith an unusually narrow source founder clade B9 HIV-1 strain(12). These subjects are likely to have been infected through thesame route and in a short time frame, thereby providing an ex-ceptional setting in which most usual cohort variables are con-trolled and where the prevalence of known protective HLA allelesis very low. We investigated whether slow disease progressionassociated with 235 SNP correlates with a stronger HLA-C–re-stricted immune response in this cohort as a result of increasedHLA-C expression. As the generation of CTL escape variantsfollows stereotypic mutational pathways based on the HLA re-striction of CTL epitopes, we were able to identify HLA–HIVpolymorphism associations that reveal how much cellular immunepressure is inflicted on the virus (13). We used this method toidentify HIV mutations that were associated with immune pres-sure from HLA-C alleles and used these as markers for HLA-C–mediated responses. We report that 235 SNP correlates withdelayed disease progression in the Han Chinese population.Moreover, we observe a significant increase in the frequency ofHLA-C–associated mutations in the proviral DNA of patients withthe protective variant, which could indicate stronger HLA-C–re-stricted immune responses in vivo. To our knowledge, these arethe first functional data about the in vivo effect of 235 SNP onHIV evolution. This study provides a new perspective to under-standing the mechanisms of immune protection during HIV in-fection and could potentially lead to the identification of HLA-C–restricted immune responses associated with viral control in HIV-infected individuals.

Materials and MethodsPatients

In the mid-1990s, many Han Chinese residents of an isolated rural com-munity in China were involved in an illegal plasma donation scheme. Tenyears later, an HIV screening program revealed that 324 villagers were HIV-1infected. We estimate that 473 former plasma donors in the village ac-quired HIV-1 infection on the basis of reports of 149 premature adult deathswith symptoms compatible with AIDS. HIV-1 transmission probably oc-curred through contaminated blood collection equipment or after the returnof pooled red cells to donors. Epidemiological and phylogenetic analysesstrongly suggest that all cohort members were infected with an unusuallynarrow source virus by the same route during the same time period (12).Members of the cohort were not aware that they had been infected for thefirst 10 y of infection, therefore information on the infected subjects anduninfected former plasma donors during that period is limited. In 2005,samples from 288 former plasma donors with chronic clade B9 HIV-1infection were collected, and subsequent patient recruitments were orga-nized on a yearly basis. Because of the very narrow source virus in thecohort, genetic divergence in the subjects reveals how much viral diversityat the population level is attributable to host factors. Preliminary analysesshowed that HLA-B*57 and HLA-B*27 are not enriched in this cohort oflong-term nonprogressors (M.A. Rai et al., manuscript in preparation).Most viral sequences, CD4 count, and HLA-typing data were derived fromsamples collected in 2005, before the onset of ART. For some patients,viral load data were not collected until 2007, but are only presented fromART-naive subjects. Clinical data were collected from randomly selectedsamples. CD3+/CD4+/CD8+ T lymphocyte percentages and true countswere determined by flow cytometry as follows: 200 ml of heparin-anticoagulated fresh whole blood was analyzed using the BD MultiTestIMK kit (BD Biosciences) and MultiSet software (BD Biosciences)according to the manufacturer’s instructions. Briefly, multitest Abs weremixed with the sample and incubated for 15 min in the dark at roomtemperature before being transferred to BD True count tubes. Four hundred

fifty microliters of 13 BD Multitest lysing solution was added to the tubeand incubated for another 15 min in the dark at room temperature. Finally,CD3+/CD4+/CD8+ T lymphocyte percentages and true counts were cal-culated using MultiSet software. HIV-1 plasma viral load was quantifiedby nucleic acid sequence-based amplification in Beijing You’An Hospital.An initial enzymatic amplification of the nucleic acid targets was followedby amplicon detection. Assay lower limit of detection was 50 RNA copies/ml, and levels below detection were assigned an arbitrary value of 25copies/ml. Ethical approval was obtained from Beijing You’An Hospitaland the University of Oxford Tropical Ethics Committee. HIV-1 gag, pol,and nef sequencing and HLA genotyping was performed on 288 samples aspreviously described (14, 15). For the analysis described in Fig. 1C, 256samples from HIV-negative Han Chinese residents of a nearby village wererandomly collected and used as a control cohort. In total, 324 HIV-infectedsubjects were included in this analysis. This population is in Hardy–Weinberg equilibrium (p = 0.65).

235 SNP genotyping

The 235 SNP (rs9264942) genotyping was performed as previously de-scribed (4). Two PCR reactions (“C” and “T” reactions) were set up foreach sample, and standard amplification conditions were used. In additionto allele-specific primers, each reaction contained control DRB1-specificprimers, which allowed failed reactions to be identified. Nucleotides werepurchased from GE Healthcare (Illustra range) and Taq from Bioline.Outliers were retyped by sequencing. Primer sequences are available onrequest. Genotyping data were generated for 324 members of the cohort;64 patients showed HLA-C–associated mutations (C-mut) (seven subjectshad two or more C-mut). Subjects for which viral sequences or genotypingdata were not available were excluded from the study with the exception ofthe case-control analysis shown in Fig. 1C.

Phylogenetic stratification of HLA allele–HIV-1 polymorphismassociations

HLA associations were determined as previously described (13). Wepreviously showed that.50% of amino acid substitutions in HIV gag, pol,and nef were strongly associated with HLA class I molecules and are thusattributable to T cell pressure (12). In this study, we selected nine sub-stitutions in pol, gag, and nef exclusively associated with HLA-C. Threemutations were excluded from the analysis as they were less than threepositions away from substitutions also associated with HLA-A or HLA-Balleles or found in fewer than three subjects. The six remaining mutationswere found in individuals expressing the corresponding HLA-C allele.Only strong associations (0.100 , q-value . 0.250) were included in theanalysis (Fig. 2).

T cell assays

CTL response specific for the VL8 (VIPMSFAL) epitope was identified inpatient 043 (expressing HLA-Cw*01:02) by IFN-g ELISPOT assay using18-mer consensus peptides. Plates were read on an AID plate reader, andbackground was subtracted from wells. AVL8-specific short-term CTL linewas generated as described previously and subsequently used for intracel-lular cytokine staining (ICS) (14). ICS was performed using Abs for IFN-gand TNF-a and the Cytofix/Cytoperm kit (Becton-Dickinson Europe) ac-cording to the manufacturer’s instructions. 721.221-HLACw*01:02 trans-fectants (obtained from Peter Parham, Stanford University) were pulsedwith VL8 peptide to restimulate the CTL line when performing ICS as wellas for maintenance. Untransfected 721.221 cells were used as a negativecontrol.

Statistics

Data were analyzed using the R software (R Development Core Team,2008). The following tests were used when appropriate: t test, x2 test,Kruskal–Wallis rank sum test, Fisher’s exact test, and test for Hardy–Weinberg equilibrium. The p values ,0.05 were considered significant.

ResultsStudy population

A total of 324 HIV-1–infected individuals from a geographicallyisolated rural community in central China who were exposed toHIV-1 through contaminated blood in the early 1990s wererecruited from 2005 to 2010 (12). Members of this cohort areformer plasma donors who are likely to have been infected witha single or very narrow range of HIV-1 strains by the same in-

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fection route in a 2-y period (12). All patients enrolled in thecurrent study made frequent plasma donations in the early 1990sand have survived without ART during the first 10 y of infectionbut have since progressed to diverse disease outcomes. Very littleinformation is available about individuals who may also haveacquired HIV through plasma donation but who subsequentlyprogressed to AIDS and died before recruitment started. Bloodsamples were collected in 2005, and 235 SNP genotyping datawere generated for all cohort members and were analyzed in re-lation to clinical details, proviral sequences, and HLA typing. Thisethnically homogeneous population infected with a narrow rangeof HIV-1 clade B9 strains provides an unparalleled opportunity todiscern how immune factors drive viral evolution without theconfounding effect of ART.

235CC associates with better clinical outcome than that of235TT in the Han Chinese

Previous reports showed that the 235 SNP associates with lowviral set point and maintenance of peripheral CD4 cells, resultingin slow progression to AIDS and death (1, 4). To determinewhether 235CC was also protective in the Han Chinese, eachindividual was categorized on the basis of the genotype at 235:CC, CT, or TT (Table I). In a comparison of the three genotypes inrelation to mean plasma viral load (mVL), we observed a trendtoward lower mVLs for 235CC subjects than for 235TT indi-viduals (Fig. 1A). The effect of 235 SNP on viremia was alsoobserved when subjects were grouped according to their mVLs,where 67% of TT individuals had mVL . 10,000, whereas only38% of CC subjects had uncontrolled viremia, which is inagreement with previous studies (Fig. 2A) (4). We next deter-mined whether the variant also had an effect on peripheral CD4+

T cell numbers. We found that 235 SNP had an effect on pro-gression, as CC subjects had a median CD4 count of 430 com-pared with only 267 for TT individuals (Fig. 1B). This differencewas not statistically significant, nevertheless the effect of 235CCon CD4 counts was considerable as the majority of individualswith normal CD4 counts (.500) had the 235CC genotype,whereas most noncontrollers with counts ,200 were 235TT (Fig.2B). This was also observed when a cutoff of ,350 (the currentthreshold for ART initiation) was used (Fig. 2B). Of note, indi-viduals with low steady-state viremia exhibited significantlyhigher CD4 counts than those of subjects with high mVL, sug-gesting that AIDS progression was delayed in CC subjects (Fig.2C). To test further the inference that 235 SNP is protective, weassessed whether the CC genotype associated with slow diseaseprogression in this population by comparing the 235 genotypefrequencies in the cohort to that of an HIV-negative control cohortfrom a nearby village (Table I). We hypothesized that many HIV-infected TT subjects would have succumbed to HIV disease whenART was not available, resulting in an enrichment of the protec-tive 235CC genotype. Indeed, we found a statistically significantincrease (p = 0.049) in the relative frequency of CC subjects in theHIV-positive cohort (Fig. 1C and Table I). These results suggestthat CC individuals progress more slowly to AIDS than their TT

counterparts. As no samples were collected for the fast pro-gressors who died during the first 10 y, it is impossible to confirmthat those individuals were 235TT. This might also explain whyclinical data analyses did not reach statistical significance (Fig.1A, 1B). Nevertheless, our results are in agreement with twoprevious studies and show that the 235CC genotype associateswith better clinical outcome than that of 235TT in the Han Chi-nese population.

Individuals with the protective 235CC genotype are morelikely than 235TT subjects to show mutations associated withHLA-C in HIV proviral DNA

As 235 SNP correlates with increased HLA-C expression at thecell surface, we assessed whether the variant was also associatedwith a stronger HLA-C–restricted immune response that couldpotentially lead to viral control. We hypothesized that, as HLA-C–restricted responses contribute to HIV evolution, the frequency ofC-mut in proviral DNA should correlate with the magnitude of theimmune pressure exerted on the virus in vivo (13, 16). Therefore,a potent HLA-C–restricted immune response should result in moreC-mut in HIV DNA in CC than in TT subjects. We determinedwhich C-mut were exclusively associated with HLA-C allelesand used those C-mut as markers for HLA-C–restricted immunepressure (12). A total of six mutations in HIV gag, pol, and nefwere selected according to the following criteria: 1) significantassociation with an HLA-C allele; 2) no association with anyHLA-A or HLA-B alleles; and 3) found in at least three individ-uals expressing the corresponding HLA-C allele (Fig. 3). Notably,three mutations lay within previously defined HLA-C–restricted

Table I. Genotypes of patients at 235

Study Group % TT (n) % CT (n) % CC (n) Total, n

HIV2 25.3 (65) 48.8 (125) 25.8 (66) 256HIV+ 19.3 (63) 49.8 (162) 30.8 (99) 324A-mut 17.9 (25) 52.1 (73) 30.0 (42) 140B-mut 21.6 (27) 48.0 (60) 30.4 (38) 125C-mut 2.1 (3) 25.0 (35) 18.6 (26) 64

FIGURE 1. Protective effect of the 235 SNP in the Han Chinese pop-

ulation. (A) Mean viral load (log10 cp/ml) and (B) median CD4 counts

(cells/ml blood) in HIV-infected individuals. Patient samples were grouped

based on their genotype at 235. Statistical analyses were performed using

the Kruskal–Wallis rank sum test and a two-tailed t test. (C) Enrichment of

the 235CC genotype in the HIV-positive cohort. The HIV-negative cohort

was used as baseline, and relative frequencies of each genotype were

compared with those of the HIV-positive cohort. Statistical analysis was

performed using a x2 test. The p value applies to a comparison of CC and

TT rates in cases (HIV+) versus controls (HIV2). Numbers are listed in

Table I.

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CTL epitopes. To verify that 235CC associated with C-mut, eachindividual was categorized by 235 genotype, and the frequenciesof the six mutations were compared. Taking into account thatsome individuals showed more than one mutation in their proviralDNA, we found that the frequency of C-mut was higher in thepresence of the CC variant (29%) than with the TT genotype (4%).Only 3 C-mut occurred in 63 TT subjects as opposed to 29 in 99CC patients (29%). These results suggest that HLA-C–restrictedimmune pressure is stronger in the presence of 235CC, the ge-notype associated with high HLA-C levels (Fig. 4A). Moreover,when patients showing C-mut were compared with the rest of thecohort, CC individuals were five times more likely than TT sub-jects to exhibit C-mut (Fig. 4B). This finding could be a directresult of enhanced HLA-C expression or reflect LD with protec-

tive HLA-B or HLA-A alleles. As previously reported by Thomasand collaborators (4), we found that 235 SNP is in LD withcertain HLA-C alleles (Fig. 5A). However, we found no correla-tion between the frequencies of mutations associated with HLA-A(A-mut) or HLA-B (B-mut) alleles and235CC (Fig. 4C and TableI). All HLA class I associations were more frequent in CC than inTT subjects; however, the relationship only achieved significancefor C-mut (p = 0.01). Of note, the total numbers of A-mut and B-mut were distinctly higher than the number of C-mut (Table I).This is consistent with previously published data that showed thatCTL responses restricted by HLA-A and HLA-B induce strongerimmune pressure than that by HLA-C–restricted responses (17).Moreover, HLA-C alleles and subtypes are still poorly charac-terized, which could have resulted in some C-mut being over-looked. Nevertheless, these figures indicate that 235 SNP specifi-cally correlates with C-mut, an association that could not beindirectly attributed to the presence of protective HLA alleles suchas HLA-B*51, which is enriched in subjects showing C-mut (Fig.5B). When HLA-B*51 individuals were excluded from the anal-ysis, we found that the C-mut frequency was still significantlyhigher in patients with 235CC, indicating that LD with this allele,known to be protective in Asian populations, is unlikely to accountfor the observed association (Fig. 5C) (16). Taken together, theseresults show that C-mut are more frequent when HLA-C expres-sion is enhanced (235CC), suggesting that these mutations wereinduced by a potent HLA-C–restricted response in vivo. This isconsistent with a previous report showing that HLA-C–mediatedimmune responses may restrain the virus to some extent, as Nefvariants isolated from 235CC subjects interfere with CD4+ Thcell activation and MHC class II Ag presentation, possibly tothwart HLA-C–mediated immune control (18).

C-mut give rise to escape variants in subjects exhibitingHLA-C–restricted CTL responses

We next determined whether T cells were responsible for inducingC-mut in CC subjects. The fact that three of six C-mut (C-mut no.1, no. 2, and no. 5) were found in regions coding for CTL epitopessuggests that these substitutions have been induced by CTL

FIGURE 2. 235CC subjects have lower vire-

mia and higher CD4 counts than those of TT

individuals. Groups were defined according to

the frequencies of subjects with (A) HIV-1 viral

load ,2000 or .10,000 viral RNA copies per

milliliter plasma and (B) CD4 counts ,200,

,350, and .500 cells/ml blood, which represent

different clinical stages of HIV-1 disease pro-

gression. (C) Subjects with mVL ,2000 exhibit

higher CD4 counts than those of individuals with

mVL . 10,000. Each dot represents a patient.

Statistical analysis was performed using a two-

tailed t test. p = 0.004.

FIGURE 3. Selection of C-mut. Numbers of patients showing C-mut

(black) or not (white). Six mutations associated with HLA-C exclusively

were found in individuals expressing the corresponding HLA-C alleles.

Substitutions and LD between associated HLA-C alleles and 235 SNP are

shown below the graph. For substitutions found inside known epitopes, the

epitope sequences are shown above the corresponding bars. C-mut no. 3

(KWSKCSMIGWPRVRERMR), no. 4 (TAPPEESFRFGEETTTPSQK),

and no. 6 (WQLDCTHVEGKIILVAVH) were found in unpublished epi-

topes.

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pressure (Fig. 3) (19, 20). To verify that a T cell response waspresent in patients expressing HLA-C alleles associated with eachC-mut, we performed ELISPOT assays using consensus 18-merHIV peptides spanning regions where C-mut had been mapped(Figs. 5, 6A). Peptide-specific IFN-g responses were observed forall tested peptides, showing that these nonadapted sequences areimmunogenic and presumably contain CTL epitopes. No T cellepitopes have previously been reported in the region where C-mutno. 3, no. 4, and no. 6 were found, but, as only a few HLA-C–restricted CTL epitopes have been mapped, particularly in non-white populations, those C-mut are likely to mark new HIVepitopes subject to CTL selection (Fig. 4A). We and others havepreviously confirmed that HLA associations can be used to pre-dict new T cell epitopes (12, 21, 22). To confirm the role of HLA-Cas a T cell restriction element and mediator of CTL activation,a short-term CTL line was generated using the consensus VL8peptide (VIPMSFAL) and frozen PBMCs from a Cw*01:02 pa-tient exhibiting the S173T substitution (C-mut no. 1). Using721.221-HLACw*01:02 transfectants as APCs only able to ex-press HLA-Cw*01:02, we performed ICS and found that TNF-aand IFN-g production by CTLs is dependent on Cw*01:02 ex-pression (Fig. 6B). We next verified that this response was

peptide-specific and that the S173T substitution was an escapemutation in a peptide titration assay. The peptide containing theS173T substitution was less well recognized than the nonadaptedpeptide. Notably, a statistically significant difference was found ata peptide concentration of 0.5 mg/ml, indicating that the mutationled to an escape variant (Fig. 6C). In addition, we performeda peptide titration using PBMCs from an HLA-Cw*08 patientresponding to a consensus peptide where no CTL epitopes hadpreviously been mapped and confirmed that the peptide containingthe T470A substitution (C-mut no. 4) was more weakly recognizedthan the wild-type peptide, indicating that T470A was also anescape variant (Fig. 6D). Escape mutations in highly conservedepitopes can have a beneficial effect for the patient as shown in thecontext of HLA-B*5701 KW10 epitope (23). In contrast, escapemutations can result in viral adaptation and lead to disease pro-gression, as in the case of the immunodominant HLA-B*51 CTLresponse (24). We did not assess whether those escape mutationswere beneficial for the patients, but rather used them as markersfor the intensity of the immune response.We next assessed whether C-mut were the result of NK cell

interactions with peptide–HLA-C complexes. As KIR data are notyet available for the cohort, we categorized each individual basedon the two major KIR–ligand categories: C1 and C2 (similar to theBw4 and Bw6 determinants). A weak interaction between an in-hibitory KIR and the HLA-C molecule, such as the one betweenKIR2DL3 and C1 alleles, can easily be overcome and lead to NKcell activation (25). We found no association between the distri-bution of C1 or C2 alleles and 235 SNP, indicating that neither ofthe KIR2ligand groups is in LD with 235CC (Supplemental Fig.1). Although these data do not exclude a role for NK cells in thecontrol of viral replication in the presence of the variant (for whichKIR typing data are required), our results suggest that the C-mutare unlikely to be induced by NK cell pressure. Taken together,these results strongly suggest that C-mut are induced by HLA-C–restricted CTLs, which might therefore play a role in the protec-tive effect of the 235 SNP.

The binding site for miR-148a/miR-148b is disrupted in235CC subjects

There has been some controversy about the biological relevance of235 SNP and its association with differential cell-surface ex-pression of HLA-C alleles. It was reported that HLA-C expressionis not always significantly higher in 235CC subjects and does notsystematically correlate with lower viremia, suggesting that 235SNP is just in strong LD with protective HLA alleles and does notdirectly mediate any protective effect (26). Nonetheless, a recentreport supports the initial notion that 235 SNP associates withsurface HLA-C expression. Another variant located in the 39UTRof HLA-C promotes high surface expression of HLA-C alleles,which subsequently escapes posttranscriptional regulation by mi-croRNAs (6). A deletion in position 263 of the 39UTR (263del)results in a disrupted binding site for miR-148a/miR-148b, whichleads to increased cell-surface HLA-C expression. Therefore,HLA-C alleles can be categorized based on the presence of the263del, which correlates with increased levels at the cell surfaceand associates with low mVL (,2000). Importantly, the 39UTRvariant is in strong LD with 235 SNP as the majority of 235C-associated HLA-C alleles exhibit the 263del, whereas most 235Talleles have an insertion at position 263 (263ins). In line with this,we investigated whether the protective effect of 235 SNP alsocorrelated with the 39UTR variant in the Han Chinese. We foundthat subjects homozygous for 263del (del/del) had lower mVL andhigher CD4 counts than those of subjects homozygous for “low”HLA-C alleles with the 263ins/ins genotype (Fig. 7A, 7B).

FIGURE 4. Association between C-mut and 235 SNP. (A) Increased

frequency of C-mut in presence of the CC genotype. Subjects were cate-

gorized in three groups based on their genotype at 235 (TT, CT, and CC).

The total numbers of C-mut in each group were counted and the fre-

quencies were calculated based on numbers listed in Table I (some subjects

showed more than one C-mut). (B) Distribution of 235 SNP in the cohort.

Frequencies of subjects with C-mut are shown in black, and frequencies of

individuals not exhibiting C-mut (rest of the cohort) are shown in white

(Table I). The likelihood to show C-mut is five times higher in “CC” than

in “TT” subjects (black). (C) CC subjects exhibit an increased frequency of

substitutions in HIV proviral DNA associated with HLA-C but not with

HLA-A or HLA-B alleles. Ratios of subjects with the CC genotype versus

individuals with the TT genotype are shown for each HLA class I asso-

ciation. Statistical analyses were performed using a x2 test using numbers

from Table I. In (C), the p value applies to a comparison between the

numbers of CC versus TT subjects with A-mut, B-mut, or C-mut.

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Moreover, 263del/del individuals with the CC genotype weremore likely to exhibit C-mut (Fig. 7C, 7D). We sequenced the39UTR of individuals exhibiting C-mut and found that all “high”expressing HLA-C alleles containing 263del exhibit the 235CCgenotype, with the exception of Cw*14, which contains an in-sertion at position 263 of the 39UTR but associates with 235CC(Supplemental Fig. 2). These results confirm that CC subjects withC-mut also have a disrupted binding site for miR-148a (263del),which is likely to result in increased cell-surface HLA-C expres-sion in these individuals compared with that of TT subjectsexhibiting an intact microRNA binding site (263ins). Therefore, itis likely that C-mut were induced by enhanced HLA-C–restrictedimmune pressure in CC individuals.

DiscussionAfter more than 30 years of active research on HIV/AIDS, manychallenging questions remain unanswered. For example, someindividuals progress to AIDS within a year after HIV acquisition,whereas others never develop the disease. Host genetic factors playa role in disease progression, but the precise pathways are stillunknown. Studies aimed at deciphering this are hampered by in-terindividual variability and the high propensity of HIV to mutate:as a consequence, the host–virus interactions can vary significantlybetween different infected people. How innate and adaptive im-mune mechanisms slow HIV replication is still ill defined anddiffers substantially among different ethnic groups. As moststudies to date have focused on patients of European ancestry, ithas become a global health priority to determine how HIV controlis achieved in nonwhites (27). In the current study, we assessed theprotective effect of 235 SNP in Han Chinese subjects who were

infected in a short time frame by a very narrow range of HIV-1strains. In addition to being the first report to our knowledge onthe effect of 235 SNP in an Asian population, this study confirmsthe protective effect of the variant in a setting where confounderssuch as viral strain diversity and time after HIV acquisition arelargely controlled. We analyzed six substitutions in HIV’s proviralDNA that were associated with HLA-C (C-mut) to determinewhether immune pressure on the virus was stronger in 235CCthan in 235TT subjects. C-mut were identified using sequencingdata from PBMC proviral DNA rather than plasma RNA, asmutations that had reached fixation in the population were morerelevant to the purpose of the analysis than quasi-species from anactively replicating population (28). The key finding of this studyis that subjects with the protective CC genotype at 235 showa higher C-mut frequency than that of their TT counterparts,suggesting that the virus is exposed to a stronger immune pressurein presence of the CC genotype. We suggest that HLA-C–re-stricted CTLs play a role in the protective effect of the variant,without excluding the possibility that NK cells or some HLAalleles also contribute to viral control.Of all SNPs associated with delayed HIV disease progression,

235 SNP is consistently prominent, clearly implicating HLA-C asa key player in HIV control. The mechanisms underlying this havebeen partially elucidated with the identification of the 39UTRvariant but remain subject to considerable debate. In contrast,there are associations between HLA-C and several autoimmunediseases (29). Notably, 235 SNP is also associated with increasedsusceptibility to psoriasis where disease is thought to be mediatedby HLA-Cw*06–restricted CTLs specific for self peptides, a re-sponse that could potentially be elicited as a result of enhanced

FIGURE 5. LD between235 SNP and HLA alleles. (A)235 SNP is in LDwith someHLA-C alleles. Allelic frequencies of the different HLA-C alleles are

shown according to the genotypes at235:CC (black),CT (gray), and TT (white). Annotations above each bar indicate which of the “C” or “T” allele at235 is

associated with HLA-C alleles. Significant associations are represented by the asterisk. HLA-Cw*03 subtypes are differentially associated with 235 SNP:

Cw*03:02 associateswith “C”whereasCw*03:03 is in LDwith “T.” (B) Frequencies of themain protectiveHLAalleles in subjects showingC-mut.HLA-B*51

is the only allele enriched in patients with C-mut (black). Frequencies of subjects without C-mut are shown in white. (C) Increased frequency of C-mut in CC

subjects in absence ofHLA-B*51.Analysis described in Fig. 4Awas repeated after exclusion of all HLA-B*51 subjects showingC-mut. Statistical analysiswas

performed using a x2 test. In addition, a logistic regression analysis adjusting for HLA-B*51 showed that its presence did not affect the results.

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HLA-C expression (30, 31). HLA-C is involved in other autoim-mune disorders suggesting that it is somehow implicated in T celltolerance. HLA-C binds a restricted range of self peptides asa result of limited polymorphism in its a1 domain and prolongedassociation with chaperones in the endoplasmic reticulum. Thiscould result in HLA-C molecules being more available to bindpathogen-derived peptides, which could result in specific targetingof key viral molecules, thus countering pathogen replication.Supporting this, the molecule H-2Ld, corresponding to HLA-C inthe mouse, is poorly expressed and defective in binding endoge-nous self peptides but is the dominant CTL-restriction element forrecognition of vesicular stomatitis and lymphocytic choriome-ningitis viruses (32). Therefore, the contribution of HLA-C topathogen recognition in peripheral and lymphoid organs could bedistinct but complementary to that of HLA-A and HLA-B.HLA-C molecules could also play a role during thymic T cell

selection (33). The efficacy of thymic negative selection is pro-portional to the expression level of MHC complexes in the thymicmedulla, which means that fewer clones with a high avidity forself MHC–peptide complexes are deleted when MHC expressionis low. Assuming that HLA-C expression in medullary epithelialand dendritic cells is lower than that of HLA-A and HLA-B (as itis in other tissues), HLA-C–mediated negative selection might beless stringent. Thymic negative selection imposes specificity onthe T cell repertoire, consequently a less efficient negative selec-tion leads to the generation of a highly avid T cell repertoire (34).Therefore, it is tempting to speculate that the T cell repertoirerestricted by HLA-C comprises several high-avidity clones ableeffectively to recognize viral peptides in the periphery. Selection

of a high-avidity T cell repertoire could be a double-edged sword:it might lead to better pathogen recognition but also result in thegeneration of autoimmune pathologies. The dual association of235 SNP linking HLA-C to better HIV control and susceptibilityto psoriasis seems to corroborate this hypothesis. Moreover, dif-ferential HLA-C expression could also influence thymic positiveselection by allowing a more diverse range of T cell clones to beselected in subjects with the CC genotype where HLA-C ex-pression is thought to be higher than that in TT individuals. Thishypothesis remains to be tested.We report in this study that increased expression of HLA-C in the

235 SNP leads to the generation of escape mutations in HIV. Thisis, to our knowledge, the first function-based evidence linking235 SNP to HIV control in vivo. Whether HLA-C expressionlevels are directly responsible for the protective effect of235 SNP(or 263del) or strong LD between the variants and other protectivegenes in the HLA locus exert viral control is still unclear. This isextremely difficult to disentangle, therefore we cannot exclude thepossibility that some protective HLA alleles have an effect on

FIGURE 6. C-mut are escape mutations and markers for immunogenic

epitopes. (A) Magnitude of IFN-g responses to 18-mer HIV-1 consensus

peptides. PBMCs from patients expressing HLA-C alleles associated with

each C-mut were used in an ELISPOT assay. Each bar represents one

patient. HLA-C type of each subject is shown above the bars; the HLA-C

alleles associated with C-mut are in boldface (Fig. 2). Two to five

responding patients were identified for each peptide. (B) TNF-a and IFN-g

production is CD8+ T cell mediated, HLA-Cw*01:02 restricted, and

peptide specific. A short-term CTL line was restimulated with VL8-pulsed

0.221HLACw*01:02 transfectants (left). Untransfected VL8-pulsed 0.221

cells were used as control (right). Numbers indicate percentage of positive

cells. Gated on CD3+ T cells. (C and D) S173T and T470A substitutions are

escape mutations. Peptide titration assay with CTL line or PBMCs in

presence of decreasing concentrations of adapted (white) and nonadapted

(black) (C) VL8 or (D) TAPPEESFRFGEETTTPSQK peptides in an

ELISPOT assay. Data are representative of two independent experiments.

Statistical analyses were performed using a two-tailed t test. Significant

differences are indicated by the presence of an asterisk.

FIGURE 7. 39UTR variant associates with protection and C-mut in the

Han Chinese population. (A) Mean viral load (log10 cp/ml) and (B) median

CD4 counts (cells/ml blood) in HIV-infected individuals in relation to 263

del/ins. (C) Increased frequency of C-mut in CC subjects. Frequencies of

C-mut in the cohort in relation to 263del/ins. (D) Frequencies of subjects

with (black) or without (white) C-mut. Subjects were grouped based on the

presence of 263del/ins. Frequencies of individuals are shown. Statistical

analyses were performed using Kruskal–Wallis rank sum test and x2 test.

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delayed progression to AIDS independently or in conjunction with235 SNP. In contrast, as LD between HLA alleles in the HanChinese substantially differs from that of the whites, we were ableto partially separate some associations with 235 SNP to concludethat the protective effect of the variant can be dissociated from theeffect of the main protective HLAs (i.e., B*57, B*27). Althoughpolymorphisms in the binding site of miR-148a (HLA-C 39UTR)provide a mechanism explaining how HLA-C expression can bemodulated, we cannot exclude that HLA-C alleles in LD with235CC play a role in protection. However, a growing body ofevidence supports the concept that increased HLA-C expressionresults in a more effective immune response through mechanismssuch as NK cell licensing and activation, Ag cross-presentation toCTLs, and increased survival of APCs (35). Further studies arerequired, nevertheless our data support the concept that HIV-specific CTLs are key components of HIV control in vivo andhighlight the relevance of exploring HLA-C–restricted CTLs inthe design of a potential HIV vaccine.

AcknowledgmentsWe thank Jane Holmes, Ly-Mee Yu, Daniel Lunn, Joe Parker, and Louis-

Marie Yindom for help with statistical analyses, Peter Parham for providing

721.221-HLACw*01:02 transfectants, Smita Kulkarni and Mary Carring-

ton for help with the 39UTR PCR for sequencing protocol, and Simon

Brackenridge for help with 235 SNP genotyping.

DisclosuresThe authors have no financial conflicts of interest.

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