Comparative Analysis of T Lymphocytes Recovered from the Lungs of Mice Genetically Susceptible, Resistant, and Hyperresistant to Mycobacterium tuberculosis-Triggered Disease

Animals

I/StYCit (I/St), A/JSNYCit (A/Sn), and (A/Sn × I/St) F₁ mice were bred at the animal facilities of the Central Institute for Tuberculosis (Moscow, Russia) according to the rules of Russian Academy of Medical Sciences, with water and food provided ad libitum. Female mice, 2–4 mo of age, were used.

Infection

Mice were infected i.v. with 10⁵, 10⁴, or 10³ CFUs of mid-log phase M. tuberculosis strain H37Rv (collection of the Central Institute for Tuberculosis) in 0.5 ml of saline. The method of establishment of clump-free, mid-log phase mycobacterial preparations was described previously in detail (22, 25). To assess mycobacterial load in spleens and lungs, 0.2 ml of serial 10-fold dilutions of organ homogenates was plated onto Dubos agar, and colonies were counted after 18–20 days of incubation at 37°C.

Lung, spleen, and lymph node cell suspensions

Two, 5, and 8 wk following challenge, mice were euthanized by injection of an overdose of thiopental (Biochemie, Vienna, Austria), and suspensions of spleen, auxiliary lymph nodes, and lung cells were prepared individually. Lung cells were isolated using the method described by Holt et al. (26) with our modifications (25). Briefly, blood vessels were washed out, and repeated bronchoalveolar lavage was performed using 0.02% EDTA-HBSS solution. Lung tissue was sliced into 1- to 2-mm³ pieces and incubated at 37°C in RPMI 1640 containing 5% FCS, antibiotics, 10 mM HEPES (all from HyClone, Carlington, The Netherlands), 200 U/ml collagenase, and 50 U/ml DNase (Sigma, St. Louis, MO). Single-cell suspensions were obtained by vigorous pipetting. Cells (hereafter referred to as unseparated cells) were washed and resuspended in culture medium, i.e., RPMI 1640 supplemented with 5% FCS, 10 mM HEPES, 2 mM l-glutamine, 1% nonessential amino acids, pyruvate, 5 × 10⁻⁵ 2-ME, antibiotics (all from HyClone). T lymphocyte enrichment was achieved by sequential elimination of plastic-adherent and nylon wool-adherent cells, which resulted in approximately 75% CD3⁺ cell purity (25). The viability of cells, as determined by trypan blue exclusion, was >93%.

Cloning of lung T cells

To estimate the efficacy of lung T cell cloning, T-enriched lung cells were cloned by limiting dilutions as described previously (25). Briefly, lung T lymphocytes obtained at week 5 postinfection were enriched in mycobacteria-specific cells by stimulation in vitro with H37Rv sonicate; the latter were isolated by centrifugation on Lympholyte M gradient (Cedarlane, Ontario, Canada) and cloned in the presence of H37Rv sonicate, irradiated splenic APCs, and conditioned medium, as a source of cytokines. Positive wells were restimulated in situ or were split into new wells every 10–14 days. The efficacy of T cell cloning in response to Con A (2.5 μg/ml; Sigma) was estimated for freshly isolated T-enriched lung cells from either infected or naive mice. Cells were stimulated once with Con A, APC, and cytokines at the beginning of experiment, and the number of positive wells was estimated microscopically between days 7 and 12 of culture.

Proliferative response

T-enriched lung and spleen cells (10⁵) mixed with 3 × 10⁵ syngenic APC or 3 × 10⁵ bulk lymph node cells were cultured in a well of 96-well flat-bottom plate (Costar, Badhoevedorp, The Netherlands) at 37°C in 5% CO₂ in the presence of 10 μg/ml H37Rv sonicate. All cultures were performed in triplicate, and nonstimulated wells served as controls. Cultures were pulsed with 0.5 μCi of [³H]thymidine for the last 18 h of a 48- to 72-h incubation. The label uptake was measured in a liquid scintillation counter (Wallac, Turku, Finland) after harvesting the well’s contents onto fiberglass filters using a semiautomatic cell harvester (Scatron, Oslo, Norway).

Staining of cell surface molecules

Lung cells (3–5 × 10⁵) were washed twice in PBS containing 0.01% NaN₃ and 0.5% BSA and were incubated for 5 min at 4°C in the presence of CD16/CD32 mAbs (clone 2.4G2, PharMingen, San Diego, CA) to block Fc receptors. Cells were then double or triple stained with directly conjugated Abs according to the manufacturer’s instructions. All Abs except FITC-anti-CD11α (clone I21/7, Sigma) were purchased from PharMingen: FITC-anti-CD4 (clone H129.19), PE-anti-CD8a (clone 53-6.7), PE-anti-CD44 (clone IM7), FITC-anti-CD45RB (clone 16A), PE-anti-CD28 (clone 37.51), FITC-anti-CD152 (CTLA-4, clone UC10-4F10-11), FITC-anti-CD95 (clone Jo2), and FITC-anti-CD95 ligand (anti-CD95L; clone MFL3). Stained cells were washed twice, fixed with 1% paraformaldehyde, and analyzed by flow cytometry.

Apoptosis evaluation

Unseparated lung cells recovered from the lungs of infected mice were cultured in 24-well plates (10⁶/well) in the presence or the absence of 10 μg/ml H37Rv sonicate for 40 h. Cells were harvested and stained with PE-labeled anti-CD4 or anti-CD8 mAbs (Caltag, South San Francisco, CA), followed by staining with FITC-annexin V and 7-amino-actinomycin D (7AAD; PharMingen), according to the manufacturer’s instructions. 7AAD stain was used because, unlike propidium iodide, its fluorescent spectrum allows gating of PE-stained cell subsets.

Assessment of T cell proliferation by bromodeoxyuridine (BrdU) incorporation

At week 5 postinfection, mice were injected with 2 mg of BrdU (Sigma) i.p. Two hours later, auxiliary lymph nodes were extracted, single-cell suspensions were prepared individually, and BrdU incorporation was assessed as described previously by Esin et al. (27). Cells were washed in mouse tonicity HBSS supplemented with 5% FCS and 20 U/ml DNase I (Sigma). CD4 and CD8 membrane markers were stained with PE-labeled mAbs. Cells were washed in supplemented mouse tonicity HBSS and fixed with 1% paraformaldehyde in PBS containing 0.01% Tween 20. After 96 h of incubation at 4°C, cells were washed and incubated with DNase solution (4.2 mM MgCl₂, 10 μM HCl, and 50 U/ml DNase in 0.15 M NaCl) at 37°C for 60–120 min. After washing, cells were stained with FITC-anti-BrdU mAbs (Becton Dickinson, San Jose, CA), washed in PBS/FBS/Tween 20 (PBS supplemented with 0.5% Tween 20 and 5% FBS), resuspended in PBS, and analyzed by flow cytometry.

Flow cytometry of stained cells

An EPICS Elite flow cytometer (Coulter, Miami, FL) equipped with a Cyonics argon laser (Uniphase, San Jose, CA) with excitation at 488 nm and 15-mW power, and barrier filters at 488BK, 550DL, 525BP, 625DL, and 575BP, was used throughout the experiments. At least 10⁴ cells from each sample were analyzed, and the data were processed by means of MultiGraph software (Coulter). Unstained cell controls were analyzed at each time point.

Cytokine assays

ELISAs were used to detect IL-4, IL-5, IL-10, IL-12, TNF-α, and IFN-γ in 48-h culture supernatants. Capture and detecting (biotinylated) mAbs specific for mouse cytokines were purchased from PharMingen: for IFN-γ, clones R4-6A2 and XMG1.2 (sensitivity, 312 pg/ml); for IL-4, clones 11B11 and BVD6-24G2 (sensitivity, 62 pg/ml); for IL-5, clones TRFK5 and TRFK4 (sensitivity, 24 pg/ml); for IL-10, clones JES5-2A5 and JES5-16E3 (sensitivity, 312 pg/ml); for IL-12, clones C 17.8 and C 15.6 (sensitivity, 250 pg/ml); and for TNF-α, clone MP6-XT22 and polyclonal Abs (sensitivity, 125 pg/ml). ELISAs were performed following the manufacturer’s instructions. A standard curve for each assay was generated with known concentrations of mouse rIL-4, rIL-5, rIL-12, and rTNF-α (all from PharMingen), rIL-10 (Sigma), and rIFN-γ (Genzyme, Boston, MA).

Antimycobacterial activity of macrophages

To assess the anti-mycobacterial activity of macrophages, we used the method described by Stach et al. (28) with modifications (25). Briefly, peritoneal exudate cells (15 × 10⁶) were incubated for 1 h on 60-mm petri dishes (Costar) in 3 ml of antibiotic-free cultural medium at 37°C. Plastic nonadherent cells were removed, and plastic adherent cells were detached by incubation in 2 mM EDTA-PBS. Plastic adherent cells (6 × 10⁴; >90% of nonspecific esterase-positive cells) were put in a well of a flat-bottom 96-well plate (Costar) and 12 × 10⁴ live-filtered M. tuberculosis/well were added. Infected macrophages were cocultured with 6 × 10⁴/well lung or spleen T cells freshly isolated from infected mice. T cell-free cultures of mycobacteria-loaded macrophages, supplemented or not with 100 U/ml recombinant murine IFN-γ (Genzyme), served as positive and negative controls, respectively. Multiplication of mycobacteria was assessed by [³H]uracile uptake exactly as previously described (25, 28). Results are expressed as counts per minute.

NO production by infected macrophages

NO production was assessed as described previously (29). Briefly, peritoneal macrophages were loaded with M. tuberculosis and cultured with or without addition of T cells as described above. Thirty-six hours later, 100 μl of Griess reagent was added to 100-μl supernatant aliquots in the wells of a round-bottom plate, and plates were incubated for 10 min at room temperature. Absorbance was measured at 550 nm in a micro-ELISA reader (Sigma), using a 620-nm reference filter.

Statistical analysis

The significance of the differences was estimated by Student’s t test, Wilcoxon test, and Mann-Whitney test. p < 0.05 was considered statistically significant.

Mycobacterial growth in lungs and spleens of infected mice

Groups of I/St, A/Sn, and F₁ female mice were challenged i.v. with 10⁵ M. tuberculosis H37Rv CFUs, and individual whole-organ homogenates of lungs and spleens were plated in serial dilutions onto agar dishes to determine mycobacterial recovery. As shown in Fig. 1⇓, during the first 2 wk of infection mycobacterial multiplication was prominent in spleens of all infected mice; at least 20 times more mycobacteria were recovered compared with the number injected. An approximately 5-fold difference between susceptible I/St and two other mouse strains was observed (Fig. 1⇓A). The size of the mycobacterial population residing in lungs 2 wk postinfection was considerably smaller than that in spleens, and mycobacterial burden was 10-fold higher in I/St compared with A/Sn and F₁ mice (Fig. 1⇓B). At week 5 following challenge, the number of mycobacteria in spleens of susceptible I/St mice remained constant, whereas in spleens of both A/Sn and F₁ mice mycobacterial load decreased to a significantly lower (p = 0.03) level (Fig. 1⇓A). In the lungs, the enlargement of the mycobacterial population between weeks 2 and 5 was registered in all three mouse strains (p < 0.01 compared with week 2). Again, the mycobacterial population reached about 10-fold higher numbers in I/St compared with A/Sn (p = 0.01) and F₁ (p = 0.02) mice (Fig. 1⇓B). At week 8 postinfection, the mycobacterial population diminished in size in spleens (Fig. 1⇓A) and reduced the speed of growth in lungs (Fig. 1⇓B) of A/Sn and F₁ mice. In contrast to the generally good health of all resistant animals at week 8 postinfection, only 4 of 11 I/St mice survived (one, one, and two in three independent experiments), which did not allow us to reliably estimate the bacterial load in their organs. These results indicate that our earlier conclusions concerning interstrain differences in susceptibility to infection (20, 22) and the dominant inheritance of resistance by F₁ hybrids (24) are applicable to the dynamics of mycobacterial growth in organs of infected mice.

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FIGURE 1.

Dynamics of mycobacterial multiplication in spleens (A) and lungs (B) of infected I/St, A/Sn, and F₁ mice. Groups of mice (n = 4–6) were infected i.v. with 10⁵ CFUs of M. tuberculosis. Two, 5, and 8 wk following the challenge, serial dilutions of whole organ homogenates were plated individually onto Dubo agar for CFU assessment. The number of CFUs per organ are shown. ▪, I/St; □, A/Sn; ⋄, F₁.

Lung T cell subsets following infection

The total cell yield from the lungs of naive and infected mice of the three strains was estimated by microscopy of unseparated lung cell suspensions. The proportions of CD3⁺, CD4⁺, CD8⁺, and NK-1.1⁺ cells were assessed in T-enriched cell suspensions by flow cytometry (see the footnote to Table I⇓).

In naive mice there were no apparent interstrain differences with respect to the total number of cells recovered from lungs. The total cell yield was relatively low (8–10 × 10⁶/mouse), and the CD4/CD8 ratio in the CD3-positive population was uniformly close to 1.3:1 (data not shown). As shown in Table I⇑, the cellularity of lungs of all three strains was markedly elevated (p < 0.05–0.01) throughout the infectious course compared with that in uninfected syngenic controls. Accumulation of bulk CD3⁺ T cells following infection was more pronounced in the lungs of A/Sn and F₁ mice compared with that in I/St mice. The most notable difference between susceptible I/St and resistant A/Sn and F₁ mice was that significantly higher numbers of CD8⁺ T cells were recovered from lungs of the latter two strains throughout infection (p < 0.05). In addition, in F₁ hybrids the total lung cellularity as well as the number of CD4⁺ T cells increased earlier than in either parental strain (Table I⇑, second week, p < 0.05). NK-1.1⁺ cells were scarcely present in the lungs of all mice (∼1.5% positive cells; data not shown).

Proliferation of lung and spleen T cells in the presence of mycobacterial Ag

It was of interest to find out whether the proliferative capacity of lung T cells is essential for the expression of the resistant phenotype. We also wished to clarify whether a more pronounced accumulation of these cells in resistant animals is the consequence of their capacity to proliferate more vigorously in response to mycobacterial Ags. Thus, we assessed the level of lung T cell proliferation in the presence of syngenic splenic APCs and H37Rv sonicate as a source of Ag. Unexpectedly, I/St lung T cells started to proliferate earlier following challenge and retained a higher proliferative capacity (p = 0.02) throughout the infectious course than their A/Sn counterparts (Fig. 2⇓A). In four of five experiments A/Sn lung T cell did not proliferate at all at 2 wk postinfection. Furthermore, lung T cells from F₁ mice exhibited intermediate levels of proliferation. Similar results were obtained when the proliferation of cells from auxiliary lymph nodes at 5 wk postinfection was measured (Fig. 2⇓B). Taken together, these results suggest that neither a more pronounced accumulation of T cells in the lungs nor the ability of the host to better control the disease is due to Ag-induced local T cell proliferation in the vicinity of tuberculous foci.

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FIGURE 2.

Ag-specific proliferative response of T-enriched lung cells (A) and lymph node cells (B) to M. tuberculosis sonicate. Mice were infected with 10⁵ CFUs of H37Rv i.v. Proliferation was assessed 2, 5, and 8 (lung cells) or 5 (lymph node cells) wk following challenge and is expressed as [³H]thymidine uptake (lung cells: cpm ± SD for four independent experiments; lymph node cells: counts per minute ± SD for three individual mice). Only 4 of 11 I/St mice survived longer than 8 wk (1, 1, and 2 in three independent experiments), which did not allow us to estimate reliably their response. ∗, Significant differences between A/Sn and I/St .

A more vigorous proliferation of I/St T cells could simply reflect a higher mycobacterial load in these mice throughout infection (Fig. 1⇑) or could be due to their intrinsic capacity to develop higher levels of mycobacteria-specific and/or nonspecific T cell responses. To distinguish between these two possibilities, groups of I/St mice were infected with decreasing doses of mycobacteria and compared with A/Sn mice infected with a standard dose (10⁵ CFUs) with respect to mycobacterial load in their lungs and capacity of lung T cells to respond to the mycobacterial sonicate. As shown in Table II⇓, I/St lung T cells responded to the Ag almost regardless of the challenging dose of H37Rv. Moreover, when I/St mice were infected with 10³ CFUs and the mycobacterial load in their lungs did not differ from that of A/Sn mice challenged with 10⁵ CFUs, a dramatic interstrain difference in proliferative response was still present (Table II⇓).

The suggestion that lung T cells of infected I/St mice proliferate more vigorously in the course of infection was further confirmed in T cell cloning experiments. As shown in Tables III⇓ and IV⇓, I/St and A/Sn lung T cells extracted at 5 wk postinfection showed strikingly different patterns of growth under identical limiting dilution conditions (described previously (25)). During the initial three stimulation/rest cycles, the efficacy of cloning (the number of positive wells) for I/St lung T cells was at least 10- fold higher than that for A/Sn cells (Table III⇓). Moreover, a few initially established A/Sn T cell clones ceased to proliferate at the fifth to sixth stimulation round, while several I/St clones were successfully expanded.

To determine whether the difference in cloning efficacy was due to the inherently poor T cell clonal growth in A/Sn mice or was secondary to differently developing lung disease, we have estimated parameters of clonal growth in the presence of the T cell mitogen Con A. Lung T cells were obtained from infected and naive I/St and A/Sn mice and cloned by limiting dilutions in a single Con A stimulation round. The frequency of Con A-responding precursors was identical in the lungs of naive I/St and A/Sn mice (Table IV⇑). However, the size of all proliferating cell populations of A/Sn origin by day 12 of culture was limited to 20–40 cells, whereas the vast majority of I/St-positive wells contained several hundred blast-like cells. The difference was even more striking when T cells were obtained from infected mice. In addition to the manyfold bigger size of individual I/St clones, a higher frequency of positive wells in I/St cultures was apparent (Table IV⇑). These results suggest that I/St lung T cells are more readily activated for further proliferation both in vivo by infection and in vitro by Con A stimulation than their A/Sn counterparts.

To confirm that the interstrain difference in the proliferative capacity of T cells was not restricted to the in vitro experimental system, we assessed the proliferation of T cells in situ by BrdU incorporation. At 5 wk postinfection, I/St and A/Sn mice were injected i.p. with BrdU, and its incorporation into CD4⁺ and CD8⁺ lung and lymph node T cells was individually analyzed by flow cytometry. As shown in Fig. 3⇓, among lymph node cells extracted from I/St mice, about twice as many (p < 0.05, by Mann-Whitney t test; n₁ = 4; n₂ = 4) CD4⁺ and CD8⁺ cells incorporated BrdU compared with A/Sn cells, thus confirming the results obtained in vitro. The proportion of lung T cells incorporating BrdU was too low to draw conclusions.

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FIGURE 3.

BrdU incorporation in vivo by CD4⁺ (A and B) and CD8⁺ (C and D) cells extracted from auxiliary lymph nodes at 5 wk postinfection. Mice were given BrdU i.p. 2 h before being euthanized. Auxiliary lymph nodes cells were stained with PE-anti-CD4 or PE-anti-CD8 mAbs followed by staining with FITC-anti-BrdU mAbs and were assessed by flow cytometry. Four mice of each strain (two independent experiments) were analyzed individually, and the results of one experiment are shown.

Lung T cell surface phenotype before and after infection

Given the differences in the ability of I/St and A/Sn T cells to proliferate in vitro in response to mycobacterial Ags, we wondered whether the expression of T cell activation markers and of costimulatory molecules also differ in the lungs of these mice. T-enriched lung cell suspensions were triple stained with Abs against either CD4 or CD8 subset marker in combination with anti-(CD44, CD45RB) Abs, triple stained with the combination of anti-(CD4, CD8) Abs with either anti-CD95 or anti-CD95L, or double stained with anti-CD4 and anti-CD8 Abs in reciprocal combinations with anti-CD28, anti-CD152, or anti-CD11-α Abs.

No major interstrain differences were found in the expression of CD44/CD45RB molecules on T cells before challenge (Fig. 4⇓, plates 1–3). Regardless of the mouse strain, 30–45% CD4⁺ cells and 15–30% CD8⁺ cells expressed the CD44^low/CD45RB⁺ phenotype of resting/naive cells. Approximately 20–40% CD4⁺ and 70–80% CD8⁺ cells expressed the phenotype of activated cells (CD44⁺/CD45RB^low and CD44⁺/CD45RB⁺, respectively). The relative abundance of activated lung T cells before challenge is most likely due to routine stimulation of the respiratory tract with inhaled antigenic substances. Since the expression of CD44 and CD45 markers is known to vary naturally between mouse strains (30), it was important to show that there was no major intrinsic genetic variation with respect to this trait in the strains under study. As early as 2 wk postinfection, the percentage of CD44-positive activated lung T cells markedly increased in both major subsets (data not shown) and remained remarkably high throughout infection. At 5 wk only about 10–20% of the cells retained the CD44^low phenotype and up to 90% were expressing CD44 activation marker (Fig. 4⇓, plates 4–6 and 10–12).

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FIGURE 4.

Expression of CD44/CD45RB molecules by CD4⁺ (panels 1–6) and CD8⁺ (panels 7–12) lung T cells. Cells were isolated from lungs of control or infected mice and were analyzed by flow cytometry after gating CD4⁺ or CD8⁺ cells. Numbers represent the percentage in each quadrant. Two independent experiments gave similar results.

The most prominent difference between mouse strains was in CD45RB expression at 5 wk following challenge. Activated CD4⁺ cells are known to strongly reduce CD45RB expression (31), whereas activated CD8⁺ cells are thought to continue to express this molecule (32, 33). Between weeks 2 and 5 postinfection, there was a progressive decrease in CD45RB expression by CD4-positive cells, and by week 5 this decrease was more pronounced in I/St mice. As shown in Fig. 4⇑, in I/St lungs the total number of CD45RB⁺ CD4⁺ cells did not exceed 30% (Fig. 4⇑, plate 4) compared with 50% in A/Sn lungs (Fig. 4⇑, plate 5). Cells from F₁ mice occupied an intermediate position between those from parental strains (Fig. 4⇑, plate 6). Among CD8⁺, the CD44⁺/CD45RB⁺ activated phenotype was expressed by >70% A/Sn (Fig. 4⇑, plate 11) and F₁ (Fig. 4⇑, plate 12) cells, but only by approximately 50% I/St (Fig. 4⇑, plate 10) cells. Making an inventory of the interstrain differences in total numbers of lung CD8⁺ cells (Table I⇑) following the challenge, the number of CD8⁺ CD44⁺/CD45RB⁺ cells was about 4-fold lower in I/St compared with A/Sn and F₁ mice (0.7, 2.7, and 2.0 million/mouse, respectively).

Neither CD4⁺ nor CD8⁺ lung T cells from all three mouse strains expressed CTLA-4 (CD152) molecule, but both subsets readily and uniformly expressed CD28 molecule. In all mice, the proportion of CD28-positive cells increased from 40–50% at 2 wk to 90–95% at 5 wk postinfection. Analogously, infection was accompanied by a substantial increase in the proportion of cells expressing the α-chain of LFA-1 (CD11-α) integrin (from 45–60% before infection to 90–99% at 5 wk postinfection).

Since a significantly higher proliferative response of T cells in the lungs of infected I/St mice (Fig. 2⇑) did not lead to their accumulation in the organ (Table I⇑), we compared the expression of the most common apoptotic marker, CD95 (Fas), on lung T cells, anticipating that more I/St cells could undergo apoptosis during the infectious course. In all mice CD95 was scarcely present on the surface of lung T cells at 2 wk postinfection (data not shown). As shown in Fig. 5⇓, at 5 wk following challenge, a significantly higher proportion of I/St lung CD4⁺ T cells carried the CD95 marker compared with A/Sn (58 and 30%; p < 0.05). Interestingly, in all three mouse strains the proportion of CD8⁺ lung T cells expressing CD95 was always higher than that of CD4⁺ cells, although no interstrain differences were found (Fig. 5⇓). To ensure ourselves that Fas expression is not a simple consequence of cell activation but is functionally linked to apoptosis, we have evaluated the proportion of CD4⁺ and CD8⁺ apoptotic cells from I/St and A/Sn infected lungs. Unseparated lung cells were stained with annexinV/7AAD following stimulation in vitro with mycobacterial sonicate. As shown in Fig. 6⇓, fewer I/St CD4⁺ cells remained alive, and more underwent apoptosis compared with A/Sn CD4⁺ cells (16 vs 26% and 83 vs 64%, respectively). As with CD95 expression, a higher proportion of CD8⁺ compared with CD4⁺ cells was subjected to apoptotic death, and no interstrain differences were found in this T cell subset.

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FIGURE 5.

CD95 molecule expression by CD4⁺ (A–C) and CD8⁺ (D–F) lung T cells at 5 wk postinfection.

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FIGURE 6.

Apoptosis is induced in a higher proportion of I/St compared with A/Sn lung CD4⁺ cells. Unseparated lung cells were stimulated in vitro with H37Rv sonicate for 40 h, harvested, and triple stained with PE-anti-CD4 or PE-antiCD8 mAbs combined with FITC-annexin V and 7-AAD. CD4 and CD8 subsets were gated and analyzed separately. Quadrants 2, 3, and 4 correspond to, respectively, late apoptotic plus necrotic, early apoptotic, and live cells.

Cytokine production by lung cells

Cytokine production was determined in lung, spleen, and lymph node cell culture supernatants of two different types. First, unseparated cells alone were cultured in the presence of H37Rv sonicate, and the contents of six different cytokines in supernatants were determined. Second, T-enriched lung or spleen cells were cocultured with irradiated syngenic splenic APC and the Ag, and the contents of IFN-γ, IL-4, IL-5, and IL-10 were assessed. Since TNF-α and IL-12 are synthesized predominantly by macrophage-like cells, we did not measure the contents of these two products in the second type of cultures.

At week 2 postinfection, I/St bulk lung cells produced significantly (p < 0.05) less IFN-γ than their A/Sn and F₁ counterparts. At this time purified lung T cells from all mice produced similar levels of IFN-γ, but always lower levels compared with unseparated cells, suggesting that non-T lung cells contribute much to IFN-γ production/induction, especially in resistant mice (Table V⇓). The most potent IFN-γ inducer, IL-12, was also synthesized by unseparated lung cells of all three mouse strains. At week 2 postinfection I/St and F₁ cells exhibited identical IL-12 production, whereas at week 5 postinfection I/St cells synthesized lower amounts of IL-12. A 2-fold difference in TNF-α levels occurred at weeks 2 and 5 postinfection between I/St and resistant mice. Thus, the production of three major type 1 cytokines by the lung cells was moderately higher in resistant mice.

IL-10 was equally well produced by unseparated lung cells of all mice at 2 and 5 wk; however, its production by T-enriched lung cells differed between mouse strains. I/St T cells produced IL-10 in larger quantities than A/Sn and F₁ T cells, and this was particularly clear late in the infectious course (Table V⇑). These differences, however, were not statistically significant between parental strains due to variations in the levels of cytokine production in distinct experiments. Thus, in four experiments IL-10 levels in I/St cultures varied between 300 and 3500 pg/ml. A level of 600 pg/ml was detected in A/Sn supernatant in one experiment, but only trace quantities were spotted in three others. However, the difference between I/St and F₁ mice was significant (p < 0.01). In agreement with the results obtained in lungs, IL-10 was synthesized by auxiliary lymph node cells from infected I/St mice (500–800 pg/ml; three mice were assessed individually at week 5), but was not produced by A/Sn lymph node cells. That I/St lung cells more readily produce type 2 cytokines, as suggested by IL-10 data, was confirmed by IL-5 assessment. Unseparated lung cells from I/St mice produced measurable amounts of IL-5 at weeks 2 and 5, whereas only trace amounts were found in A/Sn supernatants (Table V⇑). F₁ mice showed intermediate inheritance of this phenotype. Interestingly, no production of IL-5 was detected in cultures of T-enriched lung cells, suggesting that non-T cells residing in infected lungs are responsible for IL-5 production and/or induction.

Taken together, these results indicate that there is a moderate bias toward activation of a type 2-like cell response in the lungs of TB-susceptible I/St mice. However, polarization of response is incomplete (i.e., the absence of IL-4 and readily detectable levels of IFN-γ in I/St supernatants) and is masked when unseparated lung cells are studied, apparently due to abundant production of several cytokines by non-T lung cells.

Stimulation of anti-mycobacterial activity of macrophages by lung and spleen cells from infected mice

Given that IFN-γ production by I/St lung T cells was somewhat lower than that by A/Sn and F₁ cells and anticipating that not only IFN-γ potentiates macrophage activation, we examined whether T cells from these mice differ with respect to their capacity to activate anti-mycobacterial macrophage function. To address this issue, peritoneal macrophages from I/St, A/Sn, and F₁ mice were cocultured in vitro with live H37Rv mycobacteria and syngenic T-enriched lung cells obtained from infected mice. Mycobacterial growth was assessed by [³H]uracil incorporation (28). When cultured in antibiotic-free culture medium, alone or within macrophages, mycobacteria readily incorporate [³H]uracil (25). As shown in Fig. 7⇓, addition of exogenous rIFN-γ to macrophage/mycobacteria cocultures (positive control) caused a profound inhibition (85–95%) of mycobacterial growth regardless of the mouse strain. Addition of lung T cells extracted 2 wk postinfection from I/St, A/Sn, and F₁ mice moderately and equally inhibited mycobacterial multiplication (20–30% inhibition in different experiments). Five weeks after challenge the ability of T cells of all three strains to stimulate anti-mycobacterial activity of syngeneic macrophages significantly increased; up to 90% inhibition of mycobacterial growth was registered. Thus, no major interstrain differences in the ability of T cells to stimulate the restriction of mycobacterial growth in vitro were found.

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FIGURE 7.

Stimulation of macrophage (mph) anti-mycobacterial activity by lung T cells from infected mice. Peritoneal macrophages (6 × 10⁴/well) were loaded with 12 × 10⁴ live mycobacteria, and 10⁵ freshly isolated lung T cells were added to cultures. Following 96 h of incubation, mycobacterial growth was measured as [³H]uracil uptake (mean cpm ± SD for triplicate determinations). The results of one of two similar experiments are shown. □, Macrophages and H37Rv; ▪, macrophages, H37Rv, and rIFN-γ; ▨, macrophages, H37Rv, and lung T cells.

It is widely accepted (34, 35, 36) that the main mechanism of intracellular mycobacterial killing by murine macrophages is production of reactive nitrogen intermediates (RNI). To determine the role of RNI in our system and to evaluate the correctness of measuring mycobacterial growth by [³H]uracil incorporation, we assessed the content of NO in supernatants of mycobacteria-loaded cell cultures. Individual NO concentrations, as measured in all types of cell cultures (with and without addition of lung T cells or rIFN-γ and with cells obtained from all three mouse strains) were plotted against the levels of [³H]uracil incorporation (counts per minute) in corresponding cultures. As shown in Fig. 8⇓, there was a good inverse correlation between the two measurements (r = −0.781), suggesting the pivotal role of RNI in mycobacterial killing in our system.

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FIGURE 8.

Reverse correlation between mycobacteria growth and RNI production by peritoneal macrophages in the presence of lung T cells. Mycobacteria, macrophages, and T cells were cocultured as described in Fig. 5⇑. Forty-eight hours later the supernatants of three wells were harvested, and the levels of NO production were determined. Mycobacterial growth was assessed by determining [³H]uracil uptake. r = −0.781.