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Region of Difference 1 Antigen–Specific CD4+ Memory T Cells Correlate with a Favorable Outcome of Tuberculosis

  1. Delia Goletti1,2,
  2. Ornella Butera2,
  3. Federica Bizzoni2,
  4. Rita Casetti3,
  5. Enrico Girardi4 and
  6. Fabrizio Poccia3
  1. 1Second Division of Infectious Diseases of the Health Department,
  2. 2Translational Research Unit, and
  3. 3Laboratory of Cellular Immunology and
  4. 4Epidemiology Unit, Department of Experimental Research, National Institute for Infectious Diseases “Lazzaro Spallanzani,” Institute for Hospitalization, Care, and Scientific Research, Rome, Italy
  1. Reprints or correspondence: Dr. Delia Goletti, Laboratorio di Collegamento tra Ricerca di Base e Clinica, “Padiglione Del Vecchio,” Istituto Nazionale per le Malattie Infettive “Lazzaro Spallanzani,” IRCCS, Roma 00149, Italy (d.goletti{at}tiscali.it)
 
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Abstract

BackgroundInterferon (IFN)–γ response to region of difference (RD) 1 proteins (culture filtrate 10 and early secreted antigenic target 6) or overlapping peptides is a novel diagnostic marker of tuberculosis (TB) infection. Because we have recently shown that the response to certain peptides selected from RD1 allows discrimination between active TB (A-TB) and successfully treated TB (T-TB), we analyzed here the effector memory T cell profile and RD1-specific responses under the same clinical conditions

MethodsT cell responses to RD1 antigens were analyzed in patients with either severe or mild A-TB (classified on the basis of radiological lesions) and in 2 sets of healthy control subjects—those who had been successfully treated (the T-TB control subjects) and those whose tuberculin skin test (TST) results were negative (the TST-negative control subjects). IFN-γ–producing CD4+ effector T cells were monitored by flow-cytometric analysis and ex vivo enzyme-linked immunospot (ELISPOT) assay, whereas a “cultured” ELISPOT assay was used to determine the frequency of memory T cells

ResultsIn the patients with severe A-TB, both CD4-mediated effector memory and central memory responses to the selected RD1 peptides were almost absent, whereas these responses were found in the majority of the patients with mild A-TB. In contrast, recognition of the selected RD1 peptides was detected in the T-TB control subjects only by expanding the central memory T cell pool

ConclusionsThese data suggest a protective role for RD1 peptide–specific CD4+ effector T cells, which undergo clonal expansion during Mycobacterium tuberculosis replication and then a contraction phase after disease resolution, culminating in the generation of CD4+ memory T cells

Individuals who harbor Mycobacterium tuberculosis in an asymptomatic, latent form (latent tuberculosis [TB] infection [LTBI]) [1] retain a lifelong risk of developing postprimary active TB (A-TB) [2]. Until recently, the tuberculin skin test (TST) has been the only tool used to detect LTBI, but this test is flawed both operationally and with respect to specificity and sensitivity [3]. In an effort to develop more-accurate tools for the immunological diagnosis of M. tuberculosis infection, several studies of the early secreted antigenic target 6 (ESAT-6) and culture filtrate 10 (CFP-10) proteins have been conducted [4–6]. These proteins, encoded by genes located within the region of difference (RD) 1 of the M. tuberculosis genome, are substantially specific to M. tuberculosis as they are not shared with M. bovis bacille Calmette-Guérin (BCG) substrains or most environmental mycobacteria

Peripheral-blood mononuclear cells (PBMCs) from patients with TB [7, 8] and from household contacts of patients with TB [9] release interferon (IFN)–γ when exposed in vitro to intact ESAT-6 or CFP-10 proteins or to overlapping peptides spanning the length of these antigens [10, 11]. These findings resulted in the development of 2 commercially available tests [6, 10, 11] that have been approved for the diagnosis of M. tuberculosis infection [12]. Although these tests provide an accurate diagnosis of infection, they do not discriminate between A-TB and successfully treated TB (T-TB) or LTBI. Interestingly, using multiepitopic peptides from the ESAT-6 and CFP-10 (RD1) proteins that were selected by quantitative implemented HLA peptide–binding motif analysis, we have recently shown a positive response to the selected RD1 peptides only in patients with A-TB [13]. This positive response was present in 70% of the patients studied. The absence of a response to the selected RD1 peptides appeared to be associated with severe TB disease, as characterized by the extent of pulmonary disease determined on the basis of radiological lesions. In those with A-TB who responded to the selected RD1 peptides, the response became undetectable after 3 months of successful therapy, suggesting that this response could be used to monitor the efficacy of therapy [14]. No response to the selected peptides was found in the control subjects, even in TST-positive, non–BCG-vaccinated individuals [13–16]. However, both groups of individuals without A-TB—that is, those who had been successfully treated and those with LTBI—responded to either the intact RD1 proteins or the overlapping peptides, depending on the reagent used [13, 15, 16]. It was fascinating to speculate about the possible immunological mechanisms underlying the different responses to the RD1 antigens in relation to the clinical status of the patients; however, just what the mechanisms were remained unclear

The role played by effector and memory T cells in the pathogenesis of several infectious diseases has been studied [17–20]. In particular, an association between an effector phenotype (as characterized by the absence of CD45RA molecules and either the lymphotropic chemokine receptor CCR7 or the costimulatory CD27 molecule) and viral replication has been shown for both HIV and hepatitis C virus (HCV) infection [17–19]. HIV-specific CD45RA−CD27−/CCR7− T cells have been shown to disappear after successful antiretroviral treatment [21]. Moreover, the presence of cells with a memory phenotype (CD45RA−CCR7+/CD27+) has been demonstrated after HCV clearance by use of a “cultured” enzyme-linked immunospot (ELISPOT) assay, which has been described as a tool to specifically expand antigen-specific CD4+ memory cells [17]. Altogether, these studies indicate that the pool of effector T cells is expanded during active viral replication, whereas only memory cells are detectable after viral control or eradication has been achieved

Thus, the objectives of the present study were to analyze M. tuberculosis–specific T cells for each different infection/disease status in terms of phenotype, in vitro generation of antigen-specific memory T cells, and the potential role played by these cells in the pathogenesis of TB. Specifically, responses of effector and memory T cells to RD1 antigens—including the selected peptides, the overlapping peptides, and the intact RD1 proteins—were analyzed in participants of differing TB status: patients with severe A-TB, patients with mild A-TB, and 2 sets of healthy control subjects, those who had been successfully treated for TB and those who were TST negative

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Materials and Methods

Patient population and study designPatients admitted to the infectious and respiratory diseases wards and outpatient clinics of the National Institute for Infectious Diseases (INMI) “Lazzaro Spallanzani” were evaluated for enrollment. The study was approved by the Ethics Committee of our institution, and all enrolled individuals provided written, informed consent. Only patients with a sputum culture positive for M. tuberculosis were included in the study. Patients were excluded if they tested positive for HIV or were receiving immunosuppressive drugs. Enrolled patients were divided into 2 groups (those with mild A-TB and those with severe A-TB), on the basis of the extent of pulmonary disease (as described below). All M. tuberculosis isolates were drug susceptible, except for those from 2 patients with severe A-TB; these isolates were resistant to both rifampicine and isoniazide. As control subjects, we included 10 healthy persons who were TST positive and who had successfully completed treatment for pulmonary TB during the previous 1–30 years (the T-TB control subjects) as well as 10 healthy persons who were TST negative (the TST-negative control subjects). Characteristics of all participants are shown in table 1

The patients with A-TB were studied within 7 days of admission and before they started anti-TB therapy. For each enrolled participant, a blood sample was drawn into a tube containing EDTA, and an ex vivo ELISPOT assay [13–16] and a cultured ELISPOT assay were performed as described below

Image examination and analysisImages were reviewed by 2 experienced, board-certified radiologists who were blinded to the ELISPOT results. Chest radiographs were assessed for the presence and distribution of parenchymal abnormalities consistent with infiltrates and/or cavities, to determine the extent and severity of disease. Pulmonary disease was defined as severe when lesions characterized by lobe infiltrate, pleural effusions, and cavities involved 2 or more lobes in 1 or both lungs [22]

Peptides and ELISPOT assaysSelection of HLA class II–restricted RD1 peptides from the ESAT-6 and CFP-10 M. tuberculosis proteins was performed by quantitative implemented HLA peptide–binding motif analysis, as described elsewhere [13–16]. Briefly, the following lyophilized peptides, diluted in dimethyl sulfoxide (DMSO) at stock concentrations of 10 mg/mL, were used: ESAT-66–28, ESAT-667–79, CFP-1018–31, CFP-1041–68, and CFP-1074–86. PBMCs were separated and stimulated as described elsewhere [13–16]. Cell cultures were treated for 24 h in duplicate, as follows: with a pool of the 2 ESAT-6 peptides at 5 μg/mL each, with a pool of the 3 CFP-10 peptides at 2 μg/mL each, with phytohemagglutinin (PHA; Sigma) at 5 μg/mL, with purified protein derivative (PPD; batch RT47; Statens Serum Institute) at 5 μg/mL, and with intact recombinant (r) ESAT-6 and rCFP-10 proteins (Lionex) at 1 μg/mL. Laboratory personnel were blinded to the clinical status of the subject. ELISPOT assays (Oxford Immunotec) were performed as indicated by the manufacturer

Results scoringResults are presented as the number of spot-forming cells per 1×106 PBMCs after subtraction of the appropriate control, according to criteria described elsewhere [13–16]

In vitro cultured ELISPOT assayThe cultured ELISPOT assay was performed using a protocol that has been described elsewhere [17, 23, 24] and that was adapted to our requirements. We stimulated 0.5×106 fresh PBMCs as described above (except that PPD was not used, because of the possibility of low cell recovery), with the addition of overlapping ESAT-6 and CFP-10 peptides (Oxford Immunotec)—which consisted of pools of 17 and 18 peptides, respectively, and which are hereafter referred to as “the RD1 panels”—at the concentration suggested by the manufacturer in 0.5 mL of complete medium in a 48-well plate. Each peptide was 15 aa long and overlapped its adjacent peptide by 10 residues. After a 3-day and a 6-day incubation period at 37°C in 5% CO2, we removed 0.25 mL of the cell culture supernatant and replaced it with fresh complete medium containing recombinant interleukin (rIL)–2 (Boehringer Mannheim) at 5 U/mL. On day 8, PBMCs were detached, washed 3 times, and left in the incubator at 37°C with fresh complete medium either in the absence of stimuli or with rIL-2. On day 9, 1×105 lymphocytes were transferred to an ELISPOT plate (Oxford Immunotec) and stimulated for 18 h with the reagents described above (PPD, PHA, intact RD1 proteins, selected RD1 peptides, and panels) in the absence of rIL-2. The ELISPOT plate (Oxford Immunotec) was then developed as indicated by the manufacturer

Phenotypic analysisPBMCs were cultured (5×105 cells/0.5 mL) in complete medium in either the presence or the absence of DMSO (final concentration, 10 μg/mL) and RD1 antigens, as indicated above. PMA (Sigma Aldrich) at 50 ng/mL plus ionomycin (Sigma Aldrich) at 1 μg/mL were used as positive controls. To detect intracellular expression of IFN-γ, brefeldin A at 10 μg/mL (Sigma Aldrich) was used, as described elsewhere [21]. Briefly, production of IFN-γ was assessed by staining with appropriate combinations of monoclonal antibodies (MAbs) directly conjugated to fluorochromes: fluorescein isothiocyanate–conjugated anti-CD4 MAb (Becton Dickinson), phycoerythrin–cyanine 5–conjugated anti-CD27 MAb (Instrument Laboratories, Coulter), and allophycocyanine-conjugated anti-CD45RA MAb (Becton Dickinson). Data acquisition and analysis were done using a FACSCalibur flow cytometer (Becton Dickinson) and CellQuest software (version 3.1; Becton Dickinson). For all staining procedures, an isotype-matched negative control was processed in parallel. Results are expressed as a percentage of IFN-γ–producing cells

Statistical analysisTo compare different groups, we used the Mann-Whitney U test for continuous variables and the McNemar test for categorical variables. Analysis was done by use of SPSS for Windows (version 11; SPSS)

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Results

Association between response to the selected RD1 peptides and mild A-TBSamples were analyzed by ex vivo ELISPOT assay with our selected RD1 peptides, as described elsewhere [13–16]. By this assay, the median level of IFN-γ–producing spot-forming cells was significantly higher in the patients with mild A-TB (175 IFN-γ–producing sfc/1×106 PBMCs) than in the patients with severe A-TB (12 IFN-γ–producing sfc/1×106 PBMCs) (P=.01), the T-TB control subjects (32 IFN-γ–producing sfc/1×106 PBMCs) (P=.002), and the TST-negative control subjects (4 IFN-γ–producing sfc/1×106 PBMCs) (P= .0001) (figure 1). As an internal control, we used the intact RD1 proteins and found that the median level of IFN-γ–producing spot-forming cells was significantly higher in the patients with mild A-TB (237 IFN-γ–producing sfc/1×106 PBMCs) than in the TST-negative control subjects (9 IFN-γ–producing sfc/1×106 PBMCs) (P=.0001) and, although the difference was smaller, the T-TB control subjects (66 IFN-γ–producing sfc/1×106 PBMCs) (P=.03); conversely, no significant difference was found between the patients with mild A-TB and the patients with severe A-TB (44 IFN-γ–producing sfc/1×106 PBMCs) (P>.05) (figure 1). Note that the response to the intact RD1 proteins, but not to the selected RD1 peptides, was significantly higher in the T-TB control subjects than in the TST-negative control subjects (P=.001), similar to previous results in TST-positive individuals [13, 15, 16]

Figure 1
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Figure 1

Interferon (IFN)–γ response to region of difference (RD) 1 antigens in patients with mild or severe active tuberculosis (A-TB) and in 2 sets of healthy control subjects (those who had been successfully treated for tuberculosis [T-TB] and those whose tuberculin skin test [TST] results were negative). IFN-γ was detected by ex vivo enzyme-linked immunospot assay. The highest response (in spot-forming cells per 1×106 peripheral-blood mononuclear cells [PBMCs]) to selected RD1 peptides and to intact RD1 proteins by specific IFN-γ–producing T cells was plotted for each participant. Horizontal bars represent the median value for each group of patients. *P<.05, for the indicated comparisons

The response to the selected RD1 peptides was scored as positive in 9 (90%) of the 10 patients with mild A-TB, 2 (20%) of the 10 patients with severe A-TB, 1 (10%) of the 10 T-TB control subjects, and 0 of the 10 TST-negative control subjects. In contrast, the response to the intact RD1 proteins was scored as positive in 9 (90%) of the 10 patients with mild A-TB, 5 (50%) of the 10 patients with severe TB, 9 (90%) of the 10 T-TB control subjects, and 0 of the 10 TST-negative control subjects. Therefore, although the proportion of positive responses to the intact RD1 proteins did not differ significantly from that to the selected RD1 peptides among the patients with mild A-TB, the T-TB control subjects were significantly more likely to respond to the intact RD1 proteins than to the selected RD1 peptides (P=.001), in agreement with previous results in TST-positive individuals [13, 15, 16]. Thus, the response to the selected RD1 peptides is associated with mild A-TB

Mediation of the response to the selected RD1 peptides byCD4+ T cells with a prevalent effector memory (EM) phenotype We further evaluated the phenotypic characteristics of the cells responding to the RD1 antigens in the patients with mild A-TB. All the patients analyzed responded to the positive control, PMA plus ionomycin (data not shown). As shown in representative results in figure 2, a significant IFN-γ response to the selected RD1 peptides and to the intact RD1 proteins was observed for CD4+ T cells (figure 2C and 2F), whereas no response was detected for CD8+ T cells (figure 2B and 2E)

Figure 2
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Figure 2

Phenotypic analysis of interferon (IFN)–γ–producing CD4+ T cells in patients with mild active tuberculosis (A-TB). Peripheral-blood mononuclear cells (PBMCs) from patients with mild A-TB were cultured in the presence of selected region of difference (RD) 1 peptides and intact RD1 proteins. Negative control staining is shown in panel A, indicating no IFN-γ production by T cells (both CD4+ and CD4−) in the absence of specific stimuli. The IFN-γ response to the selected RD1 peptides and to the intact RD1 proteins is not mediated by CD8+ T cells (B and E respectively) but is mediated by CD4+ T cells (C and E respectively). Gated IFN-γ–producing CD4+ T cells were analyzed for CD45RA and CD27 expression, and the percentages of CD4+ T cells producing IFN-γ in response to the selected RD1 peptides (D) and to the intact RD1 proteins (G) are shown. In panels D and G, effector memory (EM) cells (CD45RA−CD27−) are represented in the lower left quadrants, central memory (CM) cells (CD45RA−CD27+) are represented in the lower right quadrants, terminally differentiated (TD) effector cells (CD45RA+CD27−) are represented in the upper left quadrants, and naive (N) cells (CD45RA+CD27+) are represented in the upper right quadrants. The percentages of CD4+ T cells producing IFN-γ in response to the selected RD1 peptides and to the intact RD1 proteins among CM and EM cells are shown in panel H (each dot represents a single patient with mild A-TB). APC, allophycocyanine; FITC, fluorescein isothiocyanate; PC5, phycoerythrin–cyanine 5

To characterize this immune response, naive and memory phenotypes were studied. All the patients with mild A-TB were analyzed with one exception, for whom the analysis could not be performed because of unresponsiveness to the RD1 antigens. Most CD4+ T cells responding to the RD1 antigens in vitro presented an EM phenotype (defined as CD45RA−CD27−), as shown in representative results in figure 2D (response to the selected RD1 peptides) and in figure 2G (response to the intact RD1 proteins). A relevant but minor fraction of PBMCs presented a central memory (CM) phenotype (defined as CD45RA−CD27+) (figure 2D and 2G). Combining the results for all patients with mild A-TB analyzed, we found that, among the cells responding to the selected RD1 peptides, the percentage of CD4+ T cells with an EM phenotype was significantly higher than those with a CM phenotype (P=.0067), as shown in figure 2H. Similarly, among the cells responding to the intact RD1 proteins, the percentage of CD4+ T cells with an EM phenotype was significantly higher than those with a CM phenotype (P=.0026), as shown in figure 2H

Recovery of a response to the selected RD1 peptides in memory cells from the T-TB control subjects by the cultured ELISPOT assayNext, to further investigate the discrepancy in the IFN-γ response to the selected RD1 peptides between the active and inactive phases of TB, we used the cultured ELISPOT assay [17, 23, 24]. The protocol for this assay, unlike that for the ex vivo ELISPOT assay, allows the in vitro expansion of memory cells specific for the antigen used to perform the in vitro restimulation

T cell responses from 6 representative healthy subjects—3 T-TB control subjects and 3 TST-negative control subjects—were studied at time 0 and after 9 days of in vitro culture. As internal positive controls, stimulation with PHA, PPD, the intact RD1 proteins, and the RD1 panels [25] were used

As shown in figure 3A–3C by the ex vivo ELISPOT assay, no reactivity to the selected RD1 peptides was detected in the 3 representative T-TB control subjects, although a response to the intact RD1 proteins and to the RD1 panels was found. In contrast, by the IFN-γ cultured ELISPOT assay, a selective expansion of memory cells specifically able to respond to the selected RD1 peptides was observed in the 3 T-TB control subjects. Conversely, no expansion of memory cells responding to the RD1 antigens was found in the TST-negative control subjects (figure 3D–3F), although a positive response to the mitogen PHA was detectable

Figure 3
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Figure 3

Evolution of the effector response vs. the memory response to selected region of difference (RD) 1 peptides in CD4+ T cells in healthy control subjects. Effector responses (detected by ex vivo enzyme-linked immunospot [ELISPOT] assay after 1 day) and memory responses (detected by cultured ELISPOT assay after 9 days) to RD1 epitope–specific CD4+ memory T cells were evaluated. Results for 6 representative healthy control subjects, of whom 3 had been successfully treated for tuberculosis (T-TB) (A–C) and 3 had negative tuberculin skin test (TST) results (D–F) are shown. In panels A–C, a dichotomy between the effector response and the memory response to the selected RD1 peptides in the 3 T-TB control subjects can be seen. Neither an effector response nor a memory response was detected in the 3 TST-negative control subjects (D–F). Phytohemagglutinin (PHA), purified protein derivative (PPD), intact RD1 proteins, and RD1 panels were used as internal controls. IFN, interferon; PBMCs, peripheral-blood mononuclear cells

Association between response to the selected RD1 peptides and less-severe clinical presentation in the patients with A-TBWe then retrospectively evaluated whether the response to the selected RD1 peptides at the time of TB diagnosis was associated with the severity of clinical presentation in terms of chest radiological patterns. To this end, we compared patients who were classified as having mild A-TB with those who were classified as having severe A-TB. As shown in table 1, these 2 groups differed in terms of time to sputum culture negativity. Of the 10 patients with severe A-TB, only 2 responded to the selected RD1 peptides (figure 1). When this immune reactivity to the RD1 antigens was studied in detail in 3 representative patients, no response to the intact RD1 proteins or to the selected RD1 peptides was found by the ex vivo ELISPOT assay (measuring the effector response), although a response to the RD1 panels was detected (figure 4A–4C). By use of the IFN-γ cultured ELISPOT assay, a selective expansion of lymphocytes specifically able to respond to the intact RD1 proteins was found; however, no significant induction of response to the selected RD1 peptides was observed. In fact, in only 1 of the 3 patients analyzed was even a minor response to the RD1 selected peptides found (40 IFN-γ–producing sfc/1×106 PBMCs) (figure 4B). No change in this immune response was observed in any of the patients studied after 45 days of anti-TB therapy (data not shown). In contrast, in the patients with mild A-TB, a response to all antigens studied was found by both the ex vivo and the cultured ELISPOT assay (measuring effector and memory responses, respectively) (figure 4D–4F). These data suggest that the effector and memory responses to the selected RD1 peptides detected at the time of TB diagnosis may be associated with a good clinical outcome of TB over time

Figure 4
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Figure 4

Lack of an effector response to selected region of difference (RD) 1 peptides and the absence of evolution vs. a memory response in CD4+ T cells in patients with severe active tuberculosis (A-TB): comparison with patients with mild A-TB. Effector responses (detected by ex vivo enzyme-linked immunospot [ELISPOT] assay after 1 day) and memory responses (detected by cultured ELISPOT assay after 9 days) to RD1 epitope–specific CD4+ memory T cells were evaluated. Results for 6 representative individuals, of whom 3 had severe A-TB (A–C) and 3 had mild A-TB (D–F) are shown. Effector and memory responses to the selected RD1 peptides were not observed in the 3 patients with severe A-TB, whereas these responses were observed in the 3 patients with mild A-TB. Phytohemagglutinin (PHA) and RD1 panels were used as internal controls. For the results obtained by stimulation with the panels and selected peptides, the highest result (in spot-forming cells per 1×106 peripheral-blood mononuclear cells [PBMCs]) was plotted

Table 1
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Table 1

Epidemiological and demographic characteristics of the study participants

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Discussion

T cell responses to RD1 proteins or overlapping peptides are commonly related to M. tuberculosis infection independent of clinical status (whether T-TB, LTBI, or A-TB) [6, 10, 11, 26], whereas, in previous studies, we have demonstrated a remarkable response to our selected RD1 peptides only in patients with A-TB [13–16]. In the present study, we analyzed the immune phenotypes and the generation of memory profiles of the T cells responding to RD1 antigens in both patients with mild A-TB and patients with severe A-TB as well as in T-TB control subjects and TST-negative control subjects. The results indicated that the response to the selected RD1 peptides was mediated by CD4+ EM T cells and was significantly associated with mild A-TB, whereas recognition of the selected RD1 peptides was observed in the T-TB control subjects only after expansion of the CM cells. Moreover, in the patients with severe A-TB, the absence of both EM and CM responses to the selected RD1 peptides at the time of TB diagnosis was associated with poor control of TB over time in terms of time to sputum culture negativity, suggesting that RD1 peptide–specific CD4+ effector T cells play a protective role

In HIV infection, the presence of a high frequency of EM T cell responses has been observed during active viral replication, and this T cell reactivity slowly decreases after viral clearance by successful antriretroviral treatment [19]. This slow decrease in the rate of EM T cell responses may represent a normal memory response to the withdrawal of antigen. Accordingly, in most cases the frequencies of HIV-specific T cells appear to be positively correlated with HIV production during highly active antiretroviral therapy [18]. Moreover, this model has been confirmed in studies of HIV-infected patients undergoing structured treatment interruption, in whom HIV-specific T cell responses have been reported to be higher at the peak of viremia and then to decrease when therapy is reintroduced [21]. Similarly, in patients who had been acutely infected with HCV and who cleared the infection, high frequencies of effector T cells are found soon after infection, whereas higher frequencies of memory T cells appear later on [17]. These data are in agreement with those obtained in animal models of M. tuberculosis infection, in which a decrease in the ESAT-6–specific CD4+ effector T cell response has been reported in both the lymph nodes and the lungs after the acute phase of infection [27]. Also in agreement are data obtained in humans by Tully et al. showing that, during LTBI, the EM response to RD1 peptides may be absent in the peripheral blood but present in T cells from lung granulomas in the same subjects [28]. Interestingly, our peptides selected from ESAT-6 (ESAT-66–28 and ESAT-667–79) are partly included in those reported by Tully et al. (ESAT-61–20 and ESAT-672–95). Moreover, it has been reported that most patients with TB have a CM response to rESAT-6, as shown by comparison of a shorter (18 h) versus a longer (6 days) period of RD1 antigen–specific in vitro stimulation [29]. However, that study involved the use of rESAT-6 only, and the reason for the T cell expansion observed in healthy control subjects by the 6-day assay is unclear. In the present study, we found a protective role for RD1 peptide–specific CD4+ effector T cells, which undergo clonal expansion during active M. tuberculosis replication and a contraction phase during active disease resolution, culminating in the generation of CD4+ memory T cells. Further studies in which the same individuals could be tested before and after treatment should be performed to confirm the observations made in the present cross-sectional study

With respect to our patients with severe A-TB (characterized by multiple lung lesions and, consequently, longer time to sputum culture negativity), no EM or CM response to the selected RD1 peptides was found, although we did observe a response to the RD1 panels (consisting of a pool of overlapping peptides spanning the length of CFP-10 and ESAT-6), as we have observed in previous studies [10, 25]. The absence of mycobacteria-specific T cells among PBMCs from patients with severe A-TB may be the consequence of a massive compartmentalization of these cells in infectious foci. This hypothesis, however, conflicts with the clinical outcome of these forms of disease. It has been suggested that, in the pathogenesis of other infectious diseases, the elimination of effector T cells may occur when T cells confront high doses of antigens [30, 31]. Moreover, in severe forms of TB characterized by high numbers of replicating bacilli, M. tuberculosis may generate inefficient dendritic cells by infecting newly recruited monocytes [32], with the functional consequence of reducing the pool of specific IFN-γ–producing T cells. In line with these observations, our data suggest that continuous exposure to M. tuberculosis–infected antigen-presenting cells may cause exhaustive differentiation and consequent programmed cell death of virtually all M. tuberculosis–specific effector cells associated with the inability to restimulate CM cells and to prime naive precursors. Moreover, irrespective of the causes, the detection of an effector response to the selected RD1 peptides at the time of TB diagnosis may reflect a functional immune response not yet compromised by the immune-evasion mechanisms operated by M. tuberculosis

Altogether, the present data suggest that RD1 peptide–specific CD4+ effector T cells undergo clonal expansion during M. tuberculosis replication and then a contraction phase after disease resolution, culminating in the generation of CD4+ memory T cells. These findings may have important implications for achieving a better understanding of the pathogenesis of TB and for TB vaccine design, because they indicate that (1) EM CD4+ T cells that produce IFN-γ in response to the selected RD1 peptides are a clear hallmark of mild A-TB and (2) a memory response, rather than an effector response, should be the target of candidate TB vaccines

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Acknowledgments

We are grateful to all patients and nursing staff who took part in this study. We thank Drs. G. D’Offizi, E. Coccia, S. Carrara, D. Vincenti, and C. Nisii, for critical review of the manuscript

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Footnotes

  • ↵Potential conflicts of interest: D.G., R.C., F.P., and E.G. (along with other investigators) have a patent pending on a T cell assay that is based on the selected RD1 peptides

    Financial support: Italian Ministry of Health (grant to the study); “Fondo per gli Investimenti della Ricerca di Base”–”Ministero dell’Istruzione, dell’Università e della Ricerca” (FIRB-MIUR; grant RBNE01PPTF_003); RTD European Union Project 6th Framework Programme (project acronym, TB-VAC; grant LSHP-CT-2003-503367 to F.P.)

  • Received March 19, 2006.
  • Accepted May 26, 2006.
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  1. J Infect Dis. (2006) 194 (7): 984-992. doi: 10.1086/507427
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