CCR5 cell-surface expression was studied in relation to CCR5 genotype and clinical course of HIV-1 infection. HIV-1 infected CCR5+/+ individuals had higher percentages of CCR5-expressing CD4+ T cells as compared with HIV-1-infected CCR532/+ individuals. For both genotypic groups, the percentages of CCR5-expressing cells were higher than for the uninfected counterparts (CCR5+/+, HIV+ 28% and HIV 15% (p < 0.0001); CCR532/+, HIV+ 21% and HIV 10% (p = 0.001), respectively). In HIV-1-infected individuals, high percentages of CCR5-expressing cells were associated with low CD4+ T cell numbers (p = 0.001), high viral RNA load in serum (p = 0.046), and low T cell function (p = 0.054). As compared with nonprogressors with similar CD4+ T cell numbers, individuals who did progress to AIDS had a higher percentage of CCR5-expressing CD4+ T cells (32% vs 21% (p = 0.002). Longitudinal analysis of CCR5+/+ individuals revealed slight, although not statistically significant, increases in CCR5-expressing CD4+ T cells and CD4+ T cell subsets characterized by the expression of CD45 isoforms, during the course of HIV-1 infection. Preseroconversion, the percentage of CCR5-expressing CD4+ T cells was higher in individuals who subsequently developed AIDS (28%) than in those who did not show disease progression within a similar time frame (20%; p = 0.059). Our data indicate that CCR5 expression increases with progression of disease, possibly as a consequence of continuous immune activation associated with HIV-1 infection. In turn, CCR5 expression may influence the clinical course of infection.

Specific G-protein-coupled seven transmembrane-spanning chemokine receptors have been found to function as coreceptors for HIV-1 (1, 2). Nonsyncytium-inducing (NSI)4 HIV-1, including macrophage-tropic variants that initiate HIV-1 infection (3, 4), use CCR5 (5, 6, 7, 8, 9, 10, 11, 12). The CXC chemokine receptor 4 (CXCR4) was described as entry cofactor for T cell line-adapted and primary syncytium-inducing (SI) HIV-1 (11, 12, 13, 14, 15). In vitro, some SI variants were able to use CCR2b and/or CCR3 (12, 14, 16, 17) but the relevance of these coreceptors for in vivo infection remains unclear.

Both CXCR4 and CCR5 are expressed on PBL (18, 19, 20), but to different extents on different T cell subsets. CXCR4 is predominantly found on resting, naive (HLA-DR and CD26low, CD45RA+) T cells whereas CCR5 is expressed on activated memory (HLA-DR+ and CD26high, CD45RO+) T cells (19, 20, 21).

Healthy individuals who are heterozygous for a 32-bp deletion in the CCR5 gene (CCR532/+) showed decreased numbers of CCR5 expressing PBL and decreased mean CCR5 expression levels on PBL as compared with individuals with a CCR5 wild-type genotype (CCR5+/+) (20). Nevertheless, considerable overlap between CCR5 expression levels of both groups of individuals was observed. The mean AIDS-free survival period of HIV-1-infected CCR532/+ heterozygotes was shown to be prolonged as compared with the AIDS-free survival period of individuals with the CCR5+/+ genotype (22, 23, 24, 25, 26, 27). This might be explained by lower CCR5 expression resulting in reduced spread of the virus. Although the mean AIDS-free survival period of HIV-1-infected CCR532/+ individuals is prolonged, rapid disease progression can be observed for some CCR532/+ individuals. Recently, we have demonstrated that this is not due to the occurrence of HIV-1 variants able to use other coreceptors, because even CCR532/+ heterozygotes can develop AIDS in the sole presence of CCR5-restricted NSI HIV-1 variants (9).

Considering the large variation in CCR5 expression among CCR532/+ individuals, we analyzed whether CCR5 expression correlated with the clinical course of HIV-1 infection, both in CCR5+/+ individuals and in CCR532/+ individuals.

Cross-sectional analysis was performed on PBMC derived from 30 healthy laboratory workers (mean age at time of analysis, 30.8 years) and 55 HIV-1 seropositive participants of the Amsterdam Cohort Studies on AIDS (mean age at time of analysis, 42.3 years). The latter included 31 participants who entered the study while still seronegative for HIV-1 Abs. For these individuals, the seroconversion date was estimated to be the midpoint between the last seronegative and the first seropositive visit. The remaining 24 HIV-1-infected persons were seropositive at their first visit, and the seroconversion date for these individuals was estimated to be 18 mo before entry in the study (24, 28). Follow-up visits of the HIV-1-positive men occurred every 3 mo. At these visits, blood was collected for the determination of CD4+ T cell numbers, T cell function, and virus phenotype, and PBMC were cryopreserved (29, 30). These frozen PBMC were used for analysis of CCR5, CXCR4, CD4, CD45RA, and CD45RO expression. Twenty-five of the 55 HIV-1 seropositives developed AIDS during the study period after a mean follow-up of 98 26–172(26–172) mo after seroconversion (mean age at time of analysis, 42.9 years). Thirty HIV-1-seropositive individuals did not develop AIDS during a mean follow-up period of 136 78–179(78–179) mo after seroconversion (mean age at time of analysis, 41.9 years). In these cases, the end-point of the study was March 1998 or the start of anti-retroviral therapy with three or more drugs. The longitudinal analysis included 24 HIV-1-seropositive individuals of the Amsterdam Cohort Studies on AIDS of whom 17 participants were HIV-1 seronegative and seven were HIV-1 seropositive at their first visit. During the study period, 10 individuals developed AIDS (mean age at first moment of analysis, 39.2 years) who were analyzed on average at −25 (−33 to −11), +29 (+11 to +50), and +97 (+66 to +149) mo relative to time of seroconversion. AIDS diagnosis was on average 94 (43 to 141) mo after seroconversion. The 14 HIV-1-infected individuals who did not develop AIDS during the study period (mean age at first moment of analysis, 37.3 years) were analyzed on average at −38 (−69 to −11), +26 (+7 to +50), and +107 (+74 to +181) mo relative to time of seroconversion. Follow-up period of individuals who did not develop AIDS during the study period was on average 121 (70 to 179) mo after seroconversion.

Routine analysis of CD4+ T cell numbers was conducted by flow cytofluorometry. PBMC were stained with CD4 mAbs according to standard procedures for FACS analysis. T cell reactivity in response to stimulation with CD3 mAbs in vitro was determined in whole blood cultures (31). The proliferative response was measured after 4 days of culture by means of [3H]thymidine incorporation. The SI HIV-1 phenotype was determined by cocultivation with MT2 cells (30). RNA levels were analyzed in cryopreserved serum samples derived from the same (or at most 3 mo apart) visit as that from which the PBMC samples were obtained by use of a nucleic acid-based amplification assay (HIV-1 RNA QT; Organon Teknika, Boxtel, The Netherlands) (32).

Genomic DNA was isolated from cryopreserved PBMC (Qiagen blood kit, Chatsworth, CA). CCR5 genotyping was performed by PCR analysis using primers flanking the 32-bp deletion in CCR5 (24).

The CCR5 mAb 2D7 was kindly provided by Dr. C. Mackay. Mouse IgG2a was produced at the Central Laboratory of The Netherlands Red Cross Blood Transfusion Service (CLB, Amsterdam, The Netherlands). PE-conjugated CXCR4 (12G5) was purchased from PharMingen (La Jolla, CA), FITC-labeled CD45RO mAb (UCHL-1) was obtained from Dako (Glostrup, Denmark), and PE-conjugated CD45RA mAb (2H4-RD1) was obtained from Coulter Immunology (Hialeah, FL). Peridinin chlorophyll protein-conjugated CD4 and PE- and FITC-labeled goat anti-mouse IgG2a were purchased from Becton Dickinson (San Jose, CA).

Cryopreserved patient PBMC were thawed, washed once with PBS, and resuspended in PBS containing 0.5% BSA (staining buffer). Cells, 2 × 105, were incubated with saturating amounts of directly labeled mAb. When unconjugated mAbs were used, 5 × 105 cells were incubated with goat anti-mouse Ig-FITC. Subsequently, cells were incubated with normal mouse serum (CLB, Amsterdam, The Netherlands) to block aspecific staining during incubation with additional mAbs. All incubation steps were performed at 4°C for 20 min. After each step, cells were washed twice with staining buffer and finally 104 cells were analyzed on a FACS (Becton Dickinson).

In the cross-sectional analysis, one randomly selected postseroconversion sample per individual was used. Student’s t test was used to compare differences between two groups (i.e., HIV+ vs HIV, CCR5+/+ vs CCR532/+, and progressors vs nonprogressors). The Pearson correlation coefficient was calculated to analyze correlations between CCR5 expression, progression markers, and CD45RO expression.

In the longitudinal study, differences between the three time points were analyzed either with an ANOVA (i.e., CCR5, CXCR4, CD45RA, and CD45RO expression on CD4+ T cells) or a Kruskal-Wallis test (i.e., CCR5 and CXCR4 expression on CD4+ T cell subsets). Student’s t test was used to compare differences between two groups (i.e., progressors vs nonprogressors). Normality of groups was tested by use of normal plots and the Shapiro-Wilk’s W test for normality.

SPSS for Windows (version 7.5.2) was used to perform all statistical analyses.

The relationship between CCR5 genotype and CCR5 expression on CD4+ T cells was examined cross-sectionally in 30 HIV-1-seronegative individuals of whom eight were CCR532/+ heterozygotes and 55 HIV-1-seropositive individuals of whom 17 individuals were CCR532/+ heterozygotes. The remaining HIV-1-seronegative and -seropositive individuals had the CCR5+/+ genotype. In the HIV-1-uninfected individuals, higher numbers of CCR5-expressing CD4+ T cells were found in the individuals with the CCR5+/+ genotype as compared with the CCR532/+ individuals, 15% (7–23%) vs 10% (6–14%), respectively (p = 0.003; Fig. 1 A). The same was found for HIV-1-infected individuals with 28% (11–59%) CCR5-expressing CD4+ T cells for the individuals with the CCR5+/+ genotype as compared with 21% (7–38%) for the CCR532/+ individuals (p = 0.02). Comparison of CCR5 expression between HIV-1-infected and uninfected individuals showed that within both the CCR5+/+ (p < 0.0001) and the CCR532/+ (p = 0.001) genotypic groups the HIV-1-seropositive individuals had higher percentages of CCR5-expressing CD4+ T cells.

FIGURE 1.

CCR5 genotype and cell-surface expression. Shown are percentages of CCR5-expressing CD4+ T cells (A) or CD45RO+ CD4+ T cells (B) of HIV-1-infected and uninfected individuals with the CCR5+/+ or CCR532/+ genotype. Lines represent means with SD.

FIGURE 1.

CCR5 genotype and cell-surface expression. Shown are percentages of CCR5-expressing CD4+ T cells (A) or CD45RO+ CD4+ T cells (B) of HIV-1-infected and uninfected individuals with the CCR5+/+ or CCR532/+ genotype. Lines represent means with SD.

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Because CCR5 is expressed mainly on CD45RO+ CD4+ T cells (19), differences in CCR5 expression on this memory T cell subset between CCR5+/+ and CCR532/+ individuals were analyzed. Both in the HIV-1-seropositive and -seronegative individuals, statistically significant higher numbers of CCR5-expressing CD45RO+ CD4+ T cells were found in the CCR5+/+ individuals than in the CCR532/+ heterozygotes (Fig. 1 B). The mean percentages of CCR5+ CD45RO+ T cells in the HIV-1-seronegative persons were 30% and 19% for CCR5+/+ and CCR532/+ individuals, respectively (p < 0.0001), whereas these respective percentages were 39% (+/+) and 27% (32/+) for the HIV-1-seropositive persons (p = 0.022). Both for CCR5+/+ and CCR532/+ individuals, higher percentages of CCR5-expressing CD45RO+ T cells were observed for HIV-1-seropositive as compared with HIV-1-seronegative individuals (CCR5+/+: p = 0.001; CCR532/+: p = 0.065). Considerable variation and overlap in percentages CCR5-expressing cells could be observed on both CD4+ and CD45RO+ T cells of HIV-1-seropositive CCR5+/+ and CCR532/+ individuals.

To determine whether CCR5 expression is correlated with HIV-1 disease stage, CCR5 expression was analyzed in relation to CD4+ T cell counts, T cell function, and viral RNA load in serum. As shown in Fig. 2 A, the proportion of CCR5-expressing CD4+ T cells and the total number of CD4+ T cells were inversely correlated (Rp = −0.432, p = 0.001). This association could be observed both for individuals solely carrying NSI variants and for individuals carrying both SI and NSI HIV-1 variants.

FIGURE 2.

CD4+ T cell numbers and CCR5 and CD45RO cell-surface expression. Correlation between percentage of CCR5-expressing CD4+ T cells and CD4+ T cell numbers (A), percentage of CCR5-expressing CD4+ T cells and CD45RO+ CD4+ T cells (B), and percentage of CD45RO+ CD4+ T cells and CD4+ T cell numbers (C) of individuals carrying NSI variants only (•) or carriers of both NSI and SI variants (○).

FIGURE 2.

CD4+ T cell numbers and CCR5 and CD45RO cell-surface expression. Correlation between percentage of CCR5-expressing CD4+ T cells and CD4+ T cell numbers (A), percentage of CCR5-expressing CD4+ T cells and CD45RO+ CD4+ T cells (B), and percentage of CD45RO+ CD4+ T cells and CD4+ T cell numbers (C) of individuals carrying NSI variants only (•) or carriers of both NSI and SI variants (○).

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In agreement, it was found that low T cell function as measured by αCD3 reactivity was associated with a high percentage of CCR5-expressing CD4+ T cells (Rp = −0.292; p = 0.054; not shown). Individuals with high viral RNA load had a relatively high percentage of CCR5-expressing CD4+ T cells as compared with individuals with a low viral RNA load (Rp = 0.330, p = 0.046; not shown).

In agreement with the fact that CCR5 is mainly expressed on CD45RO+ CD4+ T cells, correlation analysis showed a higher proportion of CCR5+ CD4+ T cells at higher percentages of memory cells (Fig. 2,B; Rp = 0.508, p = 0.003). In addition, as was previously described by Roederer et al. (33), the percentage CD45RO+ T cells increased with decreasing CD4+ T cell numbers (Fig. 2 C; Rp = −0.546, p = 0.001).

Although the cross-sectional analysis showed a clear correlation between advanced disease stage and increased CCR5 expression, it remained unclear whether the increased CCR5 expression was a cause or a consequence of clinical progression. To determine the effect of increased CCR5+ CD4+ T cell counts on progression, we first compared CCR5 expression in individuals who did or did not develop AIDS within a similar study period (average: progressors, 98 mo; nonprogressors, 136 mo). At the moment of analysis, both groups had similar CD4+ T cell counts (progressors, 453 CD4+ T cells/μl; nonprogressors, 553 CD4+ T cells/μl; p = 0.145; not shown). The groups were also similar with respect to the genotypic distribution, with approximately one-third of the individuals having the CCR532/+ genotype (progressors and nonprogressors, 32% and 29%, respectively; Fig. 3), thereby excluding a possible biasing effect of genotype on the CCR5 expression in both groups.

FIGURE 3.

CCR5 genotype and cell-surface expression. Percentage of CCR5-expressing CD4+ T cells of HIV-1-infected individuals who did (progressors) or did not (nonprogressors) show progression to AIDS within the study period. CCR5+/+ (•) and CCR532/+ () individuals are indicated. Lines represent the means with SD.

FIGURE 3.

CCR5 genotype and cell-surface expression. Percentage of CCR5-expressing CD4+ T cells of HIV-1-infected individuals who did (progressors) or did not (nonprogressors) show progression to AIDS within the study period. CCR5+/+ (•) and CCR532/+ () individuals are indicated. Lines represent the means with SD.

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HIV-1-seropositive individuals who developed AIDS had, on average, 32% CCR5-expressing CD4+ T cells, whereas HIV-1-seropositive individuals who did not develop AIDS had 21% CCR5-expressing CD4+ T cells (p = 0.002) (Fig. 3). The higher percentage of CCR5-expressing cells in the CD4+ T cell population was mirrored by a higher percentage of CCR5+ cells in the CD45RO+ CD4+ T cell subset in the individuals who developed AIDS (44%) as compared with the nonprogressors (32%; p = 0.009; not shown). The same was observed for the CD45RA+ T cell subset (progressors, 24%; nonprogressors, 10%; p = 0.012; not shown) and the CD45 double-dull T cell subset (progressors, 41%; nonprogressors, 20%; p = 0.001; not shown). Because analysis was performed at similar CD4+ T cell counts, these data suggest that higher CCR5 expression is not solely a consequence of the stage of disease but that CCR5 expression may influence disease progression.

To further unravel whether increased CCR5 expression was caused by and/or induced disease progression, longitudinal analysis of 24 HIV-1 seropositives with the CCR5+/+ genotype was performed. In this study group, 14 individuals progressed to AIDS and 10 individuals did not develop AIDS during the study period. At three different time points, percentages of CCR5-expressing T cells were determined: before seroconversion (average: progressors, −25; nonprogressors, −38 mo), relatively early (average: 29 and 26 mo, respectively) and relatively late (average: 94 and 107 mo, respectively) in HIV-1 infection. Relatively late in infection, the two groups showed a clear difference in CD4+ T cell numbers (Fig. 4,A). Proportional changes in CD4+ T cell subsets over time were similar in both groups (Fig. 4,A, inset). In the progressors, the percentages of CCR5-expressing CD4+ T cells increased slightly over time from 28% (21–40%) to 31% (19–59%) to 35% (19–56%) (Fig. 4,B). The 14 individuals who did not show progression to AIDS within the study period also showed a slight increase in percentage of CCR5-expressing T cells from 20% (13–28%) to 25% (11–38%) to 28% (11–51%) (Fig. 4,B). The difference in the percentages of CD4+ T cells expressing CCR5 between time-points was not significant. As shown in Fig. 4,B (inset), analysis of CD4+ T cell subsets showed that the percentage of CCR5-expressing CD45RO+, CD45RA+, and CD45 double-dull cells within the CD4+ T cell population all increased slightly. The increases in CCR5-expressing cells of different CD4+ T cell subsets in both groups of individuals are summarized in Table I.

FIGURE 4.

Longitudinal analysis of CD4+ T cell numbers and CCR5 and CXCR4 cell-surface expression. Mean CD4+ T cell numbers (A), mean percentages of CCR5-expressing CD4+ T cells (B), and CXCR4-expressing CD4+ T cells (C) before seroconversion, relatively early, and relatively late in HIV-1 infection of HIV-1-seropositive individuals who did (progressors, black bars) or did not (nonprogressors, white bars) show progression to AIDS within the study period. Numbers within bars indicate the number of individuals analyzed. Error bars represent the SEM. ∗, Statistically significant differences between progressors and nonprogressors. Insets show mean percentage of CD45RA+ (•), CD45RO+ (○), or CD45 double dull () cells within the CD4+ T cell population (A) and mean percentage of CCR5 (B)- or CXCR4 (C)-expressing CD45RA+ (•), CD45RO+ (○), or CD45 double dull () CD4+ T cells of HIV-1-seropositive individuals who did (squares) or did not (circles) show progression to AIDS within the study period. Error bars represent the SEM.

FIGURE 4.

Longitudinal analysis of CD4+ T cell numbers and CCR5 and CXCR4 cell-surface expression. Mean CD4+ T cell numbers (A), mean percentages of CCR5-expressing CD4+ T cells (B), and CXCR4-expressing CD4+ T cells (C) before seroconversion, relatively early, and relatively late in HIV-1 infection of HIV-1-seropositive individuals who did (progressors, black bars) or did not (nonprogressors, white bars) show progression to AIDS within the study period. Numbers within bars indicate the number of individuals analyzed. Error bars represent the SEM. ∗, Statistically significant differences between progressors and nonprogressors. Insets show mean percentage of CD45RA+ (•), CD45RO+ (○), or CD45 double dull () cells within the CD4+ T cell population (A) and mean percentage of CCR5 (B)- or CXCR4 (C)-expressing CD45RA+ (•), CD45RO+ (○), or CD45 double dull () CD4+ T cells of HIV-1-seropositive individuals who did (squares) or did not (circles) show progression to AIDS within the study period. Error bars represent the SEM.

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Table I.

Statistical results for longitudinally analyzed individuals who did or did not show progression to AIDS within the study period

No AIDSAIDS
Increase over time (%)p valueIncrease over time (%)p value
CCR5+ cells in CD4+ T cellsa +8.3 0.303 +6.9 0.632 
CCR5+ cells in CD45RO+ CD4+ T cellsb +6.8 0.506 +3.1 0.799 
CCR5+ cells in CD45RA+ CD4+ T cellsb +7.3 0.567 +8.2 0.159 
CCR5+ cells in CD45 dDull CD4+ T cellsb +10.3 0.694 +12.5 0.378 
     
CXCR4+ cells in CD4+ T cellsa –27.2 0.001 –24.8 0.029 
CXCR4+ cells in CD45RO+ CD4+ T cellsb –35.0 0.011 –36.0 0.393 
CXCR4+ cells in CD45RA+ CD4+ T cellsb –3.0 0.341 –30.0 0.141 
CXCR4+ cells in CD45 dDull CD4+ T cellsb –18.2 0.012 –26.0 0.202 
No AIDSAIDS
Increase over time (%)p valueIncrease over time (%)p value
CCR5+ cells in CD4+ T cellsa +8.3 0.303 +6.9 0.632 
CCR5+ cells in CD45RO+ CD4+ T cellsb +6.8 0.506 +3.1 0.799 
CCR5+ cells in CD45RA+ CD4+ T cellsb +7.3 0.567 +8.2 0.159 
CCR5+ cells in CD45 dDull CD4+ T cellsb +10.3 0.694 +12.5 0.378 
     
CXCR4+ cells in CD4+ T cellsa –27.2 0.001 –24.8 0.029 
CXCR4+ cells in CD45RO+ CD4+ T cellsb –35.0 0.011 –36.0 0.393 
CXCR4+ cells in CD45RA+ CD4+ T cellsb –3.0 0.341 –30.0 0.141 
CXCR4+ cells in CD45 dDull CD4+ T cellsb –18.2 0.012 –26.0 0.202 
a

Significance tested with ANOVA.

b

Significance tested with Kruskal-Wallis test.

The percentage of CCR5+ CD4+ T cells analyzed at the preseroconversion time point was higher in those individuals who did develop AIDS (28%) than in those who did not show progression to AIDS within the study period (20%; p = 0.059; Fig. 4,B). Similarly, early and late in infection progressors had more CCR5-expressing CD4+ T cells (31% and 35%, respectively) as compared with those individuals who did not show progression to AIDS within the study period (early, 25%, p = 0.266; late, 28%, p = 0.296; Fig. 4 B). The different preseroconversion set points suggest that numbers of CCR5-expressing cells may influence disease progression.

Increased percentages of CD45RO+ T cells and CCR5+ T cells over time possibly reflect increased immune activation. Because the coreceptor of SI variants, CXCR4, is mainly expressed on naive resting cells (19), we analyzed whether the expression of CXCR4 decreases during HIV-1 infection. Indeed, both nonprogressors as well as progressors showed significantly decreasing percentages of CXCR4-expressing CD4+ T cells over time as shown in Fig. 4,C (nonprogressors, preseroconversion 82%, to early 66%, to 54% late in infection, p = 0.001; progressors, preseroconversion 76%, to early 65%, to 52% late in infection, p = 0.029). In analogy with increased CCR5 expression on each CD4+ T cell subset, CXCR4 expression decreased on each subset (Fig. 4,C, inset). The decreases in CXCR4-expressing cells of different CD4+ T cell subsets in both groups of individuals are summarized in Table I. Unlike CCR5 expression, no differences in preseroconversion CXCR4 expression could be observed between individuals who did or did not show disease progression.

Several factors may be responsible for the variable clinical course of HIV-1 infection among different individuals. With the identification of CCR5 as the principle coreceptor for primary NSI HIV-1, differential functioning of this coreceptor has been hypothesized to be such factor. This was substantiated by the observation that individuals heterozygous for a 32-bp deletion in CCR5 showed a delayed progression to AIDS. However, also among individuals with a wild-type CCR5 genotype, long-term nonprogressors could be identified, whereas some CCR5 heterozygotes showed rapid disease progression. Here we observed that on CD4+ T cells, and also on the memory CD4+ T cell subset, the percentage of CCR5-expressing cells was indeed lower in CCR532/+ heterozygotes as compared with CCR5+/+ individuals. The large range in the percentage of CCR5-expressing cells supported the idea that a differential expression of CCR5 could contribute to the variable clinical course of HIV-1 infection.

At a time point in the clinical course of infection when CD4+ T cell numbers were similar in the two groups, progressors indeed had higher percentages of CCR5-expressing cells as compared with individuals who remained asymptomatic. Moreover, in longitudinal analyses, the percentage of CCR5-expressing cells in PBMC samples obtained before seroconversion was higher in individuals who developed AIDS within the study period as compared with individuals who remained asymptomatic during follow-up. It can be hypothesized that higher levels of CCR5 expression allow higher levels of HIV-1 replication, resulting in higher viral RNA load in plasma and consequently accelerated disease progression as compared with individuals with low CCR5 expression. A higher proportion of CCR5-expressing cells just before seroconversion may also be associated with a more severe primary infection. Of interest in this respect is our observation that CCR5+/+ individuals as compared with CCR532/+ heterozygotes continuously had a lower absolute CD4+ T cell number of 100 cells/mm3 directly after the recovery from their primary infection onwards, when CD4+ T cell numbers had returned to subnormal levels (24). This impaired set point could indeed explain how CCR5 expression might influence the clinical course of infection.

We also obtained evidence for an increased percentage of CCR5-expressing cells as a consequence of disease progression. In a cross-sectional analysis, we could demonstrate a correlation between high CCR5 expression and low CD4+ T cell numbers, low T cell function, and high viral RNA load in serum. In agreement, we observed an increase in the percentages of CCR5-expressing CD4+ T cells with progression of disease. This could be explained by increased percentages of CCR5-expressing CD45RO+, CD45RA+, and CD45 double-dull CD4+ T cells. In addition, an increase in the number of CD45RO-expressing cells was observed, which could also account for the increase in the proportion of CCR5-expressing CD4+ T cells. Numbers of CXCR4-expressing CD4+ T cells decreased over time as did CXCR4 expression on CD4+ T cell subsets. The decrease in percentage of CD45RA+ T cells reciprocated the increase in CD45RO+ T cells, while the decrease in CXCR4+ cells in the CD4+ T cell population was much greater than the increase in CCR5+ cells (Table I). This might be explained by the relatively high decrease in CXCR4-expressing CD45RO+ cells, compared with an only slight increase in CCR5-expressing CD45RO+ T cells. Secondly, because about 80% of the CD45RA+ T cells express CXCR4 and only 30% of the CD45RO+ T cell express CCR5, the equal loss of CD45RA+ T cells and gain of CD45RO+ T cells would result in a higher loss of CXCR4 than gain of CCR5. The increase in CCR5 and CD45RO expression may reflect immune activation due to HIV-1 infection. In agreement, Ostrowski et al. showed that CCR5 expression is associated with HLA-DR expression and that expression of both surface markers increases with ongoing HIV-1 infection (21).

We observed an increasing percentage of CCR5 and a decreasing percentage of CXCR4-expressing CD4 T cells with progressive disease, both in individuals solely carrying NSI variants and in individuals carrying both SI and NSI HIV-1 variants. This indicates that despite the increasingly favorable conditions for NSI variants, SI variants can emerge and compete with the NSI variants. It may be that sufficient SI-specific target cells are left throughout infection because the percentage of CXCR4-expressing cells, although decreasing, always remained above that of CCR5-expressing cells. Additionally, elevated percentages of CCR5-expressing CD4 T cells may actually accelerate the appearance of specific SI mutations through increased NSI HIV-1 replication and therefore enhanced mutation frequency. In agreement, we previously demonstrated that SI conversion tended to be more rapid, but not more frequent, in CCR5+/+ individuals who generally have higher CCR5 expression levels than CCR532/+ individuals (24). Thus, higher CCR5 expression does not appear to prevent and may even accelerate the emergence of SI variants.

The genotype-related differences in CCR5 expression were independent of HIV-1 serostatus. However, the range in percentage of CCR5-expressing cells was much larger in HIV-1-infected individuals as compared with the noninfected subjects, confirming previous studies (20, 21). The large variability in CCR5-expressing cells is in good agreement with the variability in CD4+ T cell numbers in this group of HIV-infected individuals in combination with the here described inverse correlation between percentages of CD4-expressing and CCR5-expressing cells. Alternatively or in addition, highly variable percentages of CCR5-expressing cells may reflect differences in levels of immune activation associated with HIV-1 infection.

Interestingly, the percentage of CCR5-expressing cells in individuals that ultimately became infected with HIV-1 was higher than the percentage of CCR5-expressing cells in HIV-1-negative control subjects. This higher CCR5 expression may be explained by the notion that individuals with high-risk sexual behavior more frequently encounter other pathogenic microorganisms that may cause immune activation and consequently enhance CCR5 expression. The higher CCR5 expression may determine the host susceptibility for HIV-1 infection. Whether individuals with a relative resistance to HIV-1 infection indeed have lower percentages of CCR5-expressing cells remains to be established.

We thank the cohort participants for their continuous participation, the laboratory workers for their contribution to the study, and Dawn Clark for proofreading the manuscript.

1

This study was performed as part of the Amsterdam Cohort Studies on AIDS, a collaboration between the Municipal Health Service, The Academic Medical Centre, and the Central Laboratory of The Netherlands Red Cross Blood Transfusion Service. This work was supported by a grant from The Netherlands Foundation for Preventive Medicine (Grant 28-2547) within the Stimulation Program AIDS Research of the Dutch Program Committee for AIDS Research (Grant 95-026).

4

Abbreviations used in this paper: NSI, nonsyncytium inducing; CXCR, CXC chemokine receptor; SI, syncytium inducing; CCR532/+, heterozygous for a 32-bp deletion in CCR5; CLB, Central Laboratory of The Netherlands Red Cross Blood Transfusion Service.

1
D’Souza, M. P., V. A. Harden.
1996
. Chemokines and HIV-1 second receptors: confluence of two fields generates optimism in AIDS research.
Nat. Med.
2
:
1293
2
Premack, B. A., T. J. Schall.
1996
. Chemokine receptors: gateways to inflammation and infection.
Nat. Med.
2
:
1174
3
Van’t Wout, A. B., N. A. Kootstra, G. A. Mulder-Kampinga, N. Albrecht-van Lent, H. J. Scherpbier, J. Veenstra, K. Boer, R. A. Coutinho, F. Miedema, H. Schuitemaker.
1994
. Macrophage-tropic variants initiate human immunodeficiency virus type 1 infection after sexual, parenteral and vertical transmission.
J. Clin. Invest.
94
:
2060
4
Zhu, T., H. Mo, N. Wang, D.S. Nam, Y. Cao, R. A. Koup, D. D. Ho.
1993
. Genotypic and phenotypic characterization of HIV-1 in patients with primary infection.
Science
261
:
1179
5
Deng, H. K., R. Liu, W. Ellmeier, S. Choe, D. Unutmaz, M. Burkhart, P. Di Marzio, S. Marmon, R. E. Suttons, C. M. Hill, C. B. Davis, S. C. Peiper, T. J. Schall, D. R. Littman, N. R. Landau.
1996
. Identification of the major co-receptor for primary isolates of HIV-1.
Nature
381
:
661
6
Dragic, T., V. Litwin, G. P. Allaway, S. R. Martin, Y. Huang, K. A. Nagashima, C. Cayanan, P. J. Maddon, R. A. Koup, J. P. Moore, W. A. Paxton.
1996
. HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5.
Nature
381
:
667
7
Alkhatib, G., C. Combadiere, C. C. Broder, Y. Feng, P. E. Kennedy, P. M. Murphy, E. A. Berger.
1996
. CC CKR5: A RANTES, MIP-1a, MIP-1b receptor as a fusion cofactor for macrophage-tropic HIV-1.
Science
272
:
1955
8
Choe, H., M. Farzan, Y. Sun, N. Sullivan, B. Rollins, P. D. Ponath, L. Wu, C. R. Mackay, G. LaRosa, W. Newman, N. Gerard, C. Gerard, J. Sodroski.
1996
. The b-chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates.
Cell
85
:
1135
9
De Roda Husman, A. M., R.P. van Rij, H. Blaak, and H. Schuitemaker. 1999. Adaptation to promiscuous usage of chemokine receptors is not a prerequisite for HIV-1 disease progression. J. Infect. Dis. In press.
10
Zhang, Y.-J., T. Dragic, Y. Cao, L. Kostrikis, D. S. Kwon, D. R. Littman, V. N. Kewal Ramani, J. P. Moore.
1998
. Use of coreceptors other than CCR5 by non-syncytium-inducing adult and pediatric isolates of human immunodeficiency virus type 1 is rare in vitro.
J. Virol.
72
:
9337
11
Zhang, L., T. He, Y. Huang, Z. Chen, Y. Guo, S. Wu, K. J. Kunstman, R. C. Brown, J. P. Phair, A. U. Neumann, D. D. Ho, S. M. Wolinsky.
1998
. Chemokine coreceptor usage by diverse primary isolates of human immunodeficiency virus type 1.
J. Virol.
72
:
9307
12
Björndal, Å., H. Deng, M. Jansson, J. R. Fiore, C. Colognesi, A. Karlsson, J. Albert, G. Scarlatti, D. R. Littman, E. M. Fenyö.
1997
. Coreceptor usage of primary human immunodeficiency virus type 1 isolates varies according to biological phenotype.
J. Virol.
71
:
7478
13
Feng, Y., C. C. Broder, P. E. Kennedy, E. A. Berger.
1996
. HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor.
Science
272
:
872
14
Simmons, G., D. Wilkinson, J. D. Reeves, M. T. Dittmar, S. Beddows, J. Weber, G. Carnegie, U. Desselberger, P. W. Gray, R.A. Weiss, P. R. Clapham.
1996
. Primary, syncytium-inducing human immunodeficiency virus type 1 isolates are dual-tropic and most can use either LESTR or CCR5 as co-receptors for virus entry.
J. Virol.
70
:
8355
15
Connor, R. I., K. E. Sheridan, D. Ceradini, S. Choe, N. R. Landau.
1997
. Change in coreceptor use correlates with disease progression in HIV-1-infected individuals.
J. Exp. Med.
185
:
621
16
Zhang, L., Y. Huang, T. He, Y. Cao, D. D. Ho.
1996
. HIV-1 subtype and second-receptor use.
Nature
383
:
768
17
Scarlatti, G., E. Tresoldi, Å. Björndal, R. Fredriksson, C. Colognesi, H. K. Deng, M. S. Malnati, A. Plebani, A. G. Siccardi, D. R. Littman, E. M. Fenyö, P. Lusso.
1997
. In vivo evolution of HIV-1 co-receptor usage and sensitivity to chemokine mediated suppression.
Nature Medicine
3
:
1259
18
Loetscher, M., T. Geiser, T. O’Reilly, R. Zwahlen, M. Baggiolini, B. Moser.
1994
. Cloning of a human seven-transmembrane domain receptor, LESTR, that is highly expressed in leukocytes.
J. Biol. Chem.
269
:
232
19
Bleul, C. C., L. Wu, J. A. Hoxie, T. A. Springer, C. R. Mackay.
1997
. The HIV coreceptors CXCR4 and CCR5 are differentially expressed and regulated on human T lymphocytes.
Proc. Natl. Acad. Sci. USA
94
:
1925
20
Wu, L., W. A. Paxton, N. Kassam, N. Ruffing, J. B. Rottman, N. Sullivan, H. Choe, J. Sodroski, W. Newman, R. A. Koup, C. R. Mackay.
1997
. CCR5 levels and expression pattern correlate with infectability by macrophage-tropic HIV-1, in vitro.
J. Exp. Med.
185
:
1681
21
Ostrowski, M. A., S. J. Justement, A. Cantanzaro, C. A. Hallahan, L. A. Ehler, S. B. Mizell, P. N. Kumar, J. Mican, T.-W. Chun, A. S. Fauci.
1998
. Expression of chemokine receptors CXCR4 and CCR5 in HIV-1-infected and uninfected individuals.
J. Immunol.
161
:
3195
22
Samson, M., F. Libert, B. J. Doranz, J. Rucker, C. Liesnard, C.-M. Farber, S. Saragosti, C. Lapouméroulie, J. Cognaux, C. Forceille, et al
1996
. Resistance to HIV-1 infection in caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene.
Nature
382
:
722
23
Dean, M., M. Carrington, C. Winkler, G. A. Huttley, M. W. Smith, R. Allikmets, J. J. Goedert, S. P. Buchbinder, E. Vittinghoff, E. Gomperts, et al
1996
. Genetic restriction of HIV-1 infection and progression to AIDS by a deletion allele of the CKR5 structural gene.
Science
273
:
1856
24
De Roda Husman, A. M., M. Koot, M. Cornelissen, M. Brouwer, S. M. Broersen, M. Bakker, M. T. L. Roos, M. Prins, F. De Wolf, R. A. Coutinho, F. Miedema, J. Goudsmit, H. Schuitemaker.
1997
. Association between CCR5 genotype and the clinical course of HIV-1 infection.
Ann. Intern. Med.
127
:
882
25
Eugen-Olsen, J., A. K. N. Iversen, P. Garred, U. Koppelhus, C. Pedersen, T. L. Benfield, A. M. Sorensen, T. Katzenstein, E. Dickmeiss, J. Gerstoft, P. Skinhoj, A. Svejgaard, J. O. Nielsen, B. Hofmann.
1997
. Heterozygosity for a deletion in the CKR-5 gene leads to prolonged AIDS-free survival and slower CD4 T-cell decline in a cohort of HIV-seropositive individuals.
AIDS
11
:
305
26
Michael, N. L., G. Chang, L. G. Louie, J. R. Mascola, D. Dondero, D. L. Birx, H. W. Sheppard.
1997
. The role of viral phenotype and CCR-5 gene defects in HIV-1 transmission and disease progression.
Nat. Med.
3
:
338
27
Zimmerman, P. A., A. Buckler-White, G. Alkhatib, T. Spalding, J. Kubofcik, C. Combadiere, D. Weissman, O. Cohen, A. Rubbert, G. Lam, et al
1997
. Inherited resistance to HIV-1 conferred by an inactivating mutation in CC chemokine receptor 5: studies in populations with contrasting clinical phenotypes, defined racial background, and quantified risk.
Mol. Med.
3
:
23
28
Van Griensven, G. J. P., E. M. M. De Vroome, J. Goudsmit, R. A. Coutinho.
1989
. Changes in sexual behaviour and the fall in incidence of HIV infection among homosexual men.
Br. Med. J.
298
:
218
29
Tersmette, M., R. E. Y. De Goede, B. J. M. Al, I. N. Winkel, R. A. Gruters, H. T. M. Cuypers, H. G. Huisman, F. Miedema.
1988
. Differential syncytium-inducing capacity of human immunodeficiency virus isolates: frequent detection of syncytium-inducing isolates in patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex.
J. Virol.
62
:
2026
30
Koot, M., A. H. V. Vos, R. P. M. Keet, R. E. Y. De Goede, W. Dercksen, F. G. Terpstra, R. A. Coutinho, F. Miedema, M. Tersmette.
1992
. HIV-1 biological phenotype in long term infected individuals, evaluated with an MT-2 cocultivation assay.
AIDS
6
:
49
31
Bloemena, E., M. T. L. Roos, J. L. A. M. van Heijst, J. M. J. J. Vossen, P. T. A. Schellekens.
1989
. Whole-blood lymphocyte cultures.
J. Immunol. Methods
122
:
161
32
van Gemen, B., R. van Beuningen, A. Nabbe, D. Van Strijp, S. Jurriaans, P. Lens, T. Kievits.
1994
. A one-tube quantitative HIV-1 RNA NASBA nucleic acid amplification assay using electrochemiluminescent (ECL) labelled probes.
J. Virol. Methods
49
:
157
33
Roederer, M., J. Gregson Dubs, M. T. Anderson, P. A. Raju, L. A. Herzenberg, L. Herzenberg.
1995
. CD8 naive T cell counts decrease progressively in HIV-infected adults.
J. Clin. Invest.
95
:
2061