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Department of Medical Microbiology and Infectious Diseases, University of Manitoba, Winnipeg, Manitoba, Canada
Department of Medical Microbiology, University of Nairobi, Nairobi, Kenya
The immune response of human immunodeficiency virus (HIV)exposed seronegative (ESN) women may be qualitatively different from that in those infected with HIV (HIV+). In a cohort of female commercial sex workers in Nairobi, Kenya, we found significantly lower (P .01) levels of CD4+-specific immune activation and apoptosis in the ESN women compared with those in the HIV+ women. Compared with the HIV+ women, a lower proportion of the ESN women showed p24 peptide pool responses by the short-term, CD4+-specific, interferon (IFN) intracellular cytokine staining assay, whereas the proportion showing responses by the long-term, CD8+-depleted T cell proliferation assay was similar. Interestingly, the ESN responders had a 4.5-fold stronger proliferation response (P = .002) than the HIV+ group. These data suggest that, compared with those in HIV+ women, CD4+ T cells in ESN women have a much greater ability to proliferate in response to p24 peptides.
Those exposed to HIV but remaining persistently seronegative have been found in a number of different groups [1, 2], and a variety of mechanisms have been proposed to explain their protection against infection [3, 4]. To further delineate the cellular immune response in HIV-exposed seronegative (ESN) women, we examined the CD4+ T cells in a group of ESN women from the Majengo Commercial Sex Worker Cohort in Nairobi, Kenya [1]. We have previously demonstrated that they do not have any known chemokine-receptor polymorphisms that would prevent infection and that, functionally, their peripheral-blood mononuclear cells (PBMCs) are susceptible to in vitro infection by HIV, indicating that they do not obtain resistance through the blocking of viral entry [5]. Many of the ESN women possess HIV-specific cytotoxic T lymphocytes or T cell immune responses (68% and 41%, respectively) [6]. The present study was designed to further characterize the immune activation status and the HIV-1specific CD4+ T cell responses in the ESN women in this cohort. Our results indicate that, compared with HIV-infected (HIV+) women, the ESN women had lower levels of immune activation and apoptosis and had a 4.5-foldstronger proliferation of p24-specific CD4+ T cells.
SUBJECTS, MATERIALS, AND METHODS
The study populations consisted of ESN, HIV+, and high-risk HIV-seronegative (HIVN-high) female commercial sex workers from the Majengo Commercial Sex Worker Cohort in Nairobi, Kenya, and low-risk HIV seronegative (HIVN-low) women from Winnipeg, Manitoba, Canada. A p24-specific CD4+ T cell response study was performed on ESN (n = 18), HIV+ (n = 21), and HIVN-high (n = 9) women from the Majengo Commercial Sex Worker Cohort, and those results were compared with those in HIVN-low women (n = 17). Six months later, an immune activation and phenotyping study was performed (ESN, n = 19; HIV+, n = 46; HIVN-high, n = 29). Women who had been enrolled in the cohort for 3 years, were HIV IgG seronegative, were HIV negative by polymerase chain reaction, and were still involved in commercial sex work were placed in the ESN group (n = 18). The women in the HIVN-high group (n = 9) were commercial sex workers who did not meet our definition of being resistant to HIV (HIV seronegative for 3 years). The low-risk HIV-seronegative (HIVN-low) controls were lab workers from Winnipeg, Manitoba, Canada (n = 17). In the HIV+ group (n = 21), the CD4+ T cell counts were <200 cells/mm3 in 2 women, 200400 cells/mm3 in 7 women, and >400 cells/mm3 in 12 women; in the ESN, HIVN-high, and HIVN-low groups, except for 1 HIVN-high woman who had a count of 200400 cells/mm3, all the women had counts >400 cells/mm3. All experiments were conducted with the understanding and consent of each participant. Ethical approval was obtained from the University of Manitoba and University of Nairobi ethics review committees.
Unstimulated PBMCs were stained with peridin chlorophyll proteinlabeled anti-CD4, fluorescein isothiocyanate (FITC)labeled antiHLA-DR, and phycoerythrinlabeled anti-CD38 (all from BD Pharmingen); the cells were then analyzed on a BD FACSCalibur. Spontaneous apoptosis in freshly isolated PBMCs was measured. Stained PBMCs were then stained with phycoerythrin-labeled anti-CD4 and allophycocyanin-labeled anti-CD95 (both from BD Pharmingen), washed with 2% fetal calf serum in PBS, resuspended in annexin binding buffer (10 mmol/L of Hepes, 140 mmol/L of NaCl, and 2.5 mol/L of CaCl2 [pH 7.4]), and incubated in the dark for 15 min before analysis by flow cytometry.
The HIV-1 clade B p24 15-mer peptides overlapping by 11 amino acids and spanning the entire HIVHXB2 protein were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program. At the time of this study, clade A p24 peptides were not available. Fifty-six individual peptides (catalog numbers 5017172) were dissolved to a concentration of 2 mg/mL in either water or 20%40% dimethyl sulfoxide and aliquoted into 15 pools of 8 peptides each in a matrix format, such that each peptide was present in 2 different pools. Individual peptides were present at concentrations of 2 g/mL for the IFN- ICS assay and 1 g/mL for the CD4+ T cell proliferation assay. If 2 pools that contained an individual p24 peptide showed an immune response, indicated by proliferation or IFN- staining, the result was considered to be positive.
For the IFN- ICS assay, 500,000 PBMCs were incubated with either a peptide pool (with each of the 8 peptides at a concentration of 2 g/mL) or 1 g/mL of Staphylococcus Aureus Enterotoxin B (SEB) (0.1 g/mL; Sigma) for 90 min at 37°C, then BD Golgi Plug (BD Pharmingen) was added, and the solution was incubated for 13 h. The cells were washed, stained with phycoerythrin-labeled antihuman CD4, fixed, and stained with FITC-labeled antihuman IFN- (all from BD Pharmingen). As in other studies [7, 8], results were considered to be positive if the percentage of CD4+ T cells producing IFN- was 0.05% above the background activity of untreated cells. In general, background activity was not >0.02%, and it was never >0.06%.
For the CD4+ T cell proliferation assay, 1 × 106 PBMCs suspended in cold 2% fetal bovine serum in PBS were depleted of CD8+ T cells by use of Dynabeads (Dynal Biotech), and this process was shown to be >95% effective at depletion of CD8+ T cells (data not shown). Two hundred thousand CD8+-depleted PBMCs were added to a 96-well U-bottom plate and stimulated with either media, peptide pools (with each of the peptides at a concentration of 1 g/mL), or phytohemagglutinin (PHA) dissolved to a concentration of 5 g/mL in water. After a 5-day incubation at 37°C, 1 Ci of [3H]-thymidine was added, and the solution was incubated for 18 h. Cells were harvested onto a glass filter, and the incorporated [3H]-thymidine was measured on a Wallac MicroBeta reader. As reported previously, a 2-fold stimulation index above background activity was considered to be a positive result [6].
For a comparison of means, the t test and Mann-Whitney test were used. Categorical variables were compared by use of a Fisher exact test.
RESULTS
Compared with those in the HIV+ group, the ESN women showed significantly lower levels of the immune activation markers HLA-DR (P < .001) and CD38 (P = .012) and a correspondingly low amount of apoptosis as measured by Annexin V (P = .002) in the CD4+ T cells (figure 1A). Lower levels of CD95 were also observed on the cells of the ESN group relative to those of the HIV+ group (37.3 ± 4.4 vs. 56.0 ± 3.7, respectively; P = .01).
Fifty-seven percent of the HIV+ group showed proliferation of CD4+ T cells in response to the p24 peptide pools, versus 33% of the ESN group (figure 1B). In the IFN- ICS assay, HIV-specific responses were detected in 89% of the HIV+ group, versus 27% of the ESN group. Forty-four percent of the ESN women and 67% of the HIV+ women showed evidence of CD4+ T cell activity by responding in either the CD4+ T cell proliferation assay or the IFN- ICS assay. There was no significant difference in the proportion of p24-specific CD4+ T cell proliferation in the 2 groups (P = .19), but a significantly higher proportion of HIV+ women showed IFN- production (P = .009). In the CD4+ T cell proliferation assay, the ESN group had significantly more CD4+ T cell responses than did the HIVN-high and HIVN-low groups (P = .002 and P = .016, respectively). In the IFN- ICS assay, there was no difference in the immune responses of the HIVN-high and HIVN-low groups.
In keeping with the proportion of women showing immune responses, the breadth of the response showed that the ESN group responded well by the CD4+ T cell proliferation assay but not by the IFN- ICS assay. In the CD4+ T cell proliferation assay, the ESN women who responded did so to an average of 3.0 p24 peptides, compared with 7.9 p24 peptides for the HIV+ group (P = .13) (figure 2A). In contrast, in the IFN- ICS assay, the ESN group responded to an average of 1.3 p24 peptides per person, whereas the HIV+ group responded to an average of 17 p24 peptides per person (P = .003) (figure 2B). Aside from the breadth of the immune response to an antigen, another aspect to consider is the strength of the response. To determine the strength of the immune response in ESN, HIV+, HIVN-high, and HIVN-low groups, we compared levels of background-corrected proliferation of CD4+ T cells and levels of IFN-producing cells. The ability of the CD4+ T cells to produce IFN- in response to the superantigen SEB and to p24 peptides was examined in relation to their effector function. There was no significant difference in the abilities of the CD4+ T cells in the ESN and HIV+ groups to produce IFN- in response to SEB (P = .14) (figure 2C) or to p24 peptides (P = .08) (figure 2D). In the 2 groups, the overall ability of the CD4+ T cells to proliferate, as assessed by stimulation with PHA, was not significantly different (P = .22) (figure 2E), reflecting that 57% (12/21) of the HIV+ women had CD4+ T cell counts <400. However, the magnitude of the background-corrected proliferation of CD4+ T cells in response to p24 peptides in ESN women was 4.5-fold greater than that in the HIV+ women (P = .001) (figure 2F). This suggests that, even though the HIV+ women have the potential to proliferate CD4+ T cells, as seen in their responses to PHA, they were unable to do so effectively in response to p24 peptides, indicating a defect in antigen-specific proliferation of CD4+ T cells.
DISCUSSION
In this study we demonstrate, using 2 distinct assays, the presence of p24-specific CD4+ T cell responses, strong proliferation of CD4+ T cells, low immune activation, and little apoptosis in ESN female commercial sex workers in Nairobi, Kenya. The CD4+ T cell proliferation assay using PBMCs depleted of CD8+ T cells was chosen because proliferation of p24-specific CD4+ T cells is likely more indicative of long-term CD4+ T cell responses than of short-term CD4+ T cell responses. The IFN- ICS assay provided phenotypic data on the T cell effector function while maintaining the inherent dynamics of the overall PBMC population because the CD8+ T cells were not depleted. Because the kinetics of the 2 assays are very different, in all likelihood, they measure distinct subtypes of T cells. The benefit of using the 2 assays is that the CD4+ T cell proliferation assay, which is of a longer duration, more likely measures cells of a memory phenotype, whereas the IFN- ICS assay, which is of a shorter duration, more likely measures CD4+ T cell effectors that can rapidly respond to stimulation. There were qualitative differences in levels of immune function between the HIV+ and ESN groups. The HIV+ women proliferated CD4+ T cells in response to more p24 peptides than did the ESN group, probably because they are exposed to much greater levels of antigen because of their HIV status (figure 2A and B). Although the HIV+ group tended to have stronger immune responses according to the IFN- ICS assay, the level relative to the responses of the ESN group was not statistically significant (P = .08) (figure 2D). This is in contrast to levels of CD8+ T cells in ESN women, which, according to another study, were 10-fold less intense by ELISpot assay [9]. On the basis of the IFN- ICS assay only, one would likely conclude that the CD4+ T cell responses are of similar strengths in each group. However, the CD4+ T cell proliferation assay showed a 4.5-fold increase in proliferation in the ESN group relative to that in the HIV+ group (P < .002). This may indicate that ESN and HIV+ women respond to p24 peptides with similar levels of IFN-producing effector cells, but the ESN women have more proliferation of CD4+ T cells. This difference is not due solely to advanced immunosuppression in the HIV+ group, because (1) most had CD4+ T cell counts >400 and (2), when their PBMCs were stimulated with SEB and PHA, their levels of IFN-producing effector cells showed no significant differences when compared with those of the ESN group.
In the 2 assays, a total of 3 positive responses were found in the HIVN-high and HIVN-low groups. These positive responses could represent the false-positive background levels in the assay or, because 1 of the positive responses came from an HIVN-high woman who is a commercial sex worker, they could represent true positive responses. In the CD4+ T cell proliferation assay, the distinction between the ESN group and the HIVN-high and HIVN-low groups both in the proportion of responders (figure 1B) and in the strength of the response (figure 2F) was very clear, so this result provides confidence that the responses in the ESN women are accurate. An earlier study of this cohort, using a panel of common recall antigens, also indicated that these p24 responses are specific and are not a result of hyperresponsiveness [10].
Recently, 2 studies have examined HIV-1specific, IFN-secreting CD4+ T cells in ESN populations, with mixed results [11, 12]. In both studies, the duration of exposure to HIV was significantly shorter than that in our group, and both studies focused only on the short-term IFN- ICS assay. Therefore, we do not know whether longer-term CD4+ T cell memorylike responses occur in these groups. It is entirely possible that recent exposure to HIV antigens stimulates effector CD4+ T cells to produce IFN- and that this product of the process is being detected in both cohorts. However, among the sex workers in our cohort who have been exposed to HIV for 319 years and are HIV-seronegative, longer-term immunity, as detected by the CD4+ T cell proliferation assay, could also be playing a role in their protection from infection. Our study represents the first in which both short-term IFN- and longer-term CD4+ T cell proliferation assays were used to assess p24-specific CD4+ T cell responses in ESN women.
If both HIV+ and ESN women have p24-specific CD4+ T cell responses, what is unique about the latter that may contribute to their protection from infection In HIV+ women, apoptosis of CD4+ T cells that are not infected with HIV and elevated levels of immune activation have been observed, and both are suggested to play a major role in HIV immunopathogenesis [1315]. ESN women have no circulating HIV proteins, such as gp120, that would induce apoptosis in CD4+ T cells, and they have no infection with HIV that would produce chronic immune activation. Not unexpectedly, in the ESN group, we have observed significantly lower levels of CD4+ T cell immune activation and apoptosis, compared with those in the other groups. Therefore, it is possible that the immune systems of ESN women respond uniquely to p24, and that this different immunological context could result in their p24-specific CD4+ T cells living longer and functioning in a very different way than do the cells in HIV+ women. This study emphasizes that immune responses in ESN women are similar to those in uninfected vaccinees, so the immune environments and the p24-specific immune responses of ESN women should be considered in the design and development of an effective HIV vaccine.
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