Functional Heterogeneity of Memory CD4 T Cell Responses in Different Conditions of Antigen Exposure and Persistence

http://www.100md.com 免疫学杂志 2005年第2期

     Abstract

    Memory CD4 T cell responses are functionally and phenotypically heterogeneous. In the present study, memory CD4 T cell responses were analyzed in different models of Ag-specific immune responses differing on Ag exposure and/or persistence. Ag-specific CD4 T cell responses for tetanus toxoid, HSV, EBV, CMV, and HIV-1 were compared. Three distinct patterns of T cell response were observed. A dominant single IL-2 CD4 T cell response was associated with the model in which the Ag can be cleared. Polyfunctional (single IL-2 plus IL-2/IFN- plus single IFN-) CD4 T cell responses were associated with Ag persistence and low Ag levels. A dominant single IFN- CD4 T cell response was associated with the model of Ag persistence and high Ag levels. The results obtained supported the hypothesis that the different patterns observed were substantially influenced by different conditions of Ag exposure and persistence.

    Introduction

    During the past few years, several studies performed in both mice and humans have revealed the complexity of memory T cell responses (reviewed in Refs. 1, 2, 3, 4, 5, 6, 7, 8). The use of panels of surface markers has been instrumental in the delineation of the different subsets of memory CD4 and CD8 T lymphocytes (9, 10, 11, 12, 13, 14, 15, 16). In particular, the observation by Sallusto et al. (9) regarding the identification of two phenotypic and functionally distinct subsets of memory T cells on the basis of the expression of CCR7, e.g., CCR7+ cells (central memory, TCM)3 and CCR7– cells (effector memory, TEM), has changed substantially the way we view memory T cells. Based on Sallusto’s hypothesis (9), TEM cells reside predominantly in the periphery (blood, spleen, and nonlymphoid tissue) and are ready to intervene rapidly after Ag re-encounter by mediating effector function, while TCM reside predominantly within the secondary lymphoid organs. These latter do not mediate effector function, but may serve as precursors for TEM after Ag stimulation. However, recent studies have demonstrated that CD8 TCM also mediate effector function and are more effective in conferring protective immunity (17, 18). Along this line, studies in humans have also shown that both TCM and TEM CD8 T cells mediated effector function (16, 19). Furthermore, it has been shown that both CD4 and CD8 T cells with TEM phenotype and function accumulate within the target organ of the pathogen in mice and humans (19, 20, 21, 22).

    Studies on CD8 T cells performed in mice and humans have provided coherent models of differentiation of memory T cells (3, 4, 5, 9, 16, 17, 19, 23). These observations have provided clear evidence that following Ag encounter naive T cells generate a population of effector cells. Recent studies (24) have shown that a small proportion of effector cells expressing the IL-7R serves as precursors for memory CD8 T cells after Ag clearance. Furthermore, it has also been shown that TCM and TEM are not distinct subsets, but rather correspond to different differentiation steps of a linear pathway (17).

    Several studies have demonstrated wide heterogeneity of memory CD8 and CD4 T cells in mice and humans. Multiple phenotypes and a broad spectrum of functions have been observed in different viral infections (13, 14, 15, 16, 17). In particular, substantial differences in virus-specific CD8 and CD4 immune responses have been observed in EBV, CMV, and HIV-1 infections (13, 16, 19, 22, 25, 26). These studies have proposed that the differences in HIV-1-specific CD8 T cell responses may be the result of a skewed maturation of memory cells (16, 19), of replication senescence (25), and of the type of pathogen/virus that would specifically influence the development of distinct memory T cell populations (13). Similarly, a skewed representation of different populations of Ag-specific CD4 T cells has been demonstrated in HIV-1 infection with a selective reduction in the proportion of helper (IL-2-secreting and -proliferating) CD4 T cells (22, 27, 28) that may be due to a preferential infection with and elimination by HIV-1 (29). Furthermore, two recent studies (22, 30) indicated that high Ag load in HIV-1 infection may prevent the generation of virus-specific IL-2-secreting cells.

    The reasons, however, for the heterogeneity of the immune response against different Ag/pathogens remain unclear, and a series of factors, including Ag structure, localization, dose, Ag availability, time to Ag exposure, and diversity of APCs, have been suggested to regulate potentially the immune response (31, 32).

    In the present study, we have performed a comprehensive analysis of Ag-specific CD4 T cell responses in humans. What differentiated the models we have used was the exposure to and/or the persistence of Ag. In particular, we have used: 1) the tetanus toxoid (TT) as a model of memory T cell response in which the Ag is cleared; 2) the HSV, EBV, CMV chronic infections, and HIV-1 infection in subjects with nonprogressive disease, e.g., long-term nonprogressors (LTNP), as models of Ag persistence and protracted Ag exposure under conditions of controlled virus replication with low/undetectable levels of viral Ag (it is largely accepted that CMV undergoes transient reactivations (33, 34, 35), and that low levels of Ag expression are associated with HSV and EBV (36)); 3) chronic and progressive HIV-1 infection as a model of Ag persistence under conditions of noncontrolled virus replication and high levels of viral Ag; and 4) primary CMV and HIV-1 infections as models of acute Ag exposure and high levels of viral Ag. The changes of the memory CD4 T cell responses associated with the variation in Ag exposure and persistence within the four models were also investigated. Our results provide a complete characterization of memory CD4 T cell responses against some of the most diffuse human viruses. Furthermore, the investigation of the Ag-specific CD4 T cell responses in different models of Ag exposure and persistence indicated that the functional heterogeneity of memory CD4 T cells is strongly modulated by the Ag exposure and persistence.

    Materials and Methods

    Study groups

    The following study groups were included in the present study: 1) blood from 52 HIV-negative subjects was obtained either from the local blood bank (Lausanne, Switzerland) or from laboratory coworkers; 2) 11 subjects with progressive chronic HIV-1 infection, naive to antiviral therapy, with CD4 T cell count 250 cells/μl, and plasma viremia 5000 HIV-1 RNA copies/ml; 3) 8 HIV-1-infected patients who were treated for 1 year (37) (therapy containing two nucleoside reverse transcriptase inhibitors plus one or two protease inhibitors) and had CD4 T cell counts >500 cells/μl and plasma viremia <50 HIV-1 RNA copies/ml; 4) 6 HIV-1-infected patients with nonprogressive disease, i.e., LTNP, as defined by documented HIV-1 infection since >14 years, stable CD4 T cell counts >500 cells/μl, and plasma viremia <1000 HIV-1 RNA copies/ml; 5) 10 patients with primary HIV-1 infection; primary HIV-1 infection was diagnosed on the basis of the presence of an acute clinical syndrome, a negative HIV-1 Ab test, a positive test for HIV-1 RNA in plasma, and a presence of less than three positive bands in a Western blot; 6) 2 subjects with chronic HIV-1 infection treated with antiviral therapy for 2 years who underwent treatment interruption; and 7) 4 HIV-1-infected patients with primary CMV infection who have been previously described (38). These studies were approved by the local Institutional Review Board.

    Determination of HIV-1 RNA and CMV DNA in plasma

    HIV-1 RNA in plasma was measured using the Amplicor 1.5 HIV-1 assay. CMV viremia was measured using a modified version of the Amplicor CMV Monitor test (Roche Diagnostic Systems) with a limit of detection of 10 DNA copies/ml of plasma (39).

    Determination of EBV DNA in blood mononuclear cells

    DNA from 200 μl of EDTA-treated whole blood was purified with the whole blood DNA purification kit (Roche; catalogue no. 3 003 990) on a Magna Pure LC Instrument (Roche), and eluted in 100 μl of elution buffer. A total of 5 μl of DNA (equivalent to 10 μl of whole blood) was then analyzed in duplicate by quantitative real-time PCR on an ABI 7900 instrument (Applied Biosystems) in 25 μl reactions containing 12.5 μl of Taqman Universal Master mix (Applied Biosystems), primers, and probe at the indicated concentrations, and PCR grade water to 25 μl of negative controls was always included in the extraction procedure (at least 4 per 32 samples) and in the PCR assays. False positives were never observed. Primers and probes were either from Applied Biosystems or from MWG Biotech. Amplification was performed with the following profile: 50°C for 2 min, followed by 95°C for 9 min and 45 cycles of 95°C for 15 s and 60°C for 1 min. Fluorescence was recorded at 60°C. EBV DNA load was assessed using the 5 primers (200 nM) and FAM-labeled probe (100 nM) published by Kimura et al. (40). To normalize the EBV load and assess the presence of inhibitors in the DNA preparation, human DNA load was also determined in separate duplicate reactions containing a human -actin detection system. Results were expressed as EBV copy number per 160,000 cells. Standard curves for EBV were generated with serial 10-fold dilutions in PCR grade water of DNA from Namalwa cells known to contain two copies of integrated EBV genome per cell. Those for human DNA were generated with human genomic DNA (Roche; catalogue no. 1 691 112). Copy number of human DNA and hence of EBV DNA in Namalwa cells was calculated by considering that 1 μg of human DNA corresponds to 160,000 cells, according to Kimura et al. (40). Concentration of Namalwa DNA was assessed by spectrophotometry and confirmed by gel electrophoresis and ethidium bromide staining against known amounts of DNA standards.

    FACS analysis

    Cryopreserved cells stored in liquid nitrogen were thawed and used for flow cytometry (16, 19, 22, 38). The following Abs were used in combination: rat anti-human CCR7 (BD Biosciences), followed by goat anti-rat IgG (H + L), -FITC, or -allophycocyanin (Caltag Laboratories), CD4-PerCP Cy5.5, -Pacific Blue or purified (BD Biosciences) labeled with Zenon Pacific Blue mouse IgG1 labeling kit (Molecular Probes), CD45RA biotin, followed by anti-streptavidin-PerCP, anti-CD69 FITC or -allophycocyanin Cy7, anti-IL-2 PE, and anti-IFN- FITC or -allophycocyanin (BD Biosciences). Data were acquired on a FACSCalibur or on an LSR II cytometer and analyzed using CellQuest and DiVa software (BD Biosciences).

    Intracellular cytokine staining (ICS)

    Intracellular IFN- and IL-2 production was assessed, as previously described (22). Blood mononuclear cells (2–4 x 106 cells in 1 ml of RPMI 1640 Gutamax-1 medium containing 10% inactivated FCS) were stimulated with 5 μg/ml HIV-1-p55 gag (Protein Sciences); 1 μg/ml CMV, EBV, or HSV lysates (Applied Biosystems); 100 μg/ml TT (Aventis Pasteur); or 200 ng/ml staphylococcal enterotoxin B (Calbiochem; positive control) for 16 h at 37°C, in the presence of 0.5 μg/ml purified anti-CD28 Ab (BD Biosciences) and 1 μg/ml GolgiPlug (BD Biosciences). Cell surface staining was completed, as described, following the in vitro activation (22). Cells were then permeabilized, fixed with FACS permeabilizing solution (BD Biosciences), and labeled with anti-human IFN- and IL-2 (BD Pharmingen). Simultaneously, activation was assessed by staining with anti-CD69 (BD Biosciences). The number of lymphocyte-gated events ranged between 150,000 and 600,000 in the flow cytometry experiments shown. With regard to the criteria of positivity of ICS, the background in the unstimulated controls never exceeded 0.01–0.02%. An ICS to be considered positive had to have a background <20% of the total percentage of cytokine-positive cells in the stimulated samples.

    Statistical analysis

    Statistical significance (p values) of the results was calculated by two-tailed t test. A two-tailed p < 0.05 was considered significant.

    Results

    Functional analysis of memory CD4 T cells

    Studies performed in mice and humans have demonstrated a large functional heterogeneity of memory CD4 T cells (2, 3, 9, 15, 22, 41, 42, 43, 44, 45, 46). The factors responsible for this heterogeneity are not completely defined. To address this issue, we investigated the function of memory CD4 T cells in different in vivo conditions of Ag exposure and persistence. The four conditions included memory CD4 T cell responses under conditions of: 1) Ag clearance, 2) protracted Ag exposure with low/undetectable levels of Ag, 3) Ag persistence with high Ag levels, and 4) acute Ag exposure and high Ag levels. Immune response against TT was used as a model of memory CD4 T cell response under conditions of Ag clearance; chronic HSV, EBV, CMV infections; and chronic HIV-1 infection in LTNP as models of Ag persistence under conditions of controlled virus replication (low/undetectable viremia), chronic HIV-1 infection as a model of Ag persistence and high Ag levels, and primary CMV and HIV-1 infections as models of acute Ag exposure and high Ag levels. The function of blood memory CD4 T cells was evaluated on the basis of their ability to secrete cytokines such as IL-2 and IFN- following Ag-specific stimulation. In this regard, we have recently (22) shown that three functionally distinct populations of memory CD4 T cells can be identified on the basis of their ability to secrete IL-2 and IFN-: 1) single IL-2-secreting cells, 2) IL-2/IFN--secreting cells, and 3) single IFN--secreting cells. The results on the TT-, CMV-, EBV-, and HSV-specific CD4 T cell response shown in the present study were performed in blood mononuclear cells from healthy donors. With regard to the evaluation of virus, i.e., CMV, EBV, or HSV levels, it is well known that CMV viremia (plasma or cell associated) is not detectable in healthy donors and is measurable only during primary CMV infection or in immunocompromised patients with CMV-associated disease (47). EBV viremia, e.g., cell-associated DNA, was evaluated in 8 of 18 healthy donors investigated, and very low viremia levels were only found in 3 of 8 subjects (18, 135, and 176 EBV DNA copies per 160,000 blood mononuclear cells in subjects 272, 275, and 259, respectively). Finally, HSV DNA is generally measured in the case of early diagnosis and treatment of neonatal HSV infection, but is not measurable in the blood compartment in healthy human adults (48). The levels of viremia of the patients with chronic progressive and nonprogressive HIV-1 infection are shown in Table I. The mean ± SE of HIV-1 RNA copies/ml plasma was 60,057 ± 26,148 in patients with progressive disease and 177 ± 93 in LTNP.

    Table I. Immunological and virological characteristics of the chronically HIV-1-infected patients

    Data obtained from the investigation of TT-specific CD4 T cell responses in 30 healthy donors and of CMV-, EBV-, and HSV-specific responses in 52, 18, and 16 healthy donors, respectively, are shown in Fig. 1. In addition, data obtained from the investigation of HIV-1-specific CD4 T cell responses in nine progressors and six LTNP are also shown (Fig. 1). Blood mononuclear cells were stimulated with the specific Ag for 16 h and, at the end of the stimulation period, stained with CD4, CD69, IL-2, and IFN- Abs. Representative examples are shown in Fig. 1. The majority (80–90%) of TT-specific CD4 T cells secreted IL-2, while IL-2/IFN-- and IFN--secreting cells were almost absent (Fig. 1A). The population of IL-2-secreting CD4 T cells was the most represented population within EBV- and HSV-specific CD4 T cells (Fig. 1A), although also the IL-2/IFN- and IFN- cell populations were present. Consistently with previous studies (22), a more homogeneous representation of the proportions of IL-2-, IL-2/IFN--, and IFN--secreting CD4 T cells within CMV-specific CD4 T cells was observed (Fig. 1A). Interestingly, the pattern of HIV-1-specific CD4 T cell response in LTNP was very similar to that of CMV (Fig. 1A). Cumulative data for TT-, EBV-, HSV-, and CMV-specific CD4 T cell responses obtained from the analysis of a large number of healthy donors and of the HIV-1-specific CD4 T cell response from six LTNP are shown in Fig. 1B. Furthermore, consistently with our previous observations, the large majority (>90%) of HIV-1-specific CD4 T cells in progressors secreted IFN- and not IL-2 after stimulation with p55 gag (Fig. 1C). This comprehensive characterization allowed us to perform a detailed evaluation of the representation of the three functionally distinct populations of memory CD4 T cells within the different models of immune response.

    FIGURE 1. Analysis of the distribution of Ag-specific IL-2-, IFN-/IL-2-, and IFN--secreting memory CD4 T cell populations in five representative subjects. A, Blood mononuclear cells were stimulated either with TT, EBV, HSV, CMV, or HIV-1, and stained with anti-CD4 PerCP Cy5.5, anti-CD69 FITC, anti-IL-2 PE, and anti-IFN- allophycocyanin, and analyzed by flow cytometry. HIV-1-specific CD4 T cell responses were evaluated in LTNP. B, Mean ± SE of the cumulative data on the proportion of TT-, EBV-, HSV-, CMV-, and HIV-1 (in LTNP)-specific CD4 T cells within the different cytokine-secreting cell populations. C, Analysis of the distribution of HIV-1-specific IL-2-, IFN-/IL-2-, and IFN--secreting memory CD4 T cell populations in one representative HIV-1-infected patient with progressive disease, i.e., progressor, and cumulative data on the proportion of HIV-1-specific CD4 T cells within the different cytokine-secreting cell populations. The cluster of events shown in red corresponds to the responder CD4 T cells, i.e., expressing IL-2 and/or IFN-, while the cluster of events in blue corresponds to the nonresponder CD4 T cells. At least 1 x 106 events were analyzed.

    The relative proportion of TT-specific IL-2-secreting CD4 T cells was significantly higher (p < 0.05) compared with that of EBV-, HSV-, CMV-, and HIV-1-specific (both LTNP and progressors) CD4 T cells (Fig. 2A). The proportion of EBV- and HSV-specific IL-2-secreting cells was significantly higher compared with that of CMV- and HIV-1-specific (both LTNP and progressors) CD4 T cells (Fig. 2A). The proportion of CMV- and HIV-1 (in LTNP)-specific CD4 T cells was significantly higher compared with that of HIV-1 (in progressors). No differences were observed between HSV- and EBV-specific and between CMV- and HIV-1 (in LTNP)-specific IL-2-secreting cells (Fig. 2A).

    FIGURE 2. Comparative analysis of the average proportions of TT-, EBV-, HSV-, CMV-, and HIV-1 (both LTNP and progressors)-specific IL-2-, IFN-/IL-2-, and IFN--secreting cells in different populations of blood memory CD4 T cells. A, Mean ± SE of the proportion of IL-2-secreting CD4 T cells. B, Mean ± SE of the proportion of IFN--secreting CD4 T cells. C, Mean ± SE of the proportion of IFN-/IL-2-secreting CD4 T cells.

    The relative proportion of HIV-1-specific IFN--secreting CD4 T cells in progressors was significantly higher (p < 0.05) compared with all the other Ag-specific responses studied (Fig. 2B). The proportion of CMV-specific and HIV-1-specific (in LTNP) IFN--secreting cells was very similar, but higher (p < 0.05) compared with the others. No differences were observed in the relative proportion between HSV- and EBV-specific IFN--secreting cells, but these latter were higher compared with that of TT-specific CD4 T cells (Fig. 2B).

    Finally, the relative proportion of HIV-1 specific in LTNP and of CMV-, HSV-, and EBV-specific IL-2/IFN--secreting CD4 T cells was very similar (p > 0.05) and significantly higher (p < 0.05) compared with that of HIV-1 specific in progressors and TT-specific CD4 T cells (Fig. 2C).

    These results demonstrate the existence of great heterogeneity in the function and in the representation of distinct cytokine-secreting CD4 T cell populations within the different models of immune responses investigated.

    CD4 T cell responses under conditions of acute Ag exposure

    We then investigated CD4 T cell responses in patients with primary HIV-1 and primary CMV infection. The levels of viremia at the time of the diagnosis of primary HIV-1 infection are shown in Table II, while the criteria for the diagnosis of primary infection have been described in Materials and Methods. The flow cytometry profiles of HIV-1-specific CD4 T cell responses of one representative patient, i.e., patient 1004, and the cumulative data of nine patients are shown in Fig. 3A. The large majority (80%) of HIV-1-specific CD4 T cells were single IFN--secreting cells, while IL-2/IFN--secreting cells and single IL-2-secreting cells were poorly represented (Fig. 3A). Similar functional results were obtained in the nine patients studied (Fig. 3A). We also had the opportunity to study the CMV-specific CD4 T cell response in four patients who experienced primary CMV and HIV-1 coinfection. The levels of CMV and HIV-1 viremia are shown in Table II, and these four patients were previously described (38). The flow cytometry profiles of CMV-specific CD4 T cell responses of one representative patient, i.e., patient 4, and the cumulative data of the four patients studied are shown in Fig. 3B. Similarly to primary HIV-1 infection, the large majority (90%) of CMV-specific CD4 T cells were single IFN--secreting cells (Fig. 3B). We have also studied one HIV-negative subject with primary CMV infection, and also in this case the dominant CD4 T cell response was composed of single IFN--secreting cells (data not shown).

    Table II. Immunological and virological characteristics of the patients with primary HIV-1 and primary HIV-1 and CMV coinfection

    FIGURE 3. Analysis of the patterns of HIV-1- and CMV-specific CD4 T cell responses in conditions of acute Ag exposure and high Ag levels, i.e., during primary infection. Flow cytometry profiles of HIV-1-specific (A) and CMV-specific (B) CD4 T cells within IL-2-, IFN-/IL-2-, and IFN--secreting cell populations in two representative subjects with primary HIV-1 and CMV infection. Mean ± SE of the cumulative data of nine subjects with primary HIV-1 infection and four subjects with primary CMV infection are also shown. The cluster of events shown in red corresponds to the responder CD4 T cells, i.e., expressing IL-2 and/or IFN-, while the cluster of events in blue corresponds to the nonresponder CD4 T cells. At least 1 x 106 events were analyzed.

    In vivo manipulation of Ag levels and exposure

    The functional analysis (Fig. 1) indicated that single IL-2-secreting cells were dominant within the model of Ag clearance, e.g., the TT-specific CD4 T cell response. The polyfunctional (IL-2 plus IL-2/IFN- plus IFN-) CD4 T cell response was dominant within the model of protracted Ag persistence under conditions of controlled virus replication, e.g., EBV-, HSV-, CMV-, and HIV-1 (in LTNP)-specific CD4 T cell responses. Finally, single IFN--secreting cells were dominant within the model of Ag persistence and noncontrolled virus replication and acute Ag exposure, e.g., the HIV-1-specific CD4 T cell response in progressors and CMV- and HIV-1-specific CD4 T cell responses during primary infection. We therefore decided to determine the functional changes in CD4 T cell responses following manipulation in the levels of Ag. The conditions investigated included: 1) Ag re-exposure, e.g., TT reimmunization; 2) decrease in the levels of Ag exposure, e.g., virus suppression by antiviral therapy (ART); and 3) rapid increase in Ag exposure, e.g., ART interruption. The TT-specific model of CD4 T cell response represented the ideal in vivo model to address the effects of Ag re-exposure on the function of memory CD4 T cells because TT-specific CD4 T cells were almost all single IL-2-secreting cells. We selected four subjects (one representative is shown in Fig. 4) with exclusively TT-specific single IL-2-secreting cells. These subjects were reimmunized, and the kinetics of TT-specific CD4 immune responses was assessed in blood mononuclearcells at different time points by the determination of IL-2- and IFN--secreting cells. TT-specific CD4 T cells were exclusively composed of single IL-2-secreting cells in subject GP at baseline, i.e., before reimmunization (Fig. 4). After reimmunization, we observed a substantial increase (5-fold) in the percentage of TT-specific single IL-2-secreting CD4 T cells with a peak at day 11 (Fig. 4). Of interest, we observed the appearance of IL-2/IFN-- and single IFN--secreting cells, and the kinetics of these functionally distinct cell populations was very similar to that of single IL-2-secreting cells (Fig. 4). The three distinct cytokine-secreting cell populations decreased over time, and by day 60 after reimmunization the IL-2/IFN-- and the single IFN--secreting cells had almost disappeared (Fig. 4). A substantial percentage of single IL-2-secreting cells was, however, still present at day 60 (Fig. 4).

    FIGURE 4. Kinetics of TT-specific memory CD4 T cell responses after in vivo reimmunization. Flow cytometry profiles of the distribution of blood TT-specific CD4 T cells within IL-2-, IFN-/IL-2-, and IFN--secreting cell populations at baseline (before reimmunization) and at day 11 (peak of the response) and day 60 postimmunization in one representative subject. The cluster of events shown in red corresponds to the responder CD4 T cells, i.e., expressing IL-2 and/or IFN-, while the cluster of events in blue corresponds to the nonresponder CD4 T cells. At least 1 x 106 events were analyzed.

    The suppression of virus replication by ART was instrumental to determine the effects of substantial reduction in Ag exposure and levels on HIV-1-specific CD4 T cell responses in subjects with chronic infection. Consistently with a previous publication of our group (22), we observed after 12 mo of successful ART a change from the single IFN- response, e.g., a CD4 T cell response typical of the models of high levels of Ag persistence and acute Ag exposure, to the polyfunctional CD4 T cell response, e.g., typical of protracted and low levels of Ag exposure (Fig. 5A). Cumulative data obtained from eight patients treated with ART are also shown (Fig. 5A). Finally, we also investigated the effects of the rapid increase in Ag exposure levels. For these purposes, we monitored the changes in HIV-1-specific CD4 T cell response after treatment interruption in two patients successfully treated for 2 years with ART. At baseline, e.g., 2 years after ART, the HIV-1-specific CD4 T cell response, e.g., polyfunctional, observed also in these two patients was consistent with the data shown in Fig. 5A, and thus with the conditions of the models of virus persistence and controlled replication/low Ag exposure levels (Fig. 5B). Following treatment interruption (3 wk after interruption), there was a major increase of IFN--secreting cells associated with virus rebound and high levels of Ag exposure. A 15-fold increase in the percentage of single IFN--secreting CD4 T cells was observed in patient 2115, while the other types of cytokine-secreting populations remained unchanged (Fig. 5B). Similarly, a 23-fold increase in the percentage of single IFN--secreting CD4 T cells was observed in patient 2076 (Fig. 5B). However, there was also a slight increase in the single IL-2 (2-fold)- and in the dual IL-2/IFN--secreting cells (8-fold) (Fig. 5B). Therefore, rapid exposure to the high Ag levels resulted in a skewing toward a dominant single IFN--secreting CD4 T cell response.

    FIGURE 5. In vivo manipulation of the Ag levels and exposure. A, Flow cytometry profiles of HIV-1-specific CD4 T cells within IL-2-, IFN-/IL-2-, and IFN--secreting cell populations in a representative HIV-1-infected patient before and after treatment with ART. Mean ± SE of the cumulative data of eight treated subjects are also shown. B, Flow cytometry profiles of HIV-1-specific single IL-2-, IFN-/IL-2-, and IFN--secreting CD4 T cells in two patients treated successfully for 2 years with ART and after treatment interruption. The cluster of events shown in red corresponds to the responder CD4 T cells, i.e., expressing IL-2 and/or IFN-, while the cluster of events in blue corresponds to the nonresponder CD4 T cells. At least 1 x 106 events were analyzed.

    Relationships between function and phenotype of distinct populations of memory CD4 T cells

    On the basis of previous observations (9, 22), it is clear that CCR7–CD4 T cells are at a more advanced stage of differentiation compared with CCR7+ cells. Furthermore, we have recently identified a third population, e.g., CD45RA+CCR7–, of memory CD4 T cells that appears to be at a more advanced stage of differentiation compared with the CD45RA–CCR7+ andCD45RA–CCR7– cell populations (49). It was then important to provide insights on the phenotype of the three distinct cytokine-secreting populations. We investigated the phenotype in the CMV and chronic progressive HIV-1 infection models of memory CD4 T cell responses (Fig. 6). Following stimulation with CMV lysates or HIV-1 p55, blood mononuclear cells were stained with CD4, CD45RA, CCR7, CD69, IL-2, and IFN- Abs. The expression of surface markers such as CCR7 and CD45RA is known to provide insights in the stage of differentiation of memory T cells (9). With regard to CMV-specific CD4 T cells, single IL-2-secreting cells were mostly contained within the CD45RA–CCR7+ cell population (Fig. 6A). Single IFN--secreting cells were mostly contained within CCR7–CD45RA– and CD45RA+CCR7–CD4 T cell populations and IL-2/IFN--secreting cells within the CD45RA–CCR7– cell population (Fig. 6). These results indicated that single IL-2-secreting cells are at early, while single IFN--secreting cells at advanced stage of differentiation. Because IL-2/IFN--secreting cells were mostly contained within the CD45RA–CCR7– cell population, it is likely that these memory CD4 T cells are at intermediate stage of differentiation. The large majority (>90%) of HIV-1-specific CD4 T cells in progressors (patient 1009 is shown) were single IFN--secreting cells, and they were exclusively contained within the CD45RA–CCR7– cell population (Fig. 6B).

    FIGURE 6. Distribution of HIV-1-specific (progressors) and CMV-specific IL-2-, IFN-/IL-2-, and IFN--secreting cells in different populations of blood memory CD4 T cells defined by the expression of CD45RA and CCR7 Ags. A, Flow cytometry profiles of a representative example of the distribution of CMV-specific IL-2-, IFN-/IL-2-, and IFN--secreting CD4 T cells gated on CD45RA–CCR7+, CD45RA–CCR7–, and CD45RA+CCR7– cell populations. B, Flow cytometry profiles of a representative example of the distribution of HIV-1-specific IL-2-, IFN-/IL-2-, and IFN--secreting CD4 T cells gated on CD45RA–CCR7+, CD45RA–CCR7–, and CD45RA+CCR7– cell populations. At least 2 x 106 events were analyzed.

    Influence of HIV-1 infection on non-HIV-1-specific CD4 T cell responses

    Ag-specific CD4 T cell responses for TT, EBV, HSV, and CMV were also investigated in 20 subjects with chronic HIV-1 infection at early stage disease, e.g., CD4 T cell counts >500/μl. No significant quantitative and qualitative differences in the three functionally distinct cell populations were observed in TT-, EBV-, HSV-, and CMV-specific CD4 T cells (data not shown) between HIV-1-infected and HIV-1-negative subjects. However, single IFN--secreting CMV-specific CD4 T cells were significantly increased in HIV-1-infected subjects (data not shown).

    Discussion

    The understanding of the functional and phenotypic heterogeneity and the delineation of the differentiation pathways of memory T cells have been the objects of extensive investigation over the past few years (1, 2, 3, 4, 5, 6, 7, 8). This is the first study performing an extensive assessment and comparison of Ag-specific memory CD4 T cell responses against TT and four largely diffuse human viruses, e.g., HSV, EBV, CMV, and HIV-1, based on the recent identification of three functionally distinct populations of CD4 T cells in both healthy donors and HIV-1-infected subjects (22). The results we reported define three patterns of memory CD4 T cell response within the different models investigated and contribute to explain the functional heterogeneity of Ag-specific CD4 T cells and help to understand the differences in the immune responses against different types of Ag/pathogens.

    We have followed two strategies: 1) to study Ag-specific immune responses under different conditions of Ag exposure/persistence, and 2) to manipulate each of the in vivo experimental models of immune response studied by acting on Ag exposure and Ag load. The TT-specific CD4 response represented the model of the typical memory T cell response in which the Ag is cleared. This model was manipulated in vivo by reimmunizing the subjects and then monitoring the functional changes in TT-specific memory CD4 T cells associated with Ag re-exposure. The HIV-1-specific CD4 T cell response in subjects with nonprogressive disease and HSV-, EBV-, and CMV-specific immune responses in subjects with chronic infection were used as models of protracted Ag persistence/exposure and under conditions of controlled virus replication, e.g., low Ag levels. The HIV-1-specific immune response was used as a model of Ag persistence and high Ag levels of exposure. The HIV-1 system was manipulated in vivo by interrupting ART and causing rapid exposure to high Ag levels. Finally, primary CMV and HIV-1 infections as models of acute Ag exposure were also investigated.

    As mentioned above, a series of factors can contribute to the heterogeneity of memory CD4 T cell responses (31), and one recent study investigating antiviral memory CD8 T cell responses proposed that it is the type of pathogen that drives this heterogeneity (13). Our results on the analysis of Ag-specific CD4 T cell responses in different conditions of Ag persistence and exposure have confirmed this heterogeneity. Three distinct patterns of memory CD4 T cell responses were defined on the basis of Ag persistence and exposure. The memory CD4 T cell response in the model of Ag clearance, i.e., TT-specific response, was almost exclusively composed of single IL-2 cells, i.e., typical TCM response. The CD4 T cell response in the model of Ag persistence and protracted Ag exposure and controlled levels of virus replication/low Ag levels, i.e., chronic HSV, EBV, and CMV infection and HIV-1 infection in LTNP, was characterized by the presence of the three distinct functional (single IL-2, IL-2/IFN-, and single IFN-) cell populations, i.e., polyfunctional response. Finally, the CD4 T cell response in the models of Ag persistence and noncontrolled virus replication/high Ag levels (chronic and progressive HIV-1 infection) and acute Ag exposure (primary CMV and HIV-1 infections) is dominated by the presence of single IFN- effector cells, i.e., TEM response. The in vivo manipulation of the different conditions of Ag exposure and Ag levels indicated that the function of the CD4 Ag-specific immune responses is modulated by Ag persistence/exposure and Ag levels. In fact, a shift from the single IL-2 to the polyfunctional memory TT-specific CD4 T cell response was observed following Ag re-exposure. The substantial down-regulation of Ag load associated with ART in patients with progressive HIV-1 infection resulted in a shift of the HIV-1-specific immune response from the single IFN- to the polyfunctional memory CD4 T cell response. The rapid increase of the level of Ag exposure following treatment interruption was associated with a skewing toward a typical effector T cell response and not with the polyfunctional response observed after successful suppression of HIV-1 replication by ART.

    It is worthy to mention that other factors such as the type of virus-infected cells, the immunomodulating capacity of viral proteins, the compartmentalization of tested cells, and the TLR activation by viruses can potentially be involved in the modulation of the immune response.

    Our results also provide insightful information on the relationships between the function and the phenotype of memory CD4 T cells and their stage of differentiation. On the basis of previous studies (9, 22), we propose that the single IL-2-secreting cells that are characterized by the CD45RA–CCR7+ phenotype are at earlier stage of differentiation compared with the IL-2/IFN-- and single IFN--secreting cells. The single IL-2 cells have a typical TCM phenotype and most likely represent the memory cells that persist for an indefinite number of years after Ag clearance, and thus correspond to the population of long-lived memory cells. In support of this hypothesis, they represented the dominant cell population within the model of Ag clearance, e.g., TT-specific response. The single IFN--secreting cells are characterized by the CD45RA–CCR7– and/or CD45RA+CCR7– phenotype that corresponds to a typical TEM phenotype. IL-2/IFN--secreting cells have the CD45RA–CCR7– phenotype. These IL-2/IFN- cells may represent a transition population of memory cells in the process of differentiating from TCM to TEM. The question is why these cells have a dual function. An obvious explanation is that their ability to produce IL-2 allows them to support their own expansion and the ability to produce IFN- to be already equipped to mature into single IFN- TEM. The persistence of cells with dual function for an extended period of time in the absence of high levels of Ag may indicate that these cells represent a mechanism of rapid generation of effector cells if Ag is re-encountered shortly after clearance or in the event of frequent Ag exposure. In support of this hypothesis, CD4 T cells with dual function are indeed the dominant population of memory CD4 T cells under conditions of Ag persistence, i.e., chronic HSV, EBV, and CMV infections and HIV-1 infection in LTNP. Because IL-2/IFN- cells may represent a transition population of memory cells, we propose to term these cells as T intermediate memory.

    The present results indicate that the presence of a dominant effector IFN- CD4 T cell response is the physiological immune response to Ag persistence and to high levels of Ag, and the generation of the polyfunctional CD4 T cell response cannot occur without substantial reduction of Ag levels.

    Our results provide new insights in understanding the heterogeneity of memory T cell responses. They indicate that changes in Ag exposure/persistence or Ag levels substantially influence the functional composition of the Ag-specific CD4 T cell populations, and shed light on the understanding of the patterns of immune response associated with different virus infections.

    Acknowledgments

    We thank Dr. Roland Sahli for his kind cooperation.

    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 This work was supported by research grants from the Swiss National Foundation (FN 3100-058913/2) and the European Commission (QLK2-CT-1999-01321).

    2 Address correspondence and reprint requests to Dr. Giuseppe Pantaleo, Laboratory of AIDS Immunopathogenesis, Division of Immunology and Allergy, Centre Hospitalier Universitaire Vaudois, Rue Bugnon, 1011 Lausanne, Switzerland. E-mail address: giuseppe.pantaleo@chuv.hospvd.ch

    3 Abbreviations used in this paper: TCM, T central memory; ART, antiviral therapy; ICS, intracellular cytokine staining; LTNP, long-term nonprogressor; TEM, T effector memory; TT, tetanus toxoid.

    Received for publication April 23, 2004. Accepted for publication November 4, 2004.

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