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Division of Infectious Diseases, Johns Hopkins University School of Medicine
Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland
Division of Infectious Diseases, Vanderbilt University Medical Center, Nashville, Tennessee
Department of Statistics and Institute of Health Care Policy and Aging Research, Rutgers University, Piscataway, New Jersey
Center for Urban Epidemiologic Studies, New York Academy of Medicine, New York, New York
Sex-based differences in the levels of human immunodeficiency virus 1 (HIV-1) RNA in plasma could be associated with differences in the strength of HIV-1specific CD8+ T cell responses. CD8+ effector responses in 18 men and 15 women were measured 02 years (time A) and 57 years (time B) after seroconversion. CD8+ effector responses were seen in 7 (39%) of 18 men and 2 (13%) of 15 women at time A (P = .13) and in 12 (67%) of 18 men and 10 (67%) of 15 women at time B (P = .99). At time B, the strength of CD8+ effector responses correlated with the number of CD4+ lymphocytes in women ( = -0.68; P = .005) but not in men ( = -0.14; P = .58). The level of HIV-1 RNA was not associated with the strength of CD8+ effector responses according to sex, but there was a sex-based difference in the correlation between the strength of CD8+ effector response and the number of CD4+ lymphocytes.
Shortly after HIV-1 seroconversion, the level of HIV-1 RNA in plasma is 0.5 log10 lower in women than it is in men [1], but this difference in levels decreases as duration of time after seroconversion increases [2]. Biological mechanisms underlying the initial sex-based difference in the levels of HIV-1 RNA, and the subsequent equilibration, are unknown.
Several studies have demonstrated a close association between the levels of HIV-1 RNA and the strength of HIV-1specific CD8+ effector responses, including cytotoxic T lymphocyte responses. Shortly after HIV-1 infection, induction of CD8+ effector responses is associated with control of viral replication and lower levels of HIV-1 RNA [3]. HIV-1infected long-term nonprogressors have stronger CD8+ effector responses and lower levels of HIV-1 RNA than do persons with more-rapid progression of HIV disease [4]. Loss of CD8+ effector response is associated with increased levels of HIV-1 RNA and clinical disease progression [5], and HIV-1specific cellular immune response is inversely correlated with disease progression [6]. Other studies, however, have found increased levels of HIV-1 RNA and more-rapid disease progression in persons with CD8+ activation [7]. The more-pronounced cell-mediated and humoral immune responses in women [8] could affect the CD8+ effector response.
We hypothesized that lower initial levels of HIV-1 RNA in women are associated with a stronger CD8+ effector response and that this initial sex-based difference in the strength of CD8+ effector response decreases as duration of time after seroconversion increases, just as the sex-based difference in the levels of HIV-1 RNA decreases.
Participants, materials, and methods.
Between February 1988 and March 1989, injection-drug users from Baltimore, MD, were enrolled in a longitudinal study of HIV-1 infection [9]. Criteria for entry in this study were that participants be 18 years old, be free of AIDS, and have used injection drugs. All participants had semiannual physical exams and venipuncture for HIV serology. When participants were first identified as HIV-1 seropositive, they returned to the study facility to have blood drawn for an assessment of CD4+ lymphocyte numbers, and plasma and cells were frozen for future studies. Assessment of CD4+ lymphocyte numbers and collection of plasma and cells were repeated semiannually. The study was approved by the Johns Hopkins School of Public Health institutional review board; the human experimentation guidelines of the US Department of Health and Human Services were followed.
Criteria for inclusion in the present study were (1) documented HIV-1 seroconversion 12 months after the last seronegative test (median duration, 3.1 months); (2) no reported use of combination antiretroviral therapy during the 6 months preceding the study visit at which CD8+ effector responses were measured; (3) availability of frozen cells at both 02 years (time A) and 57 years (time B) after seroconversion.
Demographic data were obtained at the initial study visit, and participants' self-reports of use of antiretroviral therapy and injection drugs during the preceding 6 months were obtained semiannually. The date of HIV-1 seroconversion was estimated as the midpoint between the last visit at which the participant was seronegative and the first visit at which the participant was seropositive.
HIV-1 antibodies were measured with a commercial ELISA kit (Genetic Systems); positive results were confirmed by Western blot (DuPont). T cell subsets were measured by whole-blood staining and flow cytometry. Plasma and cells were stored at -70°C and at -135°C, respectively, until testing was performed. The levels of HIV-1 RNA were quantified in plasma by use of reverse-transcription polymerase chain reaction (Roche Molecular Systems), with a minimum level of detection of 400 copies/mL; a sample with undetectable HIV-1 RNA was coded as having 200 copies/mL. Through April 1997, HIV-1 RNA was obtained from heparin-treated plasma by use of silica extraction [10]. After April 1997, plasma was collected in tubes containing EDTA and was used for testing after being thawed.
CD8+ effector responses to HIV-1 consensus subtype B 15mer peptides overlapping by 11 aa were measured. Peptide pools tested in a standard interferon- enzyme-linked immunospot (ELISPOT) assay included 122 Gag (Gag1, Gag2, and Gag3 pools), 49 Nef, 27 Rev, 23 Tat, 46 Vif, 22 Vpr, 249 Pol (Pol1, Pol2, Pol3, and Pol4 pools), and 19 Vpu peptides (National Institutes of Health AIDS Research and Reference Reagent Program). All assays were performed on cryopreserved peripheral-blood mononuclear cells (PBMCs), as described in detail elsewhere [11]. Phytohemagglutinin (PHA)stimulated PBMCs were used as the positive control, and they had a mean of 375.6 spot-forming cells (sfc) per 1 × 105 cells (range, 501492 sfc/1 × 105 cells). Cell specimens were considered to be viable if the response to PHA was >50 sfc/1 × 105 cells. Mock-stimulated PBMCs were used as the negative control, and they had a mean of 1.7 sfc/1 × 105 cells (range, 023.5 sfc/1 × 105 cells). A result of an assay was considered to be positive if test wells had >5 sfc/well (after subtraction of background response) and exceeded background levels by >3-fold. All samples were run in duplicate. CD8+ cell depletion assays, in which anti-CD8 magnetic beads (Dynal) were used, were performed on PBMCs from a subset of individuals, to confirm the phenotype of the responding cell.
Categorical variables were analyzed by use of Fisher's exact test, and continuous variables were analyzed by use of either Wilcoxon's rank sum test or t test for log-transformed data. Spearman's rank correlations between the sum of CD8+ effector responses to all HIV-1 peptides, as measured by ELISPOT, and HIV-1 RNA and CD4+ lymphocyte data were performed on log-transformed data; when the sum of CD8+ effector responses was 0 (n = 5, for time A; n = 1, for time B), a value of 0.5 was assigned prior to log transformation. For the analysis of CD8+ effector responses normalized for the percentage of CD8+ lymphocytes, the CD8+ effector response was divided by the percentage of CD8+ lymphocytes at the time at which the cells were obtained. All reported P values are 2-sided.
Results.
Of the 43 participants who met the inclusion criteria, 33 had viable cells at both time A and time B. Clinical and demographic characteristics of the study population are given in table 1. The characteristics of the participants with viable cells were similar to those of the 43 participants who met the inclusion criteria (data not shown). Median levels of HIV-1 RNA were higher in men than in women at both time A and time B, but at neither time point was the difference statistically significant. Median numbers of CD4+ and CD8+ lymphocytes in men and women were similar at both time points. Of the 18 men in the study, 8 (44%) developed AIDS; of the 15 women in the study, 7 (47%) developed AIDS (P = .89).
Three study participants reported use of nucleoside monotherapy at some point during the 6 months preceding their study visit at time A. For all other study visits (time A or time B), no participant reported use of any antiretroviral therapy during the preceding 6 months.
At time A, 7 (39%) of 18 men and 2 (13%) of 15 women had 1 positive CD8+ effector response to an HIV-1specific peptide (P = .13). In men, Gag and Nef generated the most responses; there were no responses to Rev, Tat, or Pol. In women, the only responses were to Tat and Vpr. At time B, 12 (67%) of 18 men and 10 (67%) of 15 women had 1 positive response to an HIV-1specific peptide (P = .99). In men, Gag, Nef, and Vpr generated the most responses; there were almost no responses to Pol and Vpu. In women, responses were broadly distributed, and the greatest number of responses were to Gag and Nef. All participants who responded at time A also responded at time B.
The CD8+ effector responses, as measured by ELISPOT, were then compared with the levels of HIV-1 RNA and the numbers of CD4+ lymphocytes (figure 1). In all participants, there was no correlation between the strength of CD8+ effector response and either the levels of HIV-1 RNA or the numbers of CD4+ lymphocytes, at either time A or time B (data not shown). When the data were stratified by sex, women showed a negative correlation between the strength of CD8+ effector response and the numbers of CD4+ lymphocytes, particularly at time B ( = -0.68; P = .005); there tended to be a positive correlation between the strength of CD8+ effector response and the levels of HIV-1 RNA, particularly at time A ( = 0.28; P = .30). In men, there was no correlation between the strength of CD8+ effector response and the numbers of CD4+ lymphocytes at time A or time B; there tended to be a negative correlation between the strength of CD8+ effector response and the levels of HIV-1 RNA at time A ( = -0.33; P = .19). There was no correlation between the strength of CD8+ effector response and the numbers of CD8+ lymphocytes at time A or time B, in either the entire population or according to sex (data not shown).
CD8+ depletion assays were performed on PBMCs from 11 participants (7 men and 4 women) at time B. Of these 11 participants, 9 had CD8+ effector responses to peptides in the assays of nondepleted PBMCs; 8 of 9 participants had no effector response to any peptide tested in the assays of CD8-depleted PBMCs. In 1 participant there was a minimal effector response to Gag and Nef in the assays of CD8-depleted PBMCs, but the response was less than that seen in the assays of nondepleted PBMCs. These results suggest that the effector responses seen in the study population were generated primarily by CD8+ lymphocytes, rather than by CD4+ lymphocytes.
To address the possibility that differences in the proportion of CD8+ lymphocytes that were generating responses could account for differences in the overall effector response, responses were normalized on the basis of the percentage of CD8+ cells present. This did not substantially change the proportion of men or women with a CD8+ effector response at either time A or time B (data not shown).
Discussion.
The most significant finding of this study was that a correlation between immune status and CD8+ effector response was found in women but not in men, particularly at time B. In women, but not in men, a stronger CD8+ effector response was associated with a lower number of CD4+ lymphocytes. It is unclear why such a difference was observed and why women with more-advanced immunodeficiency would have stronger CD8+ effector responses. We are unaware of previous studies on sex-based differences in the strength of CD8+ effector responses.
All study participants were essentially treatment naive, so it is unlikely that inception or cessation of antiretroviral therapy would have affected CD8+ effector response. Although use of injection drugs can affect the immune response in general and could potentially affect CD8+ effector responses in particular, there was no sex-based difference in the number of participants who used injection drugs during the 6 months preceding the study visit at either time A or time B. This result could be related to a type II and/or type I error, particularly because multiple comparisons were performed. Thus, our findings should be used to formulate hypotheses to be tested in future studies rather than as definitive proof of a sex-based difference in the strength of CD8+ effector response.
Of note, our hypotheses that lower initial levels of HIV-1 RNA in women would be associated with stronger CD8+ effector responses and that the equilibration of the levels of HIV-1 RNA in women and men would be associated with an equilibration of CD8+ effector responses were not confirmed by this study. In fact, although the differences were not statistically significant, at time A, men were more likely to have a strong CD8+ effector response than were women (39% vs. 13%). Interestingly, men had higher median levels of HIV-1 RNA than women at both time A and time B; although the difference in these levels at time A was not statistically significant, it was comparable in magnitude to that described in larger studies [1].
Given the limited sample size, it was difficult to ascertain statistically significant sex-based differences in the strength of CD8+ effector responses to specific peptides. However, it appears that men more consistently generated responses to Gag, Nef, and Vpr. Women responded fairly well to Gag but also had broad, diversified responses to the peptides at time B, as did the men. Studies on larger populations would help clarify these findings and more clearly elucidate sex-based differences in specific CD8+ effector responses.
Given the relationship between higher levels of HIV-1 RNA and more-rapid disease progression [1, 12], it is important to understand why women progress from seroconversion to AIDS at the same rate as men despite their lower initial levels of HIV-1 RNA [1, 13]. Besides CD8+ effector responses, mechanisms related to hormonal differences could possibly be at work. However, despite hormonal changes in women during the menstrual cycle, there is no significant change in their levels of HIV-1 RNA during the menstrual cycle [14]. In addition, a sex-based difference in the levels of HIV-1 RNA is seen in infant boys and girls, in whom the hormonal milieu is similar [15]. The lower density of CCR5 on CD4+ lymphocytes in women could play a role [16], but more data are needed to investigate this possibility.
CD8+ effector responses in both men and women were weaker at time A than at time B. Although these weaker responses could be related to lower levels of HIV-1 RNA at time A, they could also be related to decreased cell viability, because the cells tested at time A had been stored frozen longer than those tested at time B.
Two additional limitations of the present study should be noted. First, use of antiretroviral therapy was self-reported. Although these data may not be completely accurate, the most likely error would be toward overestimating, not underestimating, use of antiretroviral therapy. Thus, error due to unreported use of antiretroviral therapy is unlikely. Second, the study was comprised entirely of injection-drug users, and participants were predominantly African American. Although these characteristics of the study population could limit generalizability, there is no difference in progression of HIV disease according to risk group or race [17].
With these caveats acknowledged, the following conclusions can be drawn from the present study. First, there was a sex-based difference in the correlation between the strength of the CD8+ effector response and the numbers of CD4+ lymphocytes, particularly 57 years after seroconversion. In addition, the lower levels of HIV-1 RNA in women did not appear to be mediated by stronger CD8+ effector responses. Further elucidation of the sex-based differences in host immune responses to HIV-1 infection will lead to a better understanding of HIV-1 disease pathogenesis.
Acknowledgments
We thank Kenrad E. Nelson, Steffanie Strathdee, and Stephen Reynolds; Joseph B. Margolick and Elva Ramirez, for quantificating the T cell subsets; and Stacy Meyerer, for maintaining the specimen repository.
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