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The Journal of Immunology, 2000, 165: 1692-1704.
Copyright © 2000 by The American Association of Immunologists

Dynamics of HIV-Specific CD8+ T Lymphocytes with Changes in Viral Load1

Lucile Mollet*, Tai-Sheng Li*, Assia Samri*, Claire Tournay{dagger}, Roland Tubiana{ddagger}, Vincent Calvez§, Patrice Debré*, Christine Katlama{ddagger}, Brigitte Autran2,* and the RESTIM and COMET Study Groups

* Laboratoire d’Immunologie Cellulaire et Tissulaire, Bâtiment Centre d’Etudes et de Recherches en Virologie et Immunologie, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7627, Paris, France; {dagger} Immunotech-Coulter-Beckman, Marseille, France; and {ddagger} Département des Maladies Infectieuses and § Département de Virologie, Hôpital Pitié-Salpétriêre, Paris, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The influence of HIV burden variations on the frequencies of Ag-specific CD8+ T cell responses was evaluated before and during highly active antiretroviral therapy by analyzing the number, diversity, and function of these cells. The frequencies of HLA-A2-restricted CD8+ PBL binding HLA-A2/HIV-epitope tetramers or producing IFN-{gamma} were below 1%. A panel of 16 CTL epitopes covering 15 HLA class I molecules in 14 patients allowed us to test 3.8 epitopes/patient and to detect 2.2 ± 1.8 HIV epitope-specific CD8+ subsets per patient with a median frequency of 0.24% (0.11–4.79%). During the first month of treatment, viral load rapidly decreased and frequencies of HIV-specific CD8 PBL tripled, eight new HIV specificities appeared of 11 undetectable at entry, while CMV-specific CD8+ PBL also appeared. With efficient HIV load control, all HIV specificities decayed involving a reduction of the CD8+CD27+CD11ahigh HIV-specific effector subset. Virus rebounds triggered by scheduled drug interruptions or transient therapeutic failures induced four patterns of epitope-specific CD8+ lymphocyte dynamics, i.e., peaks or disappearance of preexisting specificities, emergence of new specificities, or lack of changes. The HIV load rebounds mobilized both effector/memory HIV- and CMV-specific CD8+ lymphocytes. Therefore, frequencies of virus-specific CD8 T cells appear to be positively correlated to HIV production in most cases during highly active antiretroviral therapy, but an inverse correlation can also be observed with rapid virus changes that might involve redistribution, sequestration, or expansion of these Ag-specific CD8 T cells. Future strategies of therapeutic interruptions should take into account these various HIV-specific cell dynamics during HIV rebounds.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
According to Doherty (1), "a viral infection is a race: for the infected organism, cell-mediated immunity has to develop faster than the spread of the pathogen." CD8+ T lymphocytes are generally believed to play a major protective role during both the primary and chronic phases of HIV infection. Indeed, HIV- or SIV-specific CTLs appear concurrently with a dramatic fall in viral load during primary infection (2, 3, 4, 5, 6), then persist at high levels during the chronic phase of the infection, together with persistent but low HIV/SIV replication. The inverse correlation found by some authors (7, 8) between viral load and the frequencies of HIV-specific CTL precursors, as measured by limiting dilution analysis, is thought to reflect the protective efficacy of these immune defenses. This method provides a reasonable measure of the memory T cell subset size but not of the CTL effector pool size (1).

Flow cytometry methods now allow direct quantification of virus-specific CD8+ T cells (9). The number of CD8+ T cells that express a TCR specific for a particular MHC-peptide complex can accurately be monitored with HLA class I-peptide tetramers. Tetramer binding appears to correlate with functional activity, including uncultured peptide-specific cytolysis and IFN-{gamma} production, the latter evaluated by cytofluorometry or in enzyme-linked immunospot assays after a short-term specific-peptide stimulation (1, 10, 11, 12). Indeed, HIV-specific CD8+ T cell counts have been evaluated with HLA tetramers loaded with HIV-1 CTL epitopes and have again been found to be negatively correlated with HIV plasma load (13, 14). These new methods have also revealed that as many as 70% of the activated CD8+ T cells found in the spleens of lymphocytic choriomeningitis virus (LCMV)3-infected mice at peak infection were LCMV specific (10). This elegant demonstration, by clearly showing that virus-specific CD8+ T cells can undergo major expansion, explains the CD8+ cell compartment bursts during viral infection. However, these latter data come from the analysis of strong immunodominant responses in the tissues where viral replication takes place. In contrast, CD8+ cells binding HLA/HIV peptide tetramers (9, 13, 15) or HLA/hepatitis C virus peptide tetramers (16) have been found to account for a maximum of 5% at most of the total peripheral blood CD8+ lymphocytes. Two possible hypotheses explain these findings: 1) only a minimal part of the total HIV-specific CD8+ cell response is directed against these epitopes, with most of this response directed against other epitopes, or 2) bystander CD8+ cells account for most of the circulating CD8+ cells in HIV-infected individuals. Supporting the first hypothesis is the finding of a multiplicity of epitopes simultaneously recognized by HIV-specific CD8+ T cells (17, 18); this multiplicity reflects persistent HIV replication and the recurrent emergence of new variants (19, 20, 21). Therefore, analyzing the interplay between the viral burden and the size of the HIV-specific CD8+ T cell response requires simultaneous analysis of its diversity.

Highly active antiretroviral therapies (HAART) now control viral replication effectively and restore immune defenses against opportunistic infections. A rapid redistribution of memory CD4 lymphocytes is generally thought to occur during the first 2 mo of treatment; it may reflect the release into the peripheral blood of T cells previously sequestered in infected organs (22, 23). Because activated HIV-specific CD8+ T lymphocytes are known to massively infiltrate infected tissues during active replication (5, 24, 25, 26, 27), these cells might be similarly but even more strongly redistributed early in HAART. Theoretically, this phenomenon should cause the preferential release of HIV-specific CD8+ T cells into the circulation. This has not been observed up until now, although some fluctuations in HIV-specific CD8+ T cell counts have been shown during the first months of treatment (14, 17). In the long term, stable virus control reduces the number and activation of CD8+ cells (22). Declines have also been reported in the frequency of activated CD8+ T cells that bind HLA-peptide tetramers and of effector/memory or precursor HIV-specific CTL as measured by enzyme-linked immunospot assay or limiting dilution analysis. These findings suggest a positive correlation between the intensity of Ag stimulation and HIV-specific CD8+ T cell expansion (13, 17, 18, 28). However, it is not known whether this loss in immune T cells involves the multiple specificities of the CD8+ T cell response against HIV. Current antiretroviral therapies are unable to eradicate the virus, and relapses occur when drugs are stopped (29, 30, 31). The decay in HIV-specific CD8+ T cells leaves patients with low residual immune defenses against a recurrence of HIV replication and raises several questions. Do specific memory CD8+ T cells persist over time? Do HIV-specific CD8+ T lymphocytes rebound during a relapse, when HIV replication is renewed? If so, does the rebound involve only functional T cells with the preexisting specificities or also new ones? Is this phenomenon HIV-restricted or does it trigger the expansion of CD8 T cells bearing other antigenic specificities?

To answer these questions, we simultaneously analyzed the function, diversity, and numbers of HIV-specific CD8+ T cells in relation to viral load, both during stable therapeutic virus control and during interruptions of antiretroviral therapies. To that end, we directly estimated the frequency of functional CD8+ T cells, specific for 16 different HIV epitopes from four HIV proteins (Gag, Pol, Nef, and Env) and, when possible, specific for the HLA-A2-restricted CMV epitope pp65 and EBV epitope BMLF-1, by measuring peptide-induced IFN-{gamma} production and HLA-A2 peptide complexes, both by flow cytofluorometry. This study shows the multiplicity of HIV epitopes that are simultaneously recognized by activated CD8+ T lymphocytes in chronically infected patients and the influence of changes in the viral burden on the redistribution, frequency, activation, and functional differentiation of both HIV-specific and CMV-specific CD8+ T cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients

Thirteen HIV-positive patients followed up in the Department of Infectious Diseases at La Pitié-Salpétrière Hospital were studied. All had been previously enrolled in studies of CD4 T cell reconstitution and virus dynamics (22, 30, 32) and gave their written informed consent, in accordance with the regulations of the institutional ethics committee. Table IGo summarizes patient characteristics at study entry. Nine had previously been treated by two inhibitors of reverse transcriptase but not any protease inhibitor, and six had never been treated with any antiretroviral drug. Day 0 of the study corresponded to the initiation of a HAART regimen including two reverse transcriptase inhibitors and one protease inhibitor, excluding ritonavir. Patients were prospectively followed up for 12–18 mo. Four groups were distinguished according to the course of their plasma viral load. Group A contained five patients with a stable viral load below the threshold of 200 copies/ml after the third month of HAART; group B contained two patients in whom initial virus control failed at 3 or 6 mo, but for whom modification of the treatment led to subsequent virus control; group C contained four naive patients whose treatment was interrupted, as scheduled, at 1 mo after HAART initiation, when viral load was below 1000 copies/ml; the same treatment was reintroduced after 4 wk and maintained thereafter; and group D contained four patients for whom no stable control of the viral load was achieved, despite treatment modifications.


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  		Table I. Patients’ characteristics at study entrya

 
Blood samples were prospecively drawn at day 0 and every 3 mo and, for some patients, at 1 and 18 mo. PBMCs were isolated by Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) gradient centrifugation, evaluated for HIV-1 p24-specific (Transgène, Strasbourg, France) and CMV-specific (Behring, Rueil-Malmaison, France) CD4+ T cell proliferation as previously described (22, 32), frozen, and kept in liquid nitrogen until use. For each patient, all frozen samples were simultaneously thawed and analyzed in batch experiments. Cell viability, evaluated by trypan blue exclusion, was always above 75%.

HLA typing

HLA was typed with a standard complement microcytotoxicity assay by Dr. I. Théodorou and coworkers in the Laboratoire d’Immunologie Cellulaire et Tissulaire, Hôpital Pitié-Salpétrière (Paris, France).

Five HLA-A2 patients were subtyped by classical molecular biology methods according to manufacturer’s instructions (HLA-A2 PCR-SSP; Dynal, Compiégne, France)

Plasma HIV load measurement and CD4 and CD8 counts

Plasma HIV RNA load was measured on frozen acid citrate dextrose plasma samples using an Amplicor HIV-1 Monitor 1.5 (Roche Diagnostic Systems, Branchburg, NJ). Specimens for each patient were batch-tested with a negative control and two positive controls (low, 10,000–20,000 copies/ml; high, 300,000–400,000 copies/ml). The number of HIV-1 viral RNA copies was calculated on the basis of the manufacturer’s reference standards. The threshold of detection for 26 amplification cycles was 200 HIV-1 RNA copies/ml (log 2.3).

Absolute CD4 and CD8 cell counts were directly measured by flow cytometry, with the flow count method and an XL-flow cytometer (Coulter-Immunotech, Margency, France). Briefly, whole-blood samples were simultaneously incubated with a combination of mAbs directed against the CD3, CD4, CD8, and CD45 molecules and fixed concentrations of beads as internal controls. Absolute counts of CD3+CD4+ and CD3+CD8+ cells were determined following the manufacturer’s recommendations. Each blood sample was assayed twice, and reproducibility was 95.5%. Normal values in our laboratory for CD3+CD4+ cells are 858 ± 260 cells/ml and for CD3+CD8+ cells are 482 ± 164 cells/ml.

HLA-A2/peptide tetrameric complexes

HLA-A*0201 tetramers were synthesized as previously described (9), with the following peptides: SLYNTVATL (HIV-1 Gag 77–85) (33, 34), ILKEPVHGV (HIV-1 Pol 476–484) (34, 35), GLCTLVAML (EBV BMLF-1 280–288) (36, 37), and NLVPMVATV (CMV pp65 495–503). The HLA heavy chain was expressed in Escherichia coli with an engineered C-terminal signal sequence containing the 15-aa site for biotinylation by the enzyme BirA. After refolding the heavy chain with ß2-microglobulin and peptide, the complex was biotinylated on a single lysine by BirA. Following purification by gel filtration and ion-exchange chromatography, tetramer formation was induced by adding streptavidin-PE (Sigma, St. Louis, MO).

CTL epitopes

CTL epitopes were selected according to patients’ HLA alleles from the Los Alamos HIV molecular immunology database on the following web site: http://hiv-web.lanl.gov/immunology/advancedctl.html. We focused on the four HIV proteins, Gag, Pol, Env, and Nef, and selected 16 epitopes with the following characteristics: known HLA-binding capacities, a 9–11 aa length (except for N2), and <10% variability when their sequence was aligned with all published HIV-1 sequences. The peptides were purchased from Syntem (Nîmes, France), except for G1, N1, and N3, kindly provided by Agence Nationale de Recherches sur le SIDA. All CTL epitopes are presented in Table IIGo.


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  		Table II. Panel of HIV-1, CMV, and EBV CTL epitopesa

 
IFN-{gamma} production assay

We incubated 106 PBMC with and without the peptide of interest (10 µM) in 96-well plates for 1 h in RPMI 1640 culture medium (ICN Biomedicals, Orsay, France) supplemented with 1% antibiotics, 1 mM sodium pyruvate, 2 mM L-glutamine, and 1% MEM nonessential amino acids (all from Life Technologies, Paisley, U.K.). Over the next 4 h, 5 U/ml of rIL-2 (Boehringer-Mannheim, Mannheim, Germany), 5% FCS (Life Technologies), 3 µg/ml of brefeldin A (Sigma-Aldrich, Saint-Quentin, France), and 2 µg/ml of monensin (Sigma) were added. As a negative control, spontaneous IFN-{gamma} release was evaluated for each patient at each time point, and these values were subtracted from the IFN-{gamma} secretion values after peptide-specific stimulation. As a positive control, cells were stimulated using 0.5 µg/ml PHA-P (Life Technologies), and maximun IFN-{gamma} release was evaluated. Some samples were assayed twice, with 95.5% reproducibility.

Cell sorting of tetramer-positive CTL

A Gag-specific CTL line was obtained from the PBMC of an HIV-positive donor after stimulation with autologous irradiated PHA blasts, as previously described (19). After a 3-wk culture with periodic additions of IL-2, the CTL line cytotoxic activity was assayed against an autologous B-EBV cell line infected with a recombinant vaccinia virus encoding for HIV-1 LAI Gag protein in a standard chromium release assay. At the same time, cells were stained with HLA-A2 Gag (SLYNTVATL) or HLA-A2 BMLF-1 (GLCTLVAML) tetramers and sorted by an ELITE flow cytometer (Coultronics, Miami, FL). Tetramer-positive and -negative cells were then assayed 12 h later against autologous B-LCL target cells loaded with HLA-A2 Gag peptide (SLYNTVATL) in a standard chromium release assay.

Immunofluorescence analysis

Ags were characterized with a standard staining method with the following mAbs: CD8-APC (Becton Dickinson-PharMingen, Pont de Claix, France) or CD8-PE-Cy5 (Immunotech-Coulter, Marseille, France) and anti-IFN-{gamma}-PE or FITC (PharMingen). Unless otherwise indicated, all steps were performed at room temperature. Cells were washed in PBS-0.5% BSA and resuspended in 100 µl of PBA-BSA for extracellular staining (20 min) with CD8-APC, HLA-DR-PECy5 (Immunotech), and CD38-FITC (Immunotech). When indicated below, HLA-A2-peptide tetramers (0.5 mg/ml) alone were preincubated 20 min before this extracellular staining.

For intracytoplasmic staining, cells were incubated in 4% PFA for 5 min at 37°C, then washed with PBS-0.5% BSA-0.1% saponin (Sigma), resuspended in 100 µl of the latter solution for permeabilization, and reincubated with anti-IFN-{gamma} for 25 min. They were washed and resuspended in PBS-0.5% BSA.

In both series of experiments, CD8+ T lymphocytes (50,000) were analyzed on a flow cytometer (FACScalibur; Becton Dickinson) equipped with an argon ion laser (excitation, 488 nm), a laser diode (excitation, 635 nm), four filters (FL1, BP 530 nm ± 15 nm; FL2, BP 585 ± 21 nm; FL3, LP 670 nm; and FL-4, BP 661 ± 8 nm) and CellQuest software. As in other studies, we defined a CD8 T cell response as significant compared with background when it exceeded 0.1% of CD8 T cells (38, 39, 40).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Diversity of the anti-HIV CD8 cell responses

To investigate the diversity, functional status, and frequency of HIV-specific CD8+ T cells, we assayed IFN-{gamma} production in response to a panel of previously published HIV CTL epitopes. We focused our attention on the four major targets of HIV-specific CTL, i.e., Gag, Pol, Env, and Nef. We selected from the Los Alamos database 16 epitopes restricted by nine HLA-A and six HLA-B molecules carried by the 15 patients included in the study (Table IIGo). Overall, 23 combinations of HLA-class I molecules and HIV epitopes were tested. Fig. 1Go recapitulates the HIV-specific responses at HAART initiation. Each peptide tested was recognized at least once. A mean of 3.8 peptides were tested per patient, and the mean number of peptides recognized by the CD8+ lymphocytes was 2.2 ± 1.8 (Fig. 1Go). We observed 31 anti-HIV-positive responses from 53 tests (58%) with frequencies of peptide-specific CD8+ cells ranging from 0.11 to 4.79% (median, 0.24%). The Nef and Env peptides were more frequently recognized (70% and 64%, respectively) than the Gag (55%) or Pol (50%) peptides. For each of the six peptides tested at least four times, the frequency of specific CD8+ cells differed among patients (Fig. 1Go), independent of viral load and CD4 count.



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  		FIGURE 1. Frequencies of ex vivo functional HIV-1-specific CD8+ T cell at initiation of HAART. The diversity of HIV-specific CD8 cell responses was evaluated in 14 patients at HAART initiation with the IFN-{gamma} production assay, as described in Materials and Methods. Significant peptide-specific CD8 T cell frequencies were defined above the threshold of 0.1% (dashed line). A summary per patient is given under the graph: numbers of peptides tested (*) and numbers of detectable peptide-specific CD8 subset per patient ({dagger}). A summary of the peptide evaluation is given in the legend with their rapported HLA restriction ({ddagger}) and peptide recognition ratio, i.e., number of frequencies over the 0.1% threshold to number of overall peptide evaluation (§).

 
Taken together, these results demonstrate the diversity of HIV epitopes recognized in each HIV-infected individual. These results suggest that using a single or a limited panel of selected HIV epitopes may lead to underestimation of the actual number of HIV-specific CD8+ T cells.

Anti-HIV CD8 cell responses restricted by HLA-A2 before HAART

When considering the six HLA-A2 patients tested in the IFN-{gamma} production assay (Fig. 1Go), we evidenced three positive responses against P1 (476–484) and G3 (77–85). One responder was HLA*0201 (D1), while another was HLA*0205 (D4). Among the three donors in whom no CD8 cells could be detected against any of the two HLA-A*0201-restricted peptides, two donors could be subtyped, indicating the presence of the HLA-A*0201 allele (B2, D2).

The low frequencies of P1 and G3 epitope recognition in the IFN-{gamma} production assay prompted us to evaluate whether this reflected discrepancies between the number and the functional status of CD8+ T cells directed against a given epitope. To that purpose, the HIV-specific CD8+ T cells were evaluated by using two HLA-A2 tetramers loaded by the HLA-A2-restricted epitopes Gag (SLYNTVATL, G3) (33, 34) and Pol (ILKEPVHGV, P1) (34, 53). The tetramer specificity was first checked on an HIV-specific CTL line with 2.2% Gag tetramer-positive CD8+ T cells (Fig. 2Go). The tetramer-positive and -negative CD8+ T cells were sorted and tested in a standard chromium release assay. The sorted CD8+ Gag tetramer-positive cells but not the CD8+ Gag tetramer-negative cells were able to lyse an autologous EBV-cell line coated with the G3 Gag peptide (77–85, SLYNTVATL) with 40% specific lysis at a 4:1 ratio, while no lysis was detected against the EBV target cells alone (Fig. 2Go).



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  		FIGURE 2. HLA-A2 Gag (77–85) tetramer binding dissects epitope-specific and nonspecific cytotoxic CD8 T cells. Left, A whole HIV-specific CTL line was stained with HLA-A*0201/Gag (77–85) tetramer-PE and CD8-FITC. The CD8+tetramer+ (CD8+Tet+) cells and CD8+tetramer- (CD8+Tet-) cells were then sorted according to the gates mentioned. Right, The specificity of tetramer recognition was then assayed 12 h after the sorting on an autologous B-EBV cell line loaded (black symbols) or not (white symbols) with Gag (77–85) peptide in a standard chromium release assay.

 
It was possible to study tetramer binding and IFN-{gamma} production in parallel in two HLA-A2*0201 patients (D2, B2). At treatment day 0, the percentage of CD8+ T cells binding the two HIV tetramers was below 1% in all samples tested (Table IIIGo). Similarly, the frequencies estimated in the IFN-{gamma} production assay were in the same ranges, though slightly lower, independent of viral load and CD4 count. To check further whether the low frequencies of these specific cells might reflect the patients’ advanced disease stage, we studied samples drawn 4–8 years earlier. The frequency range was similar at each time point, although some Gag and Pol responses were detectable at early time points for three patients and disappeared thereafter (D2, 1991; B2, 1994; D3, 1989).


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  		Table III. HLA-A2-restricted anti-HIV and anti-CMV CD8 T cell responses before HAART initiation as measured with tetramers or the intracellular IFN-{gamma} assay

 
To investigate whether these low frequencies were representative of the overall HLA-A2-restricted specific CD8+ T cells, we tested in parallel the recognition of another HIV-1-specific HLA-A2-restricted CTL epitope located in HIV-1 Nef 180–189 (VLEWRFDSRL, N3) (19) in two HLA-A2 patients. In all four samples analyzed, the mean frequency of Nef-specific CD8+ T cells was in the same ranges when compared with the P1- and G3-specific cell frequencies. The HLA-A2-restricted CMV peptide pp65 (495–503) was also evaluated in the six HLA-A2 patients tested, using the IFN-{gamma} production assay. Interestingly, the frequencies of CMV-specific CD8+ T lymphocytes were higher than that of Nef-, Gag-, or Pol-specific CD8+ T cells as a whole in eight of the 11 samples tested.

The similar ranges obtained when comparing HIV-specific CD8+ T cell frequencies using either the HLA-A2/peptide tetramers or the IFN-{gamma} production assays suggest that, when present in the periphery, the anti-HIV specific CD8+ T lymphocytes were functional.

Early increase of HIV-specific and CMV-specific CD8+ T lymphocytes at HAART initiation

We then studied the evolution over time of such diverse HIV-specific CD8 T cell responses as a function of viral load changes after introduction of treatment. Fig. 3Go illustrates the early dynamics of plasma viral load and HIV-specific and CMV-specific CD8+ cells in the seven patients evaluated at 1 mo after HAART began. Fig. 3GoA shows the major viral load reduction (median reduction, -5 log) obtained during this period of therapy. The percentage of CD8+ T cells that were HIV-specific increased for 15 of the 23 (65%) HIV epitope/HLA combinations tested (Fig. 3GoB, left). This phenomenon was observed for at least one epitope per patient of the 3.33 ± 0.82 epitopes tested per patient (Fig. 3GoB, left). Even more important, eight of the 11 epitope-specific CD8+ T cells that were undetectable at day 0 became detectable at significant levels after 1 mo of treatment. Overall, a 2- to 4-fold increase in the absolute number of HIV-peptide specific T cells was observed (data not shown). However, this observation was not restricted to the HIV-specific CD8+ T lymphocytes because CD8+ T cells spe-cific for the HLA-A2 restricted epitope from CMV-pp65 did also increase in three of the four patients tested (Fig. 3GoB, right).



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  		FIGURE 3. Early redistribution of HIV-specific CD8 T cells during the first month of HAART. A, Drastic plasmatic HIV RNA reduction during the first month of HAART occurred for the seven patients tested. B, Changes for CD8+ T cell counts for the various HIV-1 peptides tested in the same seven patients and represented in Fig. 1Go (left). Counts of CD8+ T cells specific for HLA-A2 CMV pp65 epitope in four HLA-A2 patients during the first month of HAART (right). Frequencies of HIV- and CMV-specific CD8+ T cells were evaluated using the IFN-{gamma} production assay.

 
Thus the rapid increase of both HIV- and non-HIV-specific CD8+ lymphocytes and the emergence of new HIV-epitope spec-ificities during the first month of therapeutic virus control are most unlikely to be a consequence of cell proliferation and may reflect a redistribution of CD8+ T lymphocytes toward the peripheral blood whatever their antigenic specificity.

Effect of efficient and stable long-term therapeutic viral reduction on HIV-specific CD8+ T cells

Individual follow-up studies help assess the influence of viral load variations on the frequency of HIV-specific CD8+ T cells. We performed such an analysis among the 15 patients over 12–18 mo of HAART. The efficacy of these treatments in controlling the virus and allowing CD4 T cell reconstitution has already been reported (22).

To investigate the influence of long-term virus reduction on the frequency of activated HIV-specific CD8+ T lymphocytes, we defined groups of patients according to the course of their plasma viral load (Table IGo). The five patients from group A had a viral load that remained stable below 200 copies/ml during the study period, despite variable levels of CD4 cell depletion at baseline. The frequency of functional HIV-specific CD8+ T lymphocytes was monitored with the peptide-specific IFN-{gamma} assay. In one patient who presented no CD8+ cell increase between day 0 and month 1, the HIV-specific CD8+ T cell count decreased very rapidly at 1 and 3 mo (Fig. 4GoA, left) and remained low thereafter. In another patient (Fig. 4GoA, middle), HIV-specific CD8+ T cells counts reached undetectable levels only at 12 mo, although viral load had dropped markedly by 3 mo and remained stable thereafter. Different epitope-specific CD8+ T cells followed different kinetics in their decrease: the P3 epitope-specific CD8+ T cells decreased rapidly by 3 mo, while the subdominant N1-specific CD8+ subset began to decline only after 6 mo (Fig. 4GoA, middle). We further investigated whether these IFN-{gamma}-producing cells expressed cell-surface markers previously linked with the effector-memory CD8 cell differentiation phenotype (54). Using four-color cytofluorometry, we analyzed the phenotype of CD8+ T cells directed against the predominant P3 epitope, i.e., the peptide that gave the highest proportions of reacting cells for a given patient. At initiation of therapy, CD8+ cells producing IFN-{gamma} after P3 stimulation were mainly CD27- CD11ahigh, a phenotype that was described to be characteristic of effector cells (54). After 6 mo of treatment, the proportion of CD27-CD11ahigh effector cells among the P3-specific CD8+ lymphocytes decreased. Concurrently, a CD8+CD27+CD11alow subset increased (Fig. 4GoB, left) with a mean fluorescence intensity for IFN-{gamma} production 1 log lower than in the CD27-CD11ahi cells (data not shown). Among the CD8+ cells that did not produce IFN-{gamma} after P3 stimulation, the CD27+CD11alow lymphocytes became the major subset as early as 3 mo (Fig. 4GoB, left).



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  		FIGURE 4. Long-term therapeutic viral reduction results in decreased frequencies of HIV-specific CD8+ T cells. A, Long-term follow-up of three patients from group A (A3, left; A4, middle; A5, right). IFN-{gamma}-producing CD8+ T cells were evaluated after peptide stimulation and are expressed as a percentage of total CD8 T cells. Histograms show the course of plasma viral load. B, Phenotypic characteristics displayed by peptide-stimulated IFN-{gamma}-producing CD8 cells and their IFN-{gamma}-negative CD8+ counterpart after predominant HIV-1 peptide stimulation: P3 for patient A4 and G5 for patient A5 ({square}, CD27-CD11a-; , CD27-CD11a+; , CD27+CD11a+; {blacksquare}, CD27+11a- CD8 cells).

 
In contrast, for a patient with a viral load already low at HAART initiation, no substantial variation in the proportion of HIV-specific CD8+ cells was observed, regardless of the peptide tested (Fig. 4GoA, right). The G5-specific CD8+ T cells were mainly constituted of CD27-CD11ahigh effector and CD27+CD11ahigh memory cells, and this phenotype remained quasi-stable over time. This was also the case for the CD8+IFN-{gamma}- cells after G5 stimulation.

In conclusion, the long-term therapeutic control of HIV results in decreased frequencies of HIV-specific effector/memory CD8+ cells that produce IFN-{gamma}. The kinetics of this phenomenon may vary between epitopes for a single patient and is usually characterized by a reduction of the CD8+CD27-CD11ahigh effector pool. These findings suggest that HIV Ag stimulation has a positive influence on the expansion and functional status of HIV-specific CD8+ effector cells.

Dynamics of HIV-specific CD8 T cells with HIV rebounds during early therapeutic interruption

To further investigate the influence of viral load on the frequencies of HIV-specific CD8+ T cells, we analyzed changes during a treat- ment interruption scheduled after 1 mo of treatment for the four patients in group C (30). All were slow progressors and had never been treated with any antiretroviral drugs at study entry. As previously reported, plasma viremia decreased during the first phase of HAART (30), then rapidly rose back up to baseline values 1 mo after treatment was interrupted. Virus levels were controlled after treatment was reintroduced, with the same kinetics as observed during the first phase. Of particular interest was the parallel between the CD8+ T lymphocyte kinetics and the course of the viral load: the former reached 2 logs above baseline value at the virus rebound peak (Fig. 5GoA). CD8 cell activation, assessed by HLA-DR and CD38 expression, also paralleled the virus kinetics (data not shown).



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  		FIGURE 5. Rebounds of dominant HIV- and CMV-specific CD8 T cell frequencies during scheduled treatment interruptions. A, Kinetics of viral load and CD4 and CD8 absolute numbers in patients from group C. Results are expressed as median variations. B, The dynamics of functional HIV-1 peptide-specific CD8+ T cells are expressed as absolute numbers of total CD8+ T cells for patient C3. C, The dynamics of functional HIV-1 peptide-specific (left) and CMV pp65-specific (right) CD8 T cells are expressed as a percentage of total CD8+ T cells for patient C1. Histograms show the course of plasma viral load, and solid bars indicate HAART. D, The dynamics of functional HIV-1 peptide-specific CD8 T cells are expressed as a percentage of total CD8+ T cells for patient C2 (left). Phenotypic characteristics displayed by N2-stimulated IFN-{gamma}-producing CD8 cells and their IFN-{gamma}-negative CD8+ counterpart for patient C2 (right).

 
We used the IFN-{gamma} production assay to examine the relative contribution of functional CD8+ T lymphocytes specific for HIV in these bursts of CD8 cell mobilization. As Fig. 5GoC shows for one patient, the absolute number of CD8+ cells directed against the predominant N2 epitope accounted for up to nearly 2.85% of the total CD8+ cells. Because no redistribution-like phenomenon had taken place during the first month in this case, we could evaluate the effect of virus rebound at 2 mo (day 60). The N2-specific CD8+ T cells rapidly returned to upper baseline values at the virus rebound peak. This increase was extremely rapid and was concurrent with the virus rebound; it was observed as early as 1 wk after treatment interruption (Fig. 5GoC). However, none of the CD8+ T cells specific for the three other subdominant epitopes rebounded when the virus peaked. In all cases, both the frequencies (data not shown) and absolute numbers (Fig. 5GoC) of these various epitope-specific CD8+ T cells decreased below their baseline values at 6 mo (day 180), after treatment was reintroduced. In a second patient, a single epitope (G3) was recognized with a low frequency at day 0, and G3-specific CD8+ T cells disappeared thereafter whatever the viral changes (Fig. 5GoD, left). In contrast, an increase of HIV-specific CD8+ T cells (P1 and N3 epitopes) was observed. The P1-specific CD8+ T cells slightly decreased at day 60 at time of virus peak to reincrease up to the day 42 level when virus became controlled again at day 120. The N3-specific cells, undetectable at day 0, rose up to day 180. The most striking changes were observed with the CMV-specifc CD8+ T cells: after an initial drop at day 42, a 5-fold increase in the frequencies of the CMV-specific CD8+ T cells was observed in parallel to HIV rebound between day 42 and day 60 (Fig. 5GoD, right).

As shown in Fig. 5GoB (left), changes of N2-specific CD8+ T cells were similar to the P1-specific frequency variations reported in Fig. 5GoD: they were negatively correlated with viral load. When analyzing the phenotype of these HIV-specific cells over time, we noted that they mostly displayed the CD27-CD11ahigh effector phenotype while a few of them were CD27+CD11ahigh memory cells. In the meantime, cells that did not produce IFN-{gamma} after N2 stimulation were mostly of the effector cell phenotype as well, though with a higher proportion of CD27+CD11alow cells (Fig. 5GoB, right).

These findings indicate that HIV-specific CD8+ T cell responses contribute significantly to the changes in the total CD8 cell count during scheduled interruption of drugs. Two different patterns of peripheral CD8+ T cell responses were observed, i.e., either a positive correlation between virus load and CD8+ cell changes or a negative one that suggests a desequestration/resequestration phenomenon. Nevertheless, CD8+ T cell variations were not restricted to the HIV-specific CD8+ lymphocytes because they also involved CMV-specific responses.

In vivo mobilization of HIV-specific CD8+ T cells during late transient therapeutic failures

The question of whether these HIV-specific CD8+ T cells can be reamplified in vivo with relapses of HIV replication was further investigated in two group B patients, whose viral load increased markedly after being initially controlled during the first 3–6 mo (Fig. 6Go, A and B). Treatment modification subsequently induced stable virus control. The proportions of Ag-specific CD8+ cells producing IFN-{gamma} are shown in Fig. 6Go. In the first case (Fig. 6GoA, top), concurrent with early virus control, the CD8+ T cells directed against the two predominant peptides recognized at day 0, E2 and P2, decreased at 3 mo but were still detectable. The proportions of these E2- and P2-specific CD8+ T cells then rebounded at 6 mo together with the viral load (Fig. 6GoA, left): the HIV-specific CD8+ T cells returned at least to initial levels while the viral load peak reached values that were 2 log lower than at day 0. Only two of the four subdominant HIV-peptides (G1, E4) underwent a similar rebound. The absolute numbers of HIV-specific CD8+ lymphocytes (Fig. 6GoA, right) also peaked at 6 mo, substantially above baseline values. The long-term virus control obtained thereafter (12 mo) was accompanied by a decrease in the proportion of HIV-specific CD8+ T cells, as previously shown in the patients whose viral control remained stable during treatment. Fig. 6GoB shows virus and CD8 cell kinetics for a patient who experienced two virus rebounds, at 3 and 6 mo. Two HIV-specific CD8+ subsets that were undetectable at day 0 increased above the threshold level with virus rebound at 3 mo, fell again at 4 mo, and disappeared thereafter even during the second virus relapse (Fig. 6GoB, middle). A peak in CMV-specific cells was also observed at each plasma HIV rebound; after a transient CMV-specific cell increase during the first month, an EBV-specific response also appeared after the first rebound and slowly decreased thereafter.



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  		FIGURE 6. Mobilization of dominant and subdominant HIV-specific CD8 T cells during transient therapeutic failures. Variations of functional peptide-specific CD8 T cells is given according to percentage of total CD8+ T cells (left) and absolute numbers (right). Histograms show the course of plasma viral load. A, Follow-up of six HIV-1 epitope-specific CD8+ T lymphocytes in patient B1 (group B). B, Patient B2 follow-up of five HLA-A2-restricted epitope-specific CD8+ T cells with three HIV-1 (solid lines), one CMV (pp65), and one EBV (BMLF-1) (dashed lines) epitopes. C, Follow-up of four HIV-I peptide-specific (solid lines) and one CMV-specific (pp65, dashed line) CD8+ T lymphocytes in patient D2 (group D) whose treatment could not control virus replication.

 
In four other patients (group D), no stable virus control could be achieved during virus rebounds. Fig. 6Go (bottom) illustrates the situation of one of them: only a CMV-specific response was observed at day 0; at 1 mo, various new epitope-specific CD8+ T cells appeared with virus reduction; at 6 mo, the CMV pp65-specific CD8+ T cells peaked with the first virus rebound while two of the three HIV-specific responses were maintained. However, in the long term, HIV-specific cells decreased despite persistence of high viral loads, a finding that suggests either exhaustion or resequestration (Fig. 6GoC). Of note, we still detected anti-CMV CD8+ T lymphocytes. Fluctuations of HIV-specific CD8+ T cells were observed in three other patients from group D (data not shown).

The virus rebounds observed after 3 or 6 mo of virus reduction induced expansion of the preexisting dominant HIV-specific CD8+ T cell responses. However, new specificities emerged rarely and did not lead to high responses. The parallel changes observed between CMV-specific CD8+ T cell responses and HIV load suggest that these Ag-specific CD8+ T cell frequency variations were not restricted to the HIV-specific CD8+ cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have evaluated the influence of HIV burden variations on the mobilization of Ag-specific CD8 T cell responses in 15 chronically HIV-infected patients by analyzing the diversity and the function of these cells immediately before and during HAART. The low frequency of both HLA-A2/HIV-Gag (77–85, SLYNTVATL) and HIV-Pol (476–484, ILKEPVHGV) tetramer-binding cells yielded only a minimum estimate of the frequencies of CD8 T cells that were HIV specific. We further evaluated that percentage with a panel of 16 CTL epitopes covering 15 HLA class I molecules in 14 patients. The peptide-specific IFN-{gamma} production assay allowed us to determine that each patient had a mean of 2.2 HIV epitope-specific CD8 subsets of the 3.8 epitopes tested per patient with epitope-specific frequencies from 0.1% to 4.7%.

Although frequencies of tetramer-binding CD8 cells were slightly higher than those of IFN-{gamma}-producing CD8+ T cells, these two methods were concordant. Our data are consistent with previously reported correlations between the frequency of tetramer-binding cells, on the one hand, and cytolytic activity in HIV infection (15) or IFN-{gamma} production in hepatitis C virus (16), LCMV (10, 55), and influenza A infections, on the other (9, 39, 40). Therefore, the circulating HIV-specific CD8+ cells that bind to tetramers might be functional activated T lymphocytes, contrary to what has been observed for T cells specific for tumor-associated Ags in melanoma patients (38). Repeated exposure to HIV Ags and various cytokines during the chronic phase of infection may maintain over time high numbers of HIV-specific CD8+ T lymphocytes that produce IFN-{gamma} without inducing anergy.

Our findings also indicate that using a broad rather than narrow or selected panel of HIV epitopes provides a better appreciation of the HIV-specific CD8 T cell count: the panel of 16 HIV epitopes selected in 23 combinations of 15 HLA class I molecules had a positive response rate of 58%, independent of viral load or CD4 count. No public dominant HIV-CTL epitope was evidenced, in contrast to the LCMV situation (10, 55). The patients studied here have different MHC haplotypes and are heterozygous for their MHC alleles, and they are representative of an unselected Caucasian population. The frequencies of epitope-specific CD8+ cells differed from patient to patient, with median values of 0.24%. Our results illustrate the limitations of using a single epitope that does not represent an immunodominant response for interpatient evaluation of the correlations existing between HIV viral load and frequency of anti-HIV CD8+ T cells. Concerning the HLA-A2-restricted epitopes, we noticed that the Pol (476–484) HLA*0201-restricted peptide can induce a significant IFN-{gamma} production by HLA*0205 CD8+ T lymphocytes.

Individual follow-up studies during antiretroviral treatment allowed us to evaluate the influence of viral load modifications on the percentage of CD8+ T cells that are HIV specific. The rapid decrease in viral load observed during the first month induced a 2- to 4-fold increase in both percentages and absolute numbers of CD8 T cells directed against 65% of the HIV epitope combinations tested. Even more important, eight of the 11 HIV-specific CD8 T cells that were undetectable at day 0 were present at significant levels after 1 mo of treatment. Moreover, a similar phenomenon was observed for CMV-specific CD8 T lymphocytes. These data suggest that during the first weeks of viral load suppression, CD8+ T lymphocytes that were trapped in infected tissues during active virus replication (25, 26) may be rapidly released into the circulation as proposed for CD4 T cells (22, 23). Suppression of viral replication by HAART may result in a decreased antigenic stimulus and a reduced inflammatory cytokine (IL-1ß, IFN-{gamma}, IL-6, and macrophage inflammatory protein-1{alpha}) production in lymphoid tissues (56, 57). Adhesion molecules that mediate lymphocyte-endothelial cell interactions and promote the sequestration of circulating lymphocytes in tissues (58) have been found to decrease in lymph node tissues after HAART (57). These various processes could lead to a net redistribution from previously inflamed tissues into the blood of both HIV-specific and CMV-specific CD8+ T cells. Although we cannot exclude that other mechanisms can take place, such as decreased apoptosis or proliferation of HIV-specific CD8 T cells, the redistribution phenomenon initially proposed for the total CD4 T cells (22, 23) might also involve CD8 T cells and reroute some previously sequestered CD8 T cells to the circulation (17, 59), regardless of their antigenic specificity.

As expected from previous studies using either tetramers or cytolytic activity, the long-term therapeutic reduction of HIV below detectable levels decreased the frequency of activated HIV-specific CD8 cells producing IFN-{gamma}, regardless of their epitope specificities. Furthermore, when analyzing the phenotype of HIV-specific CD8+ T cells during efficient HIV load control, we pointed out the predominance of the CD27-CD11ahigh subset in the IFN-{gamma}-producing HIV epitope-specific CD8+ population, which has been described as the effector CD8 population in blood (54). This effector subset decreased with viral load, but only when HAART induced substantial changes in viral load. Two main CD8 subsets, the CD27-CD11ahigh effector (54) and CD27+CD11ahigh memory cells (54), were observed in the IFN-{gamma}-producing HIV-specific T cells. However, such an association between functional status and phenotypic markers is still under debate, and some authors have recently proposed CD56 as a marker of CTL (60). This loss of functional HIV-specific CD8 T cells during efficient HAART may be a result of the long-term reduction of the Ag load. Alternatively, HIV-specific cells may migrate to tissular sites of persistent but low HIV replication during HAART (25). However, the latter hypothesis is undermined by the simultaneous reduction during HAART of viral burden and histological alterations characteristic of HIV infection in lymphoid tissues (27, 61, 62).

This decay in host response against HIV might leave treated patients with low residual defenses against HIV. Therefore, it was important to assess whether these immune responses could still be mobilized when the virus becomes uncontrolled and levels of Ag stimulation rise. Studies performed on a small number of patients treated at the time of primary infection suggested this was feasible in such patients (31, 63). However, the question remained for patients treated during chronic HIV infection in whom the memory CD8 T cell responses to HIV might have been exhausted by recurrent exposure to HIV before treatment. We found that some preexisting CD8 T cell responses, which had decreased quickly during the initial phase of virus control, were able to rebound when relapse caused the resumption of viral replication. Mobilization of at least one HIV peptide-specific response appears to be a constant facet of virus rebounds. Nevertheless, CD8 cell variations are not restricted to HIV-specific CD8+ lymphocytes because they involve CMV-specific responses as well. The CMV viremia was available only in one case of the four patients tested for CMV-specific CD8 T cells. It was shown to decrease rapidly after HAART initiation in parallel to HIV drop (64), concurrently to the increase in CD8 cells directed both against HIV and CMV. The CMV viremia did not vary thereafter, even during further HIV rebounds when a peak of CMV-specific CD8 T cells was observed (64). As during uninterrupted HAART, the long-term virus control reestablished afterward was eventually followed by a decrease in the percentage of CD8 T cells that were HIV specific. The absolute number of these HIV-specific CD8 T lymphocytes increased beyond their baseline levels, regardless of the amplitude of virus rebound. We were able to evaluate the lag time between virus and HIV-specific CD8 lymphocyte rebounds in one patient: both parameters increased simultaneously 7 days after drug interruption, suggesting that at least the predominant response was capable of immediate reaction to recurrence of virus replication. The viral load reached its initial level and maintained a plateau there for several days before treatment was restarted, while the actual number of HIV-specific CD8 cells peaked above their baseline levels, suggesting that CD8 cells might have some efficacy in controlling virus replication at least in that case. Altogether, these data show that the preexisting dominant CD8 cell responses to HIV are rapidly mobilized or expanded during virus relapses and can restore the quasi-equilibrium that was established between the host and the virus before treatment began. Nevertheless, some EBV- or CMV-specific responses can be amplified as well during rebounds of virus replication. However, in the majority of cases these rebounds of HIV-specific CD8 T cells response components may not be capable of controlling HIV relapses. Some minor epitope-specific subsets also reached transient peaks and disappeared thereafter, when antigenic stimulation came back under control. These observations indicate that the preexisting memory T cell responses had not been previously exhausted by continuous exposure to HIV but remained capable of expansion, even in the absence of detectable help from CD4 T cells.

In contrast, new CD8 cell specificities were rarely detected when viral load climbed back up, whether at 1 or 6 mo after treatment started and whether the patient began treatment with profound or only limited CD4 cell depletion. Another pattern of the peripheral CD8+ T cell responses was observed, i.e., a negative correlation between virus load and CD8+ cell changes, suggesting a desequestration/resequestration phenomenon as discussed above.

The intensity of the CD4 T cell help can be another explanation for the different patterns of HIV-specific and CMV-specific CD8 cell frequency variations. None of the patients showed any detectable HIV-specific CD4 T cells, despite variable levels of CD4 counts at baseline while CMV-specific CD4 T cells were evidenced in most cases during HAART (data not shown) (22). The lack of detectable help from HIV-specific CD4 T cells in all cases may limit the expansion capacity of CD8 lymphocytes. In line with this commentary are the major increment of CMV-specific CD8+ T cells and the presence of a detectable CMV-specific CD4 help.

In conclusion, HIV-specific CD8 lymphocytes may be underestimated when analysis considers only a limited number of epitopes, none of which triggers an immunodominant response to HIV. Variations of the viral burden during antiretroviral therapies influence the extent and the function of the various predominant and subdominant responses. Some redistribution of HIV-specific T cells can occur at HAART initiation that exposes specificities sequestered in infected tissues, but this phenomenon is not HIV restricted because anti-CMV CD8+ T lymphocytes can be mobilized as well. The long-term reduction in virus replication induces decay in all specificities and functions of the effector/memory CD8 cell responses to HIV, but they remain mobilizable in vivo with reexposure to Ag stimulation, whatever the mechanism, i.e., expansion or desequestration. Rebounds of HIV replication are paralleled by peaks in memory CD8 T lymphocytes that quickly restore the preexisting host-virus quasi-equilibrium. Such dynamics of virus-specific CD8 T cells should be taken into account when elaborating future strategies of scheduled antiretroviral treatment interruptions aiming at restoring specific defenses against HIV.


   Acknowledgments
 
The RESTIM and COMET study groups are composed of Guislaine Carcelain (Laboratoire d’Immunologie Cellulaire et Tissulaire, Bâtiment CERVI, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7627), Ait Mohand Hocine and Michèle Panchard (Département des Maladies Infectieuses), Henri Agut and Catherine Robert (Département de Virologie), Alain Mallet (Service de Biomathématiques, Hôpital Pitié-Salpétriêre, 83, Bvd de l’Hôpital, 75651 Paris Cedex 13, France), and Avidan Neuman (Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel). We are grateful to Pierre Grenot for his help with four-color flow cytometry, Dr. I. Théodorou and collaborators for HLA typing, Isabelle Viseux and Maryse Mergirie for absolute CD4 and CD8 cell counts, and Yasmine Dudoit for HLA-A2 subtyping. We thank Antje Necker and François Romagné (Immunotech-Coulter-Beckman, Marseille, France) who have developed the HLA-A2/peptide tetramers technology. We thank all subjects who donated blood for this study. The English text was edited by Jo-Ann Cahn. We also thank Dr. D. McIlroy, who did the last reading of the revised manuscript.


   Footnotes
 
1 This work was supported by grants from Agence Nationale de la Recherche sur le SIDA and SIDACTION. L.M. is supported by a fellowship from Agence Nationale de la Recherche sur le SIDA. Back

2 Address correspondence and reprint requests to Prof. Brigitte Autran, Bâtiment Centre d’Etudes et de Recherches en Virologie et Immunologie, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7627, Laboratoire d’Immunologie Cellulaire et Tissulaire, 83, Bvd de l’Hôpital, 75651 Paris Cedex 13, France. Back

3 Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; HAART, highly active antiretroviral therapies. Back

Received for publication February 28, 2000. Accepted for publication May 22, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Doherty, P. C.. 1998. The numbers game for virus-specific CD8+ T cells. Science 280:227.[Free Full Text]
  2. Schmitz, J. E., M. J. Kuroda, S. Santra, V. G. Sasseville, M. A. Simon, M. A. Lifton, P. Racz, K. Tenner-Racz, M. Dalesandro, B. J. Scallon, et al 1999. Control of viremia in simian immunodeficiency virus infection by CD8+ lymphocytes. Science 283:857.[Abstract/Free Full Text]
  3. Jin, X., D. E. Bauer, S. E. Tuttleton, S. Lewin, A. Gettie, J. Blanchard, C. E. Irwin, J. T. Safrit, J. Mittler, L. Weinberger, et al 1999. Dramatic rise in plasma viremia after CD8+ T cell depletion in simian immunodeficiency virus-infected macaques. J. Exp. Med. 189:991.[Abstract/Free Full Text]
  4. Gotch, F. M., D. F. Nixon, N. Alp, A. J. McMichael, L. K. Borysiewicz. 1990. High frequency of memory and effector gag specific cytotoxic T lymphocytes in HIV seropositive individuals. Int. Immunol. 2:707.[Abstract/Free Full Text]
  5. Hoffenbach, A., P. Langlade-Demoyen, G. Dadaglio, E. Vilmer, F. Michel, C. Mayaud, B. Autran, F. Plata. 1989. Unusually high frequencies of HIV-specific cytotoxic T lymphocytes in humans. J. Immunol. 142:452.[Abstract]
  6. Moss, P. A., S. L. Rowland-Jones, P. M. Frodsham, S. McAdam, P. Giangrande, A. J. McMichael, J. I. Bell. 1995. Persistent high frequency of human immunodeficiency virus-specific cytotoxic T cells in peripheral blood of infected donors. Proc. Natl. Acad. Sci. USA 92:5773.[Abstract/Free Full Text]
  7. Koup, R. A., C. A. Pikora, K. Luzuriaga, D. B. Brettler, E. S. Day, G. P. Mazzara, J. L. Sullivan. 1991. Limiting dilution analysis of cytotoxic T lymphocytes to human immunodeficiency virus gag antigens in infected persons: in vitro quantitation of effector cell populations with p17 and p24 specificities. J. Exp. Med. 174:1593.[Abstract/Free Full Text]
  8. Klein, M. R., C. A. Van Baalen, A. M. Holwerda, S. R. Kerkhof Garde, R. J. Bende, I. P. Keet, J. K. Eeftinck Schattenkerk, A. D. Osterhaus, H. Schuitemaker, F. Miedema. 1995. Kinetics of Gag-specific cytotoxic T lymphocyte responses during the clinical course of HIV-1 infection: a longitudinal analysis of rapid progressors and long-term asymptomatics. J. Exp. Med. 181:1365.[Abstract/Free Full Text]
  9. Altman, J. D., P. A. H. Moss, P. J. R. Goulder, D. H. Barouch, M. G. McHeyzer-Williams, J. I. Bell, A. J. McMichael, M. M. Davis. 1996. Phenotypic analysis of antigen-specific T lymphocytes. Science 274:94.[Abstract/Free Full Text]
  10. Murali-Krishna, K., J. D. Altman, M. Suresh, D. J. Sourdive, A. J. Zajac, J. D. Miller, J. Slansky, R. Ahmed. 1998. Counting antigen-specific CD8 T cells: a reevaluation of bystander activation during viral infection. Immunity 8:177.[Medline]
  11. Ogg, G. S., A. J. McMichael. 1998. HLA-peptide tetrameric complexes. Curr. Opin. Immunol. 10:393.[Medline]
  12. Butz, E. A., M. J. Bevan. 1998. Massive expansion of antigen-specific CD8+ T cells during an acute virus infection. Immunity 8:167.[Medline]
  13. Ogg, G. S., X. Jin, S. Bonhoeffer, P. R. Dunbar, M. A. Nowak, S. Monard, J. P. Segal, Y. Cao, S. L. Rowland-Jones, V. Cerundolo, et al 1998. Quantitation of HIV-1-specific cytotoxic T lymphocytes and plasma load of viral RNA. Science 279:2103.[Abstract/Free Full Text]
  14. Ogg, G. S., X. Jin, S. Bonhoeffer, P. Moss, M. A. Nowak, S. Monard, J. P. Segal, Y. Cao, S. L. Rowland-Jones, A. Hurley, et al 1999. Decay kinetics of human immunodeficiency virus-specific effector cytotoxic T lymphocytes after combination antiretroviral therapy. J. Virol. 73:797.[Abstract/Free Full Text]
  15. Gray, C. M., J. Lawrence, J. M. Schapiro, J. D. Altman, M. A. Winters, M. Crompton, M. Loi, S. K. Kundu, M. M. Davis, T. C. Merigan. 1999. Frequency of class I HLA-restricted anti-HIV CD8+ T cells in individuals receiving highly active antiretroviral therapy (HAART). J. Immunol. 162:1780.[Abstract/Free Full Text]
  16. He, X. S., B. Rehermann, F. X. Lopez-Labrador, J. Boisvert, R. Cheung, J. Mumm, H. Wedemeyer, M. Berenguer, T. L. Wright, M. M. Davis, H. B. Greenberg. 1999. Quantitative analysis of hepatitis C virus-specific CD8+ T cells in peripheral blood and liver using peptide-MHC tetramers. Proc. Natl. Acad. Sci. USA 96:5692.[Abstract/Free Full Text]
  17. Kalams, S. A., P. J. Goulder, A. K. Shea, N. G. Jones, A. K. Trocha, G. S. Ogg, B. D. Walker. 1999. Levels of human immunodeficiency virus type 1-specific cytotoxic T-lymphocyte effector and memory responses decline after suppression of viremia with highly active antiretroviral therapy. J. Virol. 73:6721.[Abstract/Free Full Text]
  18. Dalod, M., M. Dupuis, J. C. Deschemin, D. Sicard, D. Salmon, J. F. Delfraissy, A. Venet, M. Sinet, J. G. Guillet. 1999. Broad, intense anti-human immunodeficiency virus (HIV) ex vivo CD8+ responses in HIV type 1-infected patients: comparison with anti-Epstein-Barr virus responses and changes during antiretroviral therapy. J. Virol. 73:7108.[Abstract/Free Full Text]
  19. Haas, G., U. Plikat, P. Debré, M. Lucchiari, C. Katlama, Y. Dudoit, O. Bonduelle, M. Bauer, H. G. Ihlenfeldt, G. Jung, et al 1996. Dynamics of viral variants in HIV-1 Nef and specific cytotoxic T lymphocytes in vivo. J. Immunol. 157:4212.[Abstract]
  20. Wilson, C. C., S. A. Kalams, B. M. Wilkes, D. J. Ruhl, F. Gao, B. H. Hahn, I. C. Hanson, K. Luzuriaga, S. Wolinsky, R. Koup, et al 1997. Overlapping epitopes in human immunodeficiency virus type 1 gp120 presented by HLA A, B, and C molecules: effects of viral variation on cytotoxic T-lymphocyte recognition. J. Virol. 71:1256.[Abstract]
  21. Wolinsky, S. M., B. T. Korber, A. U. Neumann, M. Daniels, K. J. Kunstman, A. J. Whetsell, M. R. Furtado, Y. Cao, D. D. Ho, J. T. Safrit. 1996. Adaptive evolution of human immunodeficiency virus-type 1 during the natural course of infection. Science 272:537.[Abstract]
  22. Autran, B., G. Carcelain, T. S. Li, C. Blanc, D. Mathez, R. Tubiana, C. Katlama, P. Debré, J. Leibowitch. 1997. Positive effects of combined antiretroviral therapy on CD4+ T cell homeostasis and function in advanced HIV disease. Science 277:112.[Abstract/Free Full Text]
  23. Pakker, N. G., D. W. Notermans, R. J. De Boer, M. T. Roos, F. De Wolf, A. Hill, J. M. Leonard, S. A. Danner, F. Miedema, P. T. Schellekens. 1998. Biphasic kinetics of peripheral blood T cells after triple combination therapy in HIV-1 infection: a composite of redistribution and proliferation. Nat. Med. 4:208.[Medline]
  24. Koup, R. A., J. T. Safrit, Y. Cao, C. A. Andrews, G. McLeod, W. Borkowsky, C. Farthing, D. D. Ho. 1994. Temporal association of cellular immune responses with the initial control of viremia in primary human immunodeficiency virus type 1 syndrome. J. Virol. 68:4650.[Abstract/Free Full Text]
  25. Cheynier, R., S. Henrichwark, F. Hadida, E. Pelletier, E. Oksenhendler, B. Autran, S. Wain-Hobson. 1994. HIV and T cell expansion in splenic white pulps is accompanied by infiltration of HIV-specific cytotoxic T lymphocytes. Cell 78:373.[Medline]
  26. Plata, F., B. Autran, L. P. Martins, M. W.-H. S., C. Raphael, M. Mayaud, J. M. Denis, J. M. Guillon, P. Debré. 1987. AIDS virus-specific cytotoxic T lymphocytes in lung disorders. Nature 328:348.[Medline]
  27. Tenner-Racz, K., H. J. Stellbrink, J. van Lunzen, C. Schneider, J. P. Jacobs, B. Raschdorff, G. Grosschupff, R. M. Steinman, P. Racz. 1998. The unenlarged lymph nodes of HIV-1-infected, asymptomatic patients with high CD4 T cell counts are sites for virus replication and CD4 T cell proliferation: the impact of highly active antiretroviral therapy. J. Exp. Med. 187:949.[Abstract/Free Full Text]
  28. Dalod, M., M. Harzic, I. Pellegrin, B. Dumon, B. Hoen, D. Sereni, J. C. Deschemin, J. P. Levy, A. Venet, E. Gomard. 1998. Evolution of cytotoxic T lymphocyte responses to human immunodeficiency virus type 1 in patients with symptomatic primary infection receiving antiretroviral triple therapy. J. Infect. Dis. 178:61.[Medline]
  29. Finzi, D., J. Blankson, J. D. Siliciano, J. B. Margolick, K. Chadwick, T. Pierson, K. Smith, J. Lisziewicz, F. Lori, C. Flexner, et al 1999. Latent infection of CD4+ T cells provides a mechanism for lifelong persistence of HIV-1, even in patients on effective combination. Nat. Med. 5:512.[Medline]
  30. Neumann, A. U., R. Tubiana, V. Calvez, C. Robert, T. S. Li, H. Agut, B. Autran, C. Katlama. 1999. HIV-1 rebound during interruption of highly active antiretroviral therapy has no deleterious effect on reinitiated treatment: Comet Study Group. AIDS. 13:677.[Medline]
  31. Ortiz, G. M., D. F. Nixon, A. Trkola, J. Binley, X. Jin, S. Bonhoeffer, P. J. Kuebler, S. M. Donahoe, M. A. Demoitie, W. M. Kakimoto, et al 1999. HIV-1-specific immune responses in subjects who temporarily contain virus replication after discontinuation of highly active antiretroviral therapy. J. Clin. Invest. 104:R13.[Medline]
  32. Li, T. S., R. Tubiana, C. Katlama, V. Calvez, H. Ait Mohand, B. Autran. 1998. Long-lasting recovery in CD4 T-cell function and viral-load reduction after highly active antiretroviral therapy in advanced HIV-1 disease. Lancet 351:1682.[Medline]
  33. Parker, K. C., M. A. Bednarek, L. K. Hull, U. Utz, B. Cunningham, H. J. Zweerink, W. E. Biddison, J. E. Coligan. 1992. Sequence motifs important for peptide binding to the human MHC class I molecule, HLA-A2. J. Immunol. 149:3580.[Abstract]
  34. Van der Burg, S. H., M. R. Klein, C. J. Van de Velde, W. M. Kast, F. Miedema, C. J. Melief. 1995. Induction of a primary human cytotoxic T-lymphocyte response against a novel conserved epitope in a functional sequence of HIV-1 reverse transcriptase. AIDS 9:121.[Medline]
  35. Tsomides, T. J., B. D. Walker, H. N. Eisen. 1991. An optimal viral peptide recognized by CD8+ T cells binds very tightly to the restricting class I major histocompatibility complex protein on intact cells but not to the purified class I protein. Proc. Natl. Acad. Sci. USA 88:11276.[Abstract/Free Full Text]
  36. Scotet, E., J. David-Ameline, M. A. Peyrat, A. Moreau-Aubry, D. Pinczon, A. Lim, J. Even, G. Semana, J. M. Berthelot, R. Breathnach, M. Bonneville, E. Houssaint. 1996. T cell response to Epstein-Barr virus transactivators in chronic rheumatoid arthritis. J. Exp. Med. 184:1791.[Abstract/Free Full Text]
  37. Steven, N. M., N. E. Annels, A. Kumar, A. M. Leese, M. G. Kurilla, A. B. Rickinson. 1997. Immediate early and early lytic cycle proteins are frequent targets of the Epstein-Barr virus-induced cytotoxic T cell response. J. Exp. Med. 185:1605.[Abstract/Free Full Text]
  38. Lee, P. P., C. Yee, P. A. Savage, L. Fong, D. Brockstedt, J. S. Weber, D. Johnson, S. Swetter, J. Thompson, P. D. Greenberg, M. Roederer, M. M. Davis. 1999. Characterization of circulating T cells specific for tumor-associated antigens in melanoma patients. Nat. Med. 5:677.[Medline]
  39. Flynn, K. J., G. T. Belz, J. D. Altman, R. Ahmed, D. L. Woodland, P. C. Doherty. 1998. Virus-specific CD8+ T cells in primary and secondary influenza pneumonia. Immunity 8:683.[Medline]
  40. Flynn, K. J., J. M. Riberdy, J. P. Christensen, J. D. Altman, P. C. Doherty. 1999. In vivo proliferation of naive and memory influenza-specific CD8+ T cells. Proc. Natl. Acad. Sci. USA 96:8597.[Abstract/Free Full Text]
  41. Goulder, P. J., A. K. Sewell, D. G. Lalloo, D. A. Price, J. A. Whelan, J. Evans, G. P. Taylor, G. Luzzi, P. Giangrande, R. E. Phillips, A. J. McMichael. 1997. Patterns of immunodominance in HIV-1-specific cytotoxic T lymphocyte responses in two human histocompatibility leukocyte antigens (HLA)- identical siblings with HLA-A*0201 are influenced by epitope mutation. J. Exp. Med. 185:1423.[Abstract/Free Full Text]
  42. Brander, C., B. Walker. 1996. The HLA-class I restricted CTL response in HIV-1 infection: identification of optimal epitopes. , , , , , , ed. In HIV-1 Molecular Immunology Database vol. 3: Theoretical Biology and Biophysics, Los Alamos National Laboratory, Los Alamos, NM, Part III.
  43. Nixon, D. F., A. R. Townsend, J. G. Elvin, C. R. Rizza, J. Gallwey, A. J. McMichael. 1988. HIV-1 gag-specific cytotoxic T lymphocytes defined with recombinant vaccinia virus and synthetic peptides. Nature 336:484.[Medline]
  44. Zhang, Q. J., R. Gavioli, G. Klein, M. G. Masucci. 1993. An HLA-A11-specific motif in nonamer peptides derived from viral and cellular proteins. Proc. Natl. Acad. Sci. USA 90:2217.[Abstract/Free Full Text]
  45. Threlkeld, S. C., P. A. Wentworth, S. A. Kalams, B. M. Wilkes, D. J. Ruhl, E. Keogh, J. Sidney, S. Southwood, B. D. Walker, A. Sette. 1997. Degenerate and promiscuous recognition by CTL of peptides presented by the MHC class I A3-like superfamily: implications for vaccine development. J. Immunol. 159:1648.[Abstract]
  46. Sipsas, N. V., S. A. Kalams, A. Trocha, S. He, W. A. Blattner, B. D. Walker, R. P. Johnson. 1997. Identification of type-specific cytotoxic T lymphocyte responses to homologous viral proteins in laboratory workers accidentally infected with HIV-1. J. Clin. Invest. 99:752.[Medline]
  47. Johnson, R. P., A. Trocha, T. M. Buchanan, B. D. Walker. 1992. Identification of overlapping HLA class I-restricted cytotoxic T cell epitopes in a conserved region of the human immunodeficiency virus type 1 envelope glycoprotein: definition of minimum epitopes and analysis of the effects of sequence variation. J. Exp. Med. 175:961.[Abstract/Free Full Text]
  48. Koenig, S., T. R. Fuerst, L. V. Wood, R. M. Woods, J. A. Suzich, G. M. Jones, V. A. De la Cruz, R. T. J. Davey, S. Venkatesan, B. Moss, W. E. Biddison, A. S. Fauci. 1990. Mapping the fine specificity of a cytolytic T cell response to HIV-1 Nef protein. J. Immunol. 145:127.[Abstract]
  49. Culmann, B., E. Gomard, M. P. Kieny, B. Guy, F. Dreyfus, A. G. Saimot, D. Sereni, D. Sicard, J. P. Levy. 1991. Six epitopes reacting with human cytotoxic CD8+ T cells in the central region of the HIV-1 NEF protein. J. Immunol. 146:1560.[Abstract]
  50. Hadida, F., A. Parrot, M. P. Kieny, B. Sadat-Sowti, C. Mayaud, P. Debré, B. Autran. 1992. Carboxyl-terminal and central regions of human immunodeficiency virus-1 NEF recognized by cytotoxic T lymphocytes from lymphoid organs: an in vitro limiting dilution analysis. J. Clin. Invest. 89:53.
  51. Wills, M. R., A. J. Carmichael, K. Mynard, X. Jin, M. P. Weekes, B. Plachter, J. G. Sissons. 1996. The human cytotoxic T-lymphocyte (CTL) response to cytomegalovirus is dominated by structural protein pp65: frequency, specificity, and T- cell receptor usage of pp65-specific CTL. J. Virol. 70:7569.[Abstract]
  52. Solache, A., C. L. Morgan, A. I. Dodi, C. Morte, I. Scott, C. Baboonian, B. Zal, J. Goldman, J. E. Grundy, J. A. Madrigal. 1999. Identification of three HLA-A*0201-restricted cytotoxic T cell epitopes in the cytomegalovirus protein pp65 that are conserved between eight strains of the virus. J. Immunol. 163:5512.[Abstract/Free Full Text]
  53. Tsomides, T. J., A. Aldovini, R. P. Johnson, B. D. Walker, R. A. Young, H. N. Eisen. 1994. Naturally processed viral peptides recognized by cytotoxic T lymphocytes on cells chronically infected by human immunodeficiency virus type 1. J. Exp. Med. 180:1283.[Abstract/Free Full Text]
  54. Hamann, D., P. A. Baars, M. H. Rep, B. Hooibrink, S. R. Kerkhof-Garde, M. R. Klein, R. A. Van Lier. 1997. Phenotypic and functional separation of memory and effector human CD8+ T cells. J. Exp. Med. 186:1407.[Abstract/Free Full Text]
  55. Gallimore, A., A. Glithero, A. Godkin, A. C. Tissot, A. Pluckthun, T. Elliott, H. Hengartner, R. Zinkernagel. 1998. Induction and exhaustion of lymphocytic choriomeningitis virus-specific cytotoxic T lymphocytes visualized using soluble tetrameric major histocompatibility complex class I-peptide complexes. J. Exp. Med. 187:1383.[Abstract/Free Full Text]
  56. Lederman, M. M., E. Connick, A. Landay, D. R. Kuritzkes, J. Spritzler, M. St. Clair, B. L. Kotzin, L. Fox, M. H. Chiozzi, J. M. Leonard, et al 1998. Immunologic responses associated with 12 weeks of combination antiretroviral therapy consisting of zidovudine, lamivudine, and ritonavir: results of AIDS Clinical Trials Group Protocol 315. J. Infect. Dis. 178:70.[Medline]
  57. Bucy, R. P., R. D. Hockett, C. A. Derdeyn, M. S. Saag, K. Squires, M. Sillers, R. T. Mitsuyasu, J. M. Kilby. 1999. Initial increase in blood CD4+ lymphocytes after HIV antiretroviral therapy reflects redistribution from lymphoid tissues. J. Clin. Invest. 103:1391.[Medline]
  58. Nakajima, H., H. Sano, T. Nishimura, S. Yoshida, I. Iwamoto. 1994. Role of vascular cell adhesion molecule 1/very late activation antigen 4 and intercellular adhesion molecule 1/lymphocyte function-associated antigen 1 interactions in antigen-induced eosinophil and T cell recruitment into the tissue. J. Exp. Med. 179:1145.[Abstract/Free Full Text]
  59. Tough, D. F., P. Borrow, J. Sprent. 1996. Induction of bystander T cell proliferation by viruses and type I interferon in vivo. Science 272:1947.[Abstract]
  60. Pittet, M. J., D. E. Speiser, D. Valmori, J. C. Cerottini, P. Romero. 2000. Cutting edge: cytolytic effector function in human circulating CD8+ T cells closely correlates with CD56 surface expression. J. Immunol. 164:1148.[Abstract/Free Full Text]
  61. Wong, J. K., H. F. Gunthard, D. V. Havlir, Z. Q. Zhang, A. T. Haase, C. C. Ignacio, S. Kwok, E. Emini, D. D. Richman. 1997. Reduction of HIV-1 in blood and lymph nodes following potent antiretroviral therapy and the virologic correlates of treatment failure. Proc. Natl. Acad. Sci. USA 94:12574.[Abstract/Free Full Text]
  62. Cavert, W., D. W. Notermans, K. Staskus, S. W. Wietgrefe, M. Zupancic, K. Gebhard, K. Henry, Z. Q. Zhang, R. Mills, H. McDade, et al 1997. Kinetics of response in lymphoid tissues to antiretroviral therapy of HIV-1. Science 276:960.[Abstract/Free Full Text]
  63. Lisziewicz, J., E. Rosenberg, J. Lieberman, H. Jessen, L. Lopalco, R. Siliciano, B. Walker, F. Lori. 1999. Control of HIV despite the discontinuation of antiretroviral therapy. N. Engl. J. Med. 340:1683.[Free Full Text]
  64. Li, T. S., R. Tubiana, A. M. Fillet, B. Autran, C. Katlama. 1999. Negative result of cytomegalovirus blood culture with restoration of CD4+ T-cell reactivity to cytomegalovirus after HAART in an HIV-1-infected patient. J. Acquir. Immune Defic. Syndr. Hum. Retrovirol. 20:514.[Medline]



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