Persistent viral load in patients on HAART is associated with a higher level of activation-induced apoptosis of CD4+ T cells

Pierre-Marie Rogera,*, Jean-Philippe Breittmayerb, Jacqueline Cottalordac, Michel Ticchionib, Véronique Mondaina, Pierre Dellamonicaa and Alain Bernardb

a Service des Maladies Infectieuses et Tropicales, b Laboratoire d'Immunologie – INSERM U343, Hôpital de l'Archet and c Laboratoire de Virologie, Hôpital Pasteur, Nice, France


    Abstract
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Apoptosis is one of the mechanisms involved in the persistent CD4+ T cell lymphopenia occurring in human immunodeficiency virus (HIV)-infected patients treated with highly active antiretroviral therapy (HAART). The aim of this study was to look at the relationship between the level of T cell apoptosis in patients on HAART and their HIV viral load measurement. Forty-five patients receiving HAART for a median time of 5 months were included: 13 had an undetectable viral load and 32 had a viral load >=200 copies/mL. No relationship was observed between the level of spontaneous T cell apoptosis and the viral load measurement. Patients with a viral load >=200 copies/mL exhibited a significantly higher level of CD4-induced apoptosis of CD4+ T cells, compared with patients with undetectable viraemia (38 versus 15%, respectively, P = 0.03). Activation-induced apoptosis may be involved in the paucity of the immune reconstitution observed in HIV-infected patients treated with HAART.


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
The use of highly active antiretroviral therapy (HAART) in patients infected with the human immunodeficiency virus (HIV) is associated with a marked decrease in plasma viraemia.1 However, CD4+ T cell lymphopenia, which is the characteristic immune defect of AIDS, can be persistent despite a prolonged undetectable viral load.2 The reasons for this incomplete immune reconstitution are still not clearly understood. Apoptosis has been proposed as a mechanism to account for the CD4+ lymphopenia.3–6

T cell apoptosis appears to be upregulated using agonistic monoclonal antibodies to CD4 or Fas surface molecules.3,5 The gp120 HIV protein can stimulate the CD4 pathway and promote apoptosis of HIV-negative CD4+ T cells, and soluble gp120 has been detected in patients' blood.3,4 Recently, we have demonstrated that spontaneous T cell apoptosis was reduced by HAART, compared with combination treatments with two nucleoside analogues.6 We did not observe any correlation between the level of spontaneous T cell apoptosis and HIV plasma viraemia, suggesting that viral factors, and not the amount of virus present, could be responsible for T cell apoptosis. In this study, we have examined the relationship between the HIV viral load measurement and CD4-induced apoptosis as this pathway is thought to mimic the in vivo stimulation of CD4+ T cells via the gp120 molecule.


    Patients and methods
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
HIV-infected population studied

Patients were recruited from a large cohort of healthy donors and HIV-infected individuals, in whom spontaneous apoptosis and stimulation-induced apoptosis via surface molecules were studied.6,7 Only HIV-infected patients on HAART with two nucleoside analogues and a protease inhibitor were included in order to avoid the impact of different treatments on T cell apoptosis. Epidemiological and biological data were obtained with a computerized medical record.6 Determinations of peripheral T cell populations and T cell apoptosis by cytofluorometric techniques, and the HIV viral load measurement were performed on the same blood sample. HIV-1 RNA plasma levels were measured using commercially available assays, according to the instructions of the manufacturers. An undetectable viral load was defined as being less than the lowest quantification limit for each type of assay used at that time.

Apoptosis assay

Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood by centrifugation on a Ficoll– Hypaque gradient, washed and resuspended in RPMI 1640 complemented with 20 mM HEPES, 2 mM l-glutamine, 10% heat-inactivated fetal calf serum, penicillin and streptomycin (Life Technologies, Cergy Pontoise, France). Cell viability was evaluated by Trypan Blue exclusion.

The mouse mAbs specific for human surface antigens used included: CD4 (clone O516), CD8 (clone L533) and CD3 (clone X3), produced in our laboratory. mAb coating used 200 µL of O516 (10 mg/L final concentration) in 48-well plates incubated for 1 h at 37°C. For the apoptosis assay, PBMC (5 x 105 cells/well) were cultured overnight in the medium or on coated plates.

Apoptotic cells were quantified using Hoechst 33342 dye (H342) which detects apoptosis-associated DNA degradation (Sigma Laboratory, l'Isle d'Abeau, France). Cultured cells were first incubated for 30 min at 4°C with CD3 and CD8 mAb (10 µg/mL) coupled with fluorescein isothiocyanate and phycoerythrin, respectively. Cells were thereafter washed in RPMI-FCS 10% and stained by incubation for 30 min at 37°C with H342 (1 mg/L final concentration). For each sample, 104 double-stained lymphocytes were analysed with Lysis II software in a FACStar flow cytometer (Becton Dickinson, Mountain View, CA, USA).

Spontaneous and CD4-induced T cell apoptosis were determined in the same experiment for all patients. Each was tested in duplicate. To assess the impact of viral load on T cell activation-induced apoptosis, we compared patients with a detectable viral load with patients with an undetectable one. We used Student's t-test or the Pearson correlation test as appropriate. A P value <0.05 was considered significant.


    Results
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Forty-five patients were included; none of them presented with an acute illness. They had received HAART for a median time of 5 months at the time of T cell apoptosis determinations. Thirteen patients had an undetectable viral load (<200 copies/mL) and 32 a plasma viraemia above the lowest limit of quantification. As shown in the TableGo, there was no epidemiological or biological difference between these two groups of patients. Similarly, there was no correlation either between viral load and CDC staging at baseline or between T cell apoptosis and CDC staging.


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Table. Characteristics of the study patients
 
No relationship was observed between the level of spontaneous apoptosis and the HIV viral load (Pearson test, P = 0.33). There was a weak correlation between activation-induced apoptosis and viral load. Thus, we have compared patients with a detectable viral load, regardless of its magnitude, with patients with an undetectable viraemia: a significantly higher level of CD4-induced apoptosis of CD3+ CD8 (which are CD4+ T cells) was observed, 38 versus 15%, respectively (P = 0.03, see the FigureGo). In contrast, CD4 pathway stimulation did not upregulate CD8+ T cell apoptosis.



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Figure. Detectable viral load is associated with activation-induced apoptosis of CD4+ T cells. T cell apoptosis level from 13 patients presenting an undetectable viral load was compared with the level of apoptosis observed in 32 patients presenting a plasma HIV RNA level over the limit of detection of the current assays. Activation-induced apoptosis of CD4+ T cells is associated with an active viral replication. Results are presented as median with 25th and 75th percentiles. Attached horizontal bars represent 10th and 90th percentiles. Circles represent values from individual patients. Shading indicates patients with detectable viral load. Unfilled bars represent patients with undetectable viral load.

 

    Discussion
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Here we show that activation-induced apoptosis of CD4+ T cells via the CD4 pathway is associated with a detectable plasma viraemia in HIV-infected patients. This is in contrast to the absence of a demonstrable relationship between the level of spontaneous apoptosis of T cell subsets and the viral load.

Our results are in accordance with a previous study showing an association between the HIV viral load and activation-induced apoptosis of T cells in HIV-infected patients.8 However, in this latter work, antiretroviral treatments and their durations were not studied. Because antiretroviral treatments modulate apoptosis, we studied patients receiving two nucleosides and a protease inhibitor for a similar duration.

These results highlight the role of active viral replication on CD4+ T cell death. Even a low level of viral replication is able to upregulate activation-induced apoptosis of CD4+ T cells. Experimental studies have shown that HIV replication and/or HIV proteins such as gp 120 or Tat are needed to induce apoptosis.4,5 In addition, it has been shown that HIV-infected antigen-presenting cells (APC) were able to induce apoptosis of uninfected T cells.3,9 Thus, because infected APC are persistently infected over the natural course of the disease, we suggest that local bursts of viral replication, as described in lymphoid tissues,10 induce deleterious interactions with uninfected CD4+ T cells, leading to activation-induced apoptosis. Further studies are needed to confirm this hypothesis.

CD4-induced apoptosis has been reported to be dependent on APC, leading to an upregulation of CD8+ T cell death.3 However, we found no relationship between spontaneous or CD4-induced apoptosis of CD8+ T cell and viral load.

Taken together, our results and others have important implications for the understanding of the partial immune reconstitution in HIV-infected patients on HAART. The precise understanding of cellular interactions occurring in HIV infection could lead to new therapeutic interventions.


    Acknowledgments
 
This work was supported by a grant from the Fondation pour la Recherche Médicale and from the Association pour la Recherche contre le Cancer.


    Notes
 
* Correspondence address. Service des Maladies Infectieuses et Tropicales, Hôpital de l'Archet, Route St Antoine de Ginestière, BP79, 06202, Nice, France. Tel: +33-4-92-03-54-52; Fax: +33-4-93-96-54-54. Back


    References
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
1 . Gulick R. M., Mellors, J. W., Havlir, D., Eron, J. J., Gonzales, C., McMahon, D. et al. (1997). Treatment with indinavir, zidovudine and lamivudine in adults with human immunodeficiency virus infection and prior antiretroviral therapy. New England Journal of Medicine 337, 734–9.[Abstract/Free Full Text]

2 . Pakker, N. G., Kroon, E., Roos, M., Otto, S. A., Hall, D., Wit, F. W. et al. (1999). Immune restoration does not invariably occur following long-term HIV-1 suppression during antiretroviral therapy. AIDS 13, 203–12.[ISI][Medline]

3 . Oyaizu, N., McCloskey, T. W., Than, S., Kalyanaraman, V. S., Pahwa, S. & Hu, R. (1994). Cross-linking of CD4 molecules upregulates Fas antigen expression in lymphocytes by inducing interferon-gamma and tumor necrosis factor-alpha secretion. Blood 84, 2622–31.[Abstract/Free Full Text]

4 . Foster, S., Beverley, P. & Aspinall, R. (1995). gp120-induced programmed cell death in recently activated T cells without subsequent ligation of the T cell receptor. European Journal of Immunology 25, 1778–82.[ISI][Medline]

5 . Westendorp, M. O., Frank, R., Ochsenbauer, C., Stricker, K., Dhein, J., Walczak, H. et al. (1995). Sensitization of T cells to CD95-mediated apoptosis by HIV-1 Tat and gp120. Nature 375, 497–500.[ISI][Medline]

6 . Roger, P. M., Breittmayer, J. P., Arlotto, C., Pugliese, P., Pradier, C., Bernard-Pomier, G. et al. (1999). Highly active anti-retroviral therapy (HAART) is associated with a lower level of CD4+ T cell apoptosis in HIV-infected patients. Clinical and Experimental Immunology 118, 412–6.[ISI][Medline]

7 . Bernard, G., Breittmayer, J. P., De Matteis, M., Trampont, P., Hofman, P., Senik, A. et al. (1997). E2/CD99 mediates programmed cell death of CD4+ CD8+ huamn thymocytes with an intermediate CD3 density. Journal of Immunology 158, 2543–5.[Abstract]

8 . Karmochkine, M., Parizot, C., Calvez, V., Coutellier, A., Herson, S., Debre, P. et al. (1998). Susceptibility of peripheral blood mononuclear cells to apoptosis is correlated to plasma HIV load. AIDS Research and Human Retroviruses 17, 419–23.

9 . Cottrez, F., Manca, F., Dalgleish, A. G., Arenzana-Seisdedos, F., Capron, A. & Groux, H. (1997). Priming of human CD4+ antigen-specific T cells to undergo apoptosis by HIV-infected monocytes. Journal of Clinical Investigation 99, 257–66.[Abstract/Free Full Text]

10 . Grossman, Z., Feinberg, M., Kuznetsov, V., Dimitrov, D. & Paul, W. (1998). HIV infection: how effective is drug combination treatment? Immunology Today 19, 528–32.[ISI][Medline]

Received 19 April 2000; returned 30 August 2000; revised 18 September 2000; accepted 11 October 2000