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HAART Does Not Reverse Premature T-Cell Aging Caused by HIV
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"Expression of CD28, CTLA-4 and perforin by CD4 lymphocytes remain dysregulated in HIV-infected patients with previous severe immunodeficiency, despite increased CD4 T-cell counts and control of HIV viraemia by HAART....
...The results were compared with data from healthy controls to assess whether HAART restores normal proportions of lymphocytes expressing these markers......Here we provide evidence that CD4 lymphocytes from previously severely immunodeficient HIV-infected patients have increased expression of the co-inhibitory protein CTLA-4 and decreased expression of the co-stimulatory protein CD28, despite long-term HAART and complete suppression of HIV replication. T-cell expression of these molecules in HIV-infected patients on HAART may help to define the degree of immune dysfunction and to predict the efficacy of therapeutic vaccines or other immune-based therapies in late-presenting HIV-infected patients with very low nadir CD4 T-cell counts.....HIV-infected patients with higher proportions of CTLA-4-expressing CD4 lymphocytes and lower proportions of CD28-expressing CD4 lymphocytes may be at greater risk of impaired immune responses [7,9]. However, similar numbers of CD28-expressing and perforin-expressing CD8 lymphocytes from HIV-infected patients and controls may indicate that HAART can restore normal CD8 T-cell function in HIV-infected patients with nadir CD4 T-cell counts<50 cells/μL."
Dysregulation of CD28 and CTLA-4 expression by CD4 T cells from previously immunodeficient HIV-infected patients with sustained virological responses to highly active antiretroviral therapy
HIV Medicine July 2005 Vol 6 pg 278
SF Stone, P Price and MA French
Department of Clinical Immunology and Biochemical Genetics, Royal Perth Hospital and School of Surgery and Pathology, University of Western Australia, Perth, Australia
Objectives
Current guidelines recommend commencing highly active antiretroviral therapy (HAART) in HIV-infected patients when CD4 T-cell counts reach 350 cells/μL. However, late-presenting HIV-infected patients with CD4 T-cell counts<50 cells/μL are still common. The ability of long-term HAART to normalize immune dysregulation in severely immunodeficient HIV-infected patients remains unclear. Here we address indices of immune dysregulation in previously severely immunocompromised HIV-infected patients treated with long-term HAART who had achieved increased CD4 T-cell counts and complete suppression of HIV viraemia.
Methods
We examined expression of CD28, cytotoxic T-lymphocyte antigen-4 (CTLA-4) and intracellular perforin by CD4 and CD8 lymphocytes from 25 highly selected HIV-infected patients [nadir CD4 T-cell counts <50 cells/μL, >4 years on HAART and >6 months of complete viral suppression (<50 HIV-1 RNA copies/mL)] and 18 HIV-seronegative age- and sex-matched controls.
Results
HIV-infected patients had lower percentages of CD28-expressing CD4 lymphocytes and higher percentages of CTLA-4-expressing CD4 lymphocytes than controls. The percentage of CTLA-4-expressing CD4 lymphocytes correlated inversely with that of CD28-expressing CD4 lymphocytes. The proportion of CD4 lymphocytes expressing perforin was generally low. However, more HIV-infected patients than controls had >1% of CD4 lymphocytes expressing perforin [11 of 25 (44%) vs. one of 18 (5.5%)]. The percentage of CD8 lymphocytes expressing perforin did not differ between HIV-infected patients and controls. Amongst HIV-infected patients, the percentage of perforin-expressing CD8 lymphocytes correlated inversely with nadir but not current CD4 T-cell count.
Conclusions
Expression of CD28, CTLA-4 and perforin by CD4 lymphocytes remain dysregulated in HIV-infected patients with previous severe immunodeficiency, despite increased CD4 T-cell counts and control of HIV viraemia by HAART.
Introduction
Current guidelines recommend commencing highly active antiretroviral therapy (HAART) in HIV-infected patients when CD4 T-cell counts reach 350 cells/μL to achieve maximal immune reconstitution in a majority of patients [1]. However, late-presenting HIV-infected patients with very low CD4 T-cell counts at diagnosis are still common. For example, two recent studies found that 15-18% of HIV-infected patients presented with CD4 T-cell counts<50 cells/μL [2,3]. The ability of long-term HAART to normalize immune dysregulation in severely immnunodeficient HIV-infected patients remains unclear.
CD28 and cytotoxic T-lymphocyte antigen (CTLA)-4 play a crucial role in regulating the activation of T cells after engagement by CD80/CD86 on antigen-presenting cells [4]. CD28 is constitutively expressed on the surface of T cells and provides a stimulatory signal. CTLA-4 is maintained as an intracellular pool and is transiently expressed on the cell surface, where it provides an inhibitory signal [5]. Chronic untreated HIV infection results in decreased expression of CD28 [6] and increased expression of CTLA-4 by CD4 T cells [7], where both may contribute to impaired T-cell function. The ability of HAART to completely normalize expression of CD28 and CTLA-4 remains unclear. HIV-infected patients who successfully produce antigen-specific lymphoproliferative and humoral responses after HAART have higher numbers of CD28-expressing CD4 T cells than HIV-infected patients who do not recover normal immune responses [8,9]. Increased CTLA-4 expression by T cells from HIV-infected individuals receiving HAART correlated inversely with CD4 T-cell number and lymphoproliferative responses to anti-CD3, glycoprotein 120 (gp120)-depleted HIV immunogen (Remune; Immune Response Corp., Carlsbad, CA) and HIV-1 p24 stimulation [7]. However, the proportion of HIV-infected patients with viral loads <50 HIV-1 RNA copies/mL and the length of time these patients had been aviraemic were not discussed, so the results cannot be interpreted relative to treatment success [7].
Perforin is an effector molecule that plays an important role in cytotoxic T-cell activity. Although few HIV-specific CD8 T cells express high levels of perforin [10], patients with chronic HIV infection have higher levels of total perforin-expressing CD8 T cells than healthy controls, possibly because of persistent immune activation [11]. The percentages of total and HIV-specific CD8 T cells expressing intracellular perforin are significantly lower in HAART-treated patients than in untreated HIV-infected patients [12,13]. Increased expression of CTLA-4 may directly limit expression of perforin by CD8 T cells from HIV-infected patients, because blockade of CTLA-4 can enhance cytotoxic CD8 T-cell responses in mice [14]. However, expression of CD28, expression of CTLA-4 and expression of perforin have not been addressed together in HIV-infected patients with nadir CD4 T-cell counts<50 cells/μL with both virological and immunological responses to HAART.
Here, we assessed cell surface CD28, total CTLA-4 and intracellular perforin expression by CD4 and CD8 lymphocytes from HIV-infected patients with nadir CD4 T-cell counts<50 cells/μL, at least 4 years on HAART and 6 months of complete viral suppression (<50 copies/mL). The results were compared with data from healthy controls to assess whether HAART restores normal proportions of lymphocytes expressing these markers.
Discussion
This study has shown that severely immunodeficient HIV-infected patients who achieved complete suppression of HIV replication and increased CD4 T-cell counts after 4 years of HAART had a higher percentage of CD4 lymphocytes expressing CTLA-4 and a lower percentage of CD4 lymphocytes expressing CD28, compared with healthy controls. In addition, the percentage of CTLA-4-expressing CD4 lymphocytes correlated inversely with the percentage of CD28-expressing CD4 lymphocytes in HIV-infected patients on HAART, supporting evidence of the counter-regulation of these molecules [15]. HIV-infected patients with higher proportions of CTLA-4-expressing CD4 lymphocytes and lower proportions of CD28-expressing CD4 lymphocytes may be at greater risk of impaired immune responses [7,9]. However, similar numbers of CD28-expressing and perforin-expressing CD8 lymphocytes from HIV-infected patients and controls may indicate that HAART can restore normal CD8 T-cell function in HIV-infected patients with nadir CD4 T-cell counts<50 cells/μL. This is supported by evidence that previously severely immunodeficient HIV-infected patients and controls have similar numbers of interferon (IFN)γ-_-producing cytomegalovirus (CMV)-specific CD8 T cells [16].
A significantly higher proportion of HIV-infected patients had>1% CD4 lymphocytes expressing intracellular perforin compared with controls. Perforin-expressing CD4 lymphocytes exist at low levels in healthy controls but are expanded from the early stages of HIV infection [17]. Expansion of perforin-expressing CD4 lymphocytes appears to be related to chronic viral infections such as infection with HIV, CMV and Epstein-Barr virus (EBV), but whether they confer protective immune responses is unknown. The two HIV-infected patients with the highest percentages of perforin-expressing CD4 lymphocytes (14% and 22%) in our study were co-infected with hepatitis C virus, but the antigen specificity of their perforin-expressing CD4 lymphocytes was not assessed.
Immune-reconstituted HIV-infected patients and controls had similar percentages of perforin-expressing CD8 lymphocytes. The percentage of perforin-expressing CD8 lymphocytes did not correlate with the percentage of CD4 lymphocytes expressing CTLA-4 or CD28 in HIV-infected patients or controls, suggesting that expression of these molecules on CD4 T cells may not affect the maturation of cytotoxic effector cells. Our observation that the percentage of perforin-expressing CD8 lymphocytes correlated with nadir CD4 T-cell count rather than current CD4 T-cell count in HIV-infected patients on HAART may indicate that very severe HIV-induced immunodeficiency (i.e. CD4 T-cell count<10 cells/μL) induces a defect of perforin-expressing CD8 lymphocyte maturation that is not corrected by HAART.
Previous studies have shown increased expression of activation markers (HLA-DR and CD38) [18,19] and decreased in vitro lymphoproliferative and IFN-γ responses to mitogens and antigens [20,21] in HIV-infected patients receiving HAART compared with normal controls. Here we provide evidence that CD4 lymphocytes from previously severely immunodeficient HIV-infected patients have increased expression of the co-inhibitory protein CTLA-4 and decreased expression of the co-stimulatory protein CD28, despite long-term HAART and complete suppression of HIV replication. T-cell expression of these molecules in HIV-infected patients on HAART may help to define the degree of immune dysfunction and to predict the efficacy of therapeutic vaccines or other immune-based therapies in late-presenting HIV-infected patients with very low nadir CD4 T-cell counts.
Subjects and methods
Subjects
Twenty-five HIV-infected patients with nadir CD4 T-cell counts<50 cells/μL, at least 4 years on HAART and a minimum of 6 months of complete viral suppression (<50 copies/mL) were selected from the HIV database of the Department of Clinical Immunology and Biochemical Genetics, Royal Perth Hospital, Perth, Australia. Eighteen HIV-seronegative age- and sex-matched controls were also included in the study. Informed consent was obtained from all HIV-infected patients and controls, and the human experimentation guidelines of Royal Perth Hospital and the University of Western Australia were followed.
CD4 and CD8 T-cell counts and viral load
T-cell subsets were quantified by staining ethylenediaminetetraacetic acid (EDTA)-treated whole blood with CYTO-STAT triCHROME (CD8-FITC/CD4-RD1/CD3-PCy5; Coulter, Miami, FL). Analyses were performed on a Coulter EPICS-XL flow cytometer (Coulter). Plasma viral load was assayed by Amplicor Version 1.5 (Ultrasensitive Protocol; 50-100 000 copies/mL; Roche Diagnostic Systems, Branchburg, NJ).
Lymphocyte phenotype analysis
Red blood cells were lysed by adding lysis buffer (distilled water containing 0.15 m ammonium chloride, 0.01 m potassium bicarbonate and 0.08 mm tetrasodium EDTA, pre-warmed to 37°C) to 500 μL EDTA-treated whole blood, mixing continuously for 5 min. Cells were washed twice and reconstituted in 500 μL flow buffer [1% bovine serum albumin (BSA)/phosphate-buffered saline (PBS)]. Monoclonal antibodies directed against CD4, CD8 or CD28 (Immunotech, Marseilles, France) conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE) or phycoerythrin cyanin 5.1 (PCy5) and 50 μL cells were added to flow tubes. Tubes were vortexed and incubated for 15 min at room temperature. A volume of 100 μL IntraPrep Permeabilization Reagent 1 (Immunotech) was then added and tubes were vortexed and incubated for 15 min at room temperature.. Cells were washed in flow buffer and 100 μL IntraPrep Permeabilization Reagent 2 was added without mixing. Tubes were incubated for 5 min at room temperature, then gently agitated before addition of monoclonal antibodies directed against CTLA-4 (Immunotech) and/or perforin (BD Pharmingen, San Diego, CA) conjugated with either FITC or PE. Tubes were vortexed and incubated for 15 min at room temperature. Cells were washed, then resuspended in 0.5 mL flow buffer before analysis on a Coulter EPICS-XL flow cytometer. Lymphocytes were distinguished from monocytes on the basis of their forward versus side light scatter. A minimum of 10 000 lymphocytes per sample were analysed and gates were set using appropriate isotype controls. Cells expressing CTLA-4, CD28 or perforin are presented as a percentage of CD4 or CD8 lymphocytes. Representative flow cytometry scatter plots of CD28, CTLA-4 and perforin expression by CD4 lymphocytes from an HIV-infected patient are shown in Fig. 1.
Statistical analysis
All results are presented as median values (with range). Statistical significance was assessed by the nonparametric Wilcoxon rank sum test (SPSS 12..0.1 for Windows; SPSS Inc., Chicago, IL) or Fisher's exact test (Epistat Statistical Package, version 2.1; Tracy L. Gustafson MD, Round Rock, TX). Correlation coefficients were determined using Spearman's rank correlation test (Epistat). For all tests, P<0.05 was considered to represent a significant difference.
Results
Characteristics of patients and controls
Patients and controls were age- and sex-matched. HIV-infected patients had been on HAART for a median of 71 months (range 51-75 months). All patients had plasma viral loads <50 copies/mL for at least 6 months prior to the day of assay. One patient experienced a small increase in plasma viral load to 129 copies/mL on the day of assay. The majority of patients (80%) had viral load <1000 copies/mL for over 2 years (median 48 months; range 8.4-70 months). From a median nadir CD4 T-cell count of 13 cells/μL (range 0-48 cells/μL), every patient had experienced significant recovery of their CD4 T-cell counts on HAART (Fig. 2), but retained lower CD4 and higher CD8 T-cell counts than controls [480 (132-930) vs. 901 (555-1344) cells/μL and 1122 (432-2880) vs. 482 (255-729) cells/μL, respectively; P<0.001 for both comparisons; data not shown].
Immune-reconstituted HIV-infected patients had higher proportions of CTLA-4-expressing CD4 lymphocytes and lower proportions of CD28-expressing CD4 lymphocytes than controls
Patients had higher percentages of CTLA-4-expressing CD4 lymphocytes than controls [3.3% (2.3-7.4%) vs. 1.9% (1.4-4.6%), respectively; P<0.001; Fig. 3a]. The percentage of CTLA-4-expressing CD8 lymphocytes was low for both HIV-infected patients and controls [0.2% (0-1.1%) vs. 0.2% (0-1.0%), respectively; P>0.5; data not shown]. HIV-infected patients also had lower percentages of CD28-expressing CD4 lymphocytes than controls [97% (69-99%) vs. 98% (88-99%); P=0.005; Fig. 3b], whilst percentages of CD28-expressing CD8 lymphocytes were similar [49% (21-82%) vs. 51% (32-80%), respectively; P>0.5; data not shown]. The percentage of CTLA-4-expressing CD4 lymphocytes correlated inversely with the percentage of CD28-expressing CD4 lymphocytes in HIV-infected patients (r=_0.67, P<0.001; Fig. 3c), but not in controls (data not shown). In HIV-infected patients, the percentage of CD4 lymphocytes expressing CD28 or CTLA-4 did not correlate with current or nadir CD4 T-cell counts (data not shown)..
CD4 lymphocytes expressing perforin were more frequently detected in HIV-infected patients than in controls
HIV-infected patients had significantly more perforin-expressing CD4 lymphocytes than controls, with 11 of 25 (44%) HIV-infected patients and one of 18 (5.5%) controls having >1% of CD4 lymphocytes expressing perforin (P=0.005).
The percentage of perforin-expressing CD8 lymphocytes did not differ between HIV-infected patients and controls [23% (2.7-27%) vs. 23% (6.1-44%), respectively; P>0.5; data not shown]. The percentages of perforin-expressing CD8 and CD4 lymphocytes did not correlate with the percentage of CD4 lymphocytes expressing CTLA-4 or CD28 in HIV-infected patients or controls (data not shown). In HIV-infected patients, the percentage of perforin-expressing CD8 lymphocytes correlated with nadir (r=0.51, P=0.009; Fig. 4a) but not current CD4 T-cell counts (r=0.12, P>0.5; Fig. 4b).
No parameter measured correlated with total time on HAART, time on current HAART regimen, current CD4 T-cell count or time for which patients had plasma viral load <1000 copies/mL (data not shown).
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