Lack of control of T cell apoptosis under HAART. Influence of therapy regimen in vivo and in vitro
Pinto, Luzia Maria de Oliveiraa; Lecoeur, Hervéa; Ledru, Erica; Rapp, Christopheb; Patey, Olivierc; Gougeon, Marie-Lisea
From the aURA CNRS 1930, Department of AIDS and Retroviruses, Institute Pasteur, Paris, the bBégin Military Hospital, Saint Mandé and cCHI Villeneuve St Georges, France.
Requests for reprints to: Dr M.-L. Gougeon, Département SIDA et Rétrovirus, Institut Pasteur, 28 Rue du Dr Roux, 75724 Paris Cedex 15, France.
Received: 10 August 2001;
revised: 1 October 2001; accepted: 3 October 2001.
Sponsorship: this work was supported by grants from the Agence Nationale de Recherche sur le SIDA (ANRS), the Fondation pour la Recherche Médicale (Sidaction), Pasteur Institute, the Centre National de la Recherche Scientifique (CNRS) and the European Union (contracts BMH4-CT 97-2055 and ERB-IC15-CT97-O901). LMOP was supported by a grant from Conselho Nacional de Pesquisa e Desenvolvimento (CNPq) of the Brazilian government.
Background: Increased and premature T cell apoptosis is recognized as a feature of HIV infection, and its normalization during highly active antiretroviral therapy (HAART) is thought to contribute to quantitative CD4 T cell restoration.
Design: Cross-sectional study of spontaneous, CD3- and CD95-mediated apoptosis in lymphocytes from 53 HIV-infected individuals taking HAART.
Methods: Overnight stimulation of peripheral blood mononuclear cells (PBMC) with coated anti-CD3 or anti-CD95 monoclonal antibodies or incubation overnight in medium. Apoptosis in CD4 and CD8 T cells was measured by flow cytometry. For in vitro assay of antiretroviral drugs, normal PBMC were prestimulated with anti-CD3 monoclonal antibodies and apoptosis was induced by ligation of CD95. The expression of active caspase-8 and caspase-3 was examined by flow cytometry.
Results: We report for the first time that important levels of T cell apoptosis may persist under HAART, in spite of a rise in CD4 T cells from baseline and a sustained suppression of plasmatic viral load. Spontaneous CD3- or CD95-induced apoptosis levels were inversely correlated with the in vivo number of CD4 T cells and the CD4/CD8 ratio, but not with the viral load or duration of antiretroviral therapy. Regimens including lamivudine are associated with persistent T cell apoptosis, particularly following CD95 ligation. Lamivudine was also found to stimulate in vitro CD95-induced apoptosis and caspase activation in pre-activated T lymphocytes from healthy donors.
Conclusion: The immunomodulatory effect of lamivudine may be one of the contributing factor to increased levels of T cell apoptosis under HAART. The data suggest that there is a requirement for physiological apoptosis during HAART.
Highly active antiretroviral therapy (HAART) including HIV protease inhibitors (PI) and reverse transcriptase inhibitors allows, in the majority of patients, dramatic suppression of plasma and tissue HIV RNA load, with a concomitant rise in CD4 T cells. Therapy results in a significant reduction in AIDS-related mortality and morbidity . In vivo mechanisms governing CD4 T cell increase during HAART are complex and still controversial  and suppression of premature T cell apoptosis is thought to contribute to this quantitative restoration . Increased lymphocyte apoptosis is recognized as a feature of HIV infection; it has been attributed to direct and indirect effects of HIV on the immune system, and it contributes to the loss of CD4 T cells [3,4]. Spontaneous  or CD95-induced rates of T cell apoptosis are normalized during HAART [6–9] and this is associated with downregulation of the in vivo immune activation state [6,10] and decreased production of HIV proteins with proapoptotic effects, such as gp120 [11,12]. As a result, global survival of lymphocytes from the HIV-positive patients is restored.
Recent studies have suggested that antiapoptotic effects of PI may contribute to the increase in CD4 T cells and account for discordant responses in some patients, i.e. immunological benefits of HAART despite virological failure [8,13–15]. Similarity between HIV protease and the cellular caspases involved in apoptosis could result in PI-mediated anticaspase activity; this has been shown for ritonavir, which inhibits in vitro caspase-1 , caspase-3 and apoptosis in uninfected T cells . In other studies, indinavir was reported to induce cell cycle arrest in lymphocytes from healthy donors but to have no effect on apoptosis and caspase activation , and both saquinavir and indinavir were found to enhance T cell survival and restore proliferation of T cells with anti-CD3 stimulation in HIV-positive patients . However, these immunomodulatory effects of PI may interfere with physiological cell death, leading to alterations in T cell homeostasis. This appears to be the case for peripheral T cells primed for tumour necrosis factor α synthesis, which may accumulate in HAART-treated patients because of their escape from activation-induced apoptosis and, thus, contribute to the development of the lipodystrophy syndrome .
In the present study, the in vivo effect of HAART on spontaneous, CD3- and CD95-induced apoptosis in CD4 and CD8 T cells was systematically examined in a cohort of HIV-infected patients. In those patients who still had an increased level of T cell apoptosis, this priming for apoptosis was examined for any association with virological and immunological parameters or with the antiretroviral regimen. The influence of nucleoside reverse transcriptase inhibitors (NRTI) and PI on apoptosis and caspase activation of normal CD4 and CD8 T cells was assessed to determine the mechanisms responsible for this failure to control apoptosis in some HAART-treated patients.
Heparinized blood samples were obtained from 53 asymptomatic HIV-1-infected individuals attending the Service for Infectious Diseases of the Bégin Military Hospital, St Mandé, and the CHI at Villeneuve St Georges (France). Study subjects gave informed consent. All the patients received antiretroviral treatment including a PI. HIV RNA was measured by the Amplicor HIV-1 Monitor (Roche Molecular Systems, Branchburg, New Jersey, USA) and bDNA (Chiron) assays. The lower limit of detection was 2.3 log10 copies/ml. The level of plasma HIV RNA was below the limit of detection in 52% of the patients and an increase in CD4 T cells was observed in 84% of the patients, rising from 236 (± 188) × 106 cells/l at baseline to 398 (± 205) × 106 cells/l during HAART. Control blood samples (n = 12) were drawn from HIV-seronegative healthy donors.
Reagents and antibodies
The mouse anti-human surface monoclonal antibodies used in this study included phycoerythrin (PE)- or allophycocyanin (APC)-conjugated anti-CD4 monoclonal antibody (IgG1k, clone SK3) and anti-CD8 monoclonal antibody (IgG1k, clone SK1) (Becton Dickinson, San Jose, California, USA). Staining of apoptotic cells was performed with 7-aminoactinomycin D (Sigma, St Louis, Missouri, USA). Active caspase detection was performed with the CaspaTag kit for caspase-8 (FAM-LETD-FMK) and caspase-3 (FAM-DEVD-FMK) (Intergen, Oxford, UK). Induction of apoptosis was performed with the agonistic anti- CD3 (IgG2a, clone X35) and anti-CD95 (IgG1, clone UB2) monoclonal antibodies (Immunotech, Marseille, France). Appropriate conjugated isotype-matched monoclonal antibodies were used as controls (R&D Systems Abingdon, UK). Ritonavir (RTV) was a gift of Dr G. Peytavin and lamivudine (3TC) was obtained from Glaxo Wellcome (Uxbridge, UK).
Cell culture and apoptosis induction
Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood by centrifugation on Ficoll-Hypaque gradient (Pharmacia, Uppsala, Sweden). Spontaneous apoptosis was induced by culturing overnight 1 × 106 freshly isolated PBMC in 1 ml complete medium composed of RPMI 1640 (Sigma), supplemented with 10% (v/v) heat-inactivated fetal calf serum (Institut Jacques Boy, Reims, France), 2 mmol/l l-glutamine (Life Technologies, Paisley, UK) and 10 IU/ml penicillin/streptomycin (Life Technologies), at 37°C in a 5% CO2-humidified atmosphere. CD3- or CD95-mediated apoptosis was induced by overnight incubation of 1 × 106 PBMC in 1 ml complete medium in 24-well plates pre-coated with anti-CD3 or anti-CD95 monoclonal antibodies. Coating was performed for 1 h at 4°C with 50 and 100 μg/ml suspension of monoclonal antibody in complete medium.
In experiments testing the influence of RTV and 3TC on PBMC, 106 cells were first stimulated for 3 days with anti-CD3 monoclonal antibodies (100 μg/ml) or incubated in medium. Cells were then washed, resuspended at 106/ml in complete medium and transferred to a 96-well plate in the absence or in the presence of the antiretroviral drugs at various concentrations (RTV was pre-dissolved in methanol at 0.1 g/l and 3TC powder was dissolved in water at 500 mmol/l). Three days later, cells were transferred to a new 96-well plate pre-coated with anti-CD95 monoclonal antibodies (50 μg/ml) and incubated for 18 h. Cultures were maintained at 37°C at 5% CO2 and apoptosis was evaluated by flow cytometry.
Quantification of apoptosis
At the end of the culture, cells were washed in phosphate-buffered saline (PBS) pH 7.2, supplemented with 1% (w/v) bovine serum albumin (Sigma), and 0.1% azide (NaN3) (PBS-BSA-NaN3). Cells were then co-stained for 30 min at 4°C with PE-conjugated CD4 or CD8 monoclonal antibodies (1:100 dilution) and 7-aminoactinomycin D (20 μg/ml), to detect apoptotic cells. Stained cells were then washed in PBS-BSA-NaN3 containing non-fluorescent actinomycin D (Sigma), as described , and fixed in PBS-BSA-NaN3 containing 1% paraformaldehyde for 15 min at 4°C. Stained cells were immediately applied to a FACScalibur flow cytometer (Becton Dickinson). For each sample, 10 000–20 000 events were acquired and analyses were performed with the Cell Quest software (Becton Dickinson).
Intracellular detection of active caspase-8 and caspase-3 by flow cytometry
Cells were first co-stained with 7-aminoactinomycin D and APC-conjugated anti-CD4 or anti-CD8 monoclonal antibodies and further incubated with LETD-FMK or DEVD-FMK (peptides inhibitor of caspase-8 and caspase–3, respectively) coupled with the FAM carboxyfluorescein dye for 1 h at 37°C in PBS (CaspaTag kit, Intergen). Cells were then washed with washing buffer and fixed according to the manufacturer's procedure. Stained cells were immediately applied on a FACScalibur flow cytometer and analyses performed with the Cell Quest software.
Univariate analysis included Spearman, Mann–Whitney and Wilcoxon matched pairs test. A P value < 0.05 was considered as significant. For multivariate analysis, forward stepwise logistic regression using Statistica software was utilized. Parameters taken into consideration included the number and percentage of CD4 T lymphocytes, plasma HIV-1 RNA load and parameters for which univariate analysis indicated P ≤ 0.3 when correlation with apoptosis levels was searched. For CD4 T cell parameters at baseline and during HAART, patients were classified into three groups based on cell count: group 0, 1 and 2 having > 500, 201–500 and < 200 × 106 cells/l, respectively. There were 6, 20 and 25 patients at baseline and 16, 27 and 10 patients analysed during HAART in groups 0, 1 and 2, respectively. For plasma HIV RNA levels, patients were stratified again into groups 0, 1 and 2 on the basis of viral load of < 2 (n = 27), 2.1–3.5 (n = 11) and > 3.5 log10 copies/ml (n = 14), respectively. For CD4/CD8 ratio, classification was > 1.0 (n = 09), 0.5–0.99 (n = 12) and < 0.49 (n = 32) for groups 0, 1 and 2, respectively. Lifetime exposure to HAART was also considered and the patients were stratified into groups 0, 1 and 2 based on length of time receiving HAART: < 6 months (n = 13), 7–18 months (n = 14) and > 19 months (n = 25), respectively.
The clinical characteristics of the patients are shown in Table 1.
Long-term suppression of T cell apoptosis by HAART
Figure 1 shows a 2-year follow-up of apoptosis in T cells from seven HIV-infected donors receiving triple drug therapy, including two NRTI and one PI, indinavir. At baseline, mean CD4 T cell count was 282 (± 208) × 106 cells/l (range, 33–622) and mean plasma viral load was 4.95 ± 0.73 log10 copies/ml (range, 3.55–5.71). HAART induced a dramatic decrease in plasma viral load to below the limit of detection (< 2.3 log10 copies/ml) in all the patients; concomitantly, the mean CD4 T cell count increased to 468 (± 303) × 106 cells/l (range, 138–855). The mean change in CD4 cell count at 6 months was 186 (± 164) × 106 cells/l. At baseline, the rate of apoptosis in CD4 and CD8 T cells, following overnight culture of PBMC in medium with or without anti-CD3 or anti-CD95 monoclonal antibodies, was increased compared with that of healthy donors, reaching in some patients very high values (up to 50% of total CD4 or CD8 T cells following ligation of CD3 or CD95 receptors) (Fig. 1). Rapidly after initiation of potent antiviral therapy (after 1 to 2 weeks), the level of apoptosis in both CD4 and CD8 T cells decreased to levels comparable to those of T cells from healthy donors. In most of the patients, this suppression occurred during the first 2 months of therapy and was long lasting, as far as the suppression of viral load was concerned (Fig. 1). If HAART was interrupted, there was a rebound of the viral load accompanied by a dramatic increase in spontaneous and activation-induced apoptosis (data not shown).
HAART does not suppress T cell apoptosis in all patients
Some patients still showed high levels of spontaneous, CD3- or CD95-induced T cell apoptosis (Fig. 2) even while taking HAART. Indeed, analysis of a cohort of HAART-treated patients, described in Table 1, revealed that more than 60% of them showed increased levels of spontaneous apoptosis [given as median percentage increase (25–75 percentile range)] compared with that in healthy donors in both CD4 T cells [5.2% (3.6–8.8) versus 2.3% (1.75–3.0;P = 0.00001] and CD8 T cells [4.2% (3.1–7.4) versus 2% (1.8–2.8);P = 0.00006]. Similarly, more than 70% of these HAART-treated patients showed increased CD3-induced apoptosis compared with that in control donors in both CD4 T cells [15.4% (8–25) versus 4.75% (3.8–7.5);P = 0.00007] and CD8 T cells [20.5% (8.5–31.7) versus 4.1% (2.5–8.0);P = 0.00002] and increased CD95-induced apoptosis [21.4% (9.5–32.5) versus 5.1% (3.7–7.6);P = 0.0017 for CD4 T cells and 26.9% (7.2–37.7) versus 3.5% (2.3–5.4);P = 0.000012 for CD8 T cells] (Fig. 2a). The lack of control of apoptosis during HAART was still observed when specific CD3- and CD95-mediated apoptosis was calculated as the difference between receptor-induced apoptosis and spontaneous apoptosis (ΔCD3 and ΔCD95, respectively;Fig. 2b). Indeed, ΔCD3 [given as median percentage increase (25–75 percentile range)] in HAART-treated patients was significantly higher than in healthy controls for both CD4 T cells [8% (3.6–15.2) versus 2.6% (1.2–5.2);P = 0.0068] and CD8 T cells [12% (5–19) versus 1.6% (0.6–4.3);P = 0.0005]. Similarly, ΔCD95 in HAART-treated patients was significantly higher than in healthy controls for both CD4 T cells [11.6% (3.9–24.0) versus 2.1% (1.4–6.0);P = 0.004] and CD8 T cells [17.4% (3.3–31.0) versus 1% (0.3–4.0);P = 0.0002] (Fig. 2b). It is noteworthy that different susceptibilities to apoptosis were observed in different T cell subsets from patients, since the rate of apoptosis was significantly higher in CD8 T cells than in CD4 T cells (P = 0.00004 and P = 0.001 for ΔCD3 and ΔCD95, respectively). In contrast, no significant difference was observed between CD4 and CD8 T cell subsets in the level of spontaneous apoptosis (P = 0.07) in control donors.
Correlation of CD4 cell apoptosis during HAART with infection parameters
In order to determine which factors influence the level of T cell death during HAART, an univariate analysis was performed with the biological parameters listed in Table 1 (Spearman's test). This analysis revealed several interesting correlations in HAART-treated patients (Fig. 3). First, a negative correlation was observed between the levels of spontaneous, CD3- and CD95-induced apoptosis in CD4 T cells and the in vivo percentage of CD4 T cells (r = −0.712, P < 0.000001; r = −0.305, P = 0.033; and r = −0.493, P = 0.0004, respectively), as previously found in untreated HIV-positive patients [6,7]. Second, the increase in CD4 T cells during HAART (ΔCD4) was negatively correlated with the level of spontaneous apoptosis in this subset (r = −0.331;P = 0.018), suggesting that suppression of inappropriate CD4 T cell apoptosis contributes to CD4 T cell increase under potent antiretroviral therapy. Third , levels of spontaneous, CD3- and CD95-induced apoptosis in CD4 T cells were negatively correlated with the CD4/CD8 ratio, arguing for a role of apoptosis in the control of CD4/CD8 T cell homeostasis during HAART. Fourth, no correlation could be found between the three types of apoptosis in CD4 and CD8 T cells and either plasma viral load, extending data from previous studies , or lifetime exposure to HAART, except for CD3-induced apoptosis in CD8 T cells. However, plasma viral load was negatively correlated with CD4 T cell number at baseline (r = −0.375;P = 0.006) and with ΔCD4 T cells during HAART (r = −0.395;P = 0.004) but not with CD4/CD8 ratio during HAART (P = 0.1). Fifth, the level of spontaneous apoptosis in CD8 T cells was negatively correlated with the in vivo number (r = −0.429;P = 0.0013) and percentage (r = −0.378;P = 0.0068) of CD4 T cells; CD3-induced apoptosis was positively correlated with the in vivo number of CD8 T cells (r = 0.441;P = 0.001) and negatively correlated with lifetime exposure to HAART (r = −0.340;P = 0.015); and the CD95-induced apoptosis was positively correlated with the in vivo number of CD8 T cells (r = 0.349;P = 0.014) and negatively correlated with the in vivo percentage of CD4 T cells (r = −0.338;P = 0.02). Altogether, these data argue for a role of T cell apoptosis in the control of CD4 and CD8 T cell homeostasis during HAART, independently of the viral load and duration of therapy.
Influence of antiretroviral regimen on in vitro susceptibility to apoptosis
To identify a possible relationship between the level of T cell apoptosis and drug regimen, a multivariate analysis of parameters associated with spontaneous, CD3- or CD95-induced apoptosis during HAART was performed. This analysis showed that spontaneous apoptosis in CD4 and CD8 T cells was correlated with the absolute number of CD4 T cells (F = 22.8;P < 0.00002 and F = 6.0 ;P = 0.018, respectively). CD3- and CD95-induced apoptosis in both CD4 and CD8 T cell subsets were correlated with the in vivo CD4/CD8 ratio (ΔCD3: F = 5.0;P = 0.03 for CD4 T cells and F = 4.3;P = 0.044 for CD8 T cells; ΔCD95: F = 8.6;P = 0.0054 for CD4 T cells and F = 5.6;P = 0.022 for CD8 T cells). The level of T cell apoptosis was not directly related to the viral load.
Analysis of the possible in vivo influence of drug type on T cell apoptosis revealed that some of these drugs might contribute to the increased priming of CD4 and CD8 T cells for apoptosis. Spontaneous apoptosis may be influenced by RTV, which has a strong effect on CD8 T cells (F = 23.4;P < 0.00002); patients receiving RTV showed significantly increased spontaneous apoptosis compared with that in patients who did not receive RTV in both CD4 (15% versus 7%;P = 0.04) and CD8 (18.2% versus 6%;P = 0.02) T cells. This was not found when saquinavir or nelfinavir were considered. CD3-induced apoptosis in CD8 T cells was influenced by the non-nucleoside reverse transcriptase inhibitor (nevirapine or efavirenz) (F = 6.1;P = 0.017), and patients receiving this type of drug showed decreased CD3-induced apoptosis in both CD4 (6.3% versus 12%;P = 0.3) and CD8 (6% versus 17.3%;P = 0.08) T cells compared with patients who did not receive these drugs.
Interestingly, the NRTI 3TC appeared to influence CD95-induced apoptosis specifically, in both CD4 (F = 4.9;P = 0.032) and CD8 T cells (F = 5.9;P = 0.019). To determine the impact of 3TC-containing regimens on immunological and virological parameters, and on the level of apoptosis, the cohort was stratified into two groups on the basis of the antiretroviral regimen. Comparison of patients receiving 3TC (n = 40) with patients not receiving 3TC (n = 13) indicated that 3TC-treated patients showed an increased level of CD95-induced apoptosis compared with patients not receiving 3TC [18.7% versus 10.9% (P = 0.3) for CD4 T cells and 23.6% versus 12.7%; (P = 0.3) for CD8 T cells]. The same study was performed stratifying on the basis of zidovudine- or stavudine-containing regimens but these NRTI did not seem to influence the priming of T cells for apoptosis (not shown).
The effect of antiretroviral drugs on in vitro apoptosis in cells from healthy donors
Considering the in vivo contribution of antiretroviral drugs to the priming of T cells for apoptosis in HIV-positive patients, it was of interest to see if RTV and 3TC had apoptotic effects in vitro on normal pre-activated T cells. PBMC from healthy individuals (n = 05) were prestimulated for 3 days with coated anti-CD3 monoclonal antibodies, further incubated for 3 days in the presence of various concentrations of the antiretroviral drug and then stimulated for 1 day with anti-CD95 monoclonal antibodies. Figure 4a shows that 3TC significantly increased the level of CD95-induced apoptosis in both pre-activated CD4 and pre-activated CD8 T cells from healthy donors. This proapoptotic effect was observed at 2.5 mmol/l 3TC and was dose dependent. Spontaneous apoptosis was not affected at 2.5 mmol/l 3TC but was increased by 5 and 10 mmol/l. When RTV was tested under the same experimental conditions, it did not have any effect on spontaneous apoptosis of pre-activated T cells, while it slightly inhibited CD95-induced apoptosis of CD4 and CD8 T cells (Fig. 4b).
Recruitment of caspase-8 and caspase-3 in normal T cells in response to CD95
Caspase-8 recruitment is an essential event in CD95-induced apoptosis; active caspase-8 initiates a caspase cascade, recruiting effector caspases such as caspase-3, resulting in apoptosis . The influence of 3TC or RTV on caspase recruitment was tested following CD95 stimulation of T cells in separate experiments using PBMC from three healthy donors (Fig. 5). PBMC were prestimulated under the conditions described above and the intracellular expression of active caspase-8 and caspase-3 was detected by flow cytometry. Figure 5a shows that, following CD95 binding, 2.5 mmol/l 3TC increased the percentage of CD4 T cells expressing active caspase-8 and caspase-3 from 56% and 55% to 80% and 80%, respectively, while 1 μg/ml RTV had no effect. Activation by 3TC of both caspase-8 and caspase-3 was also observed in CD8 T cells (Fig. 5b). No effect was observed on spontaneous apoptosis at this concentration of 3TC.
Therapy-induced decrease in plasma HIV RNA has been shown to result in reduction in lymphocyte apoptosis, concomitant with an increase in CD4 T cells [6–9]. In this study, we show that, in almost 70% of PI-naive patients, HAART did not normalize the ex vivo levels of spontaneous, anti-CD3- and anti-CD95-induced apoptosis in both CD4 and CD8 T cell subsets. Among the 53 treated patients, 84% had a sustained CD4 cell increase while only 52% did achieve sustained viral suppression. However, no correlation was found between the level of T cell apoptosis during HAART and the viral load, while the level of T cell apoptosis was negatively correlated with the percentage of CD4 T cells and the CD4/CD8 ratio. Interestingly, a negative correlation was found between the increase in CD4 T cells from baseline and the levels of spontaneous apoptosis in CD4 T cells. A novel finding in this study was that the persistence during HAART of T cell priming for apoptosis could be associated with inclusion of the NRTI 3TC in the regimen. In vitro, 3TC increased CD95-induced apoptosis in pre-activated T cells from healthy donors and stimulated the recruitment of active caspase-8 and caspase-3. These findings, and the already reported immunomodulatory effect of PI, implicate antiretroviral regimens in modulating T cell homeostasis independently of their antiviral effect.
The possible causes of T cell disappearance in HIV infection are still a matter of debate, but they probably include movement of cells from the blood to lymphoid tissues, where the virus accumulates, accelerated destruction by apoptosis and impaired production of new T cells . Mechanisms contributing to the destruction of mature CD4 T cells include direct destruction of infected cells mediated by proapoptotic viral proteins and indirect induction of cell death in uninfected cells partly through chronic activation of the immune system [3,4]. The induction of apoptotic programmes in T cells from non-treated HIV-infected persons is supported by the in vivo detection of apoptotic cells in lymph nodes from HIV-positive patients [20,21] and the in vitro susceptibility to apoptosis of T cells from HIV-positive patients following activation of the T cell receptor or death receptor [10,22–27]. Recent studies, including ours, have shown that the increase in CD4 T cells following HAART is associated with a dramatic reduction in both spontaneous and death receptor-induced apoptosis in peripheral T lymphocytes [5,7–9] and in lymphoid tissues . The present study shows a longitudinal follow-up of drug-naive patients and demonstrates that normalization of apoptosis levels by HAART is very rapid and long lasting (Fig. 1), in line with previous observations in lymphoid tissues . We also found that the decrease of T cell priming for apoptosis was associated with the diminution of activation marker expression, although it occurred several weeks after apoptosis suppression (unpublished data). Similar observations have been reported by other groups [6,8].
We report for the first time that HAART is not systematically associated with suppression of T cell apoptosis in a cohort of patients who mostly experienced an increase in CD4 T cells (Fig. 2). As we previously reported in untreated patients [7,10], the levels of spontaneous, CD3- and CD95-induced apoptosis in this cohort were negatively correlated with the number of CD4 T cells but not correlated with the viral load, as indicated in a previous report  (Fig. 3). Interestingly, the levels of T cell apoptosis were significantly negatively correlated with the CD4/CD8 ratio during HAART, suggesting that T cell apoptosis plays a significant role in homeostasis of CD4 and CD8 T cells after initiation of potent therapy. In addition, CD4 T cell increase from baseline during HAART was negatively correlated with spontaneous apoptosis in CD4 T cells, suggesting that quantitative immunological recovery may dependent on the control of T cell survival (Fig. 3). Several mechanisms may be involved in the lack of suppression of apoptosis during HAART, including the greater proportion of T cells with the memory phenotype (CD45R0) in these patients (L. M. de Oliveira Pinto et al., unpublished data), memory cells being more susceptible to apoptotic signals than naive cells [10,30]. Survival of memory cells is dependent on growth factors, such as interleukins 2 or 15 [30,31]. Interleukin 2 synthesis is stimulated during HAART [32–34], but analysis of its production by memory and naive cells revealed a persistent functional defect in the naive T cells (E. Ledru et al., unpublished data). Therefore, persistent apoptosis in HAART-treated patients may be associated with an increased number of memory cells, the fragility of which is not efficiently prevented by cytokines.
The current study also shows that persistent increased T cell apoptosis is associated with the type of antiretroviral drug used. In particular, the NRTI 3TC may influence CD95-induced apoptosis in both CD4 and CD8 T cells. This is the first study showing that in vivo drug combinations influence the level of T cell apoptosis independently of the control of the viral load. Several recent reports suggested that PI drugs can interfere in vitro with survival of lymphocytes. RTV has been shown to inhibit apoptosis in PBMC from healthy donors  and HIV-infected patients  and in CD34 cells . It has also been shown to inhibit caspase-1 and caspase-3 activity [8,14,35]. In our study, RTV slightly inhibited CD95-induced apoptosis but had no effect on activation of caspase-8 and caspase-3 (Fig. 4). Indinavir and saquinavir have been shown to enhance survival of PBMC from HIV-positive patients , through inhibition of cell cycle progression rather than blockade of apoptosis . A novel finding was that the NRTI 3TC, associated with in vivo accelerated lymphocyte apoptosis during HAART, was a potent inducer in vitro of apoptosis mediated by CD95 in T cells from healthy donors. In addition, 3TC strongly stimulated the recruitment of caspase-8 and caspase-3 (Fig. 4), both involved in the CD95-dependent apoptotic pathway.
CD95-induced apoptosis is an essential event in the regulation of an antigen-specific T cell response; it may be necessary during HIV infection to counterbalance the chronic activation of effector T cells and to prevent accumulation of damaging cells. Suppression of activation-induced cell death by HAART may lead to a defect in physiological deletion of activated lymphocytes. Indeed, we have shown that peripheral T cells primed for tumour necrosis factor α synthesis may accumulate in patients taking HAART because of cell escape from apoptosis and that this was associated with the development of the lipodystrophy syndrome . In the present study, we found that patients receiving a regimen including 3TC tended to show a better quantitative restoration of CD4 T cells (data not shown). A recent study by Piconi et al.  showed that 3TC-containing bitherapy was more efficient in improving immune functions of PBMC from HIV-treated patients. Therefore, the maintenance of physiological levels of apoptosis during HAART may be essential for a better quantitative and qualitative immune restoration and to prevent the occurrence of the immune proinflammatory diseases reported in HIV-positive patients during HAART .
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