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Characterization of Lung T Cells Following Intranasal Infection with Mycobacterium bovis Bacillus Calmette-Guérin¹

Francesco Dieli^2,*, Juraj Ivanyi, Philip Marsh, Ann Williams, Irene Naylor, Guido Sireci^*, Nadia Caccamo^*, Caterina Di Sano and Alfredo Salerno^*

^* Department of Biopathology, University of Palermo, Palermo, Italy; King’s College London at Guy’s Dental and Medical School, London, United Kingdom; Center for Applied Microbiology and Research, Salisbury, United Kingdom; and Institute of Advanced Diagnostic Methodologies, National Research Council, Palermo, Italy

	Abstract

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The lungs are considered to have an impaired capacity to contain infection by pathogenic mycobacteria, even in the presence of effective systemic immunity. In an attempt to understand the underlying cellular mechanisms, we characterized the T cell population following intranasal infection with Mycobacterium bovis bacillus Calmette-Guérin (BCG). The peak of T cell expansion at 7 days postinfection preceded the 30 day peak of T cell expansion and bacterial count. The expanded population of T cells in the lungs of BCG-infected mice represents an expansion of the resident V2 T cell subset as well as an influx of V1 and of four different V gene-bearing T cell subsets. The T cells in the lungs of BCG-infected mice secreted IFN- following in vitro stimulation with ionomycin and PMA and were cytotoxic against BCG-infected peritoneal macrophages as well as against the uninfected J774 macrophage cell line. The cytotoxicity was selectively blocked by anti- TCR mAb and strontium ions, suggesting a granule-exocytosis killing pathway. Depletion of T cells by injection of specific mAb had no effect on the subsequent developing CD4 T cell response in the lungs of BCG-infected mice, but significantly reduced cytotoxic activity and IFN- production by lung CD8 T cells. Thus, T cells in the lungs might help to control mycobacterial infection in the period between innate and classical adaptive immunity and may also play an important regulatory role in the subsequent onset of T lymphocytes.

	Introduction

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It is believed that the majority of infected individuals control primary infection with Mycobacterium tuberculosis (M. tuberculosis) without progressing to clinical disease; nevertheless, tuberculosis causes about 3 million deaths annually (1). Recruitment and activation of T cells are critical steps for protective immunity to M. tuberculosis. Studies in human and animal models have established that CD4⁺ and CD8⁺ T cells contribute to the cellular response to M. tuberculosis (2, 3, 4, 5, 6, 7). As a result, tubercle bacilli are contained within developing granulomas, and although most bacteria are destroyed, a small number of bacilli persist and may cause reactivation disease. During the last years, anti-mycobacterial immune responses in peripheral lymphoid organs have been characterized in detail. However, the predominant natural infection route with mycobacteria is through the respiratory tract. Therefore, the characterization of immune responses occurring at the site of infection, the lungs, is of primary importance. This is necessary not only to understand the biology of host defenses to M. tuberculosis, but also for vaccine development.

Primary infection of mice with M. bovis bacillus Calmette-Guérin (BCG),³ the vaccine strain used in humans, initially results in mycobacterial growth, followed by control and near-complete clearance of organisms from the lungs (8). This model mimics the control of primary M. tuberculosis infection in humans and therefore represents a good model to analyze the development of the immune response in the lungs. Although some studies have recently begun to characterize immune responses in the lungs of mice infected with BCG (9, 10, 11, 12, 13), very few data are currently available about the possible role played by T cells (14).

In this study we have analyzed the molecular and functional properties of T cells in the lungs of mice infected with BCG.

	Materials and Methods

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Mice and bacteria

C57BL/6 mice were purchased from OLAC through Nossan (Correzzana, Italy). Mice were fed and kept under specific pathogen-free conditions and were used at 8–12 wk of age. In each experiment, age- and sex-matched mice were used. In some experiments mice were injected with 500 µg of anti-TCR mAb (UC7-13D5), anti-TCR mAb (H57-597), or hamster IgG as a control (all gifts from Dr. G. L. Asherson, Clinical Research Center, Harrow, U.K.) 2 days before BCG infection and thereafter every 2 wk.

BCG (strain Pasteur) was grown in Middlebrook 7H9 broth base (Difco, Detroit, MI) supplemented with 10% Bacto Middlebrook OADC enrichment (Difco) for 2 wk at 37°C, and aliquots were frozen at -70°C until used. The final concentration of viable bacteria was enumerated by plate counts of CFU with Middlebrook 7H10 agar (Difco) supplemented with 0.5% glycerol (Difco) and 10% Bacto Middlebrook OADC enrichment (Difco). Mice were infected intranasally (i.n.) with 10⁶ viable bacteria in 0.02 ml of saline or with saline alone as a control under light anesthesia.

Preparation of lung mononuclear cells and flow cytometry

Lungs were removed and digested in the presence of collagenase (200 U/ml; Sigma-Aldrich, St. Louis, MO), and lung mononuclear cell suspensions were obtained through Lympholyte M (Cedarlane Laboratories, Ontario, Canada) gradient centrifugation. The viability of cells, as determined by trypan blue exclusion, was >90%. Lung mononuclear cells (5 x 10⁵) were incubated for 10 min on ice with PBS containing 5% BSA. After washing, cells were incubated with FITC-conjugated anti CD3 mAb and PE-conjugated anti- or anti- mAbs (all from BD PharMingen, San Diego, CA) for 45 min on ice. Cells were then washed in PBS containing 0.1% NaN₃ and were analyzed with a FACScan flow cytometer (BD Biosciences, Mountain View, CA). Viable lymphocytes were gated by forward and side scatter, and analysis was performed on 100,000 acquired events for each sample.

In some experiments lung mononuclear cells were enriched in T cells by passage through a nylon wool column, and then CD4, CD8 or T cells were sorted (15) by specific Abs and immunomagnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) following the manufacturer’s instructions. The cells were incubated for 24 h at 37°C in complete medium to allow cells and beads to dissociate. The bead-adherent population contained >90% positive cells, and the viability of the cell population exceeded 90% as determined by trypan blue exclusion.

Proliferative response and cytokine production

T cell-enriched lung cells (10⁵) were cultured in a 96-well, flat-bottom plate (Nunc, Copenhagen, Denmark) with 5 x 10⁵ irradiated (3000 rad from a cesium source) syngeneic or allogeneic spleen cells, as APCs, at 37°C in 5% CO₂ in supplemented RPMI 1640 medium (Life Technologies, Grand Island, NY) in the presence of M. tuberculosis H37Ra sonicate (Difco; 50 µg/ml final concentration) or purified protein derivative (PPD; Statens Seruminstitut, Copenhagen, Denmark; 10 µg/ml final concentration). Alternatively, cells were stimulated by Con A (Sigma-Aldrich; 1 µg/ml final concentration). All cultures were performed in triplicate, and nonstimulated wells served as controls. [³H]Thymidine incorporation by proliferating cells was estimated 72 h later, and the results are expressed as counts per minute.

To assess cytokine production, lung T cell subsets were cultured at 1 x 10⁶/ml in 24-well plates (Nunc) with M. tuberculosis H37Ra sonicate (50 µg/ml final concentration) or PPD (10 µg/ml final concentration) in the presence of irradiated (3000 rad from a cesium source) syngeneic spleen cells (5 x 10⁶/ml). Alternatively, cells were stimulated by Con A (1 µg/ml final concentration) or PMA (Sigma-Aldrich; 25 ng/ml/10⁶ cells) plus ionomycin (Sigma-Aldrich; 250 ng/ml/10⁶ cells). Forty-eight hours later supernatants were collected, and cytokine levels were determined by a two-mAb sandwich ELISA (BD PharMingen). The lower limit of detection for each cytokine was 15 pg/ml. SD values were always <10% of the mean values.

Intracellular FACS staining

Intracellular staining was used to determine IFN- production at the single-cell level (16). Briefly, lung mononuclear cells were stimulated with PMA (25 ng/ml/10⁶ cells) and ionomycin (250 ng/ml/10⁶ cells) for 4 h at 37°C and were cultured for 5 h with brefeldin A (Sigma-Aldrich) to accumulate intracellular newly synthesized protein. Cells were harvested and fixed with 4% (w/v) paraformaldehyde in PBS for 10 min at room temperature. Fixed cells were suspended and washed twice with permeabilization buffer containing 0.1% saponin (Sigma-Aldrich), 1% heat-inactivated FCS, and 0.1% NaN₃ in PBS. The permeabilized cells were then incubated in the presence of saponin with FITC-conjugated, anti-mouse IFN- mAb (XMG1.2, rat IgG1; BD PharMingen) or an FITC-conjugated isotype control mAb (R3-34, rat IgG1; BD PharMingen) for 30 min at room temperature. After being washed at room temperature the cells were analyzed by FACS as described above.

To identify the phenotype of the IFN--producing cells, surface marker analysis was performed by staining the cells with PE-conjugated anti-TCR.

Cytotoxicity assay

Peritoneal macrophages or J774 macrophage target cells (4 x 10³/well) were infected with BCG (10/1 CFU/macrophage) for 24 h or were pulsed with PPD for 6 h at 37°C in 5% CO₂. The macrophages were washed with RPMI 1640, and then effector cells were added at a different ratio and incubated for 5 h at 37°C in 5% CO₂. Cytotoxicity was analyzed using a nonradioactive colorimetric cytotoxicity assay (CytoTox 96; Promega, Madison, WI) following the manufacturer’s recommendations. In some experiments the cytotoxicity assay was conducted in the presence of the following mAbs, all used at 20 µg/ml final concentrations (17): anti-I-A^b (AF6-120.1; BD PharMingen); anti-I-E^k,b,d (a gift from Prof. K. Tomonari, Fukui Medical School, Fukui, Japan); anti-D^b (KH95; BD PharMingen), anti-K^b (AF6-88.5; BD PharMingen); anti-TCR, anti-TCR, and anti-Fas ligand (anti-FasL; BD PharMingen); or anti-TNF- (BD PharMingen). Alternatively, T cells were degranulated by treatment with 25 mM Sr²⁺ for 20 h (18, 19) or were treated with 0.5 mM EDTA for 4 h at 37°C to inhibit degranulation.

Determination of CFUs in organs of mice

Mice were infected i.n. with BCG and were killed after 1, 15, 30, or 45 days from infection by cervical dislocation. CFU counts were determined by plating serially diluted homogenates on Middlebrook 7H10 agar plates supplemented with 0.5% glycerol and 10% Bacto Middlebrook OADC (Difco, Detroit, MI) enrichment. CFUs were determined after 4 wk of incubation at 37°C.

FACS analysis of V chain gene usage

V chain usage of lung T cells was assessed by FACS analysis using FITC-conjugated anti-V1 (a gift from Dr. P. Pereira, Institut Pasteur, Paris, France), anti-V2 (BD PharMingen) or anti-V3 (BD PharMingen) mAbs and PE-conjugated anti-TCR (UC7-13D5; BD PharMingen).

PCR analysis of V and V chain gene usage

Total RNA was extracted from lung cells of control mice or mice that had been injected i.n. with BCG 7 days early using the guanidinium thiocyanate/cesium chloride gradient centrifugation method. cDNA was synthesized with oligo(dT) (Amersham Pharmacia Biotech, Uppsala, Sweden) with reverse transcriptase using 10 µg of RNA, according to the manufacturer’s instructions. PCR was performed with a GeneAmp PCR system 9600 (PerkinElmer, Rome, Italy), using the following oligonucleotide primers: C (5'-CTTATGGAGATTTGTTTCAGC-3'), V1 (5'-ACACAGCTATACATTGGTAC-3'), V2 (5'-TGTCCTTGCAACCCCTACCC-3'), V3 (5'-TGTGCACTGGTACCAACTGA-3'), V4 (5'-GGAATTCAAAAGAAAACATTGTCT-3'), V5 (5'-AAGCTAGAGGGGTCCTCTGC-3'), C (5'-CGAATTCCACAATCTTCTTG-3'), V1 (5'-ATTCAGAAGGCAACAATGAAAG-3'), V2 (5'-GCTCATGGTGACTTCATCTC-3'), V3 (5'-TTCCTGGCTATTGCCTCTGAC-3'), V4 (5'-CCGCTTCTCTGTGAACTTCC-3'), V5 (5'-CAGATCCTTCCAGTTCATCC-3'), V6 (5'-TCAAGTCCATAGCCTTGTC-3'), and V7 (5'-CGCAGAGCTGCAGTGTAACT-3'). The primer combinations were chosen in such a way that the sizes of Vs and Vs fragments were 310 and 285 bp, respectively. The nomenclature of TCR V- and V-chains is according to Garman (20).

Each cycle consisted (21) of incubation at 92°C for 45 s, followed by 55°C for 30 s, and 72°C for 30 s. Before the first cycle, a 2-min 94°C denaturation step was included, and after the 30th cycle the extension at 72°C was prolonged for 4 min. Aliquots (20 µl) of PCR products were electrophoresed in 2% agarose and visualized using ethidium bromide staining as described. All gels were photographed similarly.

Statistics

Student’s t tests were used to compare the significance of differences between groups.

	Results

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Expansion of and T cells in the lungs during BCG infection

The phenotypes of T cells within lungs were determined by two-color flow cytometry during M. bovis BCG infection. Single-cell suspensions were prepared and analyzed for or TCR expression. The percentage of each T cell subset within the CD3⁺ population was determined. The results are shown in Fig. 1A. The percentage of T cells decreased 1 wk after infection and then increased, reaching a peak at 4 wk. Peak T cell expansion coincided with the onset of decline in M. bovis-BCG CFUs in the lungs. At the latest stages (8 wk), the percentage of T cells in the lung decreased in parallel with decreasing CFUs, although T cells percentage remained higher than controls. The expansion of T cells preceded that of T cells, with a peak (5-fold increase) 1 wk after infection. The percentage of T cells then sharply declined compared with values detected in uninfected mice and was sustained for at least 8 wk. The kinetics of T cell response were not altered in BCG-infected -depleted mice (Fig. 1B), although these mice had 2.5-fold more CD3⁺⁺ cells present in uninfected lungs than normal mice.

View larger version (18K): [in this window] [in a new window]   FIGURE 1. A, Expansion of () and () T cells and growth of BCG () in the lungs. Mice were infected i.n. with BCG, and lungs were removed at the indicated time points. B, Mice were treated with anti- () or isotype-matched control mAb () and infected with BCG. Lungs were removed at the indicated time points, and the percentages of T cells within the CD3+ population was calculated as described in Materials and Methods. B, Data are expressed as the percentages of CD3++ double-positive cells within lung mononuclear cells.

V and V gene usage by T cells in the lungs of BCG-infected mice

TCR V and V gene usage by T cells expanding in the lungs of BCG-infected mice was evaluated. Staining with V-specific mAbs (Fig. 2A) showed that while T cells in normal lungs preferentially expressed the V2-chain, both V1- and V2-positive cells were detected in the lungs of BCG-infected mice. These results were confirmed and further expanded by RT-PCR analysis. Fig. 2B shows that T cells in normal lungs preferentially expressed the V2-chain (although a faint message for the V1-chain was also detected by RT-PCR), while T cells from the lungs of BCG-infected mice expressed the V1- and V2-chains. Analysis of V-chain gene expression showed that T cells in normal lungs preferentially expressed V5- and V6-chains, whereas T cells from the lungs of BCG-infected mice had a more heterogeneous V gene expression with usage of at least five different V genes (V2, -4, -5, and -6).

View larger version (36K): [in this window] [in a new window]   FIGURE 2. V and V chain usage by T cells in the lungs of BCG-infected mice. Lungs were harvested from mice infected with BCG 7 days earlier or from control mice and were double stained with anti-pan TCR and anti-V chain-specific mAbs (A) or were used for V and V gene expression by PCR analysis.

Proliferative response and cytokine production by T cells in the lungs of BCG-infected mice

T cells were sorted from the lungs of mice infected with BCG 7 days early or from control mice and were stimulated in vitro with different Ag preparations and irradiated spleen cells as APCs. Proliferation and cytokine production by ELISA and intracellular FACS staining were determined. Fig. 3A shows that T cells from BCG-infected mice proliferated upon stimulation with H37Ra or with the mitogen Con A, while very low or no response was detected upon stimulation with PPD. In uninfected mice, T cells gave a proliferative response to Con A and a low response to H37Ra.

View larger version (19K): [in this window] [in a new window]   FIGURE 3. T cells were sorted from the lungs of mice infected with BCG 7 days early () or from control mice () and were tested for proliferation (A), IFN- production by ELISA (B), or intracellular IFN- staining (C) upon in vitro exposure to the indicated Ags or mitogens.

Cytotoxic activity of T cells in the lungs of BCG-infected mice

T cells have been shown to exert cytotoxic activity in many different experimental models (22). In the next experiments we investigated the cytotoxic activity of T cells from the lungs of BCG-infected mice toward BCG-infected or PPD-pulsed macrophage targets. As shown in Fig. 4A, T cells killed in a dose-dependent fashion BCG-infected macrophages, while failing to consistently lyse PPD- or medium-pulsed macrophages. However, when J774 macrophages were used as targets (Fig. 4B) T cells from BCG-infected mice were able to kill even uninfected cells, indicating that they exerted cytotoxic activity against both infected macrophages and a tumor macrophage cell line. Killing of BCG-infected macrophages was blocked efficiently by mAb directed against the TCR, but neither by irrelevant anti- TCR mAb nor by mAb directed against MHC class I (H-2K and H-2D) or II (I-A and I-E) molecules (Fig. 4C).

View larger version (29K): [in this window] [in a new window]   FIGURE 4. T cells were sorted from the lungs of mice infected with BCG 7 days earlier and tested for cytotoxicity toward peritoneal exudates macrophages (A) or J774 macrophage cells (B) as targets. C, Cytotoxic activity toward BCG-infected peritoneal exudate macrophages was tested in the presence of mAbs against TCRs, MHC molecules, or isotype control mAbs.

View larger version (41K): [in this window] [in a new window]   FIGURE 5. T cells were sorted from the lungs of mice infected with BCG 7 days earlier and were tested for cytotoxicity toward BCG-infected peritoneal exudate macrophages in the presence of anti-TNF-, FasL, or isotype-control mAbs. Additionally T cells were treated with EDTA or Sr ions and then tested for cytotoxic activity.

Effects of T cell depletion on induction of Ag-specific CD4 and CD8 T cells

To investigate the influence of T cells on the induction of Ag-specific CD4 and CD8 T cells, mice were depleted of T cells by mAb in vivo and were infected with BCG. Lung cells were harvested 4 wk later; CD4 and CD8 T cells were sorted and examined for proliferative and cytotoxic activities, respectively. Additionally, the ability of both cell subsets to produce IFN- was assessed.

Fig. 6A shows that both cytotoxic activity and IFN- production by lung CD8 T cells were significantly reduced in mice that had been depleted of T cells. Conversely, both proliferation and IFN- production by lung CD4 T cells upon stimulation with PPD-pulsed autologous APC were not influenced by depletion of T cells (Fig. 6B). This indicates that T cells play a role in the induction of Ag-specific CD8 T cells in the lungs.

View larger version (37K): [in this window] [in a new window]   FIGURE 6. Mice were treated with anti- () or isotype-matched control mAb () or were left untreated () and infected with BCG. Lungs were removed 4 wk later, and CD4 and CD8 cells were sorted. A, Sorted CD8 cells were tested for cytotoxic activity toward BCG-infected peritoneal macrophages or IFN- production upon in vitro culture with BCG-infected peritoneal macrophages. B, Sorted CD4 cells were tested for proliferation and IFN- production upon stimulation with PPD-pulsed spleen cells.

	Discussion

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The results reported here clearly show that the percentage of T cells increased and reached a peak 4 wk after infection. The expansion of T cells preceded that of T cells, with a peak 1 wk after infection. The kinetics of the T cell response were not altered in -depleted mice, although these mice had 2.5-fold more CD3⁺⁺ T cells present in uninfected lungs than normal mice. Additionally, the kinetics of T cell expansion did not correlate to the bacterial load in the lungs; rather, peak T cell expansion coincided with the initial decline in BCG CFUs, suggesting a primary role for T cells in BCG clearance from the lungs.

The increase in the percentage of T cells in the lungs of BCG-infected mice might be due to expansion of resident pulmonary T cells, recruitment of T cells from other anatomical locations, or both. To answer this question, we analyzed V gene usage by the expanding the T population. It was found that while V2-bearing cells were the dominant T population in normal lungs, V1-bearing cells were the most abundant population in the lungs of mice infected with BCG, although a certain degree of expansion also occurred for the V2 subset. This indicates that both expansion of pulmonary (V2) (28) T cells and the recruitment of T cells (V1) to the lungs contribute to the increased size of this T cell subset following BCG infection. Additionally, as V1-bearing cells represent the dominant T population in lymphoid organs (22), it is presumable that V1 cells are recruited from lymph nodes to the lungs by virtue of local production of inflammatory chemokines induced by BCG infection. Moreover, at least four different V genes were expressed in the lungs of BCG-infected mice, suggesting the polyclonal nature of the expanding T populations.

There are at least three possible pathways by which T cells can play a role in the immune response against M. tuberculosis infection: release of IFN-, lysis of infected target, and participation in the induction of conventional CD4 and/or CD8 T cells (22). In our study T cells from the lungs of BCG-infected mice release IFN- and TNF- upon Ag stimulation, providing a mechanism by which this T cell subset might contribute to immunity against M. tuberculosis infection. In fact, IFN- and TNF- synergize for induction of NO synthase and production of NO from macrophages, which has cytocidal effects on intracellular bacteria such as M. tuberculosis (29, 30, 31).

The second mechanism by which T cells can contribute to host defense against M. tuberculosis infection is in their ability to lyse infected target cells. T cells from the lungs of BCG-infected mice lysed BCG-infected macrophages through a mechanism involving the release of cytotoxic molecules contained in granules. Although we did not attempt to determine whether killing of infected targets also caused killing of the intracellular bacteria, it should be speculated that continued lysis of infected cells could lead to the release of bacteria from this safe intracellular harbor so they can be taken up at a low multiplicity by freshly activated macrophages and destroyed.

Ultimately, T cells might influence the generation of Ag-specific CD4 and CD8 T cells, which are regarded as principal effectors of anti-mycobacterial protective responses. In the present report we demonstrate that depletion of T cells from mice before BCG infection caused a decrease in cytotoxic and IFN- activities by CD8 cells in the lungs, but normal proliferation and IFN- production by lung CD4 cells. These results indicate that the lack of T cells results in a decrease in protective CD8 CTL against M. tuberculosis.

There are several possible explanations for the reduction of the BCG-specific CD8 response in the lungs of T cell-depleted mice. It is possible that a factor(s) induced by T cells is important in the optimal induction of CD8 CTLs. Lung T cells induced by BCG infection produce IFN- and TNF- while lacking IL-2, IL-4, IL-5, and IL-10 (our unpublished observation). Thus, it is possible that some of the cytokines produced by T cells participate in CD8 T cell induction in the lungs during the course of BCG infection, because cytokines such as IFN- and TNF- have been reported to have important roles in CTL induction (32, 33).

It is also possible that T cells participate in the induction of CD8 T cells indirectly through activation of other cell populations, such as macrophages. It was reported that macrophages produce IL-12 upon stimulation by IFN- and TNF- (34, 35), which, in turn, are produced by the BCG-induced T cells. IL-12 production, indirectly induced by T cells, may participate in the induction of CD8 T cells. Additionally, up-regulation of costimulatory molecules, such as CD80 and CD86 (36), on macrophages and/or dendritic cells by T cells would be another mechanism of supporting CD8 T cell induction (37).

Finally, another possible mechanism responsible for the reduction of CD8 T cell induction in T cell-depleted mice is exhaustion of CD8 T cells during the immune response in the absence of T cells (38). However, exhaustion is unlikely as the mechanism of reduced CD8 development in T cell-depleted mice, because these mice eliminated BCG infection as well as control mice, which may negate the possibility of exhaustion induced by persistent infection.

Different results have been reported in the mouse about the possible role played by cells during M. tuberculosis infection depending on the mycobacterial species and the route and size of inocula. In general, cells are vitally important in controlling high dose i.v. M. tuberculosis infection (39), whereas in low dose aerosol infection no difference in survival between wild-type and knockout mice is observed (40, 41). However, the histology was different in aerosol infection in mice lacking cells, in that a substantial pyogenic form of the granulomatous response was seen compared with the lymphocytic response detected in wild-type mice (41). This indicates that cells play an important protective role at high levels of mycobacteria inocula, while at lower levels of inocula they play a regulatory (anti-inflammatory) role by limiting the influx of inflammatory cells and consequently tissue damage (42).

Although our results suggest that T cells participate in the induction of CD8 T cells, the ligand specificity of the former is still unclear. There have been several reports showing that murine T cells respond to mycobacterial heat shock protein. On the other hand, human T cells have been reported to recognize non-peptidic mycobacterial Ags (reviewed in Ref. 22).

The results reported in this study showing that T cells proliferate or produce cytokines in response to H37Ra or BCG, but not to PPD, strongly suggest that they may recognize some non-peptide Ags or proteins that are not present in PPD. However, we cannot exclude the possibility that activation of T cells might be due to cytokines generated by BCG infection rather than to an Ag-driven expansion, as recently demonstrated in the Listeria model (43).

Nevertheless, our results suggest the presence of a new type of T-T cell regulation mediated by T cells. There are several reports on the regulation of T cells by T cells. T cells abrogate oral tolerance measured by Ig production (44). Other reports have shown that T cells were indispensable in successful transfer of contact hypersensitivity by T cells (45, 46). In contrast, T cells may also participate actively in the suppression of the T cell response (47, 48). Our finding of T cell-mediated CD8 induction is similar to the former type of positive regulation by T cells. However, the regulation of CD8 cells by T cells is different from the regulation of Ig production and contact hypersensitivity, because these responses are mediated by CD4 T cells.

In conclusion, the data presented here show that T lymphocytes accumulate in the lungs of BCG-infected mice 3 wk earlier than Ag-specific T lymphocytes. We postulate that the rapidly expanding T cells might play an important regulatory role in the subsequent onset of T lymphocytes and are consequently mandatory for the development of protection against the mycobacterial infection.

	Footnotes

 
¹ This work was supported by grants from the Italian National Research Council (to F.D.), the Ministry for Education and Scientific and Technologic Research (MURST 60%; to A.S. and F.D.), and the European Commission (Fifth Framework Program, Contract QLK2-1999-00367).

² Address correspondence and reprint requests to Dr. Francesco Dieli, Dipartimento di Biopatologia, Università di Palermo, Corso Tukory 211, I-90134 Palermo, Italy. E-mail address: dieli{at}unipa.it

³ Abbreviations used in this paper: BCG, Mycobacterium bovis bacillus Calmette-Guérin; FasL, Fas ligand; i.n., intranasally; PPD, purified protein derivative.

Received for publication August 14, 2002. Accepted for publication October 29, 2002.

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