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CD4⁺ and CD8⁺ T Cells Kill Intracellular Mycobacterium tuberculosis by a Perforin and Fas/Fas Ligand-Independent Mechanism¹

David H. Canaday^2,*, Robert J. Wilkinson^*,, Qing Li^*, Clifford V. Harding, Richard F. Silver^*, and W. Henry Boom^*

^* Division of Infectious Diseases, Department of Pathology, and Division of Pulmonary and Critical Care Medicine, Case Western Reserve University School of Medicine and University Hospitals of Cleveland, Cleveland, OH 44106; and Wellcome Center for Clinical Tropical Medicine, Imperial College School of Medicine, London, United Kingdom.

	Abstract

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Cytotoxic effector phenotype and function of MHC-restricted Mycobacterium tuberculosis (MTB)-reactive CD4⁺ and CD8⁺ T lymphocytes were analyzed from healthy tuberculin skin test-positive persons. After stimulation in vitro with MTB, both CD4⁺ and CD8⁺ T cells up-regulated mRNA expression for granzyme A and B, granulysin, perforin, and CD95L (Fas ligand). mRNA levels for these molecules were greater for resting CD8⁺ than CD4⁺ T cells. After MTB stimulation, mRNA levels were similar for both T cell subsets. Increased perforin and granulysin protein expression was confirmed in both in CD4⁺ and CD8⁺ T cells by flow cytometry. Both T cell subsets lysed MTB-infected monocytes. Biochemical inhibition of the granule exocytosis pathway in CD4⁺ and CD8⁺ T cells decreased cytolytic function by >90% in both T cell subsets. Ab blockade of the CD95-CD95L interaction decreased cytolytic function for both T cell populations by 25%. CD4⁺ and CD8⁺ T cells inhibited growth of intracellular MTB in autologous monocytes by 74% and 84%, respectively. However, inhibition of perforin activity, the CD95-CD95L interaction, or both CTL mechanisms did not affect CD4⁺ and CD8⁺ T cell mediated restriction of MTB growth. Thus, perforin and CD95-CD95L were not involved in CD4⁺ and CD8⁺ T cell mediated restriction of MTB growth.

	Introduction

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Acquired protective immunity to Mycobacterium tuberculosis (MTB)³ in humans is dependent on T cells and involves multiple T cell subsets (1, 2). However, the mechanisms used by T cells to restrict the growth of MTB within macrophages are poorly understood. Cytokine secretion or cytotoxic effector function has been proposed as major effector mechanisms for lymphocyte-mediated control of intracellular pathogens. In mice, IFN-, TNF-, and IL-12 play obligate roles in mycobacterial containment (3, 4, 5, 6, 7). In humans, the protective role of IFN- and IL-12 is illustrated by rare mutations in the genes for receptors for IFN- or IL-12, or for IL-12 itself. These mutations severely compromise the ability to control replication of ordinarily nonpathogenic mycobacteria (8, 9, 10, 11). Paradoxically, IFN- treatment of macrophages in vitro only moderately inhibits growth of MTB in human mononuclear phagocytes suggesting an indirect rather than direct effector role of this cytokine (12, 13, 14). This observation, together with the knowledge that both CD4⁺ and CD8⁺ MTB-specific cytolytic cells are present in healthy infected subjects (15, 16, 17, 18, 19, 20, 21), has encouraged investigation of the role of T cell cytolytic function as protective effector function against MTB.

Two major mechanisms of lymphocyte-mediated cytotoxicity are recognized, exocytosis of cytotoxic granules containing pore-forming perforin and serine esterase granzyme molecules, and induction of apoptosis by ligation of CD95 (Fas) by CD95 ligand (CD95L; Refs. 22 and 23). A study of MHC class II-restricted CD4⁺ T cell clones specific for purified protein derivative (PPD) showed expression of both perforin and CD95L as measured by semiquantitative PCR (24). Cytolytic mechanisms of MHC class I-restricted MTB-reactive CD8⁺ T cells have not been studied in detail but are likely to involve the granule exocytosis pathway (25). In gene-disrupted murine models, neither perforin-mediated lysis nor CD95 ligation alone is sufficient to provide early protection against MTB (26, 27). There are contradictory observations on the ability of human CD4⁺ cytolytic cells to inhibit growth of MTB in monocytes (MN) or macrophages. Although M. bovis-bacillus Calmette-Guérin (BCG)-infected macrophages were lysed by mycobacterial-specific CD4⁺ T cell clones and polyclonal lines, growth of M. bovis BCG was not inhibited (28, 29). Recent studies with MTB-reactive CD8⁺ T cells suggest that CTL function was associated with the inhibition of intracellular MTB growth (30, 31, 32).

Recent studies in humans have suggested that granulysin, present in cytotoxic cells, may be a mediator of mycobacterial growth inhibition (32). Granulysin was initially identified in activated T cells (33) and subsequently found in lytic granules of CTL and NK cells (34, 35). Granulysin has been recently described in mycobacterial Ag-stimulated ⁺, CD1-restricted CD8⁺ T cells, and CD4⁺ T cells (32, 36, 37).

In this study, we sought to extend our studies that demonstrated cytotoxic ability by MTB-specific CD4⁺ and CD8⁺ T cells and MTB growth restriction by peripheral blood lymphocytes from PPD⁺ individuals (17, 18). In this study, we analyzed CD4⁺ and CD8⁺ T cells to examine the role of the cytolytic molecules in CTL function and T cell-mediated control of MTB growth.

	Materials and Methods

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Cultivation and processing of mycobacteria

Broth cultures of MTB strains H37Rv and H37Ra (American Type Culture Collection (ATCC), Manassas, VA) were grown in sterile Middlebrook 7H9 medium (Difco, Detroit, MI) with 10% ADC (Difco) enrichment and 0.2% glycerol as described previously in detail (38). Plated cultures were grown on Middlebrook 7H10 agar with 10% OADC (Difco) enrichment and 0.5% glycerol. In preparation for infection of MN, mycobacteria were processed by mechanical disruption and centrifugation, based on the methods of Schlesinger (39) and as previously described in detail (38). These methods minimize clumping and provide accurate quantification of the inoculum.

PBMC and MN preparation

PBMC were isolated from healthy PPD⁺ donors by density centrifugation of whole blood diluted 1/1 with RPMI 1640 (BioWhittaker, Walkersville, MD) over Ficoll (Pharmacia, Uppsala, Sweden). MN were obtained by adherence purification on plastic plates (Falcon, Lincoln Park, NJ). The plates were washed extensively after 1 h of adherence, and MN were scraped off with a cell scraper (Costar, Cambridge, MA).

Expansion and purification of MTB-specific CD4⁺ and CD8⁺ T cells

To expand MTB-specific T cells, PBMC (2 x 10⁶ cells per 2 ml well) from healthy PPD⁺ subjects were cultured with MTB H37Ra as described above. The culture medium was RPMI 1640 supplemented with 10% pooled human serum (Gemini Bio-Products, Calabasas, CA), 20 mM HEPES, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. IL-2 (10 U/ml; Chiron, Emeryville, CA) was added after 48–72 h of culture and every 3–4 days thereafter. After 7–10 days of bulk culture, T cells were either purified as described below or restimulated. Before restimulation, the bulk culture underwent depletion of TCR⁺ cells by incubation with an anti- Ab (clone 5A6.E9; ATCC). After washing, cells then were incubated with goat-anti-mouse IgG-conjugated magnetic beads (Dynal, Oslo, Norway) and negatively selected with a magnetic particle separator (Dynal). The resulting purified cells were cultured (1 x 10⁶/ml) with fresh irradiated autologous PBMC feeders (1 x 10⁶/ml and MTB). Additional IL-2 was added 2 days after restimulation. T cells were purified 7–9 days after restimulation.

Immunomagnetic separation was performed on both resting primary PBMC or PBMC stimulated in vitro with MTB. CD8⁺ T cells were positively selected with anti-CD8-conjugated magnetic beads (Dynal) and Detachabead (Dynal) according to the manufacturer’s guidelines. The MTB-stimulated CD8⁺ T cell population was further purified by depletion of TCR⁺ cells (as described above) and CD4⁺ T cells with anti-CD4 conjugated magnetic beads (Dynal). Unstimulated positively selected CD8⁺ T cells were depleted of CD56⁺ CD8⁺ contaminating NK cells with anti-CD56 beads in a MACS magnetic column separation system (Miltenyi Biotech, Auburn, CA) according to the manufacturers instructions. CD4⁺ T cells were positively selected by anti-CD4 conjugated magnetic beads and detached by Detachabead. Further purification of this subset by negative selection was not necessary.

Cells were analyzed by two-color flow cytometry as described previously (18). Primary and activated CD4⁺ and CD8⁺ T cells were at least 95% CD3⁺/CD4⁺ or CD8⁺ and confirmed to be TCR⁺.

Proliferation assays

Purified CD8⁺ and CD4⁺ T cells (5 x 10⁴ per 200 µl) were incubated for 2 days in a round-bottom 96-well plate with irradiated autologous MN (5 x 10⁴) and MTB (multiplicity of infection (MOI) 5:1) and then pulsed overnight with 1 µCi of [³H]thymidine (ICN, Costa Mesa, CA). Plates were harvested onto glass wool filter with a Filtermate-196 harvester (Packard, Meriden, CT) and cpm counted with Matrix 96 counter (Packard). In experiments with blocking Abs, MN were preincubated at 4°C with mAbs for 45 min before addition of T cells and Ags. Abs remained in the cultures during the entire assay. Anti-MHC class I Ab, W6/32 (ATCC), anti-MHC-II, L243 (BD PharMingen, San Diego, CA), and IgG2a isotype control (Zymed, South San Francisco, CA) were used at 5 µg/ml final concentration.

RNA extraction and the multiprobe RNase protection assay

RNA extraction from T cells was performed using the RNeasy mini kit (Qiagen, Valencia, CA). RNA was frozen at -80°C until use. RNA (1–5 µg) was used in the RNase protection assay (BD PharMingen) according to the manufacturer’s instructions, and as described previously (40). Riboprobes specific for granzymes A and B, perforin, granulysin (two probes), TNF-related apoptosis-inducing ligand (TRAIL), and CD95L were synthesized by using appropriate templates. One riboprobe for granulysin was synthesized from DNA templates designed to detect both the spliced and unspliced transcript of the NKG5 gene and a second to specifically detect unspliced message (35). The difference between the expression of these mRNA was taken to indicate the presence of mRNA specific for granulysin (9 kDa) rather than the unspliced message, which gives rise to a 15-kDa product. At all points of detection, spliced and unspliced transcript was 15–42% higher than the mRNA specific for the 15-kDa product, implying the presence of a minority mRNA encoding the 9-kDa granulysin molecule form (41). All data are presented as the total (spliced and unspliced) message. Riboprobes for the housekeeping gene L32 were included in every assay. Hybridized and RNase digested products were electrophoresed on a 5% denaturing polyacrylamide gel. After drying, the gel was exposed overnight in a Geldoc 1000 (Bio-Rad, Hercules, CA). The identity of the protected bands was confirmed by reference to the unhybridized probes and quantitated by reference to bands for the housekeeping gene L32. A ratio of the density of bands for each molecule in each cell sample and at each time point was generated compared with the housekeeping gene L32. Comparison for induction of mRNA then were determined by calculating the CTL molecule mRNA/L32 mRNA ratio at each time point and comparing each back to the ratio of the cell type at t = 0 (unstimulated).

Flow cytometry for intracellular molecules

After 7 days of MTB stimulation, intracellular detection of cytolytic effector molecules was performed on purified and bulk T cell populations. Bulk cells were first stained with PE conjugated anti-CD4 or anti-CD8 (Caltag, Burlingame, CA). After one wash, cells were fixed with the PermWash kit (BD PharMingen) per the manufacturer’s instructions or for 15 min in 2% paraformaldehyde, then washed in 0.2 M lysine followed by RPMI 1640 before permeabilization (0.1% saponin) and blocking (10% pooled human serum in RPMI 1640) on ice for 15 min. Anti-perforin-FITC (Ancell, Bayport, MN) or isotype control was added to this buffer for an additional 30 min. Cells were washed twice in 0.1% saponin, RPMI 1640, 10% FCS (HyClone, Logan, UT) solution and fixed with 2% paraformaldehyde before flow cytometric analysis. At analysis, CD4⁺ and CD8⁺ cells were individually gated. Studies for granulysin protein expression used purified CD4⁺ and CD8⁺ T cells. Anti-granulysin polyclonal rabbit antiserum (provided by A. Krensky, Stanford University, Stanford, CA) or nonimmunized control serum was incubated with cells in permeabilization buffer for 30 min and then washed twice before adding goat F(ab')₂ anti- rabbit-FITC (Caltag).

⁵¹Cr release assay

MN were infected overnight with MTB H37Ra (7.5:1 MOI) in IMDM (BioWhittaker) and 10% autologous serum. Cells then were loaded with 100 µCi of ⁵¹Cr (ICN) for 2 h and washed four times. ⁵¹Cr-loaded target MN were added to round-bottom wells and preincubated with anti-CD95 (ZB4, 2 µg/ml; Immunotech, Marseille, France) or isotype control (IgG1; Zymed) for 20 min at room temperature. EGTA (2–5 µM final concentration) was added at the same time as T cells. After addition of T cells at varying E:T ratios, plates were centrifuged for 30 s at 200 x g to maximize cell contact. After 4–5 h of incubation at 37°C, 50 µl of supernatant was harvested and counted in a Beckman counter. All culture conditions were performed in triplicate.

For control perforin and granule exocytosis inhibition experiments, a long-term MTB-reactive CD8⁺ T cell line from an HLA-A2⁺ donor was preincubated with concanamycin A (CMA; Sigma, 10 nM) for 2 h or strontium chloride (25 mM; Sigma) for 20 h. Lysis activity was analyzed under two conditions for strontium: either immediately after 20 h of preincubation or during the last 4 h of a 24-h chase period. The effect of CMA on lysis was tested after 2 h preincubation and during the last 4 h of 24 h continuous CMA exposure. Treated T cells were cultured with MTB-infected ⁵¹Cr-loaded THP1 targets (HLA-A2⁺; ATCC). Maximum release was determined by incubating cells with 3% SDS, and spontaneous release was determined by incubating target cells in complete medium alone. The percentage of specific release was determined by the equation [(cpm experimental - cpm spontaneous release)/(cpm maximum release - cpm spontaneous release)] x 100%.

DNA fragmentation assay

Jurkat cells were loaded for 4 h with 1 µCi/ml of [³H]thymidine (ICN) and washed. Jurkat were preincubated with anti-CD95 (5 µg/ml final concentration), and a cell line (KFL9), transfected to express high levels of CD95L (Ref. 42 ; a gift from D. Kaplan, Case Western Reserve University), was preincubated with anti-CD95L (NOK-1; 5 µg/ml final concentration; BD PharMingen) for 30 min. Cells then were combined at a 1:1 ratio. At 24 h, cells were harvested onto a glass wool filter with a Filtermate-196 harvester (Packard). The cpm were counted on Matrix96 counter (Packard). The percentage of specific killing was determined by the equation [(cpm Jurkat alone - cpm Jurkat and KFL9)/(cpm Jurkat)] x 100%.

Intracellular MTB growth assay

Autologous MN (1 x 10⁵/well) were cultured in triplicate in round-bottom 96-well plates. After overnight culture, supernatants were removed and H37Rv added at a MOI of 1:1 in 100 µl/well of IMDM plus 30% non-heat-inactivated autologous serum. After 1 h of incubation at 37°C, supernatants were aspirated and each well was washed three times to remove noningested mycobacteria. Anti-CD95 and anti-CD95L at 5 µg/ml each or isotype IgG1 at 10 µg/ml were added to MN or T cells 20 min before addition of purified T cells. CMA was added at 10 nM to T cells for 2 h before addition to infected MN and maintained at this concentration after T cells were added. All T cells were resuspended in IMDM (no antibiotics) with 10% autologous serum and added so the total volume/well was 200 µl. After 1 and 24 h, wells were aspirated, and 100 µl of lysis buffer (0.067% SDS in Middlebrook 7H9) added to each well. Plates were incubated at 37°C for 10 min followed by neutralization of SDS with 300 µl of PBS with 20% BSA. Lysates from triplicates wells were pooled, and four 10-fold serial dilutions of the resultant 600 µl lysate in 7H9 were prepared. Similar serial dilutions of supernatants were prepared. Six 10-µl aliquots of each dilution of lysate and supernatant were plated onto Middlebrook 7H10 agar and incubated in 5% CO₂ at 37°C until colonies were large enough to be counted. Results were expressed as CFU/ml of lysate (corresponding to CFU/10⁶ cultured MN).

Statistics

All statistics were determined by paired t test analysis.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
MHC restriction of MTB-reactive CD4⁺ and CD8⁺ T cell lines

CD4⁺ and CD8⁺ lines were generated by stimulating PBMC from healthy mycobacterial Ag-sensitized individuals with MTB as described in Materials and Methods. CD4⁺ and CD8⁺ T cells were purified by Ab-coated magnetic beads and tested for Ag specificity with autologous MN infected with MTB. As shown in Fig. 1, both T cell subsets proliferated to MTB-infected MN but were inhibited (p < 0.05) by anti-MHC-I (W6/32) for CD8⁺ T cells and anti-MHC-II (L243) for CD4⁺ T cells. Consistent with our previous studies, both T cell subsets lysed autologous MTB infected MN (15, 17).

View larger version (33K): [in this window] [in a new window]   FIGURE 1. MTB specificity and MHC restriction of CD4+ and CD8+ T cell lines. MN (5 x 104) were infected for 2 h with MTB (MOI 5:1) and then washed. MN were incubated with anti-MHC I (W6/32) or anti-MHC II (L243) or isotype at 5 µg/ml. 5 x 104 MTB specific CD4+ or CD8+ T cells were added for a 3-day proliferation assay. Results are expressed as cpm and are representative of four experiments. Error bars indicate SEM.

First, the kinetics of mRNA expression of secreted and surface-associated cytotoxic effector molecules in PBMC from healthy PPD⁺ donors were determined after MTB stimulation. Cells were restimulated with MTB and autologous feeders on days 7 and 14. Cell lysates for RNA extraction were prepared on day 0, 3, 5, 7, 10, 14, and 17. mRNA levels for cytotoxic effector molecules were measured by quantitative RNase protection assay and compared with mRNA levels for the housekeeping gene L32 (Fig. 2A). mRNA for granzyme A and B, granulysin, perforin, TRAIL, and CD95L were selected for analysis because of their well-defined roles in cytotoxic effector mechanisms (43). A representative RNase protection assay gel is shown in Fig. 2B. Up-regulation of mRNA for these genes reached a steady state between days 10–20; therefore, this time period was chosen to study CD4⁺ and CD8⁺ T cells individually.

View larger version (29K): [in this window] [in a new window]   FIGURE 2. A, Kinetics of mRNA expression for cytotoxic effector molecules in PBMC stimulated with MTB. PBMC were stimulated with MTB and IL-2 (20 U/ml) was added to cultures after 48 h. At indicated time points, cells were harvested for RNA extraction. mRNA expression was determined by RNase protection assay as described in Materials and Methods. All mRNA were normalized relative to housekeeping gene L32 mRNA expression, and fold induction was calculated compared with mRNA levels at time 0, which was set at 1. CD95 and TRAIL were measured only at days 0, 3, and 10, all others at days 0, 3, 5, 7, 10, 14, and 17. Results represent fold induction of two donors. B, Representative RNase protection assay. A gel is shown from one of the donors in this experiment. Lane 1, Resting CD8+ T cells. Lane 2, Resting CD4+ T cells. Lane 3, MTB stimulated PMBC at day 3. Lane 4, MTB stimulated PBMC at day 5.

Fig. 3 demonstrates ratios of mRNA in resting CD4⁺ and CD8⁺ T cells for granzyme A and B, granulysin, perforin, and CD95L compared with the housekeeping gene L32. Levels of mRNA were significantly higher in nonactivated CD8⁺ T cells than CD4⁺ T cells (p < 0.05) for granzyme B, granulysin, and perforin. Differences for granzyme A did not reach statistical significance, but the trend suggested that CD8⁺ T cells had higher baseline levels (p = 0.06). Overall, mRNA levels for cytolytic effector molecules were from 5.6 to 9.4 times higher in CD8⁺ than in CD4⁺ T cells at baseline. Fig. 2B, lanes 1 and 2, illustrates these differences in baseline mRNA levels between resting CD4⁺ and CD8⁺ T cells.

View larger version (31K): [in this window] [in a new window]   FIGURE 3. Up-regulation of cytotoxic molecule mRNA in CD4+ and CD8+ T cells by MTB. PBMC underwent two rounds of in vitro stimulation with MTB, except donors 5 and 7, who had one round. CD4+ and CD8+ T cells were purified by positive selection from these cultures and freshly isolated PBMC analyzed for mRNA expression by RNase protection assay. mRNA for each protein was determined by dividing the densitometry of that mRNA value by the housekeeping gene L32, which was set at 1. mRNA values are expressed as densitometry ratios.

Because IL-2 was added to all cultures after 48–72 h, it was necessary to determine that IL-2 alone was not responsible for the observed changes in gene expression. Resting CD4⁺ T cells were positively selected from PBMC and cultured for 7 days with autologous MN with MTB, IL-2, or both. Fig. 4 demonstrates that 20 U/ml IL-2 alone did not significantly increase mRNA expression for the molecules tested, and more importantly, IL-2 did not further enhance responses to MTB. Based on these findings, we continued to use IL-2 to expand CD8⁺ T cells to achieve adequate numbers of cells to perform our studies.

View larger version (33K): [in this window] [in a new window]   FIGURE 4. mRNA expression for mediators of cytotoxicity in MTB-stimulated, IL-2-stimulated, and MTB plus IL-2-stimulated CD4+ T cells. Positively selected CD4+ T cells from two donors were cultured for 7 days with autologous MTB-infected MN. mRNA expression was determined by RNase protection assay. As described in Materials and Methods, all mRNA were normalized relative to the housekeeping gene L32 mRNA expression and then fold induction calculated compared with mRNA levels at time 0, which was set at 1. Error bars indicate SD.

To determine whether increased mRNA expression induced by MTB stimulation correlated with increased protein expression, PBMC from four PPD⁺ donors were stimulated with MTB and analyzed after one round of in vitro stimulation by flow cytometry. After MTB stimulation, the mean fluorescence index (MFI) and percentage of positive cells increased for both perforin and granulysin above levels measured in resting CD4⁺ and CD8⁺ T cells (Fig. 5). In the four donors studied, the average MFI for perforin was 10-fold higher in unstimulated CD8⁺ than CD4⁺ T cells. After MTB stimulation, the average MFI for perforin was still sixfold higher in CD8⁺ than CD4⁺ T cells. A mean of 33% of CD4⁺ T cells and 72% CD8⁺ T cells expressed perforin after MTB stimulation. The average MFI for granulysin was 3.1-fold higher in unstimulated CD8⁺ than CD4⁺ T cells. After MTB stimulation, the average MFI was 1.7-fold higher in CD8⁺ than CD4⁺ T cells. A mean of 40% of CD4⁺ T cells and 66% CD8⁺ T cells expressed granulysin after MTB stimulation. These data demonstrate that perforin and granulysin protein expression is higher in both resting and MTB-activated CD8⁺ T cells compared with CD4⁺ T cells. These results indicate that up-regulation of mRNA was associated with increased protein expression. Low levels of perforin in resting CD4⁺ T cells have been observed by others (44, 45). Western blotting of cell lysates from both CD4⁺ and CD8⁺ T cells with the polyclonal anti-granulysin antiserum confirmed that there were 15- and 9-kDa bands corresponding to the unprocessed and bioactive forms of granulysin (data not shown; Ref. 35).

View larger version (47K): [in this window] [in a new window]   FIGURE 5. Increased expression of perforin and granulysin protein in MTB activated CD4+ and CD8+ T cells. PBMC were cultured for 7 days with MTB. Intercellular staining for perforin (top) and granulysin (bottom) was performed as described in the Materials and Methods. The thin line represents unstimulated cells, and the thick line MTB-activated cells. Staining with isotype control or control rabbit anti-sera (data not shown) was performed for each condition and cell type, and used individually for each cell type and condition to calculate percent positive MFI. Control staining was slightly higher in activated T cells compared with unstimulated cells. This figure is representative of four experiments.

Next we determined the mechanisms used by CD4⁺ and CD8⁺ T cells to lyse MTB-infected MN. CD4⁺ and CD8⁺ T cell lines were generated from PBMC from PPD⁺ subjects (n = 5). Fig. 6 demonstrates the mean lytic activity of these purified T cells for MTB-infected MN. CD4⁺ and CD8⁺ T cells were equally cytolytic, consistent with our previous studies (17). Cytotoxicity by CD8⁺ T cells was partially inhibited by anti-CD95 Ab (25%; p 0.02). Cytolytic activity of CD4⁺ T cells was inhibited to a similar degree (26%; p 0.01). The inhibitory activity of the anti-CD95 Ab, verified by using Jurkat cells and CD95L transfected K562 cells (KFL9), inhibited DNA fragmentation of Jurkat by 60–100% in 6 h (data not shown). EGTA, which inhibits granule exocytosis, completely inhibited cytotoxicity by CD4⁺ and CD8⁺ T cells (p < 0.005 for both T cell types). In addition, another perforin inhibitor, CMA, which induces depolymerization of perforin and loss of lytic function, was used in selected experiments and was as effective as EGTA in inhibiting CTL activity (see Fig. 8B and Ref. 46).

View larger version (39K): [in this window] [in a new window]   FIGURE 6. Inhibition of lytic activity of MTB-stimulated CD4+ and CD8+ T cell lines. Results represent mean cytotoxicity of MTB-infected MN by MTB-stimulated T cell lines from 5 PPD+ donors. The highest E:T ratio for each donor is shown, which varied between 40:1 to 75:1. Targets were 51Cr-pulsed MTB-infected autologous MN, which were treated with EGTA or anti-CD95 Ab. Error bars indicate SEM.

View larger version (23K): [in this window] [in a new window]   FIGURE 8. Stability of perforin and CD95-CD95L inhibitors for 24 h MTB growth inhibition assay. A, MTB-specific CD8+ T cells were incubated initially with strontium chloride (25 mM) for 20 h and then medium for a 24-h chase period or with CMA (10 nM) for 2 h of pretreatment and then left in for the full 24 h. The cells were added immediately after the pretreatments or at the last 4 h of the 24-h incubation to 51Cr-loaded target cells. Results are expressed as percentage of lysis and are representative of two experiments. B, Purified CD4+ or CD8+ T cells (5 x 104) were incubated with 5 x 104 autologous MN ± PHA (3 µg/ml) and ± CMA. Supernatant was harvested at 24 h and IFN- measured by ELISA as described previously (18 ). This experiment is representative of two experiments. Error bars indicate SD. C, CD95L-expressing cells (KFL9) were incubated with [3H]thymidine-pulsed Jurkat cells (CD95-sensitive target) for 24 h in the presence of anti-CD95 and anti-CD95L Abs. DNA fragmentation results are expressed as percent specific killing and is representative of two experiments. Error bars indicate SEM.

Next, we sought to determine whether cytotoxic MTB-specific CD4⁺ and CD8⁺ T cell lines inhibited intracellular growth of MTB by using a CFU assay. Autologous MN were infected with 1:1 MOI virulent MTB H37Rv and incubated with no additional cells (negative control), or with MTB-activated CD4⁺ (seven donors) or CD8⁺ (six donors) T cell lines. Cells were harvested for enumeration of CFU after 24 h of coculture. Fig. 7 demonstrates that both CD4⁺ and CD8⁺ T cells significantly inhibited growth of MTB. Addition of CD4⁺ T cells at the highest T cell-MN ratio (usually 10:1) was associated with a 74% reduction in growth of MTB at 24 h (p < 0.001). CD8⁺ T cells at the highest ratio (usually 3:1 in most experiments because of limited cell number) reduced MTB CFU by 84% (p < 0.001). Comparing growth inhibition by both T cell subsets at the 3:1 ratio suggested that CD8⁺ T cells were more effective on a per cell basis than CD4⁺ T cells in controlling MTB growth. For both CD4⁺ and CD8⁺ T cells, the mean number of organisms released into the supernatant at 24 h was <10% of the total number of organisms present in the day 1-infected MN alone. Therefore, the observed decrease in intracellular MTB was not simply attributable to a shift to the extracellular compartment but represented true killing of MTB.

View larger version (34K): [in this window] [in a new window]   FIGURE 7. Growth inhibition of MTB by CD4+ and CD8+ T cell lines. Autologous MN were infected with MTB H37Rv. After washing off nonphagocytosed bacilli, MN were cultured in the absence of additional cells or in the presence of purified MTB-activated CD4+ or CD8+ T cells. Results show the mean of eight experiments from seven different donors for CD4+ T cells and seven experiments with cells from six donors for CD8+T cells. Error bars indicate SEM.

Next we determined whether inhibition of perforin or CD95-CD95L interactions affected MTB growth inhibition by CD4⁺ and CD8⁺ T cells. These experiments required that inhibition remained effective during the 24-h period of the MTB growth inhibition assay. We considered two agents to inhibit perforin function, strontium chloride, which induces degranulation, and CMA, which inhibits perforin polymerization to block perforin-mediated CTL function. As shown in Fig. 8A, strontium and CMA both inhibit lysis of MTB-infected targets by CD8⁺ T cells. EGTA was not suitable for the 24-h assay because of its toxicity.

CTL function was inhibited after 2 h of CMA pretreatment and remained inhibited even after 24 h of continuous exposure (Fig. 8A). Treatment of CD8⁺ or CD4⁺ T cells with CMA for up to 24 h was not associated with significant cell loss (trypan blue), and did not result in defective IFN- production (Fig. 8B). Strontium was preincubated with CD8⁺ T cells for 20 h to induce degranulation. T cells were washed to remove strontium, and cells were added immediately or after a 24-h chase period to ⁵¹Cr-loaded targets. As shown in Fig. 8A, strontium initially completely inhibited CTL activity, but after a 24-h chase, CTL activity recovered. Recovery of perforin protein expression during the 24 h chase period after strontium treatment was confirmed also by intracellular flow cytometry (data not shown). Strontium treatment induced significant T cell death, which precluded its use continuously during the 24-h MTB growth inhibition assay. Fig. 8C demonstrates that a combination of anti-CD95 and anti-CD95L Abs resulted in 70% inhibition of apoptosis in a 24 h assay of Jurkat cells by a CD95L-transfected cell line (KFL9) as measured by the DNA fragmentation assay.

CMA and anti-CD95-CD95L Abs were used to determine the role of perforin and CD95-CD95L interaction on CD4⁺- and CD8⁺-mediated MTB growth inhibition. Fig. 9 demonstrates that CMA, despite its ability to inhibit cytotoxicity, had no effect on T cell-mediated control of intracellular MTB growth. Similarly, anti-CD95 and CD95L Abs had no effect. Combination of CMA and anti-CD95-CD95L Abs did not reduce the CD4⁺- or CD8⁺-mediated inhibition of MTB growth. CMA and anti-CD95-CD95L Abs did not affect the growth of MTB in MN in the absence of T cells (data not shown). These results suggest that there was dissociation between the lytic and growth inhibitory functions of MTB specific CD4⁺ and CD8⁺ T cells.

View larger version (23K): [in this window] [in a new window]   FIGURE 9. The role of perforin and CD95-CD95L in T cell-mediated MTB growth inhibition. CD4+ and CD8+ T cells were preincubated for 2 h with CMA, anti-CD95L Ab, or the combination of CMA and anti-CD95L Ab. MN were pretreated with anti-CD95 Ab in all experimental groups where T cells were treated with anti-CD95L. Inhibitors remained present throughout the 24-h assay. T cell-MN ratio was usually 10:1. The data represent the results for CD4+ and CD8+ T cells from four different donors. Error bars indicate SEM.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The fold increase of mRNA relative to the housekeeping gene L32 was higher in CD4⁺ T cells because of significantly lower baseline levels of mRNA expression for the cytotoxic effector molecules studied. Because of greater mRNA induction in CD4⁺ T cells, final mRNA levels for cytotoxic effector molecules were similar for CD4⁺ but never higher than CD8⁺ T cells after MTB stimulation. Low-dose IL-2 was added to bulk cultures to increase T cell expansion to obtain sufficient numbers of cells to perform RNase protection assays and functional studies. IL-2 alone was not responsible for the observed up-regulation of cytotoxic effector molecule mRNA in CD4⁺ T cells.

After MTB stimulation, the ability of both CD4⁺ and CD8⁺ T cells to lyse MTB-infected targets was similar. Cytolysis of MTB infected MN in 4-h ⁵¹Cr release assay by CD4⁺ and CD8⁺ T cells was mediated primarily through the granule exocytosis pathway. The importance of CD95-related killing may be underrepresented in the 4- to 5-h ⁵¹Cr release assay because cell lysis mediated by the interaction of CD95-CD95L is slower than perforin-mediated lysis. Our data contradict the classical view that cell lysis by CD4⁺ T cells is initiated primarily by the CD95-CD95L pathway (47). In fact, up-regulation of CD95L was detected on MTB-activated T cells from only one of four donors using a novel highly sensitive flow cytometry enhancement technique that overcomes sensitivity limitations for detection of CD95L (data not shown; Ref. 48). Furthermore, Oddo et al. (49) found that MTB infection of macrophages caused down-regulation of CD95 on the cell surface, which also could explain why there was only a small contribution by the CD95-CD95L pathway to cytolysis of infected MN. Our study used polyclonal populations of T cells that may closely represent the mixed population of T cells stimulated by MTB in vivo. Inhibition of the CD95-CD95L pathway resulted in only partial loss of cytolytic activity by CD4⁺ and CD8⁺ T cells. This suggests that neither subset of T cells initiated cytotoxicity primarily through the CD95-CD95L pathway, or MN are less sensitive to CD95-CD95L mediated lysis. Studies of PPD-specific CD4⁺ T cell clones, which found that use of the CD95-CD95L pathway depended on the target used, showed MN were less CD95L-sensitive targets, consistent with our data (24).

To inhibit granule exocytosis, we evaluated strontium chloride, which has been used recently to explore cytotoxic and growth restriction mechanisms (31, 32). Strontium was initially shown to induce degranulation in resting NK cells in nonactivated PBMC (50). NK cells remained degranulated for up to 24 h. However, activated T cells rapidly repopulate their granules (51). In experiments with MTB-activated T cells, we found that after strontium treatment, lytic activity returned by 24 h. Therefore, we did not feel that strontium was optimal for inhibition to evaluate the role of CTL activity in the ability of T cells to control MTB growth. We chose CMA, which inhibits perforin for 24 h and was not associated with significant toxicity. These experiments demonstrated that perforin had no effect on MTB growth inhibition by CD4⁺ and CD8⁺ T cells.

These results contrast with findings where CD1- and MHC class I-restricted T cells were able to inhibit the intracellular growth of MTB through a granule exocytosis-dependent pathway (31, 32). They used strontium to inhibit the granule exocytosis pathway. There are at least three possible reasons for the differences in our results. First, CMA inhibits perforin only, whereas strontium blocks the entire granule exocytosis pathway, which includes granzymes and granulysin. These other granule contents may be able to compensate for the loss of perforin. Second, our polyclonal MHC-restricted T cell lines may use different CTL mechanisms than the CD1-restricted T cells. Third, the possibility of residual strontium toxicity to the T cells cannot be excluded. The absence of an inhibitory effect of CMA on T cell ability to restrict MTB growth suggests that MN ability to present MTB was not significantly effected.

Granulysin expression does not appear restricted to unique T cell subsets as initially thought (32, 34, 36, 37). All T cell lines tested expressed granulysin. CMA inhibits perforin activity but does not block degranulation. Granzymes in the absence of perforin have very little lytic and apoptosis-inducing activity (52). In addition, Stenger et al. (32) used purified perforin and granulysin to show that perforin was required for granulysin to kill mycobacteria. Granulysin alone incubated with infected macrophages did not effect the growth of mycobacteria. Because perforin inhibition in our studies did not effect T cell-mediated growth inhibition, the role of granulysin based on our studies is unclear.

The dissociation between T cell-mediated cytolytic activity and growth inhibition was an unexpected finding. We were able to effectively inhibit perforin-mediated cell lysis of MN without affecting intracellular growth of MTB. These results are consistent with studies in perforin and granzyme knockout mice, which indicated that loss of these granule exocytosis molecules alone were not required for early restriction of MTB growth (26, 27). Silva and Lowrie (31) found that only some murine CD4⁺ and CD8⁺ T cell clones inhibited mycobacterial growth and only apparently through cytotoxic granule release. They also used strontium to inhibit the granule exocytosis pathway. They found a direct correlation between amount of granzyme A released and degree of growth restriction. Their T cell lines were much less potent in controlling MTB growth because 50:1 T cell-APC ratios were necessary, in contrast to our studies where 5–10:1 ratios were sufficient. Similar to Silva and Lowrie’s findings (31), our experiments demonstrated that inhibition of the CD95-CD95L interaction also did not inhibit T cell-mediated control of MTB. This may be in part because MTB-activated CD4⁺ and CD8⁺ T cells expressed insufficient levels of CD95L. Others have shown that high concentration of soluble CD95L can inhibit mycobacterial growth in macrophages (49).

Our experiments do not exclude a role for apoptosis through CD95-CD95L-independent mechanisms. ATP-induced apoptosis in macrophages resulted in killing of M. bovis BCG (53, 54). In addition, mycobacterial infection itself can induce apoptosis in infected macrophages (55). Ingestion of infected apoptotic macrophages by uninfected macrophages results in increased control of mycobacterial growth (56). Cytokine-secreting T cells could contribute indirectly to this mechanism by activating these uninfected macrophages. Direct cell contact between T cells and macrophages also is required for mycobacterial growth inhibition (14). Thus, combinations of cytokine secretion and cell contact may be necessary for optimal mycobacterial control by T cells.

Greater insight into the relative roles of cell contact, cytolytic effector function mechanisms, and cytokine secretion by T cells will determine how protective immunity successfully controls intracellular growth of MTB in the majority of healthy individuals.

	Acknowledgments

 
We are grateful to Dr. A. Krensky of Stanford University for the kind gift of anti-granulysin Ab, Zahra Toossi and Fred Heinzel from Case Western Reserve University for reviewing the manuscript, and David Kaplan from Case Western Reserve University for helping us with flow cytometry analysis for CD95L and providing us with KFL9 and Jurkat cells.

	Footnotes

 
¹ This work was funded by grants from the National Institutes of Health to D.H.C. (K08 AI 01581), W.H.B. (AI 27243), the Tuberculosis Research Unit (AI 95383), and R.F.S. (HL 59858), and from Wellcome Trust Fellowship in Clinical Tropical Medicine and British Lung Foundation to R.J.W. (049525).

² Address correspondence and reprint requests to Dr. David H. Canaday, Division of Infectious Disease, Case Western Reserve University and University Hospitals, 10900 Euclid Avenue, BRB 1010B, Cleveland, OH 44106-4984. E-mail address: dxc44{at}cwru.edu

³ Abbreviations used in this paper: MTB, Mycobacterium tuberculosis; CD95L, CD95 ligand; PPD, purified protein derivative; MN, monocyte; MOI, multiplicity of infection; TRAIL, TNF-related apoptosis-inducing ligand; CMA, concanamycin A; MFI, mean fluorescence index; BCG, bacillus Calmette-Guérin.

Received for publication April 21, 2000. Accepted for publication June 6, 2001.

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