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Infect Immun. 2009 November; 77(11): 5025–5034.
Published online 2009 August 31. doi:  10.1128/IAI.00224-09
PMCID: PMC2772532

Patients with Multidrug-Resistant Tuberculosis Display Impaired Th1 Responses and Enhanced Regulatory T-Cell Levels in Response to an Outbreak of Multidrug-Resistant Mycobacterium tuberculosis M and Ra Strains[down-pointing small open triangle]

Abstract

In Argentina, multidrug-resistant tuberculosis (MDR-TB) outbreaks emerged among hospitalized patients with AIDS in the early 1990s and thereafter disseminated to the immunocompetent community. Epidemiological, bacteriological, and genotyping data allowed the identification of certain MDR Mycobacterium tuberculosis outbreak strains, such as the so-called strain M of the Haarlem lineage and strain Ra of the Latin America and Mediterranean lineage. In the current study, we evaluated the immune responses induced by strains M and Ra in peripheral blood mononuclear cells from patients with active MDR-TB or fully drug-susceptible tuberculosis (S-TB) and in purified protein derivative-positive healthy controls (group N). Our results demonstrated that strain M was a weaker gamma interferon (IFN-γ) inducer than H37Rv for group N. Strain M induced the highest interleukin-4 expression in CD4+ and CD8+ T cells from MDR- and S-TB patients, along with the lowest cytotoxic T-lymphocyte (CTL) activity in patients and controls. Hence, impairment of CTL activity is a hallmark of strain M and could be an evasion mechanism employed by this strain to avoid the killing of macrophages by M-specific CTL effectors. In addition, MDR-TB patients had an increased proportion of circulating regulatory T cells (Treg cells), and these cells were further expanded upon in vitro M. tuberculosis stimulation. Experimental Treg cell depletion increased IFN-γ expression and CTL activity in TB patients, with M- and Ra-induced CTL responses remaining low in MDR-TB patients. Altogether, these results suggest that immunity to MDR strains might depend upon a balance between the individual host response and the ability of different M. tuberculosis genotypes to drive Th1 or Th2 profiles.

Human interventions, namely, mistreatment of tuberculosis (TB) and poor patient compliance, selectively favor the multiplication of drug-resistant Mycobacterium tuberculosis mutants over drug-susceptible bacilli in tuberculous lesions. M. tuberculosis isolates are considered to be multidrug resistant (MDR) when showing resistance to isoniazid and rifampin (rifampicin), the most effective drugs for TB treatment; they become extensively drug resistant when showing additional resistance to key second-line drugs (32, 36). MDR-TB and extensively drug-resistant TB are very difficult to treat, their prognosis is somber, and mortality is high (14, 27).

In Argentina, a total of 11,464 new cases of TB were reported in 2006, with an incidence of 29.1 per 100,000 inhabitants. MDR-TB occurred in 4.5% of the cases. MDR-TB outbreaks emerged in Argentina among hospitalized patients with AIDS in the early 1990s (1, 45) and thereafter disseminated to immunocompetent individuals (37-39). Epidemiological, bacteriological, and genotyping data allowed the identification of certain MDR M. tuberculosis outbreak strains, such as the so-called strain M of the Haarlem family and strain Ra of the Latin America and Mediterranean (LAM) family. Each of these two strains managed to perpetuate in its geographical niche, i.e., Buenos Aires and the Rosario City area, respectively. In particular, strain M appears to be highly prosperous in the country and is able to build up further drug resistance without impairing its ability to spread (29).

TB development depends not only on the host immune response and on its natural resistance/susceptibility to M. tuberculosis infection but also on differences in transmissibility, virulence, and immunogenicity among M. tuberculosis strains, determined by the genetic background of the organisms. It is becoming evident that certain strains of M. tuberculosis with special transmission potential are able to manipulate host immunity by inducing Th1 or Th2 responses which could impact disease outcome and/or evolution (5, 28, 30, 31, 40-42, 51). Protective immunity against TB is mediated by a Th1-type immune response characterized by high levels of interleukin-12 (IL-12) from infected macrophages and gamma interferon (IFN-γ) from antigen-specific T cells, which control and contain infection in the lungs (24). Peripheral blood mononuclear cells (PBMC) from MDR-TB patients have been shown to poorly respond in vitro to H37Ra whole bacilli, purified protein derivative (PPD), and specific antigens, such as ESAT-6 and the 30-kDa protein (16, 25, 26, 33). Furthermore, increased IL-4 secretion by CD4+ T cells after H37Rv total lipid stimulation was observed in MDR-TB patients (50).

Although a Th1 profile is necessary for a protective response, it may also cause immunopathologic damage; for this reason, either regulatory T cells (Treg cells) or a Th2 response might play important regulatory functions protecting the patient from collateral host tissue damage. Nevertheless, an excessive Th1 downregulation might favor disease progression. In this context, increased levels of CD4+ CD25high Foxp3+ Treg cells have been detected in PBMC from drug-susceptible tuberculosis (S-TB) patients compared to those from PPD-positive (PPD+) healthy donors (18-20, 46). In addition, the results of studies involving in vitro depletion of CD4+ CD25high T lymphocytes suggest a role of Treg cells in TB pathogenesis (18-20, 43).

In the current study, we examined immune profiles induced by two MDR M. tuberculosis strains disseminated in Argentina, namely, strains M and Ra. Our results demonstrate that strain M is a weak inducer of IFN-γ and elicits a remarkably low cytotoxic T-cell (CTL) activity. Also, in vitro expansion of Treg cells in PBMC from TB patients is not M. tuberculosis strain dependent and efficiently suppresses antigen-induced IFN-γ and CTL responses.

MATERIALS AND METHODS

Patients and isolates.

Blood samples were obtained from MDR-TB and S-TB patients hospitalized in the Phthisio-Pneumonology Institute University of Buenos Aires, placed in the F. J. Muñiz Hospital, Buenos Aires, Argentina. Informed consent was obtained from patients according to the guidelines of the ethics committee of the F. J. Muñiz Hospital. All patients were diagnosed by the presence of recent clinical respiratory symptoms, abnormal chest radiography, a positive sputum smear test for acid-fast bacilli, and the identification of M. tuberculosis in culture. Exclusion criteria included a positive test for human immunodeficiency virus (HIV) and the presence of concurrent infectious diseases or noninfectious conditions (cancer, diabetes, or steroid therapy).

Sputum smear examination, mycobacterial culture, species identification, and drug susceptibility testing were performed according to standard procedures. Susceptibility to isoniazid, rifampin, ethambutol, and streptomycin was determined according to World Health Organization standards. Susceptibility to kanamycin, p-aminosalicylic acid, and cycloserine was tested according to the Canetti, Rist, and Grosset method, whereas the pyrazinamidase test was used to infer pyrazinamide susceptibility (59). Available MDR M. tuberculosis isolates were genotyped by IS6110 DNA fingerprinting and spoligotyping, using standardized protocols (23, 58).

A total of 25 MDR-TB patients (8 males and 17 females; median age [25th to 75th percentiles], 28 [24 to 39] years) and 20 S-TB patients (14 males and 4 females; median age [25th to 75th percentiles], 26 [21 to 54] years) were included. All MDR-TB and S-TB patients had radiological advanced pulmonary disease and were sputum smear positive at the time of the study (median number of acid-fast bacilli/field [25th to 75th percentiles] for MDR-TB, 5 [1 to 10]; that for S-TB, 5 [0.5 to 10]). Percentages of different M. tuberculosis lineages among MDR-TB patients in this study were as follows: LAM, 54%; Haarlem, 33% (70% of whom were infected with strain M); T, 8%; and other, 4%. Those among S-TB patients were as follows: Haarlem, 35%; T, 30%; LAM, 27%; and other, 8%. Ten PPD+ healthy volunteers (4 males and 6 females; median age [25th to 75th percentiles], 30 [27 to 46] years) (group N) were included as controls.

Mononuclear cells.

PBMC were isolated from heparinized blood by Ficoll-Triyosom gradient centrifugation (3) and suspended in RPMI 1640 (HyClone; Thermo Scientific, UT) containing 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% heat-inactivated fetal calf serum (Invitrogen, Gibco) (complete medium).

CD25 cell depletion.

PBMC (1 × 107) were incubated with 1 μg anti-CD25 monoclonal antibody (MAb; eBioscience, San Diego, CA) for 30 min at 4°C, washed with phosphate-buffered saline (PBS), and mixed with goat anti-mouse immunoglobulin G-coated magnetic beads (Invitrogen Dynal, Oslo, Norway) by gentle rolling at 4°C for 30 min. Nonrosetted cells (CD25-depleted PBMC) were enriched using a magnet. Generally, one cycle of treatment was sufficient for an effective depletion, as assessed by flow cytometry: approximately 80 to 98% of CD4+ CD25high T cells were eliminated after depletion, and a reduction of CD4+ CD25low T cells was observed (27 to 45%) in CD25-depleted PBMC. CD8+ CD25+ T cells were slightly reduced (5 to 28%) after depletion. CD25-depleted PBMC were suspended in complete medium, ensuring that the number of cells/ml of each subset was the same as in total cultured PBMC in order to compare their cytokine production and CD107 expression.

Antigens.

MDR outbreak strains M (Haarlem family) and Ra (LAM family), as well as laboratory strain H37Rv (T family), were grown in Middlebrook 7H9 broth (Difco Laboratories, Detroit, MI) at 37°C in 5% CO2 until log phase. Strain 410, an MDR strain of the Haarlem family that is highly related but not identical to strain M, was included in some experiments (Fig. (Fig.1).1). Mycobacteria were harvested, washed three times, and suspended in PBS that was free of pyrogens. Bacteria were killed by heating at 80°C for 1 h, suspended in PBS at an optical density at 600 nm of 1 (≈108 bacteria/ml), and stored at −20°C until use. These mycobacterial suspensions contained soluble as well as particulate antigens.

FIG. 1.
Spoligotyping and IS6110-restriction fragment length polymorphism pattern profiles of M. tuberculosis strains used as antigens in this study, including reference virulent strain H37Rv and local MDR strains M, 410, and Ra.

PBMC cultures.

Total or CD25-depleted PBMC (2 × 106 cells/ml) were cultured in polystyrene tubes (BD Falcon, Franklin Lakes, NJ) at 37°C in a humidified 5% CO2 atmosphere in complete medium in the presence of heat-killed bacilli (suspensions of M. tuberculosis strain M, Ra, 410, or H37Rv at a 2:1 ratio of M. tuberculosis to PBMC).

51Cr release cytotoxic assay.

The ability of PBMC stimulated by M, Ra, 410, or H37Rv killed bacilli (effector cells) to lyse autologous M. tuberculosis-pulsed macrophages was examined using a standard chromium release cytotoxicity assay as described previously (12).

Immunofluorescence analysis. (i) Surface membrane expression.

The following MAbs were used to evaluate surface marker expression in fresh and 5-day-cultured PBMC: anti-CD4 (Cy5-phycoerythrin [Cy5-PE] or fluorescein isothiocyanate [FITC] conjugated), anti-CD8 (Cy5-PE or PE conjugated) (both from BD Bioscience, San Jose, CA), and PE-conjugated anti-CD25 (eBioscience).

(ii) Intracellular expression of Foxp3 transcription factor.

Foxp3 expression was detected using a FITC-conjugated anti-human Foxp3 staining set (eBioscience) according to the manufacturer's instructions. An isotype-matched antibody was used as a control (eBioscience).

(iii) CD107 surface expression.

To evaluate the frequency of CD4 and CD8 T cells undergoing recent degranulation, PBMC (2 × 106 cells/ml, in complete medium) were cultured for 5 days, with or without M. tuberculosis (2:1 ratio of M. tuberculosis to PBMC). FITC-labeled anti-CD107 MAb (BD Pharmingen) was then added directly to tubes, and cells were incubated for a further 4 h at 37°C in a 5% CO2 incubator. After that, cells were washed and stained for CD4 and CD8 expression.

(iv) Intracellular cytokine expression.

Intracellular IL-10, IL-4, and IFN-γ expression was determined in 5-day-old PBMC cultures. Briefly, brefeldin A (5 μg/ml; Sigma Chemical Co., St. Louis, MO) was added for the last 4 h of culture to block cytokine secretion, and cells were surface stained with anti-CD4 and anti-CD8. They then were fixed with 0.5% paraformaldehyde and permeabilized with fluorescence-activated cell sorter permeabilizing solution 2 (BD Bioscience) before FITC- or PE-conjugated anti-IL-10, anti-IL-4 (both from BD Bioscience), or anti-IFN-γ (Caltag, Burlingame, CA) was added.

Stained cells were analyzed by flow cytometry. Twenty thousand events were acquired for each cell preparation, using a FACSCan flow cytometer (BD Bioscience) with CellQuest. FCS Express software (De Novo Software, Los Angeles, CA) was used for the analysis. Lymphocyte gates were set according to forward- and side-scatter parameters, excluding cell debris and apoptotic cells. Results were expressed as percentages of positive cells in a lymphocyte population or percentages of positive cells within CD4+ or CD8+ T cells.

Statistical analysis.

Data were expressed as medians and 25th to 75th percentiles. The analysis was performed using the nonparametric Kruskal-Wallis test to compare responses of MDR-TB and S-TB patients and healthy individuals, followed by the Mann-Whitney U test to compare two groups. The Friedman test was performed to compare responses to different treatments within each group, followed by the Wilcoxon test. Correlations between CD107 and cytokine expression were analyzed by the nonparametric Spearman's rank test. All statistical analyses were two sided, and the significance level adopted was for P values of <0.05. The analysis was performed using the statistical software SPSS 15.0 for Windows (SPSS Inc., IL) and Graphpad Prism 4.0 (Graphpad Software Inc., CA).

RESULTS

Strains M and Ra induce differential IL-10, IL-4, and IFN-γ expression in CD4+ and CD8+ T cells.

To assess the immune profiles induced by M. tuberculosis, intracellular expression of IL-10, IL-4, and IFN-γ was determined in PBMC from MDR-TB and S-TB patients and healthy controls (group N) after stimulation with or without strain H37Rv, M, or Ra for 5 days. As shown in Fig. Fig.2,2, higher spontaneous IL-10 expression in CD8+ cells was observed in MDR-TB patients than in group N controls. In addition, although the percentage of IL-10-positive (IL-10+) cells increased above basal levels upon H37Rv, M, and Ra stimulation in CD4+ and CD8+ cells from TB patients and group N controls, no significant differences were observed among them. In healthy PPD+ controls, none of the strains induced IL-4 in CD4+ cells; however, H37Rv did induce CD8+ IL-4+ cells. In TB patients, IL-4 was induced in CD4+ and CD8+ cells upon stimulation with M. tuberculosis strains (Fig. (Fig.3),3), with strain M being the highest IL-4 inducer. Remarkably, when MDR-TB patients were grouped according to the infecting M. tuberculosis genotype, those patients infected with Haarlem strains showed a higher proportion of M-induced IL-4+ cells (mean [25th to 75th percentiles] for CD4, 3.0 [1.6 to 3.2] [P < 0.05]; that for CD8, 2.5 [2.0 to 3.2] [P < 0.05]) than did patients infected with LAM strains (mean [25th to 75th percentiles] for CD4, 1.5 [0.8 to 1.6]; that for CD8, 1.8 [1.4 to 2.3]). Conversely, no differences were observed between both groups of MDR-TB patients for H37Rv- or Ra-induced IL-4+ cells. Thus, the ability to induce high IL-4 levels in TB patients seems to be an intrinsic characteristic of strain M.

FIG. 2.
H37Rv, M, and Ra induce IL-10 in CD4+ and CD8+ T cells. PBMC from 25 MDR-TB and 20 S-TB patients and from 10 PPD+ healthy individuals (N) were cultured for 5 days alone (white bars) or with H37Rv (lightest gray bars), M (light ...
FIG. 3.
H37Rv, M, and Ra induce different proportions of IL-4 in CD4+ and CD8+ T cells from TB patients. PBMC from 25 MDR-TB and 20 S-TB patients and from 10 group N controls were cultured for 5 days alone (white bars) or with H37Rv (lightest ...

CD4+ and CD8+ cells expressing IFN-γ were increased above basal levels upon PBMC stimulation with H37Rv, M, and Ra in S-TB patients and group N controls, whereas in MDR-TB patients, CD8+ IFN-γ+ cell enhancement was achieved only by H37Rv and M stimulation (Fig. (Fig.4).4). Moreover, a reduction in the number of M. tuberculosis-induced IFN-γ+ cells was observed in TB patients compared to that in PPD+ healthy controls. Furthermore, strain H37Rv was the best IFN-γ inducer in CD4+ cells from healthy PPD+ controls, whereas the strain made no difference in the induction of CD8+ IFN-γ+ cells.

FIG. 4.
H37Rv, M, and Ra induce different proportions of IFN-γ in CD4+ and CD8+ T cells in TB patients and group N controls. PBMC from 25 MDR-TB and 20 S-TB patients and from 10 group N controls were cultured for 5 days alone (white bars) ...

Strain M induces low CD107 expression in CD8+ T cells.

CD107a expression on the cell surface has been described as a marker of cytotoxic CD8+ T-cell degranulation/activation (2). CD107a and -b are intracellular proteins normally found in lysosomes that are transiently expressed on CTL surfaces upon exocytosis of cytotoxic granules (60). Considering that in our system CD8+ T cells are in close contact with antigen-presenting cells, we evaluated the presence of CD8+ T cells having undergone recent granule exocytosis in 5-day-old PBMC cultures by employing a CD107 mobilization assay. As shown in Fig. Fig.5A,5A, the proportion of CD107+ cells in CD8+ T cells increased in TB patients and group N controls upon H37Rv, M, and Ra stimulation. However, TB patients showed fewer CD107+ cells than did group N controls. Moreover, strain M induced the lowest CD107 expression not only in TB patients but also in group N controls, suggesting that this strain has an impaired ability to evoke a CTL response. These results were confirmed in group N controls by employing a standard 51Cr release cytotoxicity assay (Fig. (Fig.5B5B).

FIG. 5.
CD8+ T cells from TB patients express low levels of CD107 surface molecules. PBMC from 20 MDR-TB and 16 S-TB patients and 10 group N controls were cultured for 5 days alone (C; white bars) or with H37Rv (lightest gray bars), M (light gray bars), ...

Considering that CD107 upregulation has been demonstrated to be in concordance with perforin loss and enhanced IFN-γ production (2), we compared the proportions of IFN-γ and CD107 on antigen-stimulated CD8+ T cells from MDR-TB and S-TB patients and group N controls. A significant correlation between both markers was observed, with that for strains H37Rv and M being weaker. This suggests that impaired CD107 expression could be related to diminished IFN-γ expression in CD8+ T cells from TB patients (Fig. (Fig.5C).5C). In contrast, an inverse correlation between the proportions of CD107+ and IL-4+ or IL-10+ cells in the CD8+ cell subset was found.

Having observed that strain M induced weak IFN-γ expression in group N controls, low CTL activity in group N controls and TB patients, and strong IL-4 expression in TB patients, we wondered whether these characteristic patterns were strain specific or, in contrast, were shared with other related MDR strains of the Haarlem family. For this purpose, we evaluated the same effector functions, but using strain 410, a Haarlem strain that is highly related to strain M but has rarely caused disease since the latter started clonal expansion. A decreased percentage of CD4+ IL-4+ cells was indeed detected with strain 410 in MDR-TB patients (median [25th to 75th percentiles], 2.6% [1.7 to 5.6%] [P < 0.05]), while neither the IL-10 nor IFN-γ level was modified. Remarkably, strain 410 induced a higher % CD107+ cells among CD8+ T cells in group N controls (6.9% [6.4 to 7.6%]) and MDR-TB (4.8% [2.8 to 6.4%]) and S-TB (7.0% [5.2 to 7.4%]) (P < 0.05) patients than did strain M. These results were confirmed in group N controls by a 51Cr release assay (% cytotoxicity for strain M, 38 [14 to 42]; that for strain 410, 61 [51 to 70] [P < 0.05]).

CD4+ and CD8+ Treg cells are increased ex vivo in PBMC from MDR-TB patients.

Considering that Treg cells affect Th1 and Th2 responses (21) and that CD4+ CD25+ Foxp3+ T cells are increased in S-TB patients (18, 19, 46), we wondered whether Treg cells were involved in the impaired M. tuberculosis-induced T-cell response observed in MDR-TB patients. We first evaluated the percentages of CD4+ and CD8+ T cells ex vivo in PBMC from MDR-TB and S-TB patients and group N controls. As shown in Table Table1,1, a lower percentage of CD4+ T cells was detected in TB patients than in group N controls, while no differences were observed in the proportion of CD8+ cells. Although the absolute number of total CD4+ T cells from MDR-TB patients (856 [725 to 1,218] cells/mm3) was similar to that from group N controls (1,061 [943 to 1,166] cells/mm3), a lower value was found for S-TB patients (527 [436 to 646] cells/mm3) (P < 0.05). In addition, absolute CD8+ T-cell counts in MDR-TB and S-TB patients (for MDR-TB patients, 462 [404 to 668] cells/mm3 [P < 0.05]; for S-TB patients, 405 [282 to 556] cells/mm3 [P < 0.01]) were lower than those in group N controls (688 [633 to 788] cells/mm3). The percentage of CD25+ cells was significantly increased within the total CD4+ T-cell population from TB patients compared to that from group N controls. In contrast, the proportions of CD25+ cells within CD8+ T cells were similar among TB patients and group N controls (Table (Table11).

TABLE 1.
Increased percentage of CD25+ Foxp3+ cells in CD4+ and CD8+ T cells from MDR-TB patients

The proportions of conventional activated CD4+ T cells and natural Treg cells were analyzed by discriminating low or high CD25 expression on the basis of CD25 mean fluorescence intensity within the CD4+ cell population. As shown in Table Table1,1, higher proportions of both CD25low and CD25high cells within CD4+ cells were detected in TB patients than in group N controls. The Foxp3 transcription factor is considered a molecular marker of natural Treg cells (15); hence, we evaluated its intracellular expression in CD4+ CD25high and CD8+ CD25+ T cells. A higher proportion of CD4+ CD25high Foxp3+ cells was observed in TB patients (Table (Table1);1); however, Foxp3 mean fluorescence intensities were similar for all groups (data not shown). Furthermore, the proportion of ex vivo Treg cells strongly correlated with antigen load in S-TB patients (Spearman test; r = 8,198; P = 0.0119), while no correlation was observed for MDR-TB patients. This difference may be ascribed to the fact that MDR-TB patients constituted a much more heterogeneous population in terms of previous duration of disease and anti-TB treatment. Recently, CD8+ Treg cells that are able to inhibit T-cell proliferation and cytokine production have also been described, and human CD8+ CD25+ Foxp3+ cells have been observed in adult PBMC and in neonatal thymus (8, 52). In line with this, we detected an increased percentage of CD8+ CD25+ Foxp3+ cells only in MDR-TB patients (Table (Table1).1). Altogether, our results indicate that CD4+ and CD8+ Treg cells are expanded in vivo in MDR-TB patients and confirm previous reports that detected CD4+ Treg cell expansion in S-TB patients (6, 18, 19, 46).

High levels of CD4+ CD25+ Foxp3+ Treg cells are induced by M. tuberculosis strains in MDR-TB patients.

We next wanted to determine whether local MDR strains were able to induce an in vitro expansion of CD4+ Treg cells. To do this, PBMC from MDR-TB and S-TB patients and group N controls were stimulated for 5 days with strain H37Rv, M, or Ra, and the proportion of CD25high Foxp3+ cells in the CD4+ T-cell population was evaluated. As shown in Fig. Fig.6,6, H37Rv, M, and Ra enhanced CD4+ CD25high Foxp3+ cells in TB patients and group N controls; however, MDR-TB patients showed higher percentages of H37Rv- and M-induced Treg cells than did group N controls. Thus, outbreak MDR strains M and Ra induce Treg cells, like H37Rv does, with the highest Treg cell expansion observed in MDR-TB patients.

FIG. 6.
Expansion of CD4+ CD25+ Foxp3+ cells is not dependent on M. tuberculosis strain stimulation. PBMC from 25 MDR-TB and 20 S-TB patients and 10 group N controls were cultured for 5 days alone (white bars) or with H37Rv (lightest gray ...

Treg cells suppress effector functions of PBMC from TB patients.

Finally, we evaluated whether circulating Treg cells suppressed M. tuberculosis-induced responses, such as cytokine production and CD107a degranulation, in TB patients. Depletion of CD25+ cells enhanced the percentages of H37Rv-, M-, or Ra-induced CD4+ IFN-γ+ and CD8+ IFN-γ+ cells (Fig. (Fig.7)7) and decreased the proportions of CD4+ IL-10+ and CD8+ IL-10+ cells (Fig. (Fig.8)8) in TB patients, whereas CD25 depletion did not modify the level of IL-4+ cells (data not shown). In addition, CD25 depletion also increased M. tuberculosis-induced CD8+ CD107+ T cells in TB patients (Fig. (Fig.9),9), suggesting that CD25+ Treg cells are functionally active in TB, upregulating IL-10 and inhibiting IFN-γ expression and CTL degranulation. In spite of CD25 depletion, degranulation induced by the M and Ra strains was still lower in MDR-TB patients than in S-TB patients and group N controls. Moreover, strain M induced lower CD107 expression than did H37Rv in MDR-TB patients (P < 0.05) and group N controls (P < 0.05), and Ra also induced less degranulation than did H37Rv in MDR-TB patients (P < 0.05), even in the absence of Treg cells.

FIG. 7.
CD25 depletion enhances IFN-γ expression on antigen-stimulated CD4+ and CD8+ T cells. PBMC from 10 MDR-TB and 8 S-TB patients and 6 group N controls were depleted of CD25+ cells by magnetic methods. PBMC (white bars) and ...
FIG. 8.
CD25 depletion decreases IL-10 expression on antigen-stimulated CD4+ and CD8+ T cells. PBMC (white bars) and CD25-depleted cells (gray bars) from 10 MDR-TB and 8 S-TB patients and 6 group N controls were cultured for 5 days with M. tuberculosis ...
FIG. 9.
CD25 depletion enhances CD107 expression on antigen-stimulated CD4+ and CD8+ T cells. PBMC from 10 MDR-TB and 8 S-TB patients and 6 group N controls were depleted of CD25+ cells by magnetic methods. PBMC (white bars) and CD25-depleted ...

DISCUSSION

The development of TB disease depends on a subtle balance between host genetic factors involved in susceptibility and resistance to TB following infection (9) and the ability of different M. tuberculosis genotypes to induce a strong or a weak Th1 protective immune response (30, 41, 42, 51). Th1 responses, such as IFN-γ production and CTL activity, have been associated not only with bacterial growth control and lysis of infected macrophages but also with tissue damage, as observed in TB.

Herein we showed that our outbreak MDR strains M and Ra, and even the nonprosperous MDR strain 410, induced lower IFN-γ expression than H37Rv did in CD4+ cells from healthy PPD+ individuals. This result may be related to the lack of host selective pressure suffered by the laboratory strain H37Rv. Low in vitro Th1 responses to different M. tuberculosis antigens have been observed in MDR-TB patients, such as impaired IFN-γ production (4, 16, 25, 26, 33) and increased IL-4 production by CD4+ T cells stimulated by a lipid extract of H37Rv (50). Likewise, in our study, both MDR-TB and S-TB patients showed an impaired expression of IFN-γ in CD4+ and CD8+ T cells, irrespective of the tested strain; therefore, this might be ascribed to the altered Th1/Th2 profile characteristic of advanced disease (13). In contrast to the work of McDyer et al. (33), we found diminished IFN-γ expression in MDR-TB patients, in spite of their normal CD4+ counts of >500/μl. Moreover, high IL-4 levels were observed in CD4+ and CD8+ cells from MDR- and S-TB patients, with M being the best inducer, suggesting that this strain exploits and enhances the preexisting tendency of TB patients to mount a Th2 response. Moreover, the fact that patients infected with strain M showed more CD4+ and CD8+ IL-4+ cells highlights the peculiar ability of this strain to bias the Th1 response through IL-4 induction. High IL-4 levels have been associated with progression from latent infection to active disease (35, 54), advanced radiological disease, and cavitary TB (49, 57). In this context, IFN-γ deficiency in TB patients could be due to a shift to a Th2 cytokine profile, undermining the efficacy of Th1-mediated immunity and causing immunopathology (47). Similarly, IL-4 and IL-13 were found to be induced in human monocytes by virulent Beijing strains (30), and differences in Th1/Th2 profile have been observed in healthy individuals with single-copy IS6110-carrying strains from South India (41, 42) and in TB patients infected with non-Beijing or Beijing strains (56). Hence, the overall T-cell response to M. tuberculosis strains seems to be dependent on the host ability to mount Th1/Th2 responses and the potential of each strain to increase the Th2 cytokine profile in susceptible individuals.

CTL activity has been associated with lysis of M. tuberculosis-infected macrophages and direct killing of mycobacteria (12, 55). Here we observed that MDR- and S-TB patients failed to induce degranulation of CD8+ cells and to evoke a CTL response, likely due to the high Th2 profile induced in CD4+ and CD8+ cells from these patients. In line with this, a gradual loss of CD8-mediated CTL activity against autologous H37Rv-pulsed macrophages dependent on IL-10 production has been observed in patients with active S-TB (10-12), and modulation of Mycobacterium leprae Hsp65-induced CTL activity by IL-10 and IL-4 has also been observed in leprosy patients (11). In addition, IL-4 leads to development of a CD8+ T-cell subset that fails to upregulate granzyme B, a potent apoptosis-inducing protease of CTLs (44). A Th2 cytokine profile is a suitable explanation for the weak cytotoxic activity detected in MDR- and S-TB patients but does not account for the inability of strain M to induce cytotoxicity in healthy controls. To shed further light on this finding, we examined another MDR-TB strain with a close genetic link with M, but which is epidemiologically incompetent, namely, strain 410. In healthy controls, these two highly related Haarlem strains showed similar IL-10, IL-4, and IFN-γ expression patterns; however, they differed in cytotoxic activity. Thus, CTL impairment is a hallmark of strain M, and deficient lysis of infected macrophages could be one of multiple factors involved in strain M fitness. Current studies are being performed to evaluate the mechanisms employed by strain M to hamper the CTL response.

It has been speculated that Treg cells might play important regulatory functions in the immune response (48). Herein, we observed an increase in circulating CD4+ CD25high Foxp3+ cells in MDR-TB and S-TB patients, as previously demonstrated (6, 18, 19, 46). In addition, we also showed an increase in CD8+ CD25+ Foxp3+ cells in MDR-TB patients, although their levels were threefold lower than those of CD4+ Treg cells. Accordingly, a CD8+ regulatory T-cell subset was recently described as capable of mediating suppression of Mycobacterium bovis BCG-induced PBMC proliferation in PPD+ healthy individuals and in M. tuberculosis-infected lymph nodes and tonsils (22). Furthermore, as demonstrated with heat-killed strain Erdman and BCG (17, 20), strains M and Ra expanded Treg cells in PBMC from MDR-TB and S-TB patients and healthy tuberculin reactors, like H37Rv did, showing that this expansion is not M. tuberculosis genotype dependent. The marked Treg cell expansion observed in MDR- and S-TB patients could be related to the high IL-4 levels induced by M. tuberculosis, which is in accordance with CD4+ CD25+ Treg cell induction from CD25 peripheral blood T cells by IL-4 (53). Treg cell depletion decreased IL-10 and enhanced IFN-γ levels in CD4+ and CD8+ T cells from TB patients, suggesting that Treg cells may suppress effector functions. Indeed, Treg cell depletion also enhanced CD107+ CD8+ cells in MDR- and S-TB patients, as also observed in mice (34). Furthermore, low M- and Ra-induced CD107 expression and IFN-γ upregulation were still observed in MDR-TB patients in the absence of Treg cells, suggesting that additional factors could inhibit the Th1 response. Despite the presence of Treg cells, high proportions of CD4+ and CD8+ IL-4+ cells were induced by M. tuberculosis strains in MDR-TB patients; therefore, it is likely that this cytokine could interfere with CTL differentiation or functionality. In line with this, Th2 T-cell clones have been shown to be less susceptible to the suppressive activity of CD25+ regulatory thymocytes (7). The fact that Treg cell depletion did not restore either Ra- or M-induced CD107 to group N levels in MDR-TB patients suggests that the impairment of CTL activity and cytokine production could not be ascribed exclusively to Treg cell suppression.

In summary, we have demonstrated an increased frequency of circulating Treg cells in MDR-TB patients that are expanded in vitro by M. tuberculosis stimulation independent of the strain and that suppress antigen-induced IFN-γ and CTL responses. Outbreak MDR strain M is a weak IFN-γ inducer. Remarkably, the inability to develop CTL activity is a hallmark of strain M, regardless of its ability to induce IFN-γ in CD8+ cells in healthy PPD+ controls. Hence, impairment of CTL activity may be an evasion mechanism employed by strain M to avoid macrophage lysis by antigen-specific CTLs. Altogether, these results suggest that immune responses to M and Ra outbreak strains are the result of the host's ability to mount an efficient immune response and the potential of different M. tuberculosis genotypes to drive Th1 or Th2 profiles.

Acknowledgments

This work was supported by grants from the Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT; grant 05-38196), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET; grant PIP 6170/05), the European Commission (FP7 grant 201690), and Fundación Alberto J. Roemmers.

Notes

Editor: J. L. Flynn

Footnotes

[down-pointing small open triangle]Published ahead of print on 31 August 2009.

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