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Blood, Vol. 96 No. 1 (July 1), 2000:
pp. 250-258
IMMUNOBIOLOGY
From the Laboratory of Molecular Oncology and Virology, Necker
Faculty of Medicine at Laennec Hospital, René Descartes (Paris V)
University, Paris, France.
In 99 adults infected with human immunodeficiency virus type 1 (HIV-1) who received highly active antiretroviral therapy (HAART) (including 2 nucleoside analogues and 1 or 2 protease inhibitors) for 1 year, CD4+ and CD8+ T cells (including
memory and naive subsets) increased similarly among patients with
sustained plasma viral load decrease, transient decrease, or no
decrease. A linear correlation was observed between the decrease
in serum
Treatment of individuals infected with human
immunodeficiency virus type 1 (HIV-1) with highly active antiretroviral
therapy (HAART), including at least 1 protease inhibitor (PI) allows
dramatic decrease in plasma and tissue HIV RNA load, as well as
quantitative and functional recovery of peripheral CD4+ T
cells in the majority of patients,1-4 resulting in a
significant reduction in AIDS-related morbidity and
mortality.5 However, in vivo mechanisms governing
CD4+ T-cell improvement under HAART are still
controversial.6-9 Although several recent studies have
reported the lack of association between increase in CD4 count and
decline in viral load under HAART,10-12 a systematic study
addressing the recovery of CD4+ and CD8+ T
cells with regard to different patterns of virologic response under
HAART is still lacking.
In this study, we examined the kinetics of T-cell recovery in relation
to the patterns of viral load response in patients treated with HAART
and the in vitro effects of HIV PIs on the survival, proliferation, and
apoptosis of HIV-infected patient's peripheral blood mononuclear cells
(PBMCs) to immune stimuli.
Patients
Flow cytometry
Plasma human immunodeficiency virus RNA quantitation assay Plasma HIV RNA concentration was measured in EDTA-treated plasma samples (frozen only once) by a commercial Quantiplex HIV-1 RNA quantitation kit 3.0 (detection threshold of 50 Eq copies/mL) (Bayer) performed according to the manufacturer's instructions.Serum  20°C until use. The  2-microglobulin concentrations were measured by
a commercial radioimmunoassay kit (2-Microglobulin RIA,
Immunotech, Marseille, France). Serum 2-microglobulin
values in healthy people ranged from 1.10 to 2.40 mg/L.
Human immunodeficiency virus inhibition and cell survival Peripheral blood mononuclear cells (PBMCs) freshly isolated from whole blood of untreated HIV-1-infected patients or healthy donors were resuspended at 106 per milliliter in RPMI 1640 human lymphocyte culture medium (HLCM) containing 20% heat-inactivated human AB serum, nonessential amino acid, 100 U/mL penicillin, 100 µg/mL streptomycin, 2 mmol/L L-glutamine, 1 mmol/L sodium pyruvate, and 10 mmol/L HEPES buffer. Then, 2 × 106 PBMCs were seeded in 24-well tissue culture plates (Nunc, Roskilde, Denmark) and were stimulated with 0.5 µg/mL anti-CD3 monoclonal antibodies (Becton Dickinson) plus 5 ng/mL phorbol myristate acetate (PMA) (Sigma, St Louis, MO) in the absence or the presence of serial dilutions (0.1-1000 nmol/L) of indinavir (obtained from Merck Research Laboratories, West Point, PA), saquinavir (obtained from Roche Products, Welwyn Garden City, Hertfordshire, UK), zalcitabine (Roche), didanosine (obtained from Bristol-Myers Squibb, Fontenay sous Bois, France), or stavudine (Bristol-Myers Squibb). Indinavir and saquinavir powders were initially dissolved in 50:50 ethanol/water (10 mmol/L at 25°C) as indicated by suppliers. Further drug dilutions were performed with water and culture medium, whereas the dissolving agent (50:50 ethanol/water) alone was diluted in parallel and served as the control. Cultures were maintained at 37°C in humidified air at 5% CO2. After 48 hours, stimulated PBMCs were washed and resuspended at 106 per milliliter in HLCM containing the same concentrations of the drugs plus 20 IU/mL interleukin 2 (Boehringer, Mannheim, Germany) for an additional 14-day culture. The culture mediums were changed every 2 to 3 days keeping the cultures at a viable cell density 2 × 106 per well in a
constant volume of 2 mL per well. At each time point, viable cell
counts were determined by trypan blue exclusion and supernatants were
harvested for testing HIV-1 p24 antigen by enzyme-linked immunosorbent
assay (ELISA) (Organon Teknika, Boxtel, The Netherlands). The
percentage viral inhibition was determined by the reduction of p24
production (at peak time point) in patients' PBMC cultures in the
presence of different concentrations of each of the drugs compared with
patients' PBMC cultures alone. Cell survival was determined by net
gain of total expanded viable cells in the presence of different
concentrations of each of the drugs compared with PBMC cultures alone.
Proliferation assay Freshly isolated patients' or donors' PBMCs were resuspended at a cell density of 5 × 105/mL in HLCM and seeded in quadruplicate in 96-well round-bottomed microtitre plates (Nunc) containing 105 PBMCs per well. Cells were stimulated with 0.5 µg/mL anti-CD3 monoclonal antibodies plus 5 ng/mL PMA in the absence or the presence of serial dilutions (0.1-1000 nmol/L) of indinavir or saquinavir as described above. The microtitre plates were placed in a cell incubator in a humidified atmosphere containing 5% CO2 for 4 days. Cells were pulsed with 0.037 MBq (1 µCi) per well [3H]-thymidine (Amershan, Aylesbury, UK) for the final 16 hours of the 4-day culture. Then, cells were harvested on glass fiber filters by an automated multisample harvester and filters were put in tubes with liquid scintillation fluid and counted on a-scintillation counter.
Apoptosis assay To test TCR/CD3-ligation-induced apoptosis, freshly isolated patients' or donors' PBMCs were stimulated overnight with 0.5 µg/mL anti-CD3 antibodies in the absence or the presence of indinavir or saquinavir, as indicated, concentrations. To measure Fas-ligation-triggered apoptosis, anti-CD3/PMA-stimulated patients' or donors' PBMCs were cultured in the absence or the presence of indinavir or saquinavir for 10 days. Cells were then stimulated overnight with 100 ng/mL anti-Fas monoclonal antibodies (clone 7C11, Immunotech). Apoptosis measurement was also performed in freshly isolated PBMCs and cultured PBMCs stimulated with anti-CD3/PMA (see cell survival section). Apoptosis was measured by propidium iodide and fluorescein isothiocyanate (FITC)-labeled annexin V, a phospholipid binding protein that preferentially binds to phosphatidylserine exposed at cell surface in the early phase of apoptosis, using a commercially available kit (Immunotech). Cells that are negative for prodidium iodide and positive for annexin V will be identified as apoptotic cells, whereas those positive for both prodidium iodide and annexin V are secondary necrotic cells.Statistical analysis Baseline and follow-up data of patients receiving HAART were compared by the Wilcoxon signed-rank test. Data between different groups of patients were compared by the Mann-Whitney test.
Study patients A total of 99 HIV-1 seropositive patients (65 pretreated with nucleoside analogues and 34 naive) were enrolled in this study. Baseline median CD4 and CD8 counts were 167 and 810 cells/µL, respectively, and median plasma HIV RNA concentration was 4.21 log10 equivalent (Eq) copies per milliliter. Twenty patients had a clinical AIDS-defining illness before entry. All patients received HAART for a minimum of 1 year. The initial HAART regimen was made of 2 NRTIs (lamivudine, 300 mg/d and stavudine, 60 or 80 mg/d) and 1 PI (indinavir, 2400 mg/d). Indinavir was replaced by ritonavir (200 mg/d) plus saquinavir (1200 mg/d) in 17 cases and stavudine was replaced by zidovudine (500 mg/d) in 7 cases. No new AIDS event was diagnosed over this 1-year follow-up period. The characteristics of the 99 patients and their CD4 counts and viral load values at baseline and at 1 year under HAART were summarized in Table 1.
Plasma viral load suppression and CD4+ T-cell recovery CD4 counts increased and plasma viral load decreased at month 2 (P < .001) in the whole group (Figure 1A). However, no significant correlation was found between plasma viral load changes and CD4 count changes at any time point. Three subgroups of patients were identified according to their plasma viral load outcomes: virologic responders (n = 59) who had a sustained plasma viral load decrease (less than 1000 Eq copies/mL); virologic transient responders (n = 23) who had an initial decrease (less than 1000 Eq copies/mL) but had a viral load reincrease (at least 1000 Eq copies/mL) before 1 year; and virologic nonresponders (n = 17) who remained greater than 1000 Eq copies/mL throughout the study period (Figure 1B). CD4 counts increased at month 2 in virologic responders (P < .001); counts increased similarly in transient responders (P = .003) and nonresponders (P = .016) (Figure 1C). In the 59 virologic responders, CD4 counts increased (at least 20%) in 55 cases (93%), remained unchanged (less than 20% and more than20%) in 3 cases, and decreased (no more than 20%) in 1 case. In the 23 virologic transient responders, CD4 counts increased in 21 cases (91%)
and remained unchanged in 2 cases. Among the 17 virologic
nonresponders, CD4 counts increased in 15 cases (88%) and remained
stable in 2 cases.
CD8+ T-cell recovery The mean CD8+ T-cell count of the whole group increased at month 2 (P < .001) and reached a maximum gain of 238 cells/µL at 1 year (Figure 1D). The 1-year change of CD8 count was inversely correlated with baseline level of CD8 count (r =0.534, P < .001) (Figure 1E), but was
independent of both the baseline level and the 1-year change of plasma
viral load (data not shown). Among the 71 patients who had an increase
in their CD8 cells (consistent gain of at least 20%), 70 had a CD4
count increase and 1 had an unchanged CD4 count. Patients were then
partitioned according to their baseline CD8-count levels (less than
1000 cells/µL, n = 62; at least 1000 cells/µL, n = 37). Among
patients with a baseline CD8 count of less than 1000 cells/µL, CD8
cells increased at month 2 in the 35 virologic responders
(P < .001); it increased similarly in the 14 transient
responders (P = .019) and in the 13 nonresponders (P = .002) (Figure 1F). In addition, there was a significant
correlation between CD8 count changes and CD4 count changes at 1 year (r = 0.427, P < .001). In contrast, the
mean CD8 count of patients with a baseline CD8 count of at
least 1000 cells/µL remained unchanged.
Normalizations of T-cell compartment and serum soluble T-cell activation marker Memory or naive CD4+ T cells increased at 6 months (P < .01) and 1 year (P < .001) under HAART. Memory or naive CD4 cells had similar expansion in the 3 subgroups of patients with different patterns of plasma viral load response. In the patients with low baseline CD8 counts, a similar increase of memory or naive CD8 cells was also observed at 6 months (P < .05) and 1 year (P < .01) in the 3 subgroups. Serum2-microglobulin concentrations decreased at 6 months
(P < .01) and 1 year (P < .001) in virologic responders, transient responders, and nonresponders. No significant difference among the 3 subgroups was found for baseline levels of
memory/naive CD4 and CD8 cells or for baseline levels of serum 2-microglobulin concentrations (Table
2). The 1-year change in serum
2-microglobulin concentration was inversely correlated with the 1-year change of CD4 counts (r = 0.418,
P < .001) in the overall group (Figure
2A), as well as
with the 1-year change of CD8 count (r = 0.318,
P = .012) in the 62 patients with low baseline CD8 counts
(Figure 2B). Serum 2-microglobulin concentrations decreased at month 6 (mean change 0.85 mg/L,
P < .001) and at 1 year (1.04 mg/L,
P < .001) in the 91 patients who had a CD4 count increase
(persistent gain  20%) independently of their virologic response. In
contrast, there was no modification in the mean serum
2-microglobulin concentration at any time point in the 9 patients whose CD4 counts remained stable or decreased under HAART.
Among patients with a baseline CD8 count less than 1000 cells/µL,
serum 2-microglobulin concentrations also decreased at
month 6 (0.84 mg/L, P < .001) and at 1 year
(1.09 mg/L, P < .001) in the 53 patients who had a
CD8 count increase, whereas no significant modification was observed in
the 10 patients who did not increase their CD8 counts during the
follow-up period.
Viro-immunologic responses in naive and pretreated patients At entry, naive patients had a higher plasma viral load and CD4 count levels than pretreated patients (median 4.9 vs 4.4 log HIV-1 RNA Eq copies/mL, P = .013 and mean 212 vs 143 CD4 cells/µL, P = .006), whereas the baseline CD8 count and serum2-microglobulin concentration did not differ
significantly in naive and pretreated patients. Under HAART, plasma
viral load decreased more profoundly in naive patients than in
pretreated patients (6 months mean 2.7 vs 1.7 log Eq
copies/mL, P < .001; 1 year 2.3 vs 1.5 log Eq copies/mL, P = .002); on the other hand, CD4 counts, CD8
counts (of patients with low baseline CD8 counts), and serum
2-microglobulin concentrations decreased equally in
naive and pretreated patients from baseline to months 6 and 12.
Effects of human immunodeficiency virus protease inhibitors on T-cell survival and viral inhibition After isolating PBMCs from fresh blood samples of 10 untreated HIV-1-infected patients with CD4 count between 200 and 300 cells/µL and plasma viral load between 4 and 5 log10 Eq copies/mL, indinavir or saquinavir was added at the same time as 2 × 106 cells were stimulated with anti-CD3 antibodies plus PMA; the drug was then re-added each other day with fresh culture medium until day 16. In PBMCs taken from 10 noninfected donors, 2 × 106 cells at baseline grew up to mean (± SD) 151 (± 32) × 106 cells after 16 days of culture in the absence of PI. Neither the survival nor the phenotype of noninfected donors' PBMCs was affected by either of the 2 PIs (data not shown). PBMCs of the 10 untreated patients increased moderately from 2 × 106 cells at baseline increased to mean 6.2 (± 5.7) × 106 cells at day 16 of culture in the absence of PI. In contrast, under either of the 2 PIs, patients' PBMC survival increased dramatically with a net gain 50 × 106 viable cells at drug concentrations
ranging from 1 to 1000 nmol/L (peak: 10-100 nmol/L), whereas
viral replication (measured by p24 production in culture supernatants)
was suppressed by 90% at drug concentrations ranging from 30 to 300 nmol/L to 1000 nmol/L. Data from 3 representative patients (patients A,
B, and C, respectively) are shown in Figure
3. Phenotype analysis of patients' PBMCs
at the end of the culture period revealed that both CD4+
and CD8+ T cells were equally expanded under either of the
2 PIs (data not shown).
Effects of human immunodeficiency virus protease inhibitors on
T-cell proliferation and apoptosis
This study demonstrates that, under HAART, CD4+ and
CD8+ T-lymphocyte compartments were both involved in
immunogic recovery in naive patients, as well as in pretreated ones.
However, no quantitative correlation was found between T-cell recovery
and virologic response. Among the 99 treated patients, 40 (40%) failed to achieve sustained viral suppression. Paradoxically, as many as 91 (91%) cases had a sustained CD4 cell increase achieved paradoxically, whereas 7 stabilized their CD4 counts and only 1 had a minor decrease in his CD4 count. Interestingly, increases in CD4+ and
CD8+ T cells (including memory and naive subsets) were
found to be linearly correlated with the decline in serum
We thank M. Arlie, L. Cao, S. Doré, H. Gozard, R. Salerno-Goncalves, L. Ty, J. Yuan, and Y. J. Zhao for technical
assistance; M. Sala for measuring Submitted March 31, 1999; accepted February 28, 2000.
Supported by the Agence Nationale de Recherche Sur le SIDA (ANRS), the
Fondation pour la Recherche Médicale (SIDACTION), and the
Association pour la Recherche, l'Etude et le Traitement des Maladies
du Sang (AREMAS).
Reprints: Wei Lu, Laboratoire d'Oncologie et Virologie
Moléculaires, Hôpital Laennec, 42 rue de Sèvres,
75007 Paris, France; e-mail: weilu{at}intercancer.net or
cancero.laennec{at}lnc.ap-hop-paris.fr.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
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Related Letter in Blood Online:
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Top
Abstract
Introduction
) or memory
(CD45RA+/CD62L+) subsets were assessed on fresh
whole blood samples by flow cytometry analysis (FACScan, Becton
Dickinson, San Jose, CA). A panel of monoclonal antibodies to the
following T-cell surface markers was used: CD3-PerCP, CD4-FITC, CD8-PE,
CD45RO-PE, CD45RA-PE, CD62L-FITC, CD4-PerCP, and CD8-PerCP (Becton
Dickinson, Le Pont-de-Claix, France).
) and plasma viral loads (
) in the whole
group of patients (A). Evolution of plasma viral loads (B) and CD4
counts (C) in virologic responders (
), transient responders (
),
and nonresponders (
). Evolution of CD8 counts in the whole group
(D). Correlation between CD8 count change and baseline CD8 count (E).
Evolution of CD8 counts in patients with a baseline CD8 count less than
1000 cells/µL (
, nonresponders) (F). Data were expressed as the mean ± SE
of each group of patients. Threshold for plasma viral load measurement
by Quantiplex assay was 50 HIV RNA equivalent (Eq) copies per
milliliter. For the purpose of calculation, this value was assigned
when the plasma viral load became undetectable.
