1 Service des Maladies Infectieuses et Tropicales, Hôpital de lArchet 1, Centre Hospitalo-Universitaire de Nice, BP3079, 06202, Nice cedex 3; 2 Unité INSERM 343, Centre Hospitalo-Universitaire de Nice, Hôpital de lArchet 1, BP3079, 06202, Nice cedex 3; 3 Alphabio, Marseille, France
Received 12 December 2002; returned 5 March 2003; revised 22 April 2003; accepted 22 April 2003
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Abstract |
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Keywords: apoptosis, proliferation, HIV infection, therapeutic interruption
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Introduction |
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Methods |
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Sample collection
Six blood samples were collected from the date of STI, and thereafter at weeks 2, 4, 6, 8 and 12. Viral load determination, T cell subset count and apoptosis were determined on fresh cells, and peripheral blood mononuclear cell (PBMC) proliferation was measured on defrosted cells.
Quantification of plasma viraemia
HIV-1 RNA plasma levels were measured using a commercially available assay, according to the manufacturers instructions (Amplicor HIV-1 Monitor Test, Roche Laboratories), with a threshold sensitivity of 40 copies/mL.
Flow cytometry analysis
The mouse monoclonal antibodies (mAbs) specific for human surface antigens used for identification of T cell subpopulations were purchased from TEBU Laboratory (Le Perray en Yvelines, France) and included: CD45RA-PE, CD45RO-PE and CD38-PE. Antibodies CD3-FITC, CD4-PC5 and CD8-PE are produced in our laboratory. Lymphocyte subsets were determined on freshly isolated blood using monoclonal antibodies to CD3, CD4 or CD8, CD45RA or CD45RO, and CD38 in triple staining. Following acquisition of 5000 CD3+ lymphocytes, analysis was performed using a FacStar flow cytometer and Cellquest software (Becton Dickinson, Mountain View, CA, USA).
Apoptosis assay
T cell subsets and apoptosis of T cell subsets were determined using cytofluorometry, as previously reported.15 Hoechst 33342 dye, which detects programmed cell death (PCD)-associated DNA alterations, was used to quantify apoptotic cells. For the apoptosis assay, PBMC (5 ¥ 105 cells/mL) were cultured overnight, in the medium alone or with agonistic mAbs to Fas/CD95, 30 ng/mL final dilution (Euromedex, Souffel-Weyersheim, France), in 48-well plates. In our experiments (data not shown), this latter resulted in 100% death of Jurkat cells. For each sample, 5000 lymphocytes with a specific phenotype were analysed with CellQuest software in a FACSVantage cytometer (Becton Dickinson). Each condition was tested in duplicate. Remaining cells were frozen in a mixture containing 80% (RPMI + 50% FCS) + 20% dimethyl sulfoxide.
Proliferation assay
The proliferation capacity of PBMC after STI was tested using frozen cells. Cells were thawed rapidly and washed twice in medium; cell viability was evaluated by Trypan Blue exclusion. Proliferation was assessed using the CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA). This is a colorimetric method measuring the production of soluble products, which is proportional to the number of viable cells.16,17 Experiments were performed, following the manufacturers instructions, using an ELISA plate reader (iEMS Reader, LabSystem, Cergy-Pontoise, France). In preliminary experiments, we had determined that the optimal conditions for observing an absorbance increase at 490 nm were 50 000 cells per well, with a 4 day culture duration (data not shown). Each time-point was tested in triplicate. Wells in triplicate, containing medium and reagents but without cells, served as a negative control, to determine the background absorbance, which was subtracted from other absorbance values.
Genotypical resistance
HIV resistances were determined as previously described.18 Briefly, sequencing of the major part of the reverse-transcriptase gene (nucleotides 25230) and the entire protease gene were performed on plasma HIV-1 RNA extracted from patients plasma samples. The viral RNA was retrotranscribed into complementary DNA, and subsequently amplified by single tube PCR using the TruGene HIV-1 assay (Visible Genetics, Toronto, Canada), following the manufacturers instructions. Bidirectional DNA sequencing of the amplification products was performed with a sensitive sequencing method (CLIP, Visible Genetics). Each sequencing reaction was loaded into a MicroGene Clipper sequencer (Visible Genetics). The sequence of each sample was compared to a database of known drug-resistance mutations, to find out which mutations were present in the HIV-1 RNA. Classification of the mutations, associated or not with decreased drug sensitivity, was established according to the consensus statement on antiretroviral drug-resistance testing.19
Statistical analysis
In order to show the impact of STI on apoptosis and proliferation of mononuclear cells, we have calculated the percentage of variation from baseline for each patient as follows: percentage of variation = [apoptosis or proliferation level at W(x) apoptosis or proliferation level at baseline]/apoptosis or proliferation level at baseline. Statistical analysis was based on Wilcoxons non-parametric rank test. A P value of <0.05 was considered significant. The whole analysis was performed on Statview F-5 software (Abacus, CA, USA).
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Results |
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In order to explain these immunological variations, we studied concomitantly the variations in apoptosis of CD4+ and CD8+ T cells, and of mononuclear cell proliferation, as explained in the Methods section. As shown in Figure 2(a), triple staining using CD3-FITC and CD4-PE mAbs plus Hoechst dye allowed us to quantify PCD on both T cell subsets. The percentage of spontaneous apoptotic CD4+ T cells increased from 20.5% (843) at baseline to 35% (940) at week 12 (P = 0.16). The percentage of spontaneous apoptotic CD8+ T cells increased significantly from 14.5% (937) to 31% (1546) (P = 0.01). The kinetics of spontaneous apoptosis, as well as Fas-induced apoptosis of both CD3+ cell subsets and naive CD4+ T cells, are shown in Figure 2(b).
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Our mononuclear cell proliferation assay provided evidence of significant variations in spontaneous proliferation capacities of mononuclear cells (Figure 2c). Both spontaneous and phytohaemagglutinin (PHA)-induced proliferation was increased compared with baseline, but not in similar proportions. The spontaneous proliferation index increased from 0.011 (0.010.04) at baseline to 0.04 (0.250.836) at week 12 (P = 0.01). However, spontaneous proliferation appeared to occur early and only for a short time (Figure 2c). For PHA-induced proliferation, values were 0.15 (0.0010.423) and 0.103 (0.25800), respectively (P = 0.34).
Genotypical resistance and impact on T cell apoptosis
As shown in Table 2, seven patients out of 10 exhibited no persistent primary mutation in the reverse transcriptase gene, as well as in the protease gene over 8 weeks. We then compared the impact of resistance mutation disappearance on CD4 T cell apoptosis. As shown in Figure 3, patients with no persistent resistance mutation exhibited an increasing level of CD4+ T cell apoptosis from baseline to week 12, but without reaching statistical significance. In contrast, variations in CD4+ T cell apoptosis in patients without complete reverse resistant mutations showed a similar level of T cell apoptosis. Finally, whatever the time-point considered, the viral load did not appear to be correlated with the level of T cell subset apoptosis (data not shown).
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Discussion |
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A recent report indicated that the decrease in CD4+ T cell count occurring in STI was related to the re-emergence of drug-susceptible wild-type virus,8 suggesting that these immunological alterations may be caused by the increase in viral fitness. However, direct HIV cellular toxicity still needs to be demonstrated fully, and indirect mechanisms have been put forward to explain CD4 T cell lymphopenia.1315 Since STI appears to induce a period of intense immunological changes, we considered measuring apoptosis in T cell subsets and proliferation modifications.
Our results indicate that STI is associated with both an up-regulation of apoptosis in T cell subsets, and an early and transient increase in proliferation of PBMC. Spontaneous CD4+ T cell apoptosis appears to be up-regulated earlier than CD8+ T cell apoptosis, but is also biphasic with a decrease between weeks 2 and 6 and a second rise thereafter (see Figure 2b). This pattern of CD4+ T cell apoptosis is similar to that of both naive and memory CD4+ T cell counts. In contrast, the augmentation in CD8+ T cell apoptosis was linear over 12 weeks and did not fit with variations in CD8+ T cell counts. In addition, we did not observe significant variations in Fas-induced apoptosis, whatever the T cell subset considered.
Proliferation of mononuclear cells appears to rise early during STI, but only for a short period. This result is in accordance with previous studies, and is thought to represent endogenous immunization as a result of active viral replication.7,9 Indeed, we observed an early increase in CD8+ CD38+ cells and CD8+ CD45RA+ T cells (Figure 1b), which is in accordance with previous studies showing that proliferation of T cells in HIV-infected patients concerned mainly CD8+ T cells.23,24 Moreover, studies showed that STI leads to expansion of differentiated CD8+ T cells when viral rebound occurred, and that in contrast, the increase in HIV-specific CD4+ T cells was only transient and mainly observed in patients with low viral load at baseline.25,26 This expansion of CD8+ T cells may explain the increase in proliferation of mononuclear cells observed in our study (see Figure 2c). It should be noted that this putative specific immunological activation was unable to control HIV replication, as demonstrated by the viral load curve (see Figure 1a). Furthermore, this cellular proliferation was also unable to maintain the absolute number of T cells, highlighting the role of apoptosis in the occurrence of lymphopenia.
The existence of both apoptosis and proliferation of immune cells during STI is not surprising. It is noteworthy that these cellular events have been demonstrated during primary HIV syndrome, and that STI may lead to acute retroviral syndrome.10,11 However, the precise mechanisms of up-regulation of apoptosis during STI are unknown. Spontaneous apoptosis of T cells is independent of HIV load in patients receiving antiretroviral treatment;15 this observation is confirmed in the present study (data not shown). In addition, CD8+ T cells mediating antibody-dependent cellular cytotoxicity (ADCC) were shown to be deleterious in both experimental and clinical studies.2730 Thus, because STI leads to viral replication, it might be associated with increased production of viral proteins known to stimulate ADCC, such as gp120, which in turn leads to up-regulation of T cell apoptosis. Accordingly, CD4 stimulation by agonistic antibodies induces a higher level of CD4+ T cell apoptosis in patients with detectable viral load despite antiretroviral combinations, compared with patients with undetectable viral load.28,29,31 Moreover, reverse resistance mutations to antiviral drugs is associated with a higher viral fitness,8 but also with apoptosis up-regulation, as suggested by our results (see Figure 3). Further studies are needed to confirm these hypotheses.
The importance of a decrease in CD4+ T cells occurring with STI strongly suggests that STI should be limited to patients without immune defects. It is noteworthy that Deeks et al.8 reported three patients with severe adverse effects as a result of STI. Garcia et al.32 have shown that in patients with a normal immunological status at baseline, STI may lead to prolonged lymphopenia, persisting even after the re-introduction of antiretroviral treatment.32 In the same way, reports indicate that STI was not followed systematically by significant up-regulation of HIV-specific clones, but if so, only for a short period.26,32,33 As an explanation for these results, our present study suggests strongly that during STI apoptosis of T cells is an overwhelming phenomenon compared to T cell proliferation. Accordingly, immunological benefits of STI should be only transient, and this therapeutic option considered with caution in immunodepressed patients.
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Footnotes |
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References |
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