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The Journal of Immunology, 2007, 178: 788-797.
Copyright © 2007 by The American Association of Immunologists, Inc.

The Duration of Exposure to HIV Modulates the Breadth and the Magnitude of HIV-Specific Memory CD4+ T Cells

Souheil-Antoine Younes*, Lydie Trautmann*, Bader Yassine-Diab*, Lena H. Kalfayan*, Anne-Elen Kernaleguen*, Thomas O. Cameron{ddagger}, Rachid Boulassel{dagger}, Lawrence J. Stern§, Jean-Pierre Routy{dagger}, Zvi Grossman, Alain R. Dumont* and Rafick-Pierre Sekaly1,*,{dagger}

* Laboratoire d’Immunologie, Université de Montréal, Département de Microbiologie et Immunologie, Montréal, Canada; {dagger} Immunodeficiency Service and Division of Haematology, Royal Victoria Hospital, McGill University Health Centre, McGill University, Montreal, Quebec, Canada; {ddagger} Molecular Pathogenesis Program, Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York 10016; § Department of Pathology, University of Massachusetts Medical School, North Worcester, Massachusetts 01605; and Department of Physiology and Pharmacology, Tel-Aviv University, Tel-Aviv, Israel and Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The impact of exposure to Ag on the development and maintenance of human CD4+ memory T cells in general and HIV infection in particular is partially understood. In this study, we measured HIV-specific CD4+ T cell proliferative responses against HIV proteins and derived peptides one year after highly active antiretroviral therapy initiation in 39 HIV-infected patients who initiated therapy at different times following infection. We show that a brief exposure to HIV of <1 month does not allow the generation of significant detectable frequencies of HIV-specific CD4+ memory T cells. Patients having prolonged cumulative exposure to high viral load due to therapy failures also demonstrated limited HIV-specific CD4+ T cell responses. In contrast, patients exposed to significant levels of virus for periods ranging from 3 to 18 mo showed brisk and broad HIV-specific CD4+ T cell responses 1 year following the onset of therapy intervention. We also demonstrate that the nadir CD4+ T cell count before therapy initiation correlated positively with the breadth and magnitude of these responses. Our findings indicate that the loss of proliferative HIV-specific CD4+ T cell responses is associated with the systemic progression of the disease and that a brief exposure to HIV does not allow the establishment of detectable frequencies of HIV-specific memory CD4+ T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Cellular immunity to HIV-1 infection plays an important role in the control of HIV-1 infection (1). Although CD8+ T cells are considered the main cellular compartments responsible for the elimination of HIV-infected cells, mounting evidence suggests that the induction and the maintenance of a proper antiviral immune response requires functional Ag-specific CD4+ T cells (2). Several reports have shown that CD4+ T cells play an important role in maintaining effective CD8 T cell responses against lymphocytic choriomeningitis virus (LCMV)2 infection in mice (3, 4); moreover, functional memory CD4+ T cells have been associated with the control of chronic human infections such as HSV (5), CMV (6), and hepatitis C virus (7).

The influence of viral load and the duration of viral exposure on the maintenance of memory T cells has been largely explored in animal models of viral infections (8, 9, 10). Most of these studies have used the LCMV infection model to analyze the impact of duration of exposure to Ag on the establishment and maintenance of memory CD8+ T cells. Overall, these studies have indicated that the generation of memory T cells is influenced by the frequency of effector cells generated early in the infection as well as the concentration of Ags during primary infection with LCMV (11) or with the intracellular parasite Listeria monocytogenes (12). Few reports have focused on the maintenance of CD4+ T cells (13) and they have suggested that prolonged exposure to LCMV leads to the elimination of memory CD4+ T cells (10), although the impact of a short exposure to Ag has not been adequately assessed. In HIV-1 infection the presence of high viremia and, consequently, high levels of HIV Ags have made it difficult to address this question because HIV preferentially infects HIV-specific CD4+ T cells, leading to their gradual elimination (14) and thereby impeding the establishment of a HIV-specific memory CD4+ T cell pool.

The impact of highly active antiretroviral therapy (HAART) on the qualitative and quantitative properties of HIV-specific memory CD4+ T cell responses has also been difficult to study, because the frequency of HIV-specific CD4+ T cells is extremely low as reported in several studies (15, 16, 17, 18, 19, 20). However, early HAART intervention has been associated with allowing the persistence of HIV-specific CD4+ T cells, a possible consequence of the reduced cytopathy of HIV due to reduced viral load and antigenemia, a consequence of the antiviral therapy (21, 22). Subsequently, it was shown that subjects treated during the acute phase of the infection, but not those treated during chronic infection, were able to partially control viral rebounds after HAART cessation in humans (23, 24), suggesting that CD4+ T cell responses, which persist in these individuals, contribute to the overall control of viremia by helping to maintain the pool of HIV-specific effector and memory CD8+ T cells (25). Kaufman et al. (26), using an IFN-{gamma} ELISPOT assay that measures mostly effector functions, showed that the breadth and the magnitude of HIV-specific CD4+ effector T cell responses in early HAART-treated patients are reduced when compared with patients undergoing structural treatment interruption. In contrast, an earlier report provided evidence that early HAART intervention preserves Gag-specific CD4+ T cell proliferative responses (21), suggesting that IFN-{gamma} secretion and proliferation assays measure two distinct "types" of CD4+ T cell responses. In support of this hypothesis, several reports have shown that HIV-specific CD4+ T cells producing only IFN-{gamma} are impaired in their ability to proliferate ex vivo (27, 28) and do not persist as long-term memory CD4+ T cells (29). We (29) and others (30) have shown that memory CD4+ T cells are endowed with proliferative capacity and have phenotypic features of central (CCR7+CD45RA–) and effector (CCR7–CD45RA–) memory T cells in aviremic long-term HAART-treated patients after long-term therapy intervention initiated in the primary infection. Although it is still controversial whether IL-2 producing HIV-specific CD4+ T cells contribute to the control of HIV infection (31, 32), cumulative data support the notion that memory CD4+ T are defined by their ability to produce IL-2 and/or proliferate (29, 30, 33, 34). Furthermore, we established a positive correlation between the proliferation and the production of IL-2 by HIV-specific memory CD4 T cells (29).

In the present study, we analyze the impact of exposure to HIV on the persistence and maintenance of HIV-specific memory CD4+ T cells defined by their ability to proliferate. We investigated the impact of the duration of exposure to HIV Ags on the breadth, magnitude, and persistence of proliferation-competent HIV-specific CD4+ memory T cells in patients who received therapy at different times postinfection. We report a complex pattern, consistent with different levels of generation, suppression, and recovery of the proliferative memory response depending on the length of exposure to HIV before the introduction of HAART.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Study population

HIV-1 infected patients were recruited from different hospitals in Montreal, Canada between 1996 and 2001. Patients signed informed consent approved by the McGill University Health Center (Montreal, Canada) review board. Viremia and CD4+ T cell counts were analyzed on a monthly basis from the onset of infection until the time CD4+ T cell responses were analyzed. All patients had undetectable viral loads throughout the study as measured by the Amplicor HIV-1 monitor ultrasensitive method (Roche). The presumed date of infection was estimated for each individual using clinical and laboratory data as well as patient history information. Of 176 patients enrolled in Montreal clinics, we were able to select 39 patients for our study because most of the required information for presuming the date of HIV infection was available for these 39 patients. The following guidelines proposed by the Acute HIV Infection and Early Disease Research Program sponsored by the National Institutes of Allergy and Infectious Disease Division of AIDS (Bethesda, Maryland) were used to estimate the date of infection: 1) the date of the first positive HIV RNA test or p24 Ag assay available on the same day as a negative standard HIV enzyme immunoassay test minus 14 days; 2) the date of onset of symptoms of an acute retroviral syndrome minus 14 days; 3) the date of the first indeterminate Western blot minus 35 days; 4) The detuned assay as described in references (35, 36); and 5) information obtained from questionnaires addressing the timing of high-risk behavior for HIV transmission that was used when available to confirm the presumed date of infection. At least three independent criteria were used to estimate the date of infection for each patient.

Peptides

Gag (HXB2) 15-mer peptides were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program (Rockville, MD). Nef (BRU), Tat (MN), and B clade Pol, Env, Vpr, Vif, and Rev peptides were obtained from the Canadian Network for Vaccines and Immunotherapeutics (CANVAC) core facility (Toronto, Canada). The estimated purity of the peptides was >90% as measured by HPLC and mass spectroscopy. Individual peptides were diluted in DMSO at a concentration of 100 mg/ml (Sigma-Aldrich) and stored at –80°C.

CD4+ T cell proliferation assay

CFSE-based proliferation assays were performed as previously described (29). Briefly, PBMCs were isolated from peripheral blood or apheresis donor packs by sodium diatrizoate density centrifugation (Amersham Biosciences). Frozen samples were cryopreserved using patients’ plasma supplemented with 10% DMSO. Cryopreserved samples were maintained in liquid nitrogen and thawed for use in the proliferation assay. PBMCs (10–50 x 106) were labeled with a predetermined concentration of CFSE (Molecular Probes). The final concentration of CFSE used for PBMCs labeling varied between 0.7 and 1.5 µM. Cells were washed twice in PBS and resuspended in 10% human AB RPMI 1640 medium (Sigma-Aldrich). Typically, CFSE-labeled PBMCs (1 x 106/ml) were incubated in 96-deep well plates (Nunc) in the presence of Gag, Nef, or Tat peptide pools (2 µg/ml/peptide) or individual peptides (10 µg/ml). Cells were stained with anti-CD4 allophycocyanin and anti-CD3 PE (BD Biosciences) after a 6-day in vitro incubation at 37°C with 5% CO2. Events (3–5 x 105) gated on CD3+CD4+-viable lymphocytes were collected on a FACSCalibur dual-laser cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star).

Production of HLA-DR1 tetramers and tetramer staining

HLA-DR1 {alpha}- and beta-chains were produced in insect cells and refolded with the G066, G069 (Table II), and hemagglutinin (HA) influenza HA306–318 peptides as previously described (37). S2 Schneider cells were cotransfected with pchygro, a plasmid encoding the resistance hygromycin gene, and the pCV eGFP-DR{alpha}-BSP-DRbeta vector in which the HA, GO66, and G069 peptides were covalently linked to the beta-chain as described (38). After hygromycin selection, cells were grown and induced using 1 mM CuSO4. The supernatant was collected and passed through an affinity column containing beads coated with an Ab recognizing properly folded HLA-DR molecules (L243). Affinity-purified HLA-DR1 monomers were biotinylated and tetramerized as described (37).


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  		Table II. Promiscuous Gag, Nef, and Tat peptides

 
After 8 days of peptide-specific in vitro stimulation, PBMCs were labeled with PE-conjugated HLA-DR1 tetramers, PerCP-conjugated anti-CD3, and allophycocyanin-conjugated anti-CD4 Abs (BD Biosciences). Data were collected on a FACSCalibur dual-laser cytometer (BD Biosciences) and analyzed using Flow Jo software (Tree Star).

HLA typing

HLA-DR typing was performed using conventional genotyping with sequence-specific primer PCR high-resolution kits (QIAamp kit; Qiagen) as previously described (29).

Statistical analysis

The Kruskal-Wallis and Spearman correlation statistical tests were performed using Prism V2 software (GraphPad). Differences were considered statistically significant when p ≤ 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Patient classification

Thirty-nine patients were classified in five different groups according to the timing of HAART initiation at different intervals of primary HIV infection. All individuals selected successfully controlled their viremia after initiation of the treatment (Fig. 1). Group A (n = 8) includes patients who initiated the therapy between 11 and 28 days after the onset of infection (see Materials and Methods for the criteria used to date HIV infection). Groups B (n = 10), C (n = 7), and D (n = 8) include patients who initiated HAART between 30 and 90 days (group B), 90 and 180 days (group C), or 6 and 18 mo (group D) after HIV infection. Group E (n = 6) includes patients that did not stably control HIV replication for 3 to 4 years after infection due to resistance/failure of antiretroviral therapy. CD4+ T cell proliferative responses were measured in all five groups of patients when their viral load was <50 copies/ml at least one year after the initiation of treatment. All patients received comparable treatment regimens and had equivalent CD4+ T cell counts 1 year after HAART intervention, when HIV-specific CD4+ T cell responses were monitored. Patient characteristics are listed in Table I.


Figure 1
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  		FIGURE 1. Shown is a schematic representation of the study population. Patients initiated HAART at different times after the onset of infection. The estimated date of HIV infection is determined as described in Materials and Methods. CD4 T cell proliferative responses were performed 1 year after an undetectable viral load in each group as indicated. Patient characteristics are represented in Table I.

 

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  		Table I. Patient characteristics

 
Immunodominant HIV peptides are clustered in specific regions of Gag and Nef proteins

In a previous study we compared HIV-specific CD4+ T cell responses in aviremic and viremic patients using Gag and Nef peptides matrices (29). Using a CFSE-based proliferation assay, we were able to characterize that memory CD4+ T cells in long-term HAART-treated patients have the ability to proliferate and to produce IL-2. Pools from Pol, Env, Vpr, Rev, Vif, and Tat were also tested in this study. The proliferative CD4+ T cell responses in our patients were mainly directed against Gag and Nef epitopes (data not shown). These data corroborate the findings of Kaufman et al. (26), indicating the immunodominant nature of Gag and Nef proteins in HIV genome in the cohorts of different patients. The frequencies of CFSElowCD4+ T cells >0.5% were scored as positive responses, because PBMCs isolated from uninfected healthy individuals yield <0.05% of CFSElow cells when incubated with the same peptides. Analysis of Gag- and Nef-specific CD4+ T cell responses allowed us to determine the immunodominant regions in Gag and Nef proteins. As shown in Fig. 2, Gag and Nef epitopes are clustered into specific regions in Gag (GR1 to GR5) and Nef (NR1 to NR6) proteins independently of the HLA-DR alleles expressed by 5B, 4B, 1D, and 3D patients (Fig. 2). CD4+ T cell epitopes mapping from various patients (n = 10) allowed us to select 25 Gag and 14 Nef immunodominant peptides spanning various regions of these proteins (Table II). These epitopes were then used to analyze the breadth and the magnitude of HIV-specific CD4+ T cell responses in the above-described patient groups.


Figure 2
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  		FIGURE 2. Identification of immunodominant regions in Gag and Nef proteins. PBMCs from patients 5B, 4B, 1D, and 3D were labeled with CFSE and stimulated for 6 days with overlapping Gag and Nef peptides for 6 days. Cells were collected and stained with PE-conjugated anti-CD3 and allophycocyanin-conjugated anti-CD4 Abs. Fifty-thousand events gated on live lymphocytes were collected and analyzed. GR and NR indicate Gag and Nef immunodominant regions, respectively. Peptide sequences are listed in Table II.

 
First, we asked whether these responses are peptide-specific or due to bystander, unspecific proliferation. As shown in Fig. 2, peptides such as G066 and G069 induced 1 and 1.2% of CFSElow cells, respectively, in PBMCs from patient 5B. Thus, we were concerned about the specificity of the responses giving low frequencies of CFSElow cells. MHC class II tetramers having G066 and G069 peptides were generated and used to stain PBMCs from patient 5B after 8 days in vitro stimulation. The 8-day in vitro stimulation increased the number of T cells that proliferate and made the staining with the tetramers more reliable as shown in Fig. 3. In fact, almost 50% of the CFSElow CD4+ T cells were HLA-DR1 tetramer-positive when stimulated with the G066 and G069 peptides or the positive control HA308–318 influenza peptide (Fig. 3, upper panels). However, no staining was detected when these tetramers were tested on PBMCs stimulated by the negative control G076 peptide (Fig. 3, lower panels). Of note is the fact that not all CFSElow CD4+ T cells were tetramer-positive because the PBMCs used were derived from the non-HLA-DR1 homozygous HLA-DR1/DR7 patient (patient 5B).


Figure 3
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  		FIGURE 3. CFSElowCD4+ T cells are specific for the stimulating peptide as detected by MHC class II tetramers. PBMCs from patient 5B (HLA-DR1/HLA-DR7) were stimulated with the HA306–318, G066, G069, and G076 peptides for 8 days, incubated with PE-conjugated HLA-DR1/HA, HLA-DR1/G066, and HLA-DR1/G069 tetramers for 3 h at 37°C, and stained using anti-CD3-PerCP and anti-CD4-allophycocyanin Abs. Cells stimulated with HA, G066, and G069 peptides were stained with HA, G066, and G069 tetramers, respectively. CFSE-labeled PBMCs stimulated with the G076 peptide were used as negative controls for tetramer staining. Numbers in the quadrants represent the percentages of dividing and tetramer-positive T cells. Events (2 x 104 to 3 x 104) gated on live CD3+CD4+ lymphocytes were collected and analyzed. Data are representative of at least five independent experiments.

 
Altogether, the data presented above indicate that Gag and Nef promiscuous peptides are clustered within specific immunodominant regions in Gag and Nef proteins and that the proliferative responses detected by the CFSE proliferation assay are specific to the peptides as detected by MHC class II tetramers.

Duration of exposure to HIV influences the generation and/or maintenance of HIV-specific CD4+ T cells

We then assessed the breadth and magnitude of CD4+ T cell responses in HIV patients who initiated therapy at different times following primary HIV infection. Assessment of HIV-specific CD4+ T cell proliferative responses after 1 year of viral suppression revealed that patients treated between 11 and 28 days after infection (group A) recognize a very limited number of Gag- and Nef- derived peptides (median = 1; range = 2–4) (Fig. 4a). In contrast, patients treated between 30 days and 18 mo (groups B, C, and D) show diversified CD4+ T cell responses to Gag and Nef peptides (median = 10, 8, and 14 epitopes; range = 1–42, 2–35, and 4–25 in groups B, C, and D, respectively). However, persistent exposure to high HIV viremia (group E) impedes the maintenance of a broad HIV-specific CD4+ T cell repertoire, because these patients displayed proliferative CD4+ T cell responses only against a small number of HIV epitopes (median = 2.1; range = 0–5). Similar results were obtained when analyzing the magnitude of HIV-specific CD4+ T cell responses in these patients (Fig. 4b). Again, patients treated early (group A) or who were persistently exposed to high levels of virus (group E) showed very low frequencies of proliferating HIV-specific memory CD4+ T cells (median = 1.3; range = 3–5), as compared with patients treated between 1 and 18 mo postinfection (median = 13.1, 14.8, and 11.8%; range = 0.5–35, 5–30, and 2–34 in groups B, C, and D, respectively). The significantly higher HIV-specific proliferative CD4+ T cell responses in patients from groups B, C, and D vs those from groups A and E were not due to a general defect in CD4+ T cell proliferation in the latter two groups, because stimulation with a superantigen (staphylococcal enterotoxin A) yielded comparable CD4+ T cell proliferative responses in these groups (p = 0.3 and 0.79) (Fig. 4c).


Figure 4
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  		FIGURE 4. Impact of the duration of exposure to HIV on the maintenance of HIV-specific memory CD4+ T cell proliferative responses. a, Impact of the duration of exposure to HIV on the breadth of HIV-specific CD4+ T cell responses. CFSE-labeled PBMCs were stimulated with Gag and Nef single epitopes for 6 days followed by anti-CD3 and anti-CD4 staining. Each point represents the number of Gag and Nef peptides recognized by CD4+ T cells in every patient. b, Impact of the duration of exposure to HIV on the magnitude of HIV-specific CD4+ T cell responses. Each point represents the sum of percentages of CFSElowCD4+ T cells induced to proliferate by Gag and Nef peptides. c, Proliferative CD4+ T cell responses after pulsing patients’ PBMCs with the superantigen staphylococcal enterotoxin A. d, IFN-{gamma} intracellular staining on CD4+ T cells after stimulation of patients PBMCs with HIV proteins. Each point represents the sum of percentages of IFN-{gamma}-positive cells triggered by pools of peptides spanning the entire HIV genome. HIV-neg, HIV negative. e, pp65 CMV-specific CD4+ T cell proliferative responses in patients from all different patients groups. The number of patients (N), the median, and the range are shown at the bottom of each graph. Only p values of <0.05 are shown. Statistical significance was determined by Kruskal-Wallis test.

 
The low level of proliferation-competent CD4+ T cells detected in patients from groups A and E could be due to their differentiation into IFN-{gamma}-secreting CD4+ T cells, because we (29) and others (27, 39) showed previously that these cells have impaired proliferative capacity. Fig. 4d demonstrates that the magnitude of IFN-{gamma} HIV-specific CD4+ T cell responses following PBMC stimulation with peptide pools spanning the whole HIV genome are undetectable in patients from group A as compared with patients from groups B, C, and D (Fig. 4d). Patients from group E also show few IFN-{gamma} responses to the HIV peptides tested, as it is well established that HAART treatment will decrease the detection of effector cells (15, 16, 17, 18, 19, 20).

We next asked whether the poor HIV-specific responses in group A and group E patients were restricted only to the HIV virus. CMV-specific CD4+ T cell responses were analyzed in these patients. As shown in Fig. 4e, group A and group E patients showed detectable frequencies of CMV-specific CD4+ T cell responses, although a significant difference (p < 0.05) in the magnitude of CMV responses was found between group A patients vs those of groups B–E.

Altogether, the data presented in Fig. 4 indicate that, after prolonged HAART intervention, patients treated between 3 and 18 mo of HIV infection have significant detectable frequencies of HIV-specific memory CD4+ T cells. In contrast, patients treated between 11 and 28 days after the onset of infection and patients treated in the chronic phase have low proliferative and IFN-{gamma}+ (effector) HIV-specific CD4+ T cell responses to most HIV proteins.

The nadir CD4+ T cell count, but not viral load before therapy, correlates with the breadth and the magnitude of HIV-specific CD4+ T cell responses

As illustrated in Fig. 4, a and b, we observed high interpatient variability in the breadth (from 1 to 42 epitopes giving responses) and the magnitude (from 0.5 to 34% of CFSElow CD4+ T cells) of proliferative CD4+ T cell responses in patients from groups B, C, and D. Several parameters could potentially influence the breadth and magnitude of HIV-specific CD4+ T cell responses within each group; these include, among others, the nadir CD4+ T cell count and the baseline viral load before therapy initiation (20, 40). Fig. 5 illustrates the relationship between these two parameters and the breadth or the magnitude of HIV-specific CD4+ T cell responses. A significant positive correlation was established between the nadir CD4+ T cell count and both the breadth (R = 0.52; p = 0.007) (Fig. 5a) and the magnitude (R = 0.6; p = 0.0011) (Fig. 5c) of HIV-specific CD4+ T cell responses. In contrast, no correlation was found between the baseline viral load and the breadth (p = 0.38) or magnitude (p = 0.4) of HIV-specific CD4+ T cell responses (Fig. 5, b and d, respectively).


Figure 5
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  		FIGURE 5. The nadir CD4+ T cell count correlates with the breadth and the magnitude of HIV-specific CD4+ T cell responses. Correlation between the breadth of HIV-specific CD4+ T cells responses and the Nadir CD4+ T cell count (a) or the baseline viral load (d) in patients from groups B, C, and D. Correlation between the magnitude of HIV-specific CD4+ T cell responses and the nadir CD4+ T cell count (b) or the baseline viral load (c). Statistical significance of each correlation was calculated using the Spearman test. N, Number of patients; R, range; P, p value.

 
These data indicate that the large interpatient variations in the breadth and magnitude of HIV-specific CD4+ T cell responses are associated with their nadir CD4+ T cell counts, which most likely reflect the extent of damage caused to the immune system during untreated HIV infection before HAART intervention.

HIV-specific memory CD4+ T cells are detected after early HAART intervention but at low frequencies

The lack of detectable frequencies of HIV-specific CD4+ T cells in most of the patients from group A led us to investigate the mechanisms that interfere with the presence of HIV-specific CD4+ T cells. It is possible that the early therapy administration completely prevents the generation of memory HIV-specific CD4+ T cells due to insufficient exposure to Ag. Alternatively, due to their low frequency caused by their insufficient exposure to Ag, HIV-specific CD4+ T cells are undetectable in a 6-day proliferation assay in this category of patients.

To discriminate between these two possibilities, PBMCs derived from group A patients were stimulated with Gag pools (123 peptides) for 14 days followed by a restimulation for 4 h with Gag or pp65 pools, and the frequency of HIV-specific CD4+ T cells was detected by an IFN-{gamma} intracellular stimulation assay. As shown in Fig. 6, four tested patients from group A showed detectable frequencies of proliferating cells that were 10-fold lower than those observed in a group B patient (patient 5B).


Figure 6
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  		FIGURE 6. Detection of HIV-specific CD4+ T cells after 14 days of in vitro culture in PBMCs from group A patients. CFSE-labeled cells were stimulated with Gag pools (123 peptides) for 7 days. IL-2 (10 U/ml) was added on day 7 and the cells were restimulated for an additional 7 days. On day 14 cells were stimulated with Gag or pp65 pools for 4 h and the detection of HIV-specific CD4+ T cells was performed by IFN-{gamma} intracellular staining.

 
These data indicate that early HAART intervention does not abolish the generation of memory CD4+ T cells, because HIV-specific CD4+ T cells were detected in patients from group A albeit at much lower frequencies, in part due to the reduced peripheral expansion caused by limited availability of Ag.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this report we have investigated the relationship between the duration of exposure to viral Ags and the generation of memory CD4+ T cells. We have provided evidence that a minimal exposure of over a month is needed to induce the generation of a detectable repertoire of HIV-specific CD4+ T cells. Using the overlapping Gag and Nef epitopes we were able to characterize promiscuous epitopes derived from Gag and Nef proteins as listed in Table II. These epitopes were able to trigger the proliferation of CD4+ T cells in several patients, and these epitopes are naturally processed by patients’ APCs. Interestingly, conserved epitopes were not those that were most frequently recognized, as we did not observe a positive correlation between the frequency of recognition of epitopes and their degree of conservation among the variants of HIV clade B. For example, the N048 peptide induced CD4+ T cell proliferative responses in 68% of patients from groups B, C, and D despite its low degree of conservation (15%) in 266 sequences from HIV-1 clade B. By contrast, the G074 peptide is highly conserved (94.4%) but induced proliferative CD4+ T cell responses in only 16% of HIV patients (Table II). This result is probably due to the flexibility and the degeneracy of the MHC class II interactions with immunogenic peptides previously described and is most likely a consequence of the open nature of peptide binding grooves of HLA-DR complexes (41, 42, 43).

We also showed that patients treated between 11 and 28 days of the infection (group A) have almost undetectable frequencies of HIV-specific CD4+ T cells to all HIV-1 proteins when monitored 1 year after the initiation of treatment (Fig. 6). The detection of very low frequencies of HIV-specific memory CD4+ T cells in group A patients after 1 year of HAART intervention could first be explained by the lack of sufficient stimulation and the expansion of these cells due to insufficient exposure to HIV Ags. The requirement for prolonged exposure of CD4+ T cells to Ag to generate effector cells has been elegantly demonstrated in murine models (44). Using a tetracycline-inducible system to control the expression of MHC class II complexes, the authors show that persistent stimulation of OVA-specific CD4+ T cells is required for the longevity of these cells (44). In agreement with this, it is likely that continuous stimulation of CD4+ T cells is needed to generate detectable frequencies of HIV-specific memory CD4+ T cells. A consequence of early HAART initiation (group A patients) would be to limit viral replication and antigenemia, thereby preventing the stimulation and the expansion of HIV-specific CD4+ T cells. Alternatively, because HIV depletes CD4+ T cells in the acute phase of the infection (45, 46), few HIV-specific CD4+ T cells would persist as a memory pool after therapy intervention. Thus, the low frequencies of HIV-specific memory CD4+ T cells in group A patients could also be the consequence of the massive depletion of CD4+ T cells that occurs in the acute phase of the infection and the early HAART intervention that reduces Ag levels and prevents the expansion of a novel pool of HIV-specific CD4+ T cells (45, 46).

Previous reports have generated conflicting data regarding the maintenance of HIV-specific CD4+ T cells when patients are treated before seroconversion or after seroconversion (47, 48). In fact, it has been shown that strong Gag-specific CD4+ T cell proliferative responses are detected in patients treated before seroconversion (47), whereas by using a large number of patients Lacabaratz-Porret et al. (48) did not observe any differences in the magnitude of p24-specific proliferating CD4+ T cells between patients treated before or after seroconversion. In our study, patients 5A, 6A, and 8A were treated postseroconversion while all other group A patients initiated the therapy before seroconversion. However, group A patients did not maintain HIV-specific CD4+ T cells as compared with patients exposed for >28 days to HIV. These discrepancies are possibly due to the use of different seroconversion criteria to classify the patients, because seroconversion can occur up to 8 wk after the infection (49). We have been extremely rigorous in estimating the time of HIV infection because we have used at least three independent parameters to classify each patient enrolled in this study. This systematic classification should be used when comparing our results to those reported by other studies (22, 47, 48).

Recently, it has been proposed that the magnitude of HIV-specific CD4+ T cell proliferative responses is directly linked to the level of viral load (50). Our results indicate that the duration of exposure to the virus also constitutes an important criterion in establishing the pool of detectable HIV-specific memory CD4+ T cells. As shown in Fig. 5, b and d, we do not observe a correlation between the viral load before HAART and the persistence of HIV- specific proliferative responses after 1 year of therapy intervention. Our data suggest that the duration of viral persistence rather that the viral load per se could impact on the persistence of HIV-specific CD4+ T cells. Repeated and prolonged exposure to high levels of Ags leads to the exhaustion of pathogen-specific CD4+ and CD8+ T cells as demonstrated in several models of viral infections in mice and in human disease (8, 10, 51), possibly through the up-regulation of PD-1 as we and others have recently demonstrated (52, 53, 54).

It was striking to observe in our studies that the continuous exposure of untreated patients to high levels of HIV for up to 18 mo (group D; median = 58,014.67 counts/ml; range = 5,407–4,017,630) does not progressively decrease the breadth and magnitude of HIV-specific CD4+ T cell responses assessed 1 year after the initiation of successful HAART, strongly suggesting that HIV-specific CD4+ memory T cells are not rapidly and irreversibly eliminated by the virus following infection (14). In fact, recent reports have suggested that the cytopathic effect of SIV (46, 55) or HIV (45) on CD4+ T cells is detected within the first 15 days of the infection and mainly in gut memory CD4+ T cells. Furthermore, others have reported that the frequency of infected CD4+ T cells in the chronic phase of the infection is extremely low (14, 56). This suggests that the diminution of recoverable proliferative responses over a period of years herein reported is primarily due to factors other than the direct destruction of infected cells by the virus or by CTLs. Chronic immune activation, which damages both central and peripheral lymphoid tissues, is the most likely cause for the gradual irreversible suppression of HIV-specific CD4+ T cell responses (57, 58) as shown in group E patients of our study.

A higher CD4+ T cell count before therapy has been associated with a better prognosis and with stronger proliferative responses against both HIV and non-HIV Ags (59, 60, 61, 62). Our data agree with and extend these findings and strongly suggest that the preservation of broad and strong HIV-specific memory CD4+ T cell responses depends on the degree of disease progression before therapy initiation, as measured by the levels of CD4+ T cell depletion. Implications of these findings regarding the timing of HAART initiation and therapeutic vaccination should await further investigation in larger cohorts. A better definition of the factors that lead to loss of functional memory is required in the design of immunotherapies and therapeutic vaccines aiming to normalize immunological functions.


   Acknowledgments
 
We thank R. Cheynier and E. Haddad for critical review of the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Address correspondence and reprint requests to Dr. Rafick-Pierre Sékaly, Centre de Recherche du Centre Hospitalier de l’Université de Montréal, 264 René-Lévesque Boulevard East, Montréal, Canada H2X 1P1. E-mail address: rafick-pierre.sekaly{at}umontreal.ca Back

2 Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; HA, hemagglutinin; HAART, highly active antiretroviral therapy. Back

Received for publication July 3, 2006. Accepted for publication November 3, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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