HIV infection leads to the progressive loss of CD4+ T cells and their functions. More specifically, HIV-specific CD4+ T cells are thought to be preferentially depleted early after infection. 1 Actually, although HIV-specific CD4+ T-cell responses may be detected during primary infection (PI), HIV-specific proliferative responses have been reported to be lacking during chronic infection (CI), except in rare cases of long-term nonprogression. 2,3 However, studies evaluating HIV-specific interferon-γ synthesis have clearly demonstrated the presence of HIV-specific CD4+ T cells at this chronic stage of infection. 4,5 In addition, some recent studies have suggested that even HIV-specific CD4+ T-cell proliferative responses may be present at that time. 6,7
Highly active antiretroviral therapy (HAART) affects HIV-specific CD4+ T-cell responses. These responses also appear to depend on the stage at which therapy is initiated. Although most studies including subjects treated early (at the time of PI) reported a significant development of HIV-specific CD4+ T-cell responses, 8–10 the restoration of such responses was generally not observed at later stages. 2,11,12 Once again, studies are controversial because some recent data have demonstrated a partial restoration of HIV-specific CD4+ T-cell responses. 6,7,13
HAART has greatly improved the prognosis of HIV infection. However, drug toxicities and poor adherence limit its use over long periods. These limitations have lead to recent guidelines suggesting that initiation of therapy could be deferred. However, the preservation (or development) of HIV-specific immune responses, which are thought to be critical in the control of virus replication, 8,14–16 remains one of the strongest arguments for early initiation of treatment during PI.
The controversial data on the existence of HIV-specific CD4+ T-cell responses during CI and on their restoration during therapy prompted us to design a study to clarify these points. We focused more specifically on the immunologic and/or virologic parameters that could be associated with such responses in patients receiving HAART. Our findings indicate that both pre-HAART CD4 cell count and duration of virus suppression are independently associated with the existence of a CD4 cell proliferative response. The definition of such parameters could help optimize therapeutic guidelines.
This study was approved by Comité Consultatif de Protection des Personnes dans la Recherche Biomédicale de l’Hôpital Cochin (Paris, France), and all patients gave written consent. Peripheral blood samples were obtained from 74 subjects with HIV infection who were selected for having sustained virologic control after initiation of HAART. We included 34 individuals from the French Multicenter PRIMO Cohort (ANRS EP08) who received HAART during the acute infection phase and had sustained virologic control (plasma HIV RNA level, <200 copies/mL) at the time of analysis and during at least the 6 preceding months. We also included 40 consecutive patients with CI who agreed to participate and attended 1 of the referral clinical centers with sustained virus suppression at the time of analysis and during at least the 6 preceding months.
HIV-Specific T-Helper Cell Assays
Only freshly isolated peripheral blood mononuclear cells were used for the lymphocyte proliferation assay. After isolation by centrifugation over Ficoll-Paque (Pharmacia), 105 cells/well were added in round-bottomed microtiter plates in 200 μL of RPMI 1640 GlutaMAX-I medium supplemented with 10% heat-inactivated human AB serum (Valbiotech). In some experiments, purified CD4+ cells positively selected with magnetic beads (Miltenyi Biotec) were used. The cells were then treated in quadruplicate with 5 μg/mL baculovirus-derived recombinant p24 antigen (Protein Science). Positive controls consisted of cells with 5 μg/mL phytohemagglutinin, and negative controls consisted of incubating cells alone. On day 5, each well was pulsed with 1 μCi [ 3H]-thymidine (Amersham). Sixteen hours later, cells were harvested onto glass fiber filters, and radioactivity was quantitated with a β-scintillation counter (1450 Microbeta Plus, Wallac, Perkin Elmer). Data were reported as the stimulation index (SI; median [ 3 H]-thymidine incorporation of cells stimulated with antigen/median incorporation of unstimulated cells). A positive response was defined as both an SI of >3 and a median count of >3000 cpm in the stimulated wells. In some experiments, the nature of the proliferating cells was determined by flow cytometry using carboxyfluorescein diacetate succinimidyl ester (Molecular Probes) according to the manufacturer’s instructions. Briefly, peripheral blood mononuclear cells were stained with 0.35 μM carboxyfluorescein diacetate succinimidyl ester for 10 minutes at 37°C. After 2 washes, cells were suspended in RPMI 1640 GlutaMAX-I medium supplemented with 10% heat-inactivated bovine serum albumin (PAN Biotech) and stimulated for 5 days with 5 μg/mL HIV p24 antigen. After labeling with anti-CD4 PE (Immunotech), anti-CD8 ECD, and anti-CD3 PC5 antibodies (Coulter), peripheral blood mononuclear cells were washed and stored until flow cytometry with an Epics XL instrument and analyzed by Expo32 software (Beckman Coulter).
Data analyses were performed with the StatView 4.5 software (Abacus Concepts, Berkeley, CA). Comparisons between variables were performed using variance analysis or the Mann–Whitney U test. Correlations were identified using simple linear regression analysis and the Spearman rank test. P ≤0.05 was considered significant. Multivariate analysis was performed with SAS software (SAS Institute, Cary, NC) using logistic regression models.
The study first compared HIV p24–specific CD4+ T-cell responses in subjects with primary HIV infection with those in subjects treated during CI. Pre-HAART plasma HIV RNA levels were similar in both groups of subjects (5.1 and 4.9 log10 copies/mL for PI and CI subjects, respectively; P = 0.10). In contrast, and as expected, subjects with PI had a higher median pre-HAART CD4+ T-cell count (588/μL) than subjects with CI (189/μL; P < 0.0001). All subjects had been receiving HAART with controlled viral replication at the time of analysis and during at least the 6 preceding months. The median duration of virus control (ie, HIV RNA load, <200 copies/mL) was comparable in both groups (25 and 28 months for PI and CI subjects, respectively; P = 0.11), but the median CD4+ T-cell count at the time of analysis was different (871 vs. 489/μL for PI vs. CI subjects, respectively; P < 0.0001).
Seventy-nine percent of the subjects treated early during PI had detectable proliferative responses to HIV p24 antigen, but the SI remained relatively low. Surprisingly, we found evidence of a positive proliferative response to HIV p24 antigen in 50% of CI subjects during HAART. Globally, the magnitude of the response was significantly higher in subjects treated during PI than in CI subjects (median proliferation SI: 7.2 vs. 3.2, respectively; P = 0.04). However, when only positive responses were considered, the median SI was comparable in both groups of subjects (9.4 vs. 7.8 for PI vs. CI responders, respectively). For some subjects, additional experiments assessed the CD4+ cell nature of the proliferating cells. First, the use of purified cells showed that proliferative responses were observed in the CD4+ (and not the CD4−) T-cell population (Fig. 1A). Second, flow cytometry analyses of stimulated cells showed that the proliferating carboxyfluorescein diacetate succinimidyl esterlow T cells were contained in the CD4+ T cells (Fig. 1B).
We then focused on the parameters associated with the presence of CD4+ T-cell proliferative responses in CI. The characteristics of CI subjects with and without proliferative responses to HIV p24 antigen were therefore compared. As shown in Table 1, there was no significant difference in the median pre-HAART plasma HIV RNA level between proliferative responders (4.9 log10 copies/mL) and nonresponders (5.0 log10 copies/mL). In contrast, three parameters were associated with the proliferative response.
Proliferative responders had a higher median pre-HAART CD4+ T-cell count than nonresponders did (258 vs. 107/μL, respectively; P = 0.02). Therefore, the proliferative responses in CI subjects were further assessed by dividing the subjects into two strata according to the group’s median pre-HAART CD4+ T-cell count (190/μL) (Fig. 2A). Among the subjects with a higher CD4+ T-cell count before therapy (ie, >190/μL), there were both a higher median SI (4.1) and a higher rate of proliferative responders (65%) than in the group with a CD4+ T-cell count of <190/μL (median SI, 2.6; 35% of proliferative responders).
Proliferative responders had also a longer median duration of virus control than nonresponders did (41 vs. 22 months, respectively; P = 0.005) (Table 1). Again, when subjects were stratified in two groups according to the median duration of virus suppression (<28 or >28 months), there was a higher rate of responses among the group with a longer duration of virus suppression (Fig. 2B).
The CD4+ T-cell count at the time of analysis (current CD4+ T-cell count) was also associated with the presence of a proliferative response. Indeed, Table 1 shows that the current CD4+ T-cell count was higher in responder subjects than in nonresponders (756 vs. 366/μL, respectively; P = 0.001). This is also shown in Figure 2C, in which the responses were assessed after dividing the patients in two strata according to the group’s median current CD4+ T-cell count. Among the subjects with a higher current CD4+ T-cell count (ie, >490/μL), there were both a higher median SI (5.8) and a higher rate of proliferative responders (70%) than in the group with a CD4+ T-cell count of <490/μL (median SI, 2.5; 30% of proliferative responders).
It is likely that the CD4+ T-cell count at the time of analysis depends on both the CD4+ T-cell count at initiation of HAART and the duration of virus suppression. Indeed, a close relationship between the CD4+ T-cell count at the time of analysis and at initiation of HAART (r = 0.68; P < 0.0001), as well as between the CD4+ T-cell count at the time of analysis and the duration of virus suppression (r = 0.49; P = 0.001), was observed.
Because the CD4+ T-cell count at the time of analysis did not appear to be of particular relevance as a predictive clinical value for CD4+ T-cell proliferative response, we only included the two other parameters (CD4+ T-cell count at initiation of HAART and duration of virus suppression) in a multivariate analysis. The presence of a positive proliferative response was significantly and independently associated with both a higher CD4+ T-cell count at initiation of HAART and a longer duration of virus suppression. This is illustrated in Figure 3, in which the percentage of proliferative responders is shown in each quadrant. There was a very low proportion (9%) of proliferative responders among patients with both a low pre-HAART CD4+ T-cell count and a short duration of control (Fig. 3, lower left quadrant). In marked contrast, there was a high proportion (82%) of proliferative responders among patients with both a high pre-HAART CD4+ T-cell count and a long duration of control (Fig. 3, upper right quadrant). In the remaining two groups of subjects with intermediate findings, the proportion of proliferative responders was 44% and 67% (Fig. 3, upper left and lower right quadrants, respectively).
The level of HIV-specific CD4+ T-cell function that can be reached in HIV-infected subjects receiving HAART could be of major clinical importance. However, the precise relationship between proliferative response and viral load remains ambiguous, particularly concerning the causal relationship between these two parameters. 3,8,14,17 Preservation and/or restoration of HIV-specific CD4+ T-cell function may depend on the time of initiation of treatment during the course of HIV infection. The present results show that HIV-specific proliferative responses are observed in a substantial proportion of subjects treated during CI, although at a lesser degree than in subjects treated during PI. These results also underline the relevance of both the pre-HAART CD4 cell count and the duration of virus suppression to predict the existence of a CD4 cell proliferative response.
Seventy-nine percent of the subjects treated early during PI had detectable proliferative responses to HIV p24 antigen, and we did not evidence any difference between therapy initiated before or after seroconversion, as we described previously. 10 Our previous data clearly showed that the CD4+ T-cell proliferative response develops with time in most subjects, because baseline CD4+ T-cell proliferative responses were observed in only 20% of subjects at a low magnitude, 10 as has been shown by other investigators. 8,9
We found evidence of a positive proliferative response to HIV p24 antigen in one half of CI subjects during HAART. From this cross-sectional study, we cannot state whether the CD4+ T-cell response to HIV p24 antigen was either preserved or restored. Results from a study by Al Harthi et al 6 argue for both persistence and restoration of an HIV-specific CD4+ T-cell proliferative response, because the percentage of proliferative responders among a selected group of subjects with moderately advanced CI increased from 31% at baseline to 69% after 1 year of treatment.
Data from other studies examining CD4+ T-cell immune responses in subjects with CI are discordant. However, these studies were often limited by heterogeneous follow-up periods, variable control of HIV replication, and small numbers of subjects who differed in both stage of disease and treatment status. 2,7,11–13 We consequently focused on the parameters associated with the presence of CD4+ T-cell proliferative responses in CI subjects with virologic suppression to clarify and possibly explain some of these controversial data.
The characteristics of CI subjects with and without proliferative responses to HIV p24 antigen were therefore compared. As we had already found in subjects treated during PI, 10 there was no significant difference in the median pre-HAART plasma HIV RNA levels between proliferative responders and nonresponders. In contrast, we found that the pre-HAART CD4+ T-cell count had an impact on the proliferative response, which is in total agreement with the findings of Al Harthi et al, 6 who reported an increase in the percentage of proliferative responders for subjects treated with CD4+ T-cell counts of >500/μL. In contrast, and unexpectedly, Blankson et al 13 showed that half of subjects treated with a low nadir CD4 cell count (<75/μL) had proliferative responses, whereas no positive response was observed in subjects treated with a higher nadir CD4 cell count (>250/μL). The higher proliferative response among the subjects with a higher CD4+ T-cell count before therapy suggests that immune recovery may be more difficult to reach once CD4+ T cells have been depleted beyond a certain count, but it is worth noting that 35% of subjects with a very low pre-HAART CD4+ T-cell count have a positive proliferative response.
Proliferative responders also had a longer median duration of virus control than nonresponders did. This finding is in agreement with those of studies showing that 44% of subjects developed an HIV-specific p24 antigen proliferative response after 24 months of virus suppression during HAART 7 compared with only 25% after 6 months. 18
The demonstration of an association of both the pre-HAART CD4 T-cell count and the duration of virus suppression during HAART with a proliferative response may explain the discrepancies between the results of previous studies including CI subjects who differed in both stage of disease and treatment status. For instance, 3 studies described no or few HIV-specific proliferative responders. 2,11,12 In these studies, subjects had low CD4+ T-cell counts at baseline and short HAART duration and are comparable with the very low level of proliferative responders that we observed among subjects with similar profiles (Fig. 3, lower left quadrant). In contrast, other studies demonstrated a higher rate of proliferative response, but these occurred among subjects treated with high CD4+ T-cell counts at baseline (>500/μL) 6 and subjects for whom HAART duration was >1 year. 7 These results may be compared with our findings (Fig. 3, two upper quadrants and two right quadrants, respectively). Interestingly, we found that these two parameters were independently associated with proliferative response; therefore, their effects are additive. It is particularly worthy to note that among subjects with a high CD4+ T-cell count who are treated for a long period, the rate of proliferative responders can even reach that observed among subjects treated during PI (82% of proliferative responders;Fig. 3, upper right quadrant).
The optimal time to start HAART is still a matter of debate. First, concerning the usefulness of early therapy during PI, the assumption that only early treatment of HIV infection could preserve HIV-specific immune response should be reconsidered, as we previously showed in a longitudinal study. 10 Second, during CI, if the presence of a positive proliferative response is a goal to achieve after HAART, these results provide useful information about the conditions that may allow this goal to be met. Indeed, HIV-specific CD4+ T-cell proliferative responses are often detected when therapy is initiated with CD4+ T-cell counts of >200/μL, even reaching a rate of response similar to that among subjects treated during PI provided that the duration of virus suppression is long enough. These subjects could be the best candidates for HAART initiation in CI. This is strengthened by recent observations pointing to the predictive value of nadir CD4+ T-cell counts for immune reconstitution and clinical outcome after initiation of HAART. 19,20 However, because of drug toxicities and treatment adherence problems, current therapeutic guidelines recommend deferring therapy for these subjects. Therefore, initiation of HAART is now usually restricted to subjects with a low pre-HAART CD4+ T-cell count. Importantly, our results show that this approach does not preclude the presence of HIV-specific CD4+ T-cell proliferative responses, provided that the duration of HAART-induced virologic suppression is long enough. Finally, our results demonstrate that HIV-specific CD4+ T cells are not definitely deleted, and this point is of particular importance in the context of immunotherapeutic strategies aimed at inducing or boosting HIV-specific immune responses.
The authors thank Alejandra Urrutia for her expert technical assistance, Laurence Meyer for help with statistical analysis, and Jean-François Delfraissy for helpful discussion. They also thank the clinicians from all the participating centers of the PRIMO Cohort Study Group for their efficient collaboration as well as the individuals who agreed to participate in the study and their referring physicians.
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