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Infection and Immunity, August 1999, p. 3980-3988, Vol. 67, No. 8
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Early Emergence of CD8+ T Cells Primed for Production of Type 1 Cytokines in the Lungs of Mycobacterium tuberculosis-Infected Mice

Natalya V. Serbina, and JoAnne L. Flynn*

Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania

Received 4 February 1999/Returned for modification 11 March 1999/Accepted 7 May 1999


    ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Several lines of evidence suggest that CD8 T cells are important in protection against tuberculosis. To understand the function of this cell population in the immune response against Mycobacterium tuberculosis, T cells from lungs of M. tuberculosis-infected mice were examined by flow cytometry. The kinetics of the appearance of CD8 T cells in lungs of infected mice closely paralleled that of CD4 T cells. Both CD4+ and CD8+ T cells displaying an activated phenotype were found in the lungs as early as 1 week postinfection. By 2 weeks, total cell numbers in the lungs had tripled and percentages of T cells were increased two- to threefold; the percentages of CD4+ T cells were ca. twofold higher than those of CD8+ T cells. Short-term stimulation with M. tuberculosis-infected antigen-presenting cells induced cytokine production by primed CD4+ and CD8+ T cells. Intracellular cytokine staining revealed that 30% ± 5% of CD4+ and 23% ± 4% of CD8+ T cells were primed for production of gamma interferon (IFN-gamma ). However, a difference in in vivo IFN-gamma production by T cells was observed with ~12% of CD4+ T cells and ~5% of CD8+ T cells secreting cytokine in the lungs at any given time during infection. The data presented indicate that although early in infection the majority of IFN-gamma is produced by CD4+ T cells, cytokine-producing CD8+ T cells are readily available when triggered by the appropriate stimuli.


    INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

The importance of T lymphocytes in the control of Mycobacterium tuberculosis infection is well documented. The requirement of CD4+ or CD8+ T cells for establishment of protective immunity to M. tuberculosis was demonstrated in adoptive-transfer experiments (42) and in vitro T-cell subset depletion experiments (39). The importance of both T-cell subsets in the immune response to this infection was confirmed by using mice in which targeted disruptions in the genes encoding major histocompatibility complex (MHC) class II, CD4, or beta 2-microglobulin molecules rendered them highly susceptible to tuberculosis (10, 20, 55).

A major function of CD4+ T cells during murine and human M. tuberculosis infection is believed to be the production of type 1 cytokines such as gamma interferon (IFN-gamma ) (3-5, 44, 48). IFN-gamma production is essential for activation of macrophages and plays a crucial role in control of mycobacterial infection in mice and humans (13, 19, 29, 41). The function of CD8+ T cells in immunity to tuberculosis remains controversial. The role of CD8+ T lymphocytes as cytotoxic cells is supported by studies in both murine and human tuberculosis (33, 51, 52, 54, 58). Mycobacterium-specific CD8+ cytotoxic T lymphocytes (CTLs) have been isolated from infected mice and humans (16, 30, 31, 33, 50), and spleen cells from mice lacking CD8+ T cells have diminished cytotoxic activity (39). The mechanism of lysis by Mycobacterium-specific CD8+ CTLs is controversial (15, 34), although direct killing of M. tuberculosis bacilli by granulysin, a granule-associated protein of CD8+ T cells, has been reported (53).

Data are accumulating that CD8+ T cells also contribute to the cytokine pool during M. tuberculosis infection. Mycobacterial antigen-specific CD8+ T cells from infected humans and mice produced IFN-gamma after in vitro restimulation (33, 35, 43). Purified protein derivative-stimulated spleen cells from M. tuberculosis-infected beta 2-microglobulin-deficient mice produced lower levels of IFN-gamma and tumor necrosis factor alpha (TNF-alpha ) compared to wild-type mice (20). M. tuberculosis-infected mice lacking CD4+ T cells had increased numbers of CD8+ T cells in their lungs, such that the percentage of IFN-gamma -producing cells and IFN-gamma mRNA levels were similar in the mutant and wild-type mice by 4 weeks postinfection (10). Thus, in the absence of CD4+ T cells, CD8+ T cells produced compensatory IFN-gamma , although this was not sufficient to protect the CD4+ T-cell-deficient mice from tuberculosis. Analysis of cytokine production by cultured cells has certain limitations. The presence of cytokines in culture supernatants provides limited information regarding the proportion of cells engaged in cytokine production or the kinetics of the response. Also, in vitro culture of cells can skew the population, depending on the conditions of CD4+- and CD8+-T-cell stimulation, which can be complicated in M. tuberculosis infections. M. tuberculosis may use an alternative MHC class I presentation pathway (37, 38) and cause the production of suppressive cytokines by antigen-presenting cells (26, 27), both of which may be difficult to mimic or control in vitro. Recently it was demonstrated that IFN-gamma production by CD8+ T cells was necessary to mediate partial protection of athymic mice against M. tuberculosis infection (55). However, the presence of cytokine-secreting CD8+ T cells at the site of infection and their role in the establishment of protective immunity during the acute stage of disease have not been established. Such studies are an essential step in understanding the role of CD8+ T cells in the immune response against tuberculosis.

We hypothesized that CD8+ T cells contribute to protective immunity against M. tuberculosis by a combination of cytokine production and CTL activity. Here we assessed the ability of lung CD8+ T cells to produce cytokines at the initial stage of infection. We present data indicating that Mycobacterium-specific CD8+ T cells emerge early in infection with a cytokine profile similar to that of CD4+ T cells. Our results indicate that although significant percentages of both CD4+ and CD8+ T cells are primed for IFN-gamma production, CD4+ T cells are responsible for the majority of IFN-gamma production early in infection and only small percentages of CD8+ T cells are actually producing cytokines at any given time during the infection. Cytokine production by CD8+ T cells early in infection is likely to be important, but these cells probably have other functions in the early protection against tuberculosis.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Mice. Female C57BL/6 mice, 8 to 10 weeks old (Jackson Laboratories) were used. All mice were maintained in specific-pathogen-free Biosafety Level 3 facilities.

Bacteria and infections. M. tuberculosis (Erdman strain; Trudeau Institute, Saranac Lake, N.Y.) was passed through mice, grown in culture once, and frozen in aliquots. Before injection into mice, an aliquot was thawed, diluted in phosphate-buffered saline (PBS) containing 0.05% Tween 80, and sonicated for 10 s in a cup horn sonicator. Mice were infected intravenously via the tail vein with 2 × 105 live bacilli in 100 µl, as determined by viable counts on 7H10 agar plates (Difco Laboratories, Detroit, Mich.). For infection of antigen-presenting cells, frozen aliquots were used to start cultures at a concentration of 2.5 × 106/ml in liquid medium (7H9 Middlebrook; Difco); bacteria were grown in 5% CO2 at 37°C. Six- to seven-day-old cultures were used to infect cells. Bacteria were washed and resuspended in Dulbecco's minimal essential medium (DMEM) (Life Technologies, Grand Island, N.Y.) prior to infection of cell cultures.

Culture and infection of DC and Mphi s. Dendritic cells (DCs) and macrophages (Mphi s) were grown from bone marrow precursors from C57BL/6 mice. Briefly, cells were eluted from the femurs and tibias of mice in DMEM. For Mphi cultures, the cells were washed twice in DMEM and 1 × 106 to 3 × 106 cells were plated in Lab Tek PS petri dishes (Fisher Scientific, Pittsburgh, Pa.) in 25 ml of DMEM containing 10% certified fetal bovine serum, 1 mM sodium pyruvate, 2 mM L-glutamine (Life Technologies, Grand Island, N.Y.), and 33% supernatant from L-cell fibroblasts cultured for 5 to 7 days. All reagents were lipopolysaccharide free, and no antibiotics were used. The medium was changed once after 2 to 3 days of culture. On day 5, adherent cells were washed twice with ice-cold PBS (Life Technologies), incubated for 20 min on ice, and harvested with cell scrapers (Becton Dickinson Labware, Lincoln Park, N.J.). The cell concentration was adjusted to 0.5 × 106 cells/ml, and cells were dispersed in Teflon jars (Savillex, Minnetonka, Minn.) for infection.

For DC culture, eluted cells were centrifuged at 300 × g for 7 min and erythrocytes were lysed with NH4Cl-Tris solution. CD4+ and CD8+ T cells were depleted with Low-Tox-M rabbit complement (Accurate Chemical and Scientific Corporation, Westbury, N.Y.) after incubation with anti-CD4 antibody (GK1.4, 10 µg/ml) and anti-CD8 supernatant (antiCD8alpha , clone 83-15-5). The cell concentration was adjusted to 106 cells/ml, and adherent cells were depleted by overnight culture in DC medium containing DMEM, 2 mM L-glutamine, and 5% mouse serum (Sigma, St. Louis, Mo.). The resultant nonadherent cells were cultured at 0.25 × 106 cells/ml in 24-well plates (Costar, Cambridge, Mass.) in DC medium containing 1,000 U each of recombinant murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF) and recombinant murine interleukin-4 (rmIL-4) per ml. Fresh medium was added after 2 to 3 days of culture. On day 5, nonadherent cells were harvested, adjusted to 0.5 × 106 cells/ml in DC medium containing rmGM-CSF, and dispersed into 25-cm2 culture flasks (Costar, Cambridge, Mass.) for infection.

Cells were infected for 12 to 17 h at a multiplicity of infection of 2 to 5. At the end of the infection, extracellular bacteria were separated from cells by low-speed centrifugation. Infected and uninfected DCs and Mphi s were cultured in fresh medium for an additional 12 to 36 h before being used in stimulation assays. For DCs and Mphi s, the percentage of infection was estimated in each experiment by staining aliquots of cells by the Kinyoun method for acid-fast bacteria (Difco).

FACS analysis of cell surface markers. Lung cells were obtained from infected mice at various time points postinfection by crushing the organs in cell strainers (Becton Dickinson Labware, Lincoln Park, N.J.) to obtain single-cell suspensions. Erythrocytes were lysed with NH4Cl-Tris solution, and the cells were washed twice. The cells were stained for 30 min at 4°C for cell surface molecules with antibodies against T-cell receptor alpha beta (TCRalpha beta ) (anti-TCRbeta fluorescein isothiocyanate [FITC] Ab, clone H57-597), CD4 (anti-CD4 CyChrome Ab, clone H129.19), CD8 (anti-CD8 CyChrome Ab, clone 53-6.7), and CD44 (anti-CD44 FITC Ab, clone IM7) in PBS containing 20% mouse serum, 0.1% bovine serum albumin, and 0.1% sodium azide. All antibodies were used at 0.2 µg/106 cells and were obtained from PharMingen (San Diego, Calif.). The cells were fixed with 2% paraformaldehyde for 4 to 15 h and analyzed by fluorescence-activated (FACS) with cell sorting Lysis II software (Becton-Dickinson Immunocytometry Systems, San Jose, Calif.). Cells were gated on the lymphocyte population by size. Analysis of uninfected lung population revealed that it was composed mainly of CD4+, CD8+, and double-negative T cells (28%), B220+ cells (46%), and NK cells (12.5%). During the infection, increases in the percentages of CD4+ and CD8+ T cells (46%) were observed, with a concomitant reduction in the percentages of double-negative T cells (2%), B220+ cells (33%), and NK cells (3.5%).

In vivo intracellular cytokine staining. Single-cell suspensions of lung tissue obtained at various times postinfection were prepared as described above. The cells were stimulated for 5 to 6 h with anti-CD3 (clone 145-2C11; 0.1 µg/ml) and anti-CD28 (clone 37.51; 1 µg/ml) antibodies in the presence of 3 µM monensin (Sigma) to halt egress of cytokines from the cells. After being washed, the cells were stained for the cell surface molecules CD4, CD8, CD44, and TCRalpha beta as described above. The cells were washed and fixed in 4% paraformaldehyde at 4°C for 1.5 to 2 h. They were permeabilized with 0.1% saponin in PBS containing 0.1% bovine serum albumin and 0.1% sodium azide; stained for IFN-gamma , TNF-alpha , IL-10, and IL-4 (0.4 µg/106 cells; anti-IFN-gamma phycoerythrin [PE] Ab, clone XMG1.2; anti-TNF-alpha FITC Ab, clone MP6-XT22; anti-IL-10 FITC Ab, clone JES5-16E3; and anti-IL-4 PE Ab, clone 11B11, respectively) in 20% mouse serum for 30 min at 4°C; washed; and analyzed by FACS with Lysis II software (Becton-Dickinson Immunocytometry Systems). Isotype controls for each antibody were used, and lung cells from an uninfected control mouse were tested in each experiment. All antibodies were obtained from PharMingen.

Lung T-cell stimulation assays. DCs and Mphi s uninfected or infected for 24 to 48 h as described above were plated in 96-well U-bottom plates (Corning Inc., Corning, N.Y.) at 104 cells/well in RPMI 1640 medium (Life Technologies) containing 10% certified fetal bovine serum, 1 mM sodium pyruvate, 2 mM L-glutamine, 25 mM HEPES (Life Technologies) and 50 µM 2-mercaptoethanol (Sigma). Lung cells from mice infected for 2 to 3 weeks were obtained as described above and added to wells containing uninfected or infected DCs and Mphi s or medium at 1 × 105 to 2 × 105 lung cells/well. The cells were cultured for 8 to 15 h, and monensin was added at a final concentration of 3 µM for additional 5 to 6 h. At the end of the culture, the cells were harvested and intracellular cytokine staining was performed as described above.

Statistics. The paired Student t test was used to compare groups. Statistical analysis was performed with StatView (Abacus Concepts). P <=  0.05 was considered significant.


    RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Appearance of activated CD4+ and CD8+ T-cell populations in lungs of M. tuberculosis-infected mice. Mice were infected intravenously, and the T-cell populations in the lungs at various times postinfection were compared. Upon initiation of infection, total cell numbers increased in the lungs, in accordance with published data (36) (Fig. 1A; Table 1). A slight increase in cell numbers was seen as early as 7 days postinfection, with a marked increase by day 14 and a continued increase to day 21. Control age-matched mice did not show an increase in cell numbers over time (data not shown). Based on previous data, bacterial numbers in the lungs increase from 5 × 103 to 2 × 106 during the first 3 weeks of infection and then remain essentially unchanged for at least 15 weeks (references 10 and 21 and data not shown).



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  		FIG. 1.   Changes in cell number and T-cell composition in the lungs of mice following M. tuberculosis infection. C57BL/6 mice were infected intravenously with 2 × 105 viable M. tuberculosis bacilli (Erdman strain), and their lungs were harvested 0, 1, 2, 3, and 4 weeks postinfection. (A) Numbers of viable cells in lungs were counted by trypan blue exclusion. Each time point represents 4 to 10 mice. For each time point, P < 0.05 compared to uninfected mice. (B) Cells harvested from lungs were stained for CD4 (open squares) and CD8 (solid circles). The cells were gated on lymphocyte population by size and analyzed by flow cytometry. Each time point represents 4 to 10 mice; P < 0.05 for the 2-, 3-, and 4-week time points compared to day 0. Error bars show standard error. (C) Lung cells were harvested at 21 days postinfection, stained for CD4, CD8, and TCRalpha beta , fixed in paraformaldehyde, and analyzed by two-color flow cytometry after gating on lymphocytes. Shown are the results from samples from a representative mouse. The experiment was repeated at least three times.

                              
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  		TABLE 1.   Total numbers of CD4+ and CD8+ T cells producing IFN-gamma with or without stimulation in the lungs of mice during the infectiona

Upon infection with M. tuberculosis, the percentage of lung lymphocytes that were CD4+ T cells, as well as total numbers of CD4+ T cells, increased as early as 1 week postinfection (Fig. 1B; Table 1). By 2 weeks postinfection, CD4+ T cells comprised >30% of the gated lymphocyte population. The appearance of CD8+ T cells in the lungs followed slower kinetics, with no change observed at 1 week postinfection, and a fourfold increase in the numbers of CD8+ T cells by week 2. At 2 weeks postinfection, the percentages of both CD4+ and CD8+ T cells reached a plateau, with CD4+ T cells present in greater numbers (1.6 fold) than CD8+ T cells (Table 1). The majority (>90%) of T cells in lungs were TCRalpha beta + at all time points examined, including early (<1 week postinfection [data not shown]) and later (Fig. 1C and data not shown) time points.

To evaluate the kinetics of activation of T-cell subsets in the lungs, we analyzed the expression of the activation marker CD44 on T cells at various times postinfection. The enrichment of the CD44high subset during the first 4 weeks of M. tuberculosis infection has been documented previously for splenic CD4+ T cells, as well as for CD4+ T cells from popliteal lymph nodes from immune mice after rechallenge (24). The appearance of the CD44+CD45RB-/low T-cell population in the lungs of susceptible I/St mice by week 2 of infection has also been reported (36). In our studies, by week 2, >85% of both T-cell subsets in the lungs expressed the high-density CD44 molecule, and the increase in cell numbers was associated with increased CD44 expression; this was unchanged through 4 weeks postinfection (Table 2). Thus, the lungs of infected mice contained T-cell populations displaying the activation phenotype as assessed by the expression of CD44 molecule. Importantly, the influx of activated CD8+ T cells was evident in the lungs at the early stages of infection.

                              
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  		TABLE 2.   Acquisition of activated phenotype by CD4+ and CD8+ T cells in the lungs of M. tuberculosis-infected mice

IFN-gamma production potential of T-cell subsets in lungs during the course of infection. IFN-gamma production in vitro by CD4+ and CD8+ T cells from the spleens of infected mice and lung CD4+-T-cell clones has been demonstrated previously (36, 43). To assess more directly the cytokine-producing populations of T cells in vivo during infection, we stimulated lung cells harvested at different times postinfection with anti-CD3 and anti-CD28 monoclonal antibodies briefly (5 to 6 h) in the presence of monensin, to prevent egress of cytokines from the cells; at the end of the stimulation period, intracellular cytokine staining was performed. Under these conditions, T cells from uninfected mice (day 0) did not produce significant levels of cytokines. Cytokine production was only detected by activated (CD44high) T cells (Fig. 2; Table 1). This method of stimulation does not depend on the efficiency of antigen presentation to T cells and identifies CD4+ and CD8+ T cells capable of releasing cytokines upon receptor ligation. Few CD4+ and CD8+ T cells harvested 1 week postinfection produced IFN-gamma . However, 29.5% ± 5.5% of CD4+ and 22.7% ± 4.2% of CD8+ T cells harvested 2 weeks postinfection produced IFN-gamma upon stimulation, and this percentage remained stable through 4 weeks postinfection (Fig. 2; Table 1). Cytokine production by CD4+ and CD8+ T cells was characterized by a broad spectrum of fluorescence intensities (Fig. 2A). Subsets of both CD4+ and CD8+ T cells with high cytokine production (high fluorescence intensity) were observed. The percentage of total lung cells producing IFN-gamma at each time point was the sum of the percentages of IFN-gamma producing CD4+ and CD8+ T cells (data not shown), indicating that the contribution of cytokine-producing double-negative T cells was negligible.



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  		FIG. 2.   Intracellular IFN-gamma staining of lung cells from infected mice. Cells were harvested at 0, 1, 2, 3, and 4 weeks postinfection; stimulated for 5 to 6 h with anti-CD3 and anti-CD28 antibodies in the presence of monensin; stained for CD4, CD8, and CD44; fixed in paraformaldehyde; permeabilized; and stained for intracellular IFN-gamma . The cells were gated on lymphocytes by size and analyzed by two- or three-color flow cytometry. (A) Lung cells were gated on CD4 (top) or CD8 (bottom), and the expression of CD44 and IFN-gamma within each gate was analyzed. Results from samples from a representative mouse at each time point are shown. Two mice for each time point were used per experiment, and each experiment was repeated at least twice. (B) Percentages of IFN-gamma -producing CD4+ (open squares) and CD8+ (solid circles) T cells in the lymphocyte gate. (C) Cells in the lymphocyte gate were further gated on CD4 (open squares) and CD8 (solid circles), and the percentages of INF-gamma -producing cells within each gate were reported. For panels B and C, each time point represents 4 to 10 mice and at least two experiments. Error bars indicate standard error.

IFN-gamma production by CD4+ and CD8+ T cells at the site of infection. Short-term stimulation with anti-CD3 and anti-CD28 antibodies prior to intracellular cytokine staining induces IFN-gamma production in cells that are primed to produce IFN-gamma . However, it is not clear whether these cells indeed produce the cytokine in vivo. To address this issue, lung cells were harvested 2 and 3 weeks postinfection and stained for intracellular IFN-gamma for 6 h with or without anti-CD3/anti-CD28 antibody stimulation. At 2 and 3 weeks postinfection in four separate experiments, 13.5% ± 0.2% and 11.2% ± 0.9% of CD4+ T cells, respectively, produced IFN-gamma without in vitro stimulation (Fig. 3). However, at 2 weeks postinfection, IFN-gamma -producing CD8+ T cells did not exceed 5.4% ± 1.4% even though anti-CD3/anti-CD28 antibody stimulation resulted in cytokine production by 22.7% ± 4.2% of CD8+ T cells and these numbers did not change at 3 weeks postinfection (Fig. 3). Thus, 3.5- to 5-fold more CD4+ T cells than CD8+ T cells spontaneously secreted IFN-gamma at 2 and 3 weeks postinfection (Table 1). T cells from uninfected mice did not produce IFN-gamma with or without stimulation (Fig. 2 and 3B; Table 1). The CD4+ and CD8+-T-cell subsets secreting high levels of cytokine (high fluorescence intensity) were not observed without anti-CD3/anti-CD28 antibody stimulation. The unstimulated (i.e., spontaneous) IFN-gamma production may be a more accurate reflection of cytokine production by T cells at a particular time in infection. These results suggest the existence of differential regulation of CD4+- and CD8+-T-cell cytokine production at the site of infection, since T cells producing IFN-gamma in vivo would probably continue to produce cytokine for an additional 6 h after being removed from the lungs. In these experiments, cytokine production in the lungs appeared to be entirely attributed to TCRalpha beta + CD4+ and CD8+ T cells (Fig. 3A).


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  		FIG. 3.   Intracellular IFN-gamma staining of lung cells from infected mice. Lung cells from mice at 0, 2, and 3 weeks postinfection were stimulated with anti-CD3 and anti-CD28 antibodies or left unstimulated in the presence of monensin for 5 to 6 h. The cells were stained for CD4, CD8, TCRalpha beta , and intracellular IFN-gamma , as in Fig. 2. The cells were gated on CD4 or CD8, and the expression of TCRalpha beta and IFN-gamma within each gate was analyzed. (A) Production of IFN-gamma by lung TCRalpha beta + CD4+ and CD8+ T cells with or without anti-CD3/anti-CD28 antibody stimulation. Stimulation with anti-CD3 antibody results in downregulation of TCR on the T-cell surface. Results from samples from a representative mouse infected for 2 weeks are shown, and the experiment was repeated at least three times with two mice per time point. (B) Cytokine production by unstimulated lung CD4+ and CD8+ T cells. Each time point represents three to six mice.

Production of cytokines other than IFN-gamma by T-cell subsets. Since only 20 to 30% of CD4+ and CD8+ T cells in the lungs of infected mice produced IFN-gamma upon stimulation, although the majority displayed an activated phenotype, we reasoned that the T cells might be primed for production of cytokines other than IFN-gamma . Another cytokine critical for the control of M. tuberculosis infection is TNF-alpha (21). To assess the production of this cytokine by T cells, lung cells from mice either uninfected or infected for 2 to 3 weeks were analyzed by intracellular staining for IFN-gamma and TNF-alpha with or without short-term anti-CD3/anti-CD28 antibody stimulation. Upon stimulation, 13% ± 1% of CD4+ and 14% ± 2% of CD8+ T cells harvested from lungs of uninfected (day 0) mice produced TNF-alpha , although no significant IFN-gamma production by these cells was observed (Fig. 4A). Similarly, 18 to 22% of CD4+ T cells and 10 to 15% of CD8+ T cells harvested 2 and 3 weeks postinfection secreted TNF-alpha (Fig. 4A). Three different patterns of cytokine secretion by stimulated T cells were observed: 15 to 20% of CD4+ and CD8+ T cells secreted both IFN-gamma and TNF-alpha , 10 to 15% of both subsets secreted IFN-gamma only, and 2 to 5% of both subsets secreted TNF-alpha only (Fig. 4B). Unstimulated T cells did not produce TNF-alpha at any time points examined (data not shown).


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  		FIG. 4.   Intracellular IFN-gamma and TNF-alpha staining of lung cells from infected mice. Lung cells were harvested at 0, 2, and 3 weeks postinfection, stimulated with anti-CD3/anti-CD28 antibodies, and analyzed by intracellular cytokine staining, as in Fig. 2. (A) Production of IFN-gamma (solid triangles) and TNF-alpha (open circles) within the CD4 (top) and CD8 (bottom) gates. Each time point represents two to four mice. (B) Cytokine production within the CD4 and CD8 gates. Results from samples from representative uninfected and 3-week-infected mice are shown.

Other cytokines reported to be produced by CD4+ T cells from M. tuberculosis-infected mice upon stimulation with infected antigen-presenting cells include IL-4 and IL-10 (44). We did not detect IL-4- and IL-10-producing lung CD4+- and CD8+-T-cell populations by intracellular staining 2 and 3 weeks postinfection by using anti-CD3/anti-CD28 antibody stimulation (data not shown).

Cytokine production by T cells from lungs of infected mice following stimulation with APCs. One possibility for observed differences in cytokine production by unstimulated CD4+ and CD8+ T cells from infected mice may be a differential ability of M. tuberculosis-infected APCs in the lungs to stimulate T-cell subsets. Cytokine production by T cells from infected mice stimulated for several days with murine Mphi s either pulsed with mycobacterial protein fractions or infected with live bacilli has been demonstrated (36, 43, 44). In our hands, only minimal IFN-gamma production by splenic T cells stimulated for 3 days with M. tuberculosis-infected bone marrow-derived Mphi s was detected, while IFN-gamma production was readily detected upon stimulation with infected bone marrow-derived DCs (unpublished data). To minimize the impact of culture conditions on T-cell responses and gain insight into the cytokine response in the lungs during infection, we used short-term exposure of fresh lung T cells to Mphi s and DCs followed by intracellular staining for IFN-gamma and TNF-alpha production. T cells harvested from lungs 2 to 3 weeks postinfection were stimulated for 12 h with bone marrow-derived DCs or Mphi s that were previously infected with live M. tuberculosis for 24 to 48 h. Short-term stimulation with anti-CD3/anti-CD28 antibodies served as an estimation of the maximal capacity of T cells for cytokine production. CD4+ and CD8+ T cells from infected (but not uninfected) mice cultured in the medium alone produced IFN-gamma spontaneously, which was an indication of cytokine production in vivo as described above. Stimulation with uninfected DCs or Mphi s did not augment IFN-gamma production by either T-cell subset (Fig. 5). Although stimulation with DCs infected with M. tuberculosis for 24 h prior to stimulation resulted in a twofold increase in the percentage of both CD8+ and CD4+ T cells producing IFN-gamma , these percentages remained low (Fig. 5A). Although a small increase in cytokine-producing CD4+ T cells was observed after culture with infected Mphi s, CD8+ T cells failed to respond to this stimulation (Fig. 5A). Stimulation with APCs infected for 24 h induced TNF-alpha production in <2% of CD4+ and CD8+ T cells (data not shown).


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  		FIG. 5.   Intracellular cytokine staining of lung cells from infected mice after stimulation with APCs. Lung cells from 2- to 3-week-infected mice were cultured for 12 to 20 h with bone marrow-derived DCs or Mphi s which were infected with viable M. tuberculosis (M.tb) for 24 h (A) or 48 h (B) or left uninfected. T cells from uninfected mice did not produce IFN-gamma under these conditions (data not shown). Production of IFN-gamma by CD4 (top) and CD8 (bottom) T cells was analyzed by intracellular cytokine staining, as in Fig. 2. Each time point represents 3 to 12 mice. Error bars represent standard error.

The lack of stimulation of CD8+ T cells by infected APCs suggested that insufficient time was allowed for antigen processing, and so APCs infected 48 h prior to stimulation of fresh lung cells were used. DCs infected for 48 h effectively stimulated IFN-gamma production by CD8+ and CD4+ T cells (Fig. 5B). Unexpectedly, Mphi s infected for 48 h completely failed to stimulate cytokine production by CD4+ T cells (Fig. 5B), while a small percentage of CD8+ T cells did respond to Mphi stimulation. Up-regulation of TNF-alpha production was observed when T cells were stimulated with DCs infected for 48 h, with <5% of both CD4+ and CD8+ T cells secreting TNF-alpha (data not shown).


    DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

The involvement of CD8+ T cells in the protective immune response to M. tuberculosis infection has been demonstrated in several experimental systems, but the role of these cells remains controversial. We hypothesized that CD8+ T cells function as both CTL and cytokine producers during M. tuberculosis infection; the kinetics of CD8+-T-cell appearance and function may be crucial to control of the infection. The studies presented here focused on cytokine production by CD8+ T cells in the lungs during the initial stage of infection. By varying the stimulation of the cells, certain parameters of the immune response to M. tuberculosis were measured. Short-term stimulation with anti-CD3/anti-CD28 antibodies provides the percentage of primed cells capable of producing cytokines in the lungs, whereas spontaneous cytokine production by unstimulated T cells from infected mice is likely to be more reflective of the in vivo situation at a particular point in time. T cells were also stimulated more specifically, using APCs infected with M. tuberculosis; again, short-term stimulation was used to gauge the initial responses of the T cells to stimulation and avoid skewing the T-cell populations during in vitro culture. The data indicate that although 20 to 30% of CD4+ and CD8+ T cells were primed to produce IFN-gamma , the majority of IFN-gamma secretion at early time points in the infection was by CD4+ T cells. This suggests differential regulation of the two T-cell subsets, either by antigen presentation of mycobacterial antigens or by the action of immunoregulatory cytokines in the lungs. The data also suggests that activated CD8+ T cells present in large numbers in the lungs of infected mice have other functions in protection against tuberculosis. However, in the experiments presented here, the infection was initiated by the intravenous route, which does not represent the natural infection route. It has been suggested previously that expression of immunity in the lungs following the aerosol mode of infection occurs with slower kinetics than that following the intravenous mode of infection (14).

During the first 2 weeks of infection, the lungs underwent significant changes that were reflected both in increases in cell volume and in percentages of TCRalpha beta + CD4+ and CD8+ T cells. The majority (>85%) of T cells were of the CD44high activated phenotype, which reflects the requirement for high-density CD44-complex binding to hyaluronan for T-cell migration into infected organ. It has been demonstrated that CD44 expression increases upon primary T-cell stimulation (7, 8), suggesting that T cells in the lungs have been exposed to antigens, presumably mycobacterial antigens. However, chemokine-mediated migration of naive or nonspecific lymphocytes into the lungs, with up-regulation of CD44 expression due to inflammatory cytokines, may also be involved.

In viral systems, specific dominant peptides or antigens have been used to examine the percentages of responding antigen-specific T cells (2, 9, 22, 40). Although some mycobacterial T-cell antigens have been identified (30, 31, 51, 58), it is unlikely that the use of one or a few mycobacterial antigens would give an accurate representation of the T-cell-mediated response in the lungs. To circumvent these problems, we used a nonspecific method of T-cell restimulation, which allowed the quantitation of T cells capable of secreting cytokines upon TCR engagement. In the first 3 weeks of infection, 22 to 30% of T cells secreted IFN-gamma upon anti-CD3 antibody stimulation. We hypothesize that the lung cells primed to produce IFN-gamma represent activated antigen-specific cells. Interestingly, the proportion of CD8+ T cells primed for IFN-gamma secretion was similar to that of CD4+ T cells.

The presence of T cells primed for cytokine secretion in the lungs does not necessarily indicate that these cells produce cytokine in vivo at all times. Although approximately equal percentages of lung CD4+ and CD8+ T cells were capable of secreting IFN-gamma upon TCR ligation, the percentages of CD8+ T cells spontaneously producing IFN-gamma were ca. threefold lower than those of CD4+ T cells. Taking into consideration the differences in actual numbers of CD4+ and CD8+ T cells, the number of CD4+ T cells actually producing IFN-gamma at a given time was greater (3.5- to 5-fold) than that of CD8+ T cells. The primary role of IFN-gamma is to activate Mphi s to kill intracellular mycobacteria (1), and activated Mphi s produce toxic products, such as reactive nitrogen intermediates (11, 17), that can damage the tissues. That no more than one-third of cells are primed to produce cytokine may reflect the fact that cytokine secretion must be carefully balanced such that protection is achieved without inflicting significant damage on the infected organ. The factors controlling differential cytokine production are unclear. Some primed T cells may be engaged in other effector functions or may not encounter a relevant antigen presented by infected APCs. This may be especially true in the case of Mycobacterium-specific CD8+ T cells, which may be functioning as CTLs and respond to antigens that are processed differently from those that are presented to CD4+ T cells. There may be downregulatory mechanisms in the lungs that result in the unresponsiveness of certain T-cell populations. It has been suggested previously that M. tuberculosis-infected alveolar macrophages have a pronounced suppressive phenotype (26, 27, 47).

Since mycobacteria reside primarily within vacuoles inside the cell (12, 49, 57), antigens are efficiently processed and presented on MHC class II molecules to CD4+ T cells. On the other hand, the ideal conditions for processing and presenting mycobacterial antigens in the context of MHC class I molecules to CD8+ T cells are not known. Proposed mechanisms which lead to increased presentation of mycobacterial antigens by MHC class I molecules include escape of mycobacteria from the phagolysosome to the cytosol and leaking of mycobacterial proteins into the cytoplasm through Mycobacterium-induced pores (32, 37, 38, 45). DCs infected with M. tuberculosis for 48 h, as compared to 24 h, were better APCs for stimulation of CD8+ T cells, suggesting that prolonged interaction between bacteria and APCs is required to present epitopes recognized by CD8+ T cells. Expression of MHC class I and II and costimulatory molecules on the infected DCs increased over the course of infection, which also might contribute to enhanced stimulation (reference 25 and data not shown). Interestingly, infected macrophages were not efficient stimulators of cytokine production by either T-cell subset. The recent demonstration of sequestration of immature MHC class II molecules in infected Mphi s suggests that mycobacteria downregulate the presentation of antigens to CD4+ T cells (28). M. tuberculosis-infected Mphi s can also produce cytokines such as transforming growth factor beta , which downmodulate T-cell responses (26, 27). Cytotoxic activity by CD8+-T-cell clones requires much lower peptide concentrations than cytokine production does (56). Therefore, the stimulation of CTL activity by Mycobacterium-specific CD8+ T cells may occur under conditions that do not induce cytokine secretion by either CD4+ or CD8+ T cells, such as by infected Mphi s.

The possibility that lung T-cell populations are primed for production of cytokines other than IFN-gamma was investigated. TNF-alpha is an essential component of the immune response to M. tuberculosis (21) and is thought to be involved in Mphi activation and granuloma formation and function (11, 18, 23). TNF-alpha may also be required for T-cell priming (6, 46). A subset of T cells produced TNF-alpha upon stimulation; >70% of TNF-alpha -producing cells also secreted IFN-gamma . Neither IL-4 nor IL-10 production was observed in the T cells during the first 4 weeks of infection.

In this study, we have demonstrated that (i) activated CD8+ T cells were present in the lungs in large numbers early in M. tuberculosis infection; (ii) although ~23% of the CD8+ T cells were primed to produce IFN-gamma and TNF-alpha , only a fraction of the cells actually produced cytokine at any one time during infection; (iii) ~30% of CD4+ T cells were also primed for production of IFN-gamma and TNF-alpha and were the major producers of IFN-gamma in early infection; and (iv) M. tuberculosis-infected DCs were superior to Mphi s in stimulation of antigen-specific cytokine production by both CD4+ and CD8+ T cells. The presence of large numbers of activated CD8+ T cells in the lungs, with apparently only moderate cytokine production, provides support for the hypothesis that CD8+ T cells engage in other functions, e.g., that they are cytotoxic in the response against M. tuberculosis. Cytokine secretion by CD8+ T cells was stimulated only by APCs infected for at least 2 days; in the lungs, cytotoxic activity might be exerted prior to that time. Defining the contribution of CD8+ T cells to antimycobacterial immunity and the ideal stimulation conditions for these cells is essential in the design of effective vaccines against tuberculosis.


    ACKNOWLEDGMENTS

We are grateful to Walter Storkus, Michael Lotze, and Schering-Plough for the generous gift of rmGM-CSF and rmIL-4. We thank the laboratory of Susan McCarthy for providing us with anti-CD8 supernatant for cell depletions and anti-IL10 antibodies for intracellular staining. We thank John Chan and Padmini Salgame for critical reading of the manuscript and members of the Flynn laboratory for helpful discussions.

This work was supported by National Institutes of Health grant AI37859 (J.L.F.) and American Cancer Society grant JFRA-644 (J.L.F.).


    FOOTNOTES

* Corresponding author. Mailing address: Department of Molecular Genetics and Biochemistry, E1240 Biomedical Science Tower, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261. Phone: (412) 624-7743. Fax: (412) 624-1401. E-mail: joanne{at}pop.pitt.edu.

Editor:   S. H. E. Kaufmann


    REFERENCES
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

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Infection and Immunity, August 1999, p. 3980-3988, Vol. 67, No. 8
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