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Lack of CD27^-CD45RA^-V9V2⁺ T Cell Effectors in Immunocompromised Hosts and During Active Pulmonary Tuberculosis¹

Cristiana Gioia^*, Chiara Agrati^*, Rita Casetti^*, Cristiana Cairo, Giovanna Borsellino, Luca Battistini, Giorgio Mancino, Delia Goletti^*, Vittorio Colizzi, Leopoldo P. Pucillo^* and Fabrizio Poccia^2,*

^* Laboratory of Clinical Pathology-Immunopathology, "Padiglione Del Vecchio," National Institute for Infectious Diseases, "Lazzaro Spallanzani," Istituto di Ricerca e Cura a Carattere Scientifico, Rome, Italy; and Research Center, "San Pietro-Fatebenefratelli," Laboratory of Neuroimmunology, "Santa Lucia Foundation," Istituto di Ricerca e Cura a Carattere Scientifico, and Department of Biology, University of Rome, "Tor Vergata," Rome, Italy

	Abstract

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In humans, the circulating pool of mycobacteria-reactive V9V2⁺ T cells is expanded with age and may contribute to Mycobacterium tuberculosis immunosurveillance. We observed that two subsets of V9V2⁺ T cells could be identified on the basis of CD27 expression in immunocompetent adults, showing that functionally differentiated T cells have lost CD27 expression. In contrast, the CD27^-CD45RA^-V9V2⁺ T cell subset of effector cells was absent in cord blood cells from healthy newborns and lacking in the peripheral blood from HIV-infected patients. Moreover, circulating V9V2⁺ T cell effectors were significantly reduced in patients with acute pulmonary tuberculosis, resulting in a reduced frequency of IFN--producing cells after stimulation with nonpeptidic mycobacterial ligands. These observations indicate that monitoring and boosting T cell effectors could be clinically relevant both in immunocompromised hosts and during active tuberculosis disease.

	Introduction

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Tuberculosis (TB)³ is one of the leading causes of morbidity and mortality worldwide. After decades of declining incidence, the number of infected individuals is increasing once again with the spread of multidrug-resistant Mycobacterium tuberculosis (MTB). Anti-MTB immunity depends on the interaction of peptide-specific CD4 T lymphocytes with macrophages, although several studies indicate that T lymphocytes reactive with nonpeptidic compounds are important for TB immunosurveillance (1, 2, 3). The complex array of cell surface lipids and lipoglycans is characteristic of mycobacteria and triggers responses of a subpopulation of T cells capable of recognizing Ags bound to the CD1 protein on the surface of presenting cells (4). A second class of MTB nonpeptidic Ags is used by a great variety of pathogens as intermediates of isoprenoids biosynthesis (5). A variety of these natural and synthetic metabolites were described, such as TUBAg-1 and isopentenyl-pyrophosphate, that were first isolated from mycobacteria (6, 7), other natural and synthetic phosphocarbohydrates (8, 9), and alkylamines and aminobiphosphonates (10, 11). These compounds directly trigger T cells expressing the V9V2⁺ TCR without the need for Ag processing and presentation. However, functionally mature V9V2⁺ T cells display cell surface inhibitory receptors for MHC class I molecules that may control TCR-mediated reactivities against conserved self Ags and exogenous mycobacterial ligands (12).

V9V2⁺ T cells are rare in the adult thymus but increase with age in the blood, suggesting a positive selection in the periphery consecutive to a sustained antigenic stimulation (13). Accordingly, the large majority of circulating V9V2⁺ T lymphocytes express the CD45RO⁺CD95⁺ effector/memory phenotype (14). In children with TB disease, the proliferative V9V2⁺ T cell response is highly increased in comparison with age-matched tuberculin-negative controls (15). A few months after chemotherapy, the increased responsiveness of T cells sharply declines close to the levels detected in healthy tuberculin-negative children, indicating that persistent exposure to mycobacterial Ags is required for T cell hyperactivity. Moreover, a quantitative and qualitative alteration of T cell subsets was previously observed during HIV infection (14, 16, 17).

CD27 is a 120-kDa transmembrane homodimeric molecule expressed on the majority of T cells, B cells, and NK cells that belongs to the TNF/nerve growth factor receptor family. The interaction between CD27 and its ligand CD70 induces costimulatory signals in naive T cells (18). It was reported that activation of T cells induces a transient increase of CD27 expression that gradually switched off (19). In the present study, we demonstrated that CD27^-V9V2⁺ T lymphocytes are functionally differentiated cells that have lost CD27 expression. Moreover, a lack of V9V2⁺ T cell effectors was observed in immunocompromised hosts, such as newborn-derived cord blood cells or HIV-infected adults, and during active pulmonary TB disease. Altogether, our results indicate that 1) monitoring T cell effectors is clinically relevant and 2) boosting T cell effectors could be useful for vaccine strategies aimed to improve the immune response in immunocompromised hosts and during active TB disease.

	Materials and Methods

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Patients and control donors

Peripheral blood samples were obtained from a total of 15 patients with first diagnosis of pulmonary TB infection, seven patients with HIV disease and ongoing opportunistic infections, and eight patients with TB and HIV coinfection. All patients were recruited from the National Institute for Infectious Diseases, Lazzaro Spallanzani (Rome, Italy). Healthy donors (HD; n = 11) were used as normal controls. The study was approved by the local Ethical Committee of the Spallanzani Institute (Rome, Italy) and peripheral blood samples from each patient and healthy volunteer were obtained upon informed consent. Umbilical cord blood cells (n = 6), accompanied by informed consent of the mothers, were obtained from normal full-term pregnancies by venipuncture of umbilical vein immediately after delivery. Cord blood cells were collected at the "San Pietro Hospital." In TB patients, clinical presentation and chest radiographs were compatible with pulmonary TB and sputum was positive for acid fast bacilli. TB patients were treated with chemotherapy consisting of isoniazid (5 mg/kg), rifampicin (10 mg/kg), ethambutol (15–25 mg/kg), and pyrazinamide (15–30 mg/kg). In addition, no corticosteroids or immunosuppressive agents were administered. All patients responded to the treatment and after 2–6 mo had negative acid fast bacilli smear, negative TB culture, and absence of clinical symptoms. At this stage the patients were classified as TB inactive.

All the HIV⁺ patients with MTB coinfection presented pulmonary TB (n = 8). These patients were treated with the same anti-TB chemotherapy described above to treat TB patients without HIV infection. No highly active antiviral therapy was performed by these patients, with the exception of one. CD4 cell counts of HIV⁺TB⁺ patients were between 47 and 300 cells/mmc. HIV⁺ patients used as control for the HIV⁺TB⁺ patients presented the following opportunistic infections at the time of the study: CMV retinitis (four patients), neurotoxoplasmosis (one patient), CMV encephalitis (one patient), and isosporiasis (one patient). Three of the seven patients of this group were under highly active antiviral therapy and CD4 cell counts of HIV⁺ TB⁺ patients were between 4 and 219 cells/mmc.

Cell preparation and stimulation

Peripheral and cord blood mononuclear cells were isolated from heparinized blood by Ficoll-Hypaque (Pharmacia Biotech, Uppsala, Sweden) and cultured at 1.5 x 10⁶ cells/ml in complete medium (RPMI 1640, 10% v/v heat-inactivated FCS, 2 mM L-glutamine, 10 U/ml penicillin/streptomycin). PBMC from control donors or HIV⁺ patients were stimulated in vitro for 10 days in the presence of 100 µM isopentenyl pyrophosphate (IPP; Sigma-Aldrich, St. Louis, MO) and 100 U/ml rIL-2 (Boehringer Mannheim, Mannheim, Germany). After 1 wk of culture, the volume corresponding to half-culture supernatant was replaced by complete medium with rIL-2. The expansion of V9V2⁺ T cells after 10 days of culture was determined by cytometric analysis using double staining with anti-CD3 and anti-TCR-V2 mAbs coupled to PE or FITC, respectively. V2 expansion index was calculated dividing the absolute number of V2⁺ T cells in stimulated cultures by the absolute number of V2⁺ T cells in unstimulated cultures (20, 21). The IPP-driven PBMC proliferation assay was performed in triplicate in 96-well flat-bottom plates with 2.5 x 10⁵ PBMC/ml in 0.2 ml containing 5 U/ml rIL-2 with or without 100 µM IPP. Proliferation was measured after 5 days by pulsing cultures for 6 h with [³H]thymidine (1 µCi/well; Amersham, Uppsala, Sweden). Cells were then harvested and [³H]thymidine incorporation was measured with a liquid scintillation counter (Wallac 1450 Microbeta; PerkinElmer, Boston, MA). Stimulation index (S.I.) was calculated as follows: cpm from IPP plus rIL-2-stimulated cultures divided by cpm from rIL-2-stimulated cultures. S.I. > 2 was considered significant. CD27⁺ and CD27^- T cells subsets were sorted using a MoFlo cell sorter (Cytomation, Fort Collins, CO).

mAbs and flow cytometry

mAbs coupled with FITC, PE, phycoerythrin-cyanin 5.1, and allophycocyanin were combined for simultaneous staining. The anti-human Abs used in this study were the following: anti-CD27 PE (IgG1, clone M-T271); anti-CD45RA CyChrome (IgG2b, clone HI100); anti-CD45RO allophycocyanin (IgG2a, clone UCHL-1); anti-CD95 allophycocyanin (IgG1, clone DX2); purified anti-CCR7 (IgM, clone 2H4) that was detected using biotin-conjugated rat anti-mouse IgM (IgG2a, clone R6-60.2) and streptavidin PE; anti-IFN- allophycocyanin mAb (IgG1, clone B27). All the previously described mAbs were from BD Biosciences (Mountain View, CA). The anti-V2 mAb FITC (IgG1, clone IMMU1464) was purchased by Immunotech (Marseille, France). Isotype-matched control mAbs from BD Biosciences were used in all experiments.

Analysis of surface Ag expression was performed as previously described (14). Briefly, 5 x 10⁵ PBMC were washed in PBS containing 1% BSA and 0.1% sodium azide and were incubated for 15 min at 4°C with the indicated FITC-, PE-, PE-cyanin 5.1-, and allophycocyanin-conjugated mAb. Samples were fixed in PBS/1% paraformaldehyde and immediately acquired with a FACSCalibur flow cytometer (BD Biosciences). A total of 20,000 events was acquired for each sample and analyzed with CellQuest software (BD Biosciences).

Single-cell analysis of cytokine synthesis

Cytokine production was detected by flow cytometry analysis as previously described (18). Human PBMC were stimulated for 6 h with IPP (100 µM; Sigma-Aldrich) and/or 100 U/ml rIL-2 (Boehringer Mannheim). Brefeldin A (10 µg/ml) was added 1 h after stimulation to block intracellular transport allowing cytokine accumulation in the Golgi. Cells were washed twice in PBS, 1% BSA, and 0.1% sodium azide and stained with mAb specific for the membrane Ags described above for 15 min at 4°C. Samples were then fixed in 1% paraformaldehyde for 10 min at 4°C, incubated with anti-IFN- mAb diluted in 1x PBS, 1% BSA, and 0.5% saponin. The cells were finally washed twice in 1x PBS, 1% BSA, 0.1% saponin, and acquired on a FACSCalibur (BD Biosciences). Control for nonspecific staining was monitored with isotype-matched mAbs and nonspecific staining was always subtracted from specific results.

Statistical analysis

Differences among group means were evaluated by Mann-Whitney test. Values of p < 0.05 were considered significant.

	Results

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Naive and effector/memory T cell subsets in HD and TB patients

The distribution of effector/memory T cell subsets was analyzed by flow cytometry both in HD and in patients with acute pulmonary TB disease. We did not observe significant differences in the percentage of circulating V9V2⁺ T cells in HD vs TB patients (2.4 ± 1.4% vs 2.0 ± 1.4%). In HD, the frequency of V9V2⁺ T cells was lower in cord blood when compared with adult peripheral blood (0.4 ± 0.2% in cord blood vs 2.3 ± 1.5% in the mother’s blood), confirming the expansion of this subset with age (13). The phenotype of the majority of cord blood T cells was CD45RA⁺ and CCR7⁺, indicating a prevalence of naive T cells (Table I). In contrast, the prevalent phenotype of adult V9V2⁺ T cells was CD45RO⁺ and CCR7^- independently of TB disease, indicating that the circulating pool of V9V2⁺ T cells is mainly composed of memory cells. Interestingly, we observed an increased expression of CD95 (Fas Ag) on V9V2⁺ T cells from persons with TB disease, which may reflect an increased susceptibility to apoptosis (22). These observations indicate that V9V2⁺ T cells are expanded with age, acquiring a memory phenotype. Moreover, TB disease induces a priming for apoptosis of the memory T cells.

View this table: [in this window] [in a new window]   Table I. Phenotype of circulating V9V2+ T cells in HD and TB patients1

CD27 expression can discriminate between T cell effector/memory functions which are altered during TB disease

We then analyzed the expression of CD27, a costimulatory molecule belonging to the TNF/nerve growth factor receptor family which is lost following persistent antigenic stimulation marking out mature T cell effectors (19). In adult HD more than half of V9V2⁺ T cells present the CD27⁺ phenotype (56.7 ± 6.3; Fig. 1A). Interestingly, sorted CD27⁺V9V2⁺ T cells displayed a higher clonogenic potential but a reduced capacity to secrete IFN- when stimulated with the nonpeptidic mycobacterial Ag IPP (Fig. 1B). Therefore, two primed V9V2⁺ T cell subsets may develop in humans in response to Ag stimulation: a subpopulation of CD27⁺ memory T cells and a subset of CD27^- T cell effectors. In contrast, the large majority of cord blood V9V2⁺ T cells from HD are CD27⁺ (87.8 ± 2.7%; Fig. 1C), indicating that T cell effectors are absent in cord blood. Similarly, in patients with active TB disease, a main fraction of memory CD27⁺V9V2⁺ T lymphocytes was observed (72 ± 2.6%; Fig. 1D), similar to the data obtained in cord blood cells (Fig. 1C). Accordingly, the proliferative activity of V9V2⁺ T cells was significantly increased in TB patients (p < 0.05; Fig. 2). In parallel, V9V2⁺ T cell effectors were significantly reduced in TB patients, and this reduction was associated with a diminished ability to secrete IFN- in response to specific stimulation with IPP (p = 0.001; Fig. 2).

View larger version (50K): [in this window] [in a new window]   FIGURE 1. CD27 expression can discriminate between T cell effector/memory functions. The frequency of effectors (CD27-) and memory (CD27+) V2+ T lymphocytes was analyzed by flow cytometry. The percentage of these cell subsets from a representative adult HD (A), cord blood (C), and TB+ patient (D) is shown. Moreover, the ability of sorted CD27+V2+ and CD27-V2+ T cells to proliferate in response to IPP stimulation was analyzed (B). The proliferation activity was analyzed after 5 days of IPP stimulation. On the last day of culture, cells were pulsed with [3H]thymidine for 6 h and harvested. Results are shown as S.I. Production of IFN- was determined at single-cell level by intracellular staining with specific mAb after IPP stimulation. CD27+ and CD27- V2 T cell subset were discriminated by surface staining. The percentage of IFN--producing V2+ T cells in CD27+ and CD27- subsets was evaluated after 6 h of IPP stimulation in the presence of brefeldin A. Results are expressed as arithmetic mean percentage ± SE of IFN-+V2+ T cells (open bars) and V2 S.I. (hatched bars). Significance refers to Mann-Whitney test. Values of p are indicated as follows: *, p < 0.05 (CD27+ vs CD27- T cells).

View larger version (37K): [in this window] [in a new window]   FIGURE 2. Altered T cell effector/memory functions during MTB infection. The percentage of IFN--producing V2+ T cells and the V2 expansion index after IPP stimulation were evaluated in PBMC from TB patients and from HD. Results are expressed as arithmetic mean percentage ± SE of IFN-+V2+ T cells (open bars) and V2 expansion index (hatched bars). IFN- production was determined at single-cell level by flow cytometry as described in Fig. 1. The expansion of V9V2 T cells was analyzed after 10 days of culture by flow cytometry. Results are shown as expansion index (E.I.). Significance refers to Mann-Whitney test. Values of p are indicated as follows: *, p < 0.05 (TB vs HD).

Lack of IFN--producing CD27^-CD45RA^-V9V2⁺ T cell effectors in patients with active TB disease and in immunocompromised hosts

Because CD27^-V2⁺ includes not only memory CD45RA^- but also CD45RA⁺ cells, we analyzed the frequency of effector/memory CD27^-CD45RA^-V9V2⁺ T cells in patients with active pulmonary TB disease or after successful clinical treatment with chemotherapy (TB inactive). A reduced effector function was particularly evident in patients with active TB disease (Fig. 3A) where the V9V2⁺ T cell ability to secrete IFN- was drastically compromised (p = 0.03; Fig. 3B). Interestingly, this decline in cytokine production was restored in patients with inactive TB after successful chemotherapy (Fig. 3). The frequency of CD27^-CD45RA^-V9V2⁺ T cell effectors was dramatically reduced not only during acute TB disease but also in HIV-infected persons (p = 0.02; Fig. 4). Moreover, these effector T cell subsets were physiologically absent in cord blood cells from healthy newborns (p = 0.005; Fig. 4). These results indicate that a lack of CD27^-V9V2⁺ T cell effectors may contribute to the increased susceptibility to TB in the immunocompromised host.

View larger version (21K): [in this window] [in a new window]   FIGURE 3. Lack of CD27-CD45RA-V2+ T cell effectors in patients with active TB disease. A, The percentage of CD27-CD45RA-V2+ T cell effectors was monitored in PBMC from TB patients presenting active (•) or inactive () TB disease and from HD (). Finally, the frequency of IFN--producing V2+ T cells was analyzed in the same groups of patients (B). Results are expressed as arithmetic mean percentage ± SE. Each dot represents one individual. Significance refers to Mann-Whitney test. Values of p are indicated as follows: *, p < 0.05.

View larger version (35K): [in this window] [in a new window]   FIGURE 4. Lack of T cell effectors in immunocompromised hosts. The frequency of CD27-CD45RA-V2+ T cell effectors was compared in PBMC from TB patients, HIV/TB-coinfected persons, HIV+ patients, and cord blood and PBMC from HD. Results are expressed as arithmetic mean percentage ± SE. Significance refers to Mann-Whitney test. Values of p are indicated as follows: *, p < 0.05.

	Discussion

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In the murine model of TB, evidence has been provided for a role of T cells in the control of TB (23) and in the formation of granulomatous lesions (24). However, the effective role of murine T cells in TB immunity is still controversial. Human T cells are stimulated in vitro by a range of microorganisms, including mycobacteria (12, 25). Specifically, V9V2⁺ T lymphocytes were shown to reduce the viability of intracellular MTB by perforin-mediated killing of infected macrophages (26). Whereas some studies have reported an increase of T cell numbers in the peripheral blood of TB patients, other reports suggest that this population is constant during TB disease (27, 28, 29, 30, 31). Recently, B. Li et al. (28) reported a reduced number of the mycobacteria-reactive V9V2 T cell subset in both the blood and lungs of patients with pulmonary TB. In our study, we observed no significant variation in the number of circulating V9V2⁺ T lymphocytes in TB patients when compared with HD.

A higher expression of LFA-1, very late Ag-4, and ICAM-1 on T cells from TB patients has been previously shown (31). This surface expression pattern indicates activation and readiness for extravasation. Human naive and memory T cells can be distinguished according to their expression of CD45RA and CD45RO isoforms (32). However, CD45RO isoforms can back-revert to CD45RA, whereas CD45RO can be induced by cytokines (33). Thus, the lack of the CD95 (Fas) marker was recently proposed to identify the population of naive T cells (34). In healthy adults, we observed that the phenotype of circulating V9V2⁺ T cells is mainly composed of CD45RO⁺ and CD95⁺ memory cells. Among CD95⁺ T cells, some have lost the expression of CD27 costimulatory molecules and lack the proliferative potential of memory cells. In contrast, memory CD27⁺ T cells are not cytolytic without further activation but show a higher clonogenic potential than effector cells (35, 36). Accordingly, we observed that a subpopulation of CD95⁺CD27⁺ memory T cells and a subset of CD95⁺CD27^- T cell effectors may develop in response to Ag stimulation. Moreover, we observed that in vitro culture in the presence of IL-2 was able to induce a sustained down-regulation of CD27 expression on V2⁺ T cells (data not shown), confirming that primed T cells cultured in IL-2 become effector cells (37).

CMV-specific memory T cells lose the expression of CD27 glycoprotein on their surface, and this loss is thought to mark out mature effector cells (36). In contrast, T cells from TB patients retain the expression of CD27 molecules, suggesting that MTB-specific V9V2⁺ T cells in vivo may be immature rather than end-stage effectors as first thought. Accordingly, CD27⁺V9V2⁺ T lymphocytes showed a higher clonogenic potential than CD27^- effectors. In patients with acute pulmonary TB, we observed a significant increase of CD27⁺V9V2⁺ T cells resulting in enhanced proliferative activity to the stimulation with nonpeptidic mycobacterial ligands. This is consistent with previous observations indicating an increased proliferative activity of MTB-reactive T cells in TB patients with protective and resistant immunity (30), in children with primary TB infection (15, 39), and in healthy persons vaccinated with bacillus Calmette-Guérin (40). In parallel, because the pool of CD27^-CD45RA^-V9V2⁺ T cells was reduced during active TB, we found a lack of IFN--producing T cells upon stimulation with nonpeptidic mycobacterial ligands. Accordingly, several reports have shown that both in adult TB patients and in children with primary TB disease, MTB-stimulated PBMC produce reduced amounts of IFN- when compared with healthy tuberculin reactors (41, 42). We observed an increased frequency of CD95-expressing V9V2⁺ T cells in patients with active TB, suggesting the involvement of CD95/CD95 ligand (CD95L) pathway in the loss of T cell effectors during mycobacterial infection. MTB Ag stimulation rapidly induces CD95 and CD95L expression by T cells in vitro, inducing apoptosis in a large proportion of peripheral V9V2⁺ T cells from healthy subjects and TB patients (22). The engagement of the TCR by nonpeptidic mycobacterial ligands induces the expression of CD95L by chronically activated CD95⁺V9V2⁺ T cells (43). These effector cells are transiently attracted at the site of infection by the response toward the pathogen and may influence the mature T cell response and the ensuing granulomatous disease.

Altogether, our observations of disease-specific changes in T cell function demonstrate a correlation between T cell effector functions and manifestations of disease, consistent with the hypothesis that these T cells play a role in the protective immune response to MTB infection. Although CD4⁺ T cells remain the dominant and critical T cell subset in protection against TB, T cells appear to have an important complementary role which may be primarily expressed in patrolling the blood circulation. This "sentinel function" may allow the rapid recognition of microbial phosphometabolites released by pathogens entering the blood stream, promoting the inflammatory reaction and the activation of Ag-specific lymphocytes (44). HIV infection markedly increases susceptibility to TB (45), and TB in HIV-infected patients accelerates progression of immunodeficiency (46). Standard vaccination strategies are particularly problematic because they result in activation of CD4⁺ cells, which are the major reservoir of HIV. Thus, new vaccination strategies could be directed to augment the effector response of T cells, representing a novel tool to provide protection against TB in HIV-infected patients while minimizing the risk of enhancing HIV replication (47). In conclusion, monitoring T cell effectors is clinically relevant both in immunocompromised hosts and during active TB disease, indicating that targeting of T cells with nonpeptidic vaccines may be used to improve the immune response.

	Acknowledgments

 
We thank the Gynecology and Obstetric Division and the Service of Immunohematology of the "San Pietro Hospital" and G. P. Brunetti, L. De Leo, and E. Di Pietro for the help in cord blood collection.

	Footnotes

 
¹ This study was supported by grants from the Ministero della Salute and from the European Union TB Cluster (QLK2-CT-1999-01093).

² Address correspondence and reprint requests to Dr. Fabrizio Poccia, Laboratory of Clinical Pathology-Immunopathology, Padiglione Del Vecchio, National Institute for Infectious Diseases, Lazzaro Spallanzani, Institute for Cancer Research and Treatment, Via Portuense 292, 00149 Rome, Italy. E-mail address: immunol{at}spallanzani.roma.it

³ Abbreviations used in this paper: TB, tuberculosis; HD, healthy donor; IPP, isopentenyl pyrophosphate; MTB, Mycobacterium tuberculosis; S.I., stimulation index; CD95L, CD95 ligand.

Received for publication September 5, 2001. Accepted for publication November 26, 2001.

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