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J Virol. Dec 2007; 81(24): 13904–13915.
Published online Oct 17, 2007. doi:  10.1128/JVI.01401-07
PMCID: PMC2168869

Preserved Central Memory and Activated Effector Memory CD4+ T-Cell Subsets in Human Immunodeficiency Virus Controllers: an ANRS EP36 Study[down-pointing small open triangle]

Abstract

Human immunodeficiency virus (HIV) controllers are rare individuals who spontaneously control HIV type 1 replication for 10 years or more in the absence of antiretroviral treatment. In the present study, HIV controllers (n = 11) maintained potent HIV-specific CD4 responses in spite of very low antigenic loads. Their CD4+ central memory T (TCM) cells were characterized by near-normal numbers and preserved interleukin-2 (IL-2) secretion in response to HIV antigens and uniformly high expression of the survival receptor IL-7 receptor α (IL-7Rα). Controllers expressed CCR7 at higher levels than uninfected controls, suggesting differences in TCM-cell homing patterns. CD4+ effector memory T (TEM)-cell responses were polyfunctional in HIV controllers, while IL-2 secretion was lost in viremic patients. Cytokine production was three times higher in controllers than in treated patients with undetectable viral loads, suggesting an intrinsically more efficient response in the former group. The total CD4+ TEM-cell pool underwent immune activation in controllers, as indicated by increased HLA-DR expression, decreased IL-7Rα expression, a bias towards gamma interferon production upon polyclonal stimulation, and increased macrophage inflammatory protein 1β secretion associated with chronic CCR5 down-regulation. Thus, HIV controllers showed a preserved CD4+ TCM-cell compartment and signs of potent functional activation in the CD4+ TEM-cell compartment. While controllers did not show the generalized immune activation pattern associated with disease progression, they had signs of immune activation restricted to the effector compartment. These findings suggest the induction of an efficient, nondetrimental type of immune activation in patients who spontaneously control HIV.

Human immunodeficiency virus (HIV) controllers are rare individuals who spontaneously control HIV replication to undetectable levels in the absence of therapy. Controllers account for less than 1% of seropositive individuals (33) and can be distinguished from long-term nonprogressors (LTNP), who are defined on the basis of persistently elevated CD4+ T-cell counts. While LTNP have a significant risk of progressing to disease even after 10 years or more of asymptomatic infection, the HIV controller status is associated with a very low risk of progression (10, 11, 37). Controllers have HIV DNA loads significantly lower than those of LTNP, emphasizing the importance of limited viral dissemination in maintaining a healthy status in the long term (33, 52). Attenuated virus, characterized by major genetic defects such as deletions in the nef gene, has been isolated in a few cases of controlled HIV infection (15, 31). However, evidence for viral attenuation remains uncommon. Virologic analyses of eight of the HIV controllers included in the present study showed that replication-competent HIV could be isolated in each case and suggested a degree of ongoing viral replication in vivo based on phylogenetic evidence (34). Full-length sequencing of HIV isolates obtained from another cohort of controllers did not reveal inactivating deletions or mutations in the viral genome (7). In addition, viral fitness of HIV clones obtained from these isolates remained in the normal range (7). Based on these studies, HIV controllers appear to be infected with fully replication-competent virus in the vast majority of cases. Thus, host factors, genetic or immunologic in nature, most frequently account for HIV control.

While the role of CD8+ T cells in clearing HIV-infected cells has been clearly established (1, 3, 5, 6, 42, 43, 51), several factors also point to a contribution from CD4+ T cells in HIV control. A hallmark of nonprogressive HIV infection is the presence of CD4+ T cells that proliferate in response to HIV antigens, while this response is weak or absent in viremic patients (41, 50, 58). Evidence from murine models suggests that CD4+ T cells are needed to sustain efficient CD8 antiviral responses in the long term (13, 39). In rhesus macaques, attenuated simian immunodeficiency virus (SIV) infection induces a particularly high frequency of virus-specific CD4+ T cells that have direct effector function (20). Seronegative individuals who have been repeatedly exposed to HIV also show signs of efficient CD4+ T-cell-proliferative responses to HIV antigens (2). However, the causative link between strong CD4+ T-cell responses and HIV control is still under debate (38, 47). Strong CD4+ T-cell-proliferative responses in treated patients do not always predict a better virological outcome upon structured treatment interruption (28), a possible reason being the rapid and preferential infection of HIV-specific CD4+ T cells in the presence of replicating virus (18). Most patients with progressive HIV disease still exhibit significant numbers of cytokine-producing CD4+ T cells when stimulated with HIV antigens (48). The quality rather than the quantity of HIV-specific CD4+ T cells distinguishes slowly progressive infection. This type of infection is associated with a particular cytokine secretion profile, dominated by CD4+ T cells that produce interleukin-2 (IL-2), either exclusively or in conjunction with gamma interferon (IFN-γ). In contrast, high-level viremia is associated with the loss of IL-2 secretion and a response dominated by IFN-γ (8, 14, 19, 24, 25, 56, 60).

A specific subset of memory CD4+ T cells is thought to be responsible for the long-term maintenance of immune memory. These cells, termed central memory T (TCM) cells, are endowed with a self-renewal capacity which is thought to depend on autocrine IL-2 secretion (30, 35). In addition, CD4+ TCM cells express homing molecules such as CCR7 and CD62L that allow their recirculation into secondary lymphoid organs, where recall responses are initiated. Younes et al. (60) have proposed that persistent HIV antigenemia drives the differentiation of CD4+ TCM cells into effector memory T (TEM) cells, which lose IL-2 secretion and thus the self-renewal capacity. Whether the loss of IL-2 secretion is a mere consequence of exposure to high-level antigenemia or is the underlying cause of uncontrolled viral replication is not yet understood.

In this context, it is important to characterize CD4 responses in HIV controllers, since these responses are presumably optimal. Few studies have focused on CD4 responses in patients with spontaneously undetectable viral loads. We found that controllers had intrinsically higher-level cytokine responses to HIV antigens than treated patients with undetectable viral loads. HIV controllers showed a unique CD4+ T-cell activation pattern, characterized by the coexistence of a preserved central memory compartment and an activated effector memory compartment. Such activation status may account for both the persistence and potency of anti-HIV CD4 responses in these rare patients.

MATERIALS AND METHODS

Study design.

HIV controllers were defined as HIV-infected patients who had been seropositive for >10 years and had received no antiretroviral treatment for whom >90% of the plasma viral load measurements were <400 copies of HIV RNA/ml. Controllers were identified among 1,300 patients monitored at the Centre Hospitalo-Universitaire de Bicêtre (Bicêtre, France) and 1,500 patients of the French National Agency for Research on AIDS and Viral Hepatitis (ANRS) SEROCO-HEMOCO cohort, with a frequency of 0.6% in both cohorts. Ten HIV controllers included in the present study have been described elsewhere (33) and two were newly recruited (patients A9 and A10) (Table (Table1).1). In the course of the study, one patient (A8) exhibited a viremic episode associated with bronchitis but subsequently spontaneously controlled his viral load to 100 copies of HIV RNA/ml of plasma. The CCR5 Δ32 genotype was determined for all controller patients by sequencing (I. Théodorou, INSERM U543).

TABLE 1.
Characteristics of patients in the HIV controller groupa

Control groups included the following: (i) uninfected healthy blood donors from the Etablissement Français du Sang (Paris, France), (ii) untreated viremic patients with viral loads of >2,000 copies of HIV RNA/ml who had been infected with HIV for at least 6 months and had not received antiretroviral therapy, and (iii) HIV-infected patients successfully treated with antiretroviral therapy for more than 3 years who had <50 copies of HIV RNA/ml. Patients from the last two groups were monitored at the Centre Hospitalo-Universitaire de Bicêtre. Patient characteristics, CD4+ T-cell counts, and viral load data are reported in Table Table2.2. The study was approved by the Comité Consultatif de Protection des Personnes dans la Recherche Biomédicale de l'Hôpital Bicêtre. All participants gave written informed consent prior to blood sampling.

TABLE 2.
Summary of patient characteristicsa

Intracellular cytokine staining (ICS) assays.

Cryopreserved peripheral blood mononuclear cells (PBMC) stored in liquid nitrogen were thawed and left to recover at 37°C in complete medium (RPMI 1640 supplemented with 2 mM glutamine, antibiotics, and 10% human AB serum) for 8 to 9 h. HIV-specific stimulation was performed by adding baculovirus-derived recombinant p24 Gag protein (Protein Sciences) at 5 μg/ml to 2 × 106 PBMC. Cells were stimulated for 14 h, with brefeldin A (5 μg/ml; Sigma) added after the first 2 h of stimulation. Polyclonal stimulation was performed by incubating 0.6 ×106 PBMC in a 96-well plate precoated with anti-CD3 antibody (0.6 μg/ml [clone UCHT1; Immunotech]) in the presence of soluble anti-CD28 antibody (0.5 μg/ml [clone CD28.2; Immunotech]), with brefeldin A added after the first 2 h. Following stimulation, cells were harvested from wells and stained for surface antigens with the following combinations of antibodies (all purchased from BD Biosciences unless otherwise specified): CD3-peridinin chorophyll, CD45RA-phycoerythrin (PE)-Cy7, CCR7-allophycocyanin (CCR7-APC; R&D Systems), and CD4-APC-Cy7. Cells were fixed and permeabilized using an Intraprep permeabilization kit according to the instructions of the manufacturer (Beckman Coulter). Intracellular cytokine staining was detected with IFN-γ-carboxyfluorescein (clone 25723.11; R&D Systems) and IL-2-PE (clone MQ1-17H12). Fluorescence was measured on a six-color flow cytometer (FACSCanto) using the FACSDiva software (BD Biosciences). Data were analyzed with FACSDiva and FlowJo 8.1 (Tree Star).

For p24-stimulated cultures and unstimulated control cultures, a minimum of 750,000 lymphocyte-gated events were acquired. The percentage of cytokine-producing CD4+ T cells was determined after subtracting the percentage of cytokine-positive events in unstimulated controls. Responses below the level of 0.01% cytokine-producing cells were considered negative. None of the 10 HIV-seronegative donors tested showed a positive cytokine response upon p24 stimulation.

To detect the production of the chemokine macrophage inflammatory protein 1β (MIP-1β [CCL4]), PBMC were stimulated either with p24 Gag (5 μg/ml) or with the superantigen staphylococcal enterotoxin B (SEB; 1 μg/ml [Toxin Technology]). ICS was performed as described above, except that the IL-2 antibody was replaced with a MIP-1β-PE antibody (clone D21-1351; BD Biosciences).

Phenotyping.

Phenotyping was carried out with fresh PBMC samples. Blood (20 to 30 ml) was collected in heparinized tubes, and PBMC were separated by Ficoll-Paque (Pharmacia) density centrifugation. Four-color antibody staining of 106 PBMC was performed at 4°C for 30 min. CD4+ T-cell subsets were defined using the antibodies CD45RA-fluorescein isothiocyanate, CD4-PE-Cy7 (BD Biosciences), and CCR7-APC (R&D Systems). A fourth antibody was used to examine the expression of specific activation markers or receptors within each subset: HLA-DR-PE, CD25-PE, or CD69-PE (BD Biosciences) or CD127-PE (R&D Systems). A minimum of 250,000 lymphocyte-gated events were acquired on a FACSCalibur flow cytometer and analyzed with the CellQuest software (BD Biosciences). To standardize analyses of mean fluorescence intensity (MFI), the same settings were used on the same flow cytometer across experiments. Titration of each new lot of antibody was carried out on cryopreserved PBMC samples from the same donor.

The expression of CCR5 was assayed with whole-blood samples since this receptor can be down-regulated on PBMC after Ficoll gradient purification (36). Briefly, 200 μl of blood was diluted in 4 ml of PharmLyse buffer (BD Biosciences), and the dilution was incubated for 10 min at room temperature. Upon the completion of red-cell lysis, cells were washed, stained with CD45RA-fluorescein isothiocyanate, CD4-PE-Cy7, CCR7-APC, and CCR5-PE, and analyzed by flow cytometry as described above.

Proliferation assay.

Proliferation was assayed by [3H]thymidine incorporation as described previously (32). Briefly, PBMC at 105 cells per well were treated in quadruplicate with p24 Gag protein at 5 μg/ml. Positive controls were stimulated with 5 μg of phytohemagglutinin/ml, and negative controls were incubated with complete medium alone. On day 5, each well was pulsed with 1 μCi [3H]thymidine (Amersham). Sixteen hours later, cells were harvested onto glass fiber filters, and radioactivity was quantitated with a beta-scintillation counter (Wallac; Perkin Elmer). The stimulation index (SI) was computed as the ratio of the median [3H]thymidine incorporation into stimulated cells to the median [3H]thymidine incorporation into unstimulated cells. A positive response was defined by both an SI of >3 and a median count of >3,000 in the stimulated wells.

Statistical analysis.

Analyses were performed with GraphPad Prism version 4.0 software using nonparametric statistical tests in all cases. Differences in variables between patient groups were analyzed with the Mann-Whitney U test. Horizontal bars on scatter data plots indicate median values. All significant differences between groups (P < 0.05) are reported on the scatter data plots.

RESULTS

Potent HIV-specific CD4+ T-cell responses in HIC.

CD4+ T-cell cytokine responses in a group of HIV controllers (n = 11) with spontaneous control of viremia in the absence of therapy were analyzed. Detailed characteristics of these patients are reported in Table Table1.1. Control groups included untreated viremic patients (n = 10), patients responding to highly active antiretroviral therapy (HAART) (n = 10), and healthy, uninfected blood donors (n = 10).

CD4+ T-cell responses were tested by ICS of cryopreserved PBMC. In a representative experiment, unstimulated CD3+ CD4+ T cells produced very small but still detectable amounts of cytokines, while cells stimulated with p24 HIV Gag protein produced IL-2 and IFN-γ, either alone or in combination (Fig. (Fig.1A,1A, left and middle panels). Polyclonal stimulation with anti-CD3 and anti-CD28 antibodies induced massive cytokine secretion (Fig. (Fig.1A,1A, right panel). The analysis of p24 Gag-induced cytokine production indicated that HIV-specific CD4 responses were generally high in the HIC group, though individually variable. The levels of IFN-γ production in CD4+ T cells from controllers and viremic patients were comparable, even though in vivo the amount of viral antigen responsible for the induction of these responses was 2- to 3-log higher in the latter group (Fig. (Fig.1B).1B). Interestingly, IFN-γ responses were significantly higher in the controllers than in the HAART group, while both groups were characterized by undetectable viral loads. This observation suggested that the efficiency of the IFN-γ response, as measured by the level of IFN-γ secretion relative to the HIV antigenic load, was intrinsically high in HIV controllers.

FIG. 1.
Cytokine responses in CD4+ T cells. (A) Representative ICS analysis. PBMC of controller patient A4 were left unstimulated (left panel) or were stimulated with the HIV p24 Gag core protein (middle panel) or with anti-CD3/CD28 antibodies (aCD3/aCD28; ...

Differences in p24 Gag-induced IL-2 production among the different patient groups were marked (Fig. (Fig.1B).1B). IL-2 responses were abolished in viremic patients and were low in HAART-treated patients but remained as high as the IFN-γ responses in the controller group. Thus, HIV controllers were characterized by the persistence of potent IL-2 and IFN-γ CD4 responses in spite of low antigenic loads.

Proliferative responses to the p24 Gag protein were positive for all the patients in the controller group, as measured by an SI of >3 (median SI, 11.1). In contrast, proliferative responses were low or undetectable in the viremic group (median SI, 2.4) and intermediate in the HAART group (median SI, 3.2) (Fig. (Fig.2).2). Thus, CD4+ T cells from controllers retained the capacity for clonal expansion in response to HIV antigens, a finding compatible with their preserved IL-2 secretion capacity (56, 60).

FIG. 2.
Proliferative responses. PBMC were stimulated with the HIV p24 Gag protein for 5 days. Stimulation indexes obtained in the [3H]thymidine incorporation assay are reported. P values were determined by the nonparametric Mann-Whitney U test. Horizontal bars ...

Preserved central memory compartment with high CCR7 expression in HIV controllers.

CD4+ T-cell subsets were defined according to the expression of the CD45RA and CCR7 markers (Fig. (Fig.3A).3A). The percentages of naive CD4+ T cells (CD45RA+ CCR7+) were variable and did not distinguish the different groups studied (Fig. (Fig.3B).3B). The percentages of CD4+ TCM cells (CD45RA CCR7+) were heterogeneous in the controller group but did not differ significantly from those in the uninfected controls (Fig. (Fig.3C).3C). In contrast, the CD4+ TCM-cell compartment was decreased in viremic and HAART-treated patients. An opposite trend was observed in the CD4+ TEM-cell compartment (CD45RA CCR7), since controllers had significantly fewer CD4+ TEM cells than viremic and HAART-treated patients (Fig. (Fig.3D).3D). These data suggested that the differentiation state of peripheral CD4+ T cells varied among the groups, the reduction in central memory cells being associated with progressive HIV infection.

FIG. 3.
Phenotyping of naive and memory CD4+ T-cell subsets. (A) Representative gating strategy used to define CD4+ T-cell subsets. CD3+ CD4+ T cells were classified according to CD45RA and CCR7 expression. Naive cells were defined ...

CCR7 expression is a key determinant of the capacity of T cells to migrate to secondary lymphoid organs (16, 44). Interestingly, the level of expression of CCR7 per cell, as measured by the MFI, was markedly increased in CD4+ T cells of controllers (Fig. (Fig.3E).3E). This increase was observed in both naive and CD4+ TCM cells (data not shown). Of note, high CCR7 expression was not observed in uninfected donors, HAART-treated patients, or viremic patients but appeared to be specific to the controller group. High expression of this homing receptor may alter the recirculation pattern of controller CD4+ T cells and increase their chances of recognizing HIV antigens within lymphoid organs.

Polyfunctional effector memory responses and preserved central memory responses in HIV controllers.

Cytokine responses in the different CD4+ T-cell subsets were evaluated using six-color flow cytometry. HIV-specific cytokine production by naive CD4+ T cells was below the detection threshold (data not shown). Cytokine production was 6 to 10 times more abundant in CD4+ TEM than TCM cells (compare Fig. 4A and B). The percentages of TEM cells that produced IFN-γ only were equivalent in the controller and viremic groups, while HIV controllers were characterized by the presence of additional populations of TEM cells that produced IL-2 only or both IL-2 and IFN-γ. HIV-specific cytokine production was approximately three times lower in CD4+ TEM cells of HAART-treated patients than in those of controllers (Table (Table3).3). IL-2 secretion by TEM cells remained detectable in the HAART group, although the proportion of IL-2+ cells, including dual producers, was slightly reduced compared to that in the HIC group (Fig. (Fig.4B)4B) (37 versus 52%, respectively).

FIG. 4.
Cytokine responses in CD4+ T-cell memory subsets. The mean percentages of cells secreting IFN-γ only, IL-2 only, or both cytokines are reported. (A) HIV-specific response to p24 Gag stimulation in the CD4+-TCM-cell subset. (B) ...
TABLE 3.
Comparison of the percentages of cytokine-secreting CD4+ T cells following HIV-specific and polyclonal stimulationa

HIV-specific cytokine responses in the central memory compartment were markedly different among groups. CD4+ TCM cells of HIV controllers maintained the capacity to produce cytokines upon p24 Gag stimulation, with a predominant IL-2 secretion pattern (Fig. (Fig.4A).4A). In contrast, these responses were minimal in viremic and HAART-treated patients. Notably, the bulk of IL-2 secretion was carried out not by TCM cells but rather by TEM cells, the percentage of IL-2+ cells being four times higher in the TEM compartment (TEM-cell/TCM-cell ratio, 4.7 in the HIC group and 4.2 in the HAART group) (Table (Table3).3). IL-2 production by HIV-specific CD4+ TCM cells is not quantitatively important but may nevertheless contribute to HIV control due to the long half-life and self-renewing capacity of TCM cells. Thus, it was important to note that the TCM response appeared to be qualitatively different in the HIV controllers.

Skewed polyclonal responses in HIV controllers.

Polyclonal responses in CD4+ T-cell memory subsets were analyzed by measuring cytokine production upon stimulation with anti-CD3 and anti-CD28 antibodies. CD4+ TCM cells responded to polyclonal stimulation by cytokine production dominated by IL-2, while CD4+ TEM cells produced a mixed response, with both IL-2 and IFN-γ production (compare Fig. 4C and D). The intensities of polyclonal responses were of the same order of magnitude in TCM and TEM cells, indicating that TCM cells were not inherently limited in their cytokine secretion capacity.

The intensities of total polyclonal responses did not differ markedly among the HIC, HAART, and uninfected groups (Table (Table3).3). In particular, CD4+ T cells from HIV controllers did not appear to be endowed with intrinsically high IL-2 secretion capacity that could have accounted for the maintenance of HIV-specific TCM responses. Polyclonal responses were significantly decreased in the viremic group, in both the TEM and the TCM compartments (Table (Table3).3). These observations underscored a generalized defect of CD4+ T-cell-dependent immunity in viremic patients, as previously reported (54). Interestingly, the proportion of TEM cells producing IFN-γ only upon polyclonal stimulation was significantly higher in HIV controllers than in uninfected blood donors (P = 0.01) (Table (Table3).3). This finding suggested that global CD4+ T-cell responses were somehow altered in HIV controllers, even though these patients appeared to be healthy and suppressed HIV replication efficiently. A similar phenomenon was observed for viremic and HAART-treated patients, who showed an increased proportion of IFN-γ producers upon anti-CD3 and anti-CD28 stimulation compared to uninfected donors (P, <0.001 and <0.05, respectively). Thus, HIV infection primed the pool of CD4+ TEM cells for IFN-γ production in controllers as well as in other patient groups.

Activation of the effector memory compartment in HIV controllers.

The observation of priming for IFN-γ production suggested that the CD4+ T-cell compartment may not be in a resting state in HIV controllers. We therefore assessed the expression of activation markers at the surfaces of CD4+ T cells. Phenotyping studies were carried out for a subset of HIV controllers (patients A1 to A8; n = 8) and for patients from control groups (n = 8 for each group) (Table (Table2).2). As expected, the expression of HLA-DR was increased in viremic patients, in both the CD4+ TCM- and TEM-cell compartments (Fig. (Fig.5A).5A). HIV controllers showed an intermediate pattern, with an increase of HLA-DR expression in CD4+ TEM cells but no significant changes in CD4+ TCM cells. The increase of HLA-DR expression in TEM cells was heterogeneous, suggesting variable activation levels in the controller group. However, individual variability was also observed in the viremic group, and the median levels of HLA-DR expression were comparable between the two groups (12.3 versus 13.2% for HIV controllers and viremic patients, respectively). The percentage of CD25 expression was not increased in controllers or viremic patients (data not shown).

FIG. 5.
Expression of activation markers in CD4+ T-cell memory subsets. (A) Proportions of CD4+ TCM cells and CD4+ TEM cells expressing the HLA-DR marker. (B) MFIs of the early activation marker CD69 in CD69+ CD4+ TCM cells ...

The CD69 molecule is an early activation marker expressed at low levels in peripheral blood and is thought to play a role in the retention of recently activated lymphocytes in lymphoid organs (55). The percentage of CD69+ cells did not show significant changes between groups (median percentage of CD69+ cells among CD4+ TEM, 1.9% in HIV controllers, 4.3% in viremic patients, 2.4% in uninfected controls, and 1.5% in HAART-treated patients). However, it was interesting that the level of CD69 expression per positive CD4+ T cell, as measured by the MFI, was significantly higher in controllers than in viremic patients and uninfected controls (Fig. (Fig.5B).5B). This suggested a particular activation status of recently primed CD4+ T cells in the controller group.

The alpha chain of the IL-7 receptor (CD127) is lost upon antigen exposure and reexpressed in CD4+ memory T cells that survive in the long term (17). CD127 expression was uniformly high in CD4+ TCM cells of controllers, while it was decreased in those of viremic and HAART-treated patients (Fig. (Fig.5C).5C). Interestingly, CD127 showed a trend to decrease in CD4+ TEM cells of controllers as well as other HIV+ patients, suggesting a degree of ongoing immune activation in this compartment. Thus, the phenotyping analysis showed signs of a preferential activation of the TEM compartment in HIV controllers. Given that the level of T cell activation has been described as a surrogate marker for HIV disease progression (21), it was striking to find that, within the TEM compartment, levels of HLA-DR expression were as high in HIV controllers as in progressors.

HIV controller CD4+ T cells are primed for MIP-1β production.

The chemokine receptor CCR5 is considered to be an activation marker, since it is expressed at minimal levels in naive CD4+ T cells, at intermediate levels in CD4+ TCM cells, and at high levels in CD4+ TEM cells (means of 1.1, 9.5, and 48.8% CCR5+ cells, respectively, for uninfected donors). Thus, it was unexpected that CCR5 expression appeared to be decreased, rather than increased, in CD4+ TEM cells of controllers and other HIV-infected patients (Fig. (Fig.6A).6A). A similar decrease in CD4+ TCM cells was observed. The level of expression of CCR5 per cell was also decreased, as indicated by a lower CCR5 MFI in CD4+ TCM cells of the controller group than in those of the uninfected donors (P < 0.05; data not shown). A possibility was that chronic secretion of CCR5 ligands led to the down-regulation of the CCR5 receptor, a phenomenon classically associated with chemokine receptor desensitization (4).

FIG. 6.
Expression of the chemokine receptor CCR5 and production of the CCR5 ligand MIP-1β. (A) Proportions of CD4+ TCM cells and CD4+ TEM cells expressing the CCR5 chemokine receptor. Horizontal bars indicate median values. HIC, HIV controllers; ...

Further analyses were performed to evaluate the production of the chemokine MIP-1β, a high-affinity ligand for CCR5, in response to HIV-specific and polyclonal stimulation. Stimulation with p24 Gag induced MIP-1β production to comparable levels in the CD4+ TEM cells of controllers and viremic patients and to lower levels in the HAART group, again emphasizing the strength of HIV-specific responses in the controller group (Fig. (Fig.6B,6B, right panel). MIP-1β was produced predominantly by CD4+ TEM cells, the frequency of MIP-1β+ cells being about 10 times higher in the TEM than in the TCM subset. Thus, MIP-1β secretion represented an effector response, a notion supported by the fact that a significant fraction of CD4+ TEM cells that produced MIP-1β also produced IFN-γ.

Polyclonal stimulation with the superantigen SEB induced MIP-1β responses that were 20 times higher in the CD4+ TEM- than in the TCM-cell compartment (data not shown). Since the CD4+ TCM-cell contribution was minimal, total CD4+ T-cell responses are reported (Fig. (Fig.6C,6C, left panel). Of note, HIV controllers showed a twofold increase in polyclonal MIP-1β responses compared to uninfected donors. A threefold increase was observed for viremic patients. A large fraction of MIP-1β+ CD4+ T cells also produced IFN-γ, emphasizing the effector phenotype of the cells involved in the polyclonal response. We also evaluated MIP-1β responses in the CD8+ T-cell compartment, since CD8+ T cells are known to produce CCR5 ligands at high levels (57). Upon polyclonal stimulation, CD8+ T cells from all patient groups produced MIP-1β at higher levels than CD8+ T cells from uninfected donors (Fig. (Fig.6C,6C, right panel). Thus, HIV infection primed the total T-cell pool for increased MIP-1β production, a finding that may account for the down-regulation of CCR5.

It was striking that the intensities of MIP-1β responses, both HIV specific and polyclonal, were comparable between HIV controllers and viremic patients while these two groups differed markedly in terms of viral load. These findings suggested that, for certain parameters, the levels of functional T-cell activation were comparable in both groups. Taken together, the intensity of HIV-specific cytokine responses, the increased expression of HLA-DR, and the priming of the CD4+ T-cell pool for IFN-γ and MIP-1β production pointed to a potent activation of the CD4+ TEM-cell compartment in HIV controllers.

DISCUSSION

HIV controllers were characterized by efficient antiviral responses in both the central and the effector memory CD4+ T-cell compartments. CD4+ TCM cells were present at near normal numbers, maintained the capacity to produce IL-2 in response to HIV antigens, and expressed high levels of the survival and homing receptors IL-7 receptor and CCR7, respectively. CD4+ TEM cells were responsible for potent, polyfunctional HIV-specific responses, with half of the responding cells secreting IL-2. In addition, HIV controllers showed signs of ongoing activation within the whole CD4+ TEM-cell compartment, as indicated by increased expression of HLA-DR, decreased expression of CD127, and a bias in cytokine and chemokine responses towards polyclonal stimulation. These findings pointed to an involvement of the whole immune system in controlling HIV infection. Importantly, this set of characteristics was not shared by other patient groups. Viremic patients showed clearly defective CD4+ TCM-cell responses but had an activated CD4+ TEM compartment and showed HIV-specific cytokine responses of high levels but of limited functionality. CD4+ T cell responses in controllers also differed from those of patients successfully treated with antiretroviral therapy, especially in terms of the strength of HIV-specific responses. CD4+ TCM cells of HAART-treated patients showed signs of dysfunction, such as decreased expression of the survival receptor CD127. CD4+ TEM cells from the same patients showed some but not all signs of immune activation and produced small amounts of cytokines in response to HIV antigens. Thus, HIV controllers were unique in showing both a preserved CD4+ TCM-cell compartment and an activated CD4+ TEM-cell compartment. We propose that the combination of these two characteristics underlies the remarkable capacity of HIV controllers to suppress HIV replication in the long term.

It was noteworthy that levels of HIV-specific CD4+ T-cell responses were higher in HIV controllers than in HAART-treated patients. The strength of CD4 responses has been proposed to follow a bell-shaped curve, with a positive association between CD4 responses and viral load in the low-viral-load range (29, 59). HIV controllers were exposed to very low levels of HIV antigens, given their particularly low levels of viral DNA (median, 43 copies/106 PBMC) (Table (Table1)1) and the large amount of CD4+ T cells needed to isolate replication-competent virus from their PBMC (34). However, cytokine responses were three times higher in the controller than in the HAART group, pointing to a disparity between the strength of CD4 responses and viral load in the controller group. Specifically, it was the ratio of cytokine-producing cells to the amount of stimulating antigen that was high in this particular group of patients. It is possible that CD4+ T-cell responses appear comparatively high in controllers because they are actually decreased in HAART-treated patients. Studies of immune reconstitution indicate that patients who reach a low CD4+ T-cell nadir prior to treatment show poor recovery of HIV-specific CD4+ T-cell responses (59). The median CD4+ T-cell nadir in the HAART group was 200 cells/mm3 (Table (Table2),2), which is not unusually low but is still associated with a degree of long-term immune damage. On the other hand, HIV controllers may share genetic traits that promote the development of efficient CD4-specific responses or that help contain viral replication during acute infection so that CD4 responses develop optimally. Whether the potent CD4 responses observed in controllers directly contribute to viral control through cytotoxic mechanisms, as suggested for cytomegalovirus and attenuated SIV infections, warrants further investigation (12, 20, 61).

Central memory T cells are thought to ensure the long-term maintenance of antiviral responses due to their long half-life and self-renewal capacity (35, 39). CD4+ TCM cells of controllers showed characteristics associated with cell survival, particularly evident in their high-level expression of the alpha chain of the IL-7 receptor, CD127. Studies of the basis for immunological memory in murine models indicate that T cells that have encountered their cognate antigen and reexpress CD127 will preferentially survive the encounter, while CD127-negative cells will preferentially die by activation-induced apoptosis (39). According to a recent study, the expression of CD127 on antigen-exposed CD4+ T cells is dependent on the presence of IL-2 (17). Thus, the IL-2 secretion capacity of HIV-specific T cells in controllers may contribute to the high CD127 expression and long-term antiviral memory in these patients. The reduced expression of CD127 in viremic and HAART-treated patients suggests a shorter half-life of CD4+ TCM cells in these groups. Further studies aimed at directly measuring TCM-cell turnover will be needed to confirm this point. Of note, the differences in CD127 expression did not extend to the CD4+ TEM-cell population. The three groups of patients showed a trend towards low CD127 expression in CD4+ TEM cells, consistent with an activation of the effector memory compartment in all cases of HIV infection.

Another finding specific to the central memory compartment of HIV controllers was the increased expression of the homing receptor CCR7. This chemokine receptor binds ligands expressed at the surfaces of high endothelial venules (CCL19) and within lymphoid tissues (CCL19 and CCL21) and thus plays a central role in the recirculation of naive and TCM cells within lymphoid organs (44). CCR7 also drives the migration of TCM cells from peripheral tissues to draining lymph nodes via the lymphatics (16). A high level of expression of CCR7 may facilitate the entry and increase the residence time of CD4+ TCM cells within lymphoid organs. An increase in CD69 expression may also prolong their retention within lymphoid organs (55). Such a mechanism may favor the encounter with rare antigen-presenting cells loaded with HIV antigens and contribute to the efficiency of antiviral responses in HIV controllers. It was interesting that CCR7 expression levels in controller CD4+ T cells exceeded those of CD4+ T cells obtained from uninfected blood donors. As CCR7 expression is known to be regulated by the cytokine milieu (49, 53), it is possible that the cytokine expression profile particular to HIV controllers influences CCR7 expression and, hence, the recirculation pattern of CD4+ T cells. Taken together, CD4+ TCM cells of HIV controllers exhibited a set of characteristics that were compatible with a fully functional and long-lived central memory response.

The presence of signs of immune activation in the whole CD4+ TEM-cell population of HIV controllers was an unexpected finding. In the current model of HIV pathogenesis, abnormal immune activation is viewed as the major force driving progression to disease, by constantly replenishing the pool of activated CD4+ T cells that can be targeted by HIV and by progressively exhausting the renewal capacity of the immune system (23, 27). The degree of CD8+ T-cell activation correlates positively with the risk of progression to AIDS (21). It was intriguing that HIV controllers, who do not progress to disease, expressed levels of the HLA-DR activation marker similar to those expressed by viremic patients within the CD4+ TEM-cell compartment. The increase in HLA-DR expression was minimal within the CD4+ TCM-cell compartment for controllers but significant for viremic patients, suggesting that immune activation was confined to TEM cells in controllers and was generalized in viremic patients. Another element pointing to the activation of the TEM compartment in controllers was the increased production of the chemokine MIP-1β upon polyclonal stimulation, compared to responses from uninfected blood donors. The propensity of CD4+ TEM cells of controllers to produce IFN-γ rather than IL-2 after CD3/CD28 stimulation supports the notion of a bias towards effector responses. Thus, the CD4+ TEM-cell compartment in controllers was not resting and produced chemokines and cytokines to levels similar to those seen in viremic patients. These signs of potent immune activation may either reflect the bystander effects of the ongoing HIV-specific immune response or be intrinsic to HIV controllers. Determining the capacity of HIV controllers to cope with other chronic viral infections may help distinguish between these possibilities.

We cannot rule out that efficient activation of CD4 responses is a consequence, rather than an initial cause, of HIV control and that viral replication was contained early on due to other intrinsic or immune factors. However, once established, the effector CD4 response is likely to reinforce HIV control at several levels, through the secretion of chemokines that inhibit HIV replication, helper function, and possibly direct cytotoxic function. The finding of reduced CCR5 expression at the surfaces of CD4+ T cells suggests that HIV controllers are indeed exposed chronically to high levels of beta-chemokines such as MIP-1β. Alternate explanations such as genetic polymorphisms that would reduce basal CCR5 expression levels are also possible but less likely, since the CCR5 coding sequence was of the wild type in all the patients studied. Further analyses focused on CCR5 promoter polymorphisms and CCR5 ligand gene copy numbers are needed to evaluate the genetic component of the CCR5 expression level in controllers (22, 26). Preferential infection and deletion of CCR5+ cells may also contribute to decrease CCR5 expression (40) but is unlikely in the case of controller patients who by definition show very limited HIV replication. High levels of beta-chemokines can lead to chronic down-regulation of CCR5 through activation-induced receptor internalization (4, 45). Since CCR5 expression levels condition the infectibility of HIV target cells, CCR5 down-regulation may contribute to the low levels of infection in controllers. In particular, this mechanism may play a role in protecting cells that express already limiting amounts of CCR5, such as CD4+ TCM cells. In controllers, the combination of decreased CCR5 expression and low cellular activation would make CD4+ TCM cells suboptimal targets for HIV replication.

Of note, high-level MIP-1β production was also detected in the CD8+ T-cell population (Fig. (Fig.6C).6C). An analysis of CD8+ T-cell responses in the same cohort of controllers confirmed that the activation of the effector compartment, as measured by HLA-DR expression, extends to CD8+ T cells (51). An assessment of soluble lipopolysaccharide in plasma samples from this cohort showed raised levels in HIV controllers, another indication of chronic immune activation (9). These observations suggest that the activation of the effector compartment involves CD8+ T cells and possibly other immune cells. Importantly, the immune activation evident in HIV controllers was not equivalent in every aspect to that seen in viremic patients. T cells from controllers showed lower susceptibility to apoptosis in vitro than those from viremic patients (S. Perez-Patrigeon, unpublished observation). Levels of soluble CD14 in plasma, another indicator of inflammation, were intermediate in controllers and significantly higher in progressors (9). Conversely, CD69 was expressed to higher levels in recently activated CD4+ T cells of controllers than in those of viremic patients. A marked difference was noted in the activation status of HIV-specific CD8+ T cells, which expressed HLA-DR but not CD38 in controllers, while both activation markers were expressed in viremic patients (51). These findings point to differential patterns of immune activation in HIV controllers and viremic patients. Chronic immune activation may not be detrimental in controllers because it spares the central memory compartment and thus preserves the regenerative capacity of the immune system. Activation would then benefit controller patients by ensuring the efficacy of effector responses. In contrast, as shown in a recent study of SIV infection (46), chronic activation in the presence of viremia leads to a progressive loss of CD4+ TCM cells, with ensuing immune exhaustion.

In conclusion, HIV controllers were characterized by the maintenance of a functional and long-lived pool of CD4+ TCM cells, in parallel with a chronically activated pool of CD4+ TEM cells. The conjunction of these two types of immune memory may generate optimal responses against chronic HIV infection and represent a goal to be achieved by candidate HIV vaccines.

Acknowledgments

We thank Marie-Thérèse Rannou and the participating nurses from Bicêtre Hospital for their cooperation, Laurence Meyer for advice on the SEROCO-HEMOCO cohort, Ioannis Theodorou for CCR5 genotyping, Christine Rouzioux for contributing viral DNA data, and Florence Bugault for help with flow cytometry analysis. We are especially grateful to the patients who participated to the study.

This work was supported by the French National Agency for Research on AIDS and Viral Hepatitis (ANRS). S.J.P. is a recipient of a C. J. Martin postdoctoral fellowship from the Australian National Health and Medical Research Council. C.L. was supported by ANRS and Sidaction.

We declare no competing financial interests.

Footnotes

[down-pointing small open triangle]Published ahead of print on 17 October 2007.

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