Discordant CD4 T lymphocyte responses to antiretroviral... : AIDS (original) (raw)
Our purpose was to determine if changes in CD4 cell counts in HIV-infected patients with good viral suppression on stable antiretroviral regimens could be predicted by ex-vivo rates of apoptosis of peripheral blood mononuclear cells (PBMC). Patients were grouped by lowest pre-treatment and highest on-treatment CD4 cell counts and classified as complete immune responders, partial responders, or non-responders. Whole blood was collected from a subgroup of patients and controls, and rates of the ex-vivo apoptosis of PBMC were assessed. Non-responders exhibited significantly increased apoptosis, whereas good immune responses were associated with decreased apoptosis. Persistently accelerated apoptosis may contribute to persisting immune deficiency independent of the viral load.
A progressive decrease in the number and function of CD4 T lymphocytes is the primary mechanism that leads to secondary infections in AIDS, and the goal of immune reconstitution is the reversal of this process. Much of the depletion and dysfunction of CD4 cells involves uninfected cells. One potential mechanism is accelerated apoptosis [1]. Landmark studies have demonstrated that CD4 cell counts promptly increase when antiretroviral therapy suppresses HIV replication [2]. However, discordant immune responses to antiretroviral therapy occur, including poor immune responses that result in disease progression despite marked reductions in viral load [3]. The purpose of this study was to determine if immune responses in HIV patients with complete viral suppression on antiretroviral therapy could be predicted by ex-vivo rates of apoptosis.
Records were reviewed for approximately 800 HIV-infected patients. A total of 122 patients on stable antiretroviral regimens with HIV-RNA levels of less than 500 copies/ml for over 6 months were identified. Patients were grouped according to absolute CD4 lymphocyte counts before the initiation of antiretroviral therapy: group 1, CD4 cell count < 100/mm3; group 2, 100–199/mm3; group 3, 200–399/mm3; group 4, 400–699/mm3; and group 5, 700/mm3 or greater. This grouping was used rather than the three-group Centers for Disease Control and Prevention classification (normal immune status, CD4 cell count > 500/mm3; moderate immune suppression, CD4 cell count 200–500/mm3; and severe immune suppression, CD4 cell count < 200/mm3) to increase discrimination and detect more modest immune recovery. After antiretroviral therapy patients were classified as follows. Complete immune responders were defined as patients whose CD4 lymphocyte count increased to 700/mm3 or greater on antiretroviral therapy and partial responders as patients with a CD4 cell count increase of more than 50% over their lowest pre-treatment value and improved by at least one immune grouping according to the above criteria. All others were considered as non-responders.
Heparin anti-coagulated blood was collected from six HIV-negative controls and 20 HIV-infected patients with complete viral suppression. PBMC were isolated and cultured at 37°C in 5% carbon dioxide. After 72 h cells were stained with acridine orange and ethidium bromide, and then examined by fluorescent microscopy to observe the morphological changes of apoptosis and assess viability.
There were 38 complete responders (31.1%), 68 (55.7%) partial responders, and 16 (13.1%) non-responders out of the 122 patients with HIV-RNA levels of less than 500 copies/ml identified by chart review. Patients for whom ex-vivo apoptosis was evaluated were representative of this cohort, with four (20%) complete responders, 12 (60%) partial responders, and four (20%) non-responders (see Table 1). All but one patient had their viral load determined by an ultra-sensitive assay with a detection cut-off of 50 copies/ml, and only four patients had a detectable viral load at this level; one complete responder, two partial responders, and one non-responder.
Rates of peripheral blood mononuclear cell apoptosis and clinical characteristics by immune response category.
Immune non-responders exhibited significantly accelerated apoptosis compared with all other control and patient groups by analysis of variance (P < 0.01). Partial responders were also significantly different from controls. A highly significant correlation was found between immune response and PBMC apoptosis (Spearman R = −0.69;P = 0.001). Multiple regression analysis demonstrated statistically significant contributions from both male sex (P = 0.01) and from lymphocyte apoptosis (P = 0.001), but obviously the small number of female patients limited this analysis.
_t_-Test analysis comparing male and female patients showed no statistically significant difference in rates of apoptosis (P = 0.6). No other relationships were significant, including the lowest pre-treatment CD4 cell counts, pre-treatment viral load, peak viral load, previous opportunistic infections, or particular specific antiretroviral agents, but again, the number of patients were small. Data were also analysed using the three immune categories defined by the Centers for Disease Control and Prevention and there was still a highly significant correlation between immune response and apoptosis.
Our data demonstrate a strong relationship between the immune response to antiretroviral therapy and the ex-vivo rates of apoptosis of PBMC for patients on antiretroviral therapy with complete viral suppression. Even when controlling for age, duration of antiretroviral therapy, duration of viral suppression, peak viral load, baseline CD4 cell counts, antiretroviral medications, opportunistic infections or other co-infections, and other concomitant medical illnesses, this relationship is highly significant (P = 0.001).
The significance of T cell apoptosis in HIV immune pathogenesis has been debated. Our study suggests that apoptosis is an important mechanism of T cell depletion in HIV infection, independent of viral replication. Karmochkine and colleagues [4] showed that ex-vivo rates of apoptosis for PBMC correlated with the viral load and correlated negatively with the CD4 cell count. Thirty per cent of the variability in apoptosis rates after activation were predicted by the viral load and 40% by the CD4 cell count, suggesting that viral replication is not the only process leading to T cell depletion.
Data on the effects of antiretroviral therapy on T cell apoptosis are limited. Chavan _et al._[5] showed a decreased rate of lymphocyte apoptosis after the initiation of antiretroviral therapy. Regamey et al. [6] observed reduced apoptosis and increased expression for patients on antiretroviral therapy compared with patients not on therapy. Other studies, however, produced conflicting results. A recent kinetic study [7] showed decreased survival of both CD4 and CD8 T cell subsets, but highly active antiretroviral therapy had no effect on this. A more recent study by the same group [8], however, yielded different results, with improved T cell survival and a decreased rate of ex-vivo apoptosis, as well as increased T cell production resulting from effective antiretroviral therapy.
To our knowledge this is the first study showing persistent apoptosis in a subgroup of patients with complete viral suppression in association with poor immune recovery. Immune alterations independent of active viral replication may be responsible. Recent data suggest that immune responses to antiretroviral therapy depend on residual or restored thymic function. Improved CD4 cell counts in patients, despite virological treatment failure, are associated with greater thymic function, whereas poor T cell responses despite the suppression of HIV are seen with decreased thymic function [9]. Discordant immune responses may also be caused by the differential effects of particular antiretroviral agents on T cell apoptosis independent of viral suppression. For example, protease inhibitors have been shown to decrease rates of apoptosis of uninfected T cells [10]. Viral replication is never completely suppressed with highly active antiretroviral therapy, even when patients have undetectable plasma HIV-RNA levels. Therefore, varying degrees of low-level viral replication or replication in certain cellular compartments may continue to drive T cell apoptosis.
David L. Pitrak
José Bolaños
Ronald Hershow
Richard M. Novak
References
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© 2001 Lippincott Williams & Wilkins, Inc.