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Infection and Immunity, April 2003, p. 1763-1773, Vol. 71, No. 4
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.4.1763-1773.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Differential Effects of Control and Antigen-Specific T Cells on Intracellular Mycobacterial Growth

S. Worku and D. F. Hoft*

Saint Louis University Center for Vaccine Development, Department of Internal Medicine, St. Louis, Missouri 63110

Received 21 August 2002/ Returned for modification 14 October 2002/ Accepted 9 January 2003


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ABSTRACT
 
We investigated the effects of peripheral blood mononuclear cells expanded with irrelevant control and mycobacterial antigens on the intracellular growth of Mycobacterium bovis bacillus Calmette-Guérin (BCG) in human macrophages. More than 90% of the cells present after 1 week of in vitro expansion were CD3+. T cells were expanded from purified protein derivative-negative controls, persons with latent tuberculosis, and BCG-vaccinated individuals. T cells expanded with nonmycobacterial antigens enhanced the intracellular growth of BCG in suboptimal cultures of macrophages. T cells expanded with live BCG or lysates of Mycobacterium tuberculosis directly inhibited intracellular BCG. Recent intradermal BCG vaccination significantly enhanced the inhibitory activity of T cells expanded with mycobacterial antigens (P < 0.02), consistent with the induction of memory-immune inhibitory T-cell responses. Selected mycobacterial antigens (Mtb41 > lipoarabinomannan > 38kd > Ag85B > Mtb39) expanded inhibitory T cells, demonstrating the involvement of antigen-specific T cells in intracellular BCG inhibition. We studied the T-cell subsets and molecular mechanisms involved in the memory-immune inhibition of intracellular BCG. Mycobacteria-specific {gamma}{delta} T cells were the most potent inhibitors of intracellular BCG growth. Direct contact between T cells and macrophages was necessary for the BCG growth-enhancing and inhibitory activities mediated by control and mycobacteria-specific T cells, respectively. Increases in tumor necrosis factor alpha, interleukin-6, transforming growth factor ß, and vascular endothelial growth factor mRNA expression were associated with the enhancement of intracellular BCG growth. Increases in gamma interferon, FAS, FAS ligand, perforin, granzyme, and granulysin mRNA expression were associated with intracellular BCG inhibition. These culture systems provide in vitro models for studying the opposing T-cell mechanisms involved in mycobacterial survival and protective host immunity.


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INTRODUCTION
 
It is estimated that one-third of the world's population is infected with Mycobacterium tuberculosis and that tuberculosis (TB) kills more than 2 million people each year (4). Intradermal vaccination of infants with the currently available TB vaccine Mycobacterium bovis bacillus Calmette-Guérin (BCG) can induce partial protection against TB disease (7) but does not prevent initial or latent infection with M. tuberculosis. Once infected, individuals are at chronic risk for TB disease progression and secondary transmission, especially when vaccine immunity wanes or is suppressed. Therefore, the development of a more efficacious TB vaccine strategy capable of preventing or eliminating chronic infection is an important goal.

Mycobacteria replicate within macrophages in the human host, and defects in cell-mediated immunity result in increased susceptiblity to these intracellular pathogens. Therefore, it can be predicted that memory-immune T cells capable of inhibiting the intracellular growth of mycobacteria are important for in vivo protection against TB. Mycobacteria-specific CD4+ {alpha}ß T cells, CD8+ {alpha}ß T cells, and {gamma}{delta} T cells develop in individuals with partial resistance against TB disease, such as healthy purified protein derivative (PPD)-positive persons latently infected with M. tuberculosis and BCG-vaccinated individuals (19-21, 24, 28, 31, 32, 38, 50, 51). However, the biologically important direct effects of these various T cells on the intracellular viability and growth of mycobacteria, as well as their relative potencies, are not known.

Several immunological mechanisms mediated by T cells may control intracellular mycobacterial growth. Certain cytokine responses (e.g., gamma interferon [IFN-{gamma}], tumor necrosis factor alpha [TNF-{alpha}], and interleukin-12 [IL-12]) are clearly important for antimycobacterial immunity (8, 13, 25, 35, 52). In addition, activated T cells can induce apoptosis or cytolysis of mycobacteria-infected targets (36, 45). Furthermore, directly microbicidal molecules such as granulysin or nitric oxide can be secreted or induced by activated T cells (6, 8, 13, 44). In contrast, the cytokines transforming growth factor ß (TGF-ß) and epidermal growth factor (EGF) (3, 48), along with the prevention of macrophage apoptosis, have been implicated in mycobacterial survival (2, 23, 39). The biological significance of each of these negative and positive regulatory effects for intracellular mycobacterial growth in human macrophages is not known.

We developed an assay useful for studies of the interactions between human T cells and infected macrophages. We demonstrated that control and mycobacteria-specific T cells have differential effects on intracellular BCG growth and that BCG vaccination enhances T cells with inhibitory activity against intracellular mycobacteria. In addition, we investigated the antigen specificity, T-cell subsets, and molecular mechanisms involved in the negative and positive regulation of intracellular mycobacterial growth.


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MATERIALS AND METHODS
 
Antigenic reagents. Purified mycobacterial antigens Mtb8.4, Mtb40, Mtb9.9, Mtb39, 38kd, Mtb41, and Ag85B were obtained from Corixa Corp. (Seattle, Wash.) and Marila Gennaro (New York Public Health Institute). Lipoarabinomannans (LAM) purified from H37Ra and H37Rv strains of M. tuberculosis (Ra LAM and Rv LAM, respectively) were obtained from Larry Schlesinger at the University of Iowa (Iowa City). Native Ag85 complex was obtained from John Belisle (Colorado State University, Fort Collins) through a National Institute of Allergy and Infectious Diseases mycobacterial reagent contract, NO1-AI-75320. Short-term culture filtrate of M. tuberculosis (ST-CF) was obtained from Peter Andersen at the Statens Serum Institut (Copenhagen, Denmark). Tetanus toxoid (TT) was obtained from Pasteur-Merieux-Connaught Laboratories (Toronto, Canada). Isopentenyl pyrophosphate (IPP) was obtained from Sigma (St. Louis, Mo.). 4-Bromohydrine diphosphate (BrHPP) was obtained from Jean-Jacques Fournie at Institut National de la Santé et de la Recherche Médicale (Toulouse, France). Whole-cell lysates of Erdman strain M. tuberculosis and live Connaught strain BCG were prepared at Saint Louis University as previously described (19).

Human subjects and vaccination. Fresh and frozen peripheral blood mononuclear cells (PBMC) from healthy, PPD-positive individuals were used for the development of our in vitro assay of T-cell effects on intracellular mycobacterial growth. To study the inhibitory effects of human memory T cells induced by BCG vaccination, 10 healthy volunteers aged 18 to 45 years were recruited into an intradermal BCG vaccine trial conducted in the Saint Louis University Vaccine Treatment and Evaluation Unit. These volunteers had no history of TB infection, no known exposure to active TB, negative reactions to prescreening 5-tuberculin unit PPD skin tests, and negative human immunodeficiency virus serology. Volunteers were vaccinated intradermally in the left deltoid with 3 x 106 CFU of Connaught BCG (Connaught Laboratories). T-cell-mediated inhibition of BCG growth was studied before vaccination as well as 2 and 6 months postvaccination. This work was approved by the Saint Louis University Institutional Review Board, National Institute of Allergy and Infectious Diseases administrative staff, and Federal Drug Agency, and informed consent was obtained.

Preparation of monocytes and infection with BCG. Monocytes were cultured in RPMI with 10% AB pooled human serum and L-glutamine, but without antibiotics. In most experiments, adherent monocytes were prepared by directly plating 1.5 x 105 PBMC/well in 96-well U-bottomed microtiter plates and gently washing away nonadherent cells after overnight incubation at 37°C. In general, 10% of the total PBMC were found to remain as adherent monocytes (approximately 15,000/well, or >90% CD14+, by fluorescence-activated cell sorter analysis). For studies of contact requirements and cell density effects, monocytes were isolated by adherence to T75 flasks precoated with pooled human serum, detached with cold phosphate-buffered saline, and then plated in 96-well U-bottomed plates at different concentrations. In all experiments presented, monocytes were cultured for 6 days prior to overnight infection with BCG at an estimated multiplicity of infection of 3:1. Stocks of Connaught BCG used for the infection of monocytes were grown in 7H9 broth supplemented with 0.05% Tween 80 and ADC enrichment medium. Mid-log-phase cultures were pelleted, washed three times, and frozen in 7H9 broth at -70°C. Numbers of viable bacteria (CFU) recoverable from thawed aliquots were determined by growing serial dilutions on Middlebrook 7H10 sealed agar plates for 2 to 3 weeks at 37°C with 5% CO2. Vials of BCG were thawed and briefly sonicated in a cup horn attachment immediately prior to infection of monocytes/macrophages. Extracellular BCG was washed away after overnight infection, before expanded effector T cells were added.

Antigen expansion of effector T cells. PBMC were cultured for 7 days in RPMI medium alone (with 10% pooled AB sera and L-glutamine) to serve as rested T-cell-negative controls or stimulated for 7 days with various antigens. TT (10 LF U/ml; Connaught Laboratories) stimulation was used to generate T-cell controls for the nonspecific bystander effects of activated T cells. To expand the heterogeneous populations of mycobacteria-specific T cells relevant for TB immunity, lysates of Erdman strain M. tuberculosis (Mtbwl, 10 µg/ml) and live BCG (20,000 CFU/ml) were used as previously described (19). Purified mycobacterial antigens (Mtb8.4, Mtb40, Mtb9.9, Mtb39, 38kd, Mtb41, Ag85B, native Ag85 complex, Ra LAM, Rv LAM, and ST-CF) were used to expand T cells from PPD-positive persons at optimal concentrations ranging from 1 to 10 µg/ml (inducing stimulation indices of >5). IPP and BrHPP were used at a concentration of 10 µM plus 10 U of murine IL-2 (Boehringer Mannheim, Indianapolis, Ind.)/ml to expand phosphoantigen-responsive {gamma}{delta} T cells.

T-cell-subset analysis of effector cells. Expanded effector cell populations were stained with fluorescent antibodies and studied by three-color flow cytometry to enumerate the T-cell subsets present as described previously (19). The staining combinations used were (i) anti-CD8 fluorescein isothiocyanate, anti-CD4 phycoerythrin, and anti-CD3 PerCP and (ii) anti-{alpha}ß T-cell receptor (TCR) fluorescein isothiocyanate, anti-{gamma}{delta} TCR phycoerythrin, and anti-CD3 PerCP. Antibodies were purchased from Becton Dickinson (BD, San Jose, Calif.). After fixation with 1% formalin, stained cells were analyzed on a FACScalibur flow cytometer (BD). Forward and side scatters were used to gate on lymphocytes. Expansion indices (EI) of different T-cell subsets were calculated by dividing the absolute number (AN) of T cells present after antigen stimulation by the AN of these T cells present after incubation with medium alone [EI = (AN with Ag)/(AN with medium)] as previously described (19).

Purification of T-cell subsets. Purified T-cell subsets were positively selected by using immunomagnetic MicroBeads and MiniMACS columns (Miltenyi Biotec, Inc., Auburn, Calif.) as described previously (19). For CD4+ and CD8+ T cells, 20 µl of anti-CD4 or anti-CD8 MicroBeads was added per 107 total expanded cells and MicroBead-bound fractions were purified in MiniMACS columns. For {gamma}{delta} T cells, expanded cells were first incubated with unlabeled mouse anti-human pan {gamma}{delta} TCR antibody (BD) followed by goat anti-mouse immunoglobulin MicroBeads (Miltenyi Biotec) prior to the purification of MicroBead-bound cells in MiniMACS columns. Aliquots of purified populations were stained with fluorochrome-conjugated antibodies and analyzed by flow cytometry.

Assay of T-cell effects on intracellular BCG growth. In preliminary experiments, the optimal duration of effector-target cell coculture and the optimal effector/target cell ratios were determined to be 72 h and 12.5:1 to 25:1, respectively (data not shown). Rested control and antigen-expanded effector cells were washed, counted, and incubated with BCG-infected autologous targets in triplicate wells. After 72 h, supernatants were aspirated and target cells were lysed with 0.2% saponin to release intracellular BCG. Viable mycobacteria were determined by radiolabeling with tritiated uridine as previously described (12, 53). Briefly, saponin lysates were diluted 1:2 in 7H9 broth with 10% ADC enrichment medium (Difco, Detroit, Mich.) and pulsed with 1 µCi of [5,6-3H]uridine per well (Amersham, Little Chalfont, Buckinghamshire, United Kingdom). After incubation at 37°C for 72 h, BCG incorporating the label was harvested onto glass fiber filter mats (Skatron, Sterling, Va.) and quantitated by liquid scintillation counting. Saponin lysates were also plated on 7H10 Middlebrook agar for CFU determinations. The percentages of BCG growth inhibition were determined by using the following formula: percent inhibition = 100 - {100 x [(CFU or disintegrations per minute from Ag-stimulated T cells)/(CFU or disintegrations per minute from medium-rested T cells)]}.

Assays of cell contact requirements. Cell contact requirements were studied by using transwell plates containing semipermeable membranes porous to solutes but not to cells or bacteria (0.4-µm-pore-size Millicell plate inserts [Millipore Bedford, Mass.]). BCG-infected monocytes were prepared as described above in T75 flasks and added to both the top and bottom chambers (105/chamber) of 24-well transwell plates. Antigen-expanded and rested PBMC were added to the upper and lower chambers at an effector/target ratio of 12.5:1. When cell contact requirements for antigen-specific inhibitory effects were studied, medium-rested effectors were added to the bottom chambers and mycobacteria-antigen-expanded effectors were added to either the top or bottom chambers. To study the cell contact requirements for the growth-enhancing effects of control T cells, parallel cultures contained medium-rested T cells in either the upper or lower chambers. Levels of viable BCG after 72 h of coculture were studied in the lower compartments.

RNase protection assays. Total RNA was isolated from effector T cells alone, uninfected and infected monocytes incubated without T cells, and cocultures of T cells with BCG-infected monocytes by using the RNeasy mini kit (Qiagen, Valencia, Calif.). Riboquant RNase protection assays were performed according to the manufacturer's instructions to study the mRNA expression of cytokines, monokines, and molecules known to be involved in apoptosis, granule-mediated cytolysis, and direct microbicidal activity. Two sets of customized probe kits were purchased from Pharmingen (San Diego, Calif.). The first set included probes specific for human caspases 8 and 9, IL-12 p35, TNF-{alpha}, IFN-{gamma}, IL-4, IL-6, TGF-ß, and vascular endothelial growth factor (VEGF). The second set included probes specific for human granzymes A and 3, IL-10, FAS antigen, FAS ligand, granulocyte-macrophage colony-stimulating factor, bcl2, inducible nitric oxide synthase, perforin, and granulysin. 32P-Labeled RNA probes were synthesized and hybridized at 6 x 105 cpm with 4 µg of total RNA overnight. Hybridized samples then were treated with RNase A and T1 mix, and protected radiolabeled RNA probes were separated in Novex Quickpoint mini sequencing gels (Pharmingen) and detected by autoradiography. Levels of mRNA expression were quantified by densitometry and normalized for GADPH expression.

Measurement of cytokine secretion. Cytokines present in supernatants after 72 h of effector-target cell coculture were measured by enzyme-linked immunosorbent assay (ELISA). Levels of IFN-{gamma} were determined as described previously (19). Levels of TNF-{alpha} were measured with the cytokine-specific antibody pair 80-3399-01 mouse anti-human TNF-{alpha} (Genzyme, Cambridge, Mass.) and BAF-210 biotinylated anti-human TNF-{alpha} (R&D Systems, Minneapolis, Minn.). The human TGF-ß DuoSet ELISA kit (Genzyme) was used for measurements of TGF-ß. EGF levels were determined by using the Quantikine human EGF immunoassay kit (R&D).


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RESULTS
 
Composition of effector cells used in assays of intracellular BCG growth inhibition. PBMC from PPD-positive donors were rested in medium or expanded with TT, IPP plus IL-2, whole-cell lysate of M. tuberculosis, or live BCG. Viable cells recovered after 7 days of culture were stained for T-cell surface markers and analyzed by flow cytometry. In both rested and antigen-expanded cultures, >90% of the recovered cells were CD3+ T cells. Significant expansions (P < 0.01 by Wilcoxon matched-pairs tests) of {gamma}{delta} T cells were induced by IPP plus IL-2, M. tuberculosis lysate, and live BCG, from <5% at baseline to >40% of total viable cells after stimulation. In addition, increased blastogenic responses (increased forward- and side-scatter properties) were detected in CD4+ and CD8+ T cells activated by mycobacterial antigens. EI of CD4+ T cells after stimulation with M. tuberculosis lysate and live BCG were 1.4 ± 0.2 and 1.3 ± 0.1, respectively. EI of CD8+ T cells were 2.5 ± 0.5 and 2.4 ± 0.3 after M. tuberculosis lysate and live-BCG stimulation, respectively. The control antigen TT activated CD4+ and CD8+ cells but not {gamma}{delta} T cells. These results are similar to our previously published results (19, 53).

T cells expanded with mycobacterial antigens but not control antigens inhibit intracellular BCG growth. The rested and antigen-activated cells described above were cocultured with BCG-infected monocytes, and residual intracellular BCG viability was assessed by tritiated uridine uptake and CFU determinations 72 h later (Fig. 1). T cells expanded with M. tuberculosis lysate or live BCG significantly inhibited (>55%) intracellular BCG growth compared with rested T cells (n = 14 CFU for uridine incorporation experiments with P < 0.001 by the Wilcoxon matched-pairs test, n = 8 for CFU determination experiments with P < 0.05 by the Wilcoxon matched-pairs test). T cells expanded with IPP plus IL-2 or TT did not inhibit intracellular BCG growth. In separate experiments, PBMC were expanded with a more potent phosphoantigen for {gamma}{delta} T-cell activation, BrHPP plus IL-2. Stimulation with BrHPP induced marked {gamma}{delta} T-cell expansions, achieving percentages that were >50% of the total cells present postexpansion. However, intracellular BCG growth was not inhibited by BrHPP-expanded T cells (data not shown).



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  		FIG. 1. T cells expanded with mycobacterial antigens but not control antigens inhibit intracellular growth of BCG. T cells expanded for 7 days with the indicated antigens were cocultured at a ratio of 12.5:1 with BCG-infected autologous monocytes. After 72 h, CFU counts and [3H]uridine uptake were assessed in saponin lysates of these cocultures. Results shown represent means ± SEs from 14 experiments for tritiated uridine incorporation and from 8 experiments for CFU estimations (*, P < 0.05; **, P < 0.001 compared with those for TT by the Wilcoxon matched-pairs tests).

Rested T cells and T cells activated by irrelevant antigens enhance intracellular mycobacterial growth. To our surprise, we detected highly reproducible increases in BCG growth in infected monocytes cultured with control T cells compared with infected monocytes cultured alone. Figure 2A shows the results of a representative experiment in which monocyte targets (prepared with our standard method, which resulted in ~15,000 monocytes/well) were infected with increasing BCG-to-monocyte ratios and then cultured in the presence or absence of medium-rested control T cells. At multiplicity of infection ratios ranging from 1:1 to 20:1, >=4-fold increases in tritiated uridine uptake were detected in the presence of control T cells. Similar enhancements of intracellular BCG growth were seen in infected macrophages incubated with TT-expanded T cells (data not shown). Increases in recoverable BCG CFU also were associated with the addition of control T cells (data not shown). These results clearly indicate that nonspecific T cells can provide enhancing factors for intracellular mycobacterial growth.



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  		FIG. 2. Control T cells enhance intracellular BCG growth in suboptimal monolayers of macrophages, but mycobacteria-specific T cells always have dominant inhibitory activity. (A) BCG-growth-enhancing effects of medium-rested T cells are illustrated (means ± SEs of triplicate cultures). Monocytes were incubated with increasing BCG concentrations overnight. Then, after washing away of extracellular BCG, infected monocytes were cultured for 72 h more in the presence or absence of medium-rested T cells. Greater than fourfold increases in BCG growth were detected in the presence of medium-rested cells irrespective of the infecting dose of BCG. (B) Growth-enhancing effects of medium-rested T cells occur only when suboptimal numbers of macrophages are present (means ± SEs of triplicate cultures). Monocytes were prepared on serum-coated T75 tissue culture flasks, detached, and added to 96-well plates at increasing concentrations. All monocyte cultures were infected with a constant number of BCG CFU (45,000 CFU/well). Infected monocytes were cultured alone or with medium-rested PBMC (effector/target cell ratio of 10:1). (C) The inhibitory effects of antigen-specific T cells dominate over the enhancing effects of medium-rested T cells. BCG-infected monocytes (15,000/well) were incubated with medium-rested, Mtb-lysate-expanded, or live-BCG-expanded T cells alone at a 12.5:1 T-cell/macrophage ratio (black bars). Additional medium-rested T cells were added to parallel cocultures (white bars) at a 12.5:1 T-cell/macrophage ratio. Freshly prepared uninfected monocytes (15,000/well) were added to parallel cultures at the time of T-cell-infected-macrophage coculture (hatched bars). In seven additional experiments, intracellular BCG was significantly reduced by both types of mycobacteria-specific T-cell populations whether or not medium-rested T cells were present (P < 0.05 by the Wilcoxon matched-pairs tests). For all panels, intracellular mycobacterial growth was assayed by [3H]uridine uptake in residual viable BCG released from saponin-treated macrophages.

Enhancing effects of control T cells for intracellular BCG growth are detected only with suboptimal macrophage densities. Silver et al. have reported that freshly harvested lymphocytes can inhibit mycobacterial growth in monocyte targets (42). The major differences between the assay of Silver et al. and our present assay include their use of (i) fresh lymphocyte effectors, (ii) fresh monocyte-derived macrophages, and (iii) a 10-fold greater concentration of monocytes/macrophages. Therefore, we hypothesized that the growth-enhancing effects of control T cells detected in our assay were due to differences in T-cell activation and maturation and/or differences in the macrophage targets prepared by our methods. We found that freshly harvested PBMC could enhance BCG growth to the same extent as 7-day ex vivo lymphocytes in our assay, indicating that the T-cell activation or maturation state was not responsible for the enhancing phenomenon (data not shown). In addition, the intracellular growth of BCG in fresh versus 7-day in vitro-activated monocytes/macrophages was similarly enhanced by control T cells (data not shown). However, Fig. 2B demonstrates that optimal BCG intracellular growth was detected in cultures with 6 x 104 macrophages/well (four times the concentration in our standard assay) and that the addition of rested PBMC did not further enhance BCG growth in cultures with an optimal macrophage density. T cells expanded with mycobacterial antigens inhibited BCG growth compared with T cells rested in medium, even in cultures containing optimal macrophage densities (data not shown).

We have continued to routinely use lower densities of monocytes/macrophages in our assay for both theoretical and practical reasons. Mycobacteria will rarely encounter optimal macrophage densities in vivo sequestered from the potential effects of nonspecific T cells. Therefore, the conditions of our standard assay are relevant for potential conditions of mycobacterial exposure in vivo. For practical reasons, we wanted to develop an assay that could be used with paired frozen PBMC samples obtained from volunteers before and after vaccination with experimental TB vaccines. It is difficult to obtain large numbers of monocytes/macrophages from frozen PBMC. We have found that our assay works just as well with previously frozen and fresh PBMC. This is an important practical feature for studies of inhibitory T-cell responses induced in vaccine trials.

Mycobacteria-specific T cells directly inhibit intracellular BCG. We have shown the opposing effects of antigen-specific and -nonspecific T cells on intracellular BCG growth. The enhancing effects of control T cells occur only with suboptimal monocyte/macrophage densities. On the other hand, the inhibitory effects of mycobacteria-specific T cells were detected regardless of monocyte/macrophage density. These latter results suggest that Mtb lysate- and BCG-expanded T cells have direct inhibitory effects on intracellular mycobacterial growth. We next performed mixing experiments to further investigate this possibility. BCG-infected macrophages were incubated with an equal number of medium-rested T cells (to ensure optimal BCG growth) and T cells expanded with mycobacterial antigens (testing for direct inhibitory activity versus simply the absence of enhancing effects). Figure 2C presents one set of mixing experiments demonstrating that T cells expanded with either the mycobacterial lysate or live BCG markedly inhibit the intracellular growth of mycobacteria whether or not medium-rested T cells are present. The overall results of seven other mixing experiments demonstrated that T cells expanded with mycobacterial antigens significantly inhibited intracellular BCG growth (median inhibition, 36%; P < 0.01 by Wilcoxon matched-pairs test) whether or not rested PBMC were present. Therefore, antigen-specific T-cell inhibition of intracellular BCG was found to involve direct effects that were dominant over the enhancing activity of nonspecific T cells.

We considered the additional possibilities that viable BCG released by the cytolysis of infected targets was removed with culture supernatants prior to the saponin lysis of the attached macrophages or was inhibited by medium components after extracellular release (e.g., human serum). We studied the culture supernatants removed prior to saponin lysis and routinely found less than 10% of total uridine uptake or BCG CFU recoverable from all assay cultures. The addition of fresh, uninfected monocytes enhanced the levels of BCG detectable in macrophages incubated with control T cells, presumably by providing fresh targets for extracellular BCG to infect (Fig. 2C). However, the addition of fresh, uninfected targets to cultures containing mycobacteria-specific T cells did not increase levels of recoverable BCG. These combined results demonstrate that mycobacteria-specific T cells expanded from healthy PPD-positive individuals have direct inhibitory effects on intracellular BCG.

BCG vaccination up regulates antigen-specific T-cell memory responses capable of inhibiting intracellular mycobacteria in human macrophages. The preceding results demonstrated that mycobacteria-reactive T cells harvested from individuals with latent TB infections can inhibit intracellular BCG growth. However, in preliminary experiments, we also found that PBMC harvested from PPD-nonreactive persons and expanded with M. tuberculosis lysate or live BCG could inhibit intracellular BCG growth to some degree (data not shown). To determine whether our in vitro assay could measure antigen-specific memory-T-cell responses relevant for vaccine immunity, and not just innate immune mechanisms, we recruited volunteers into an intradermal BCG vaccination trial (Fig. 3A). T-cell inhibition of intracellular BCG growth was studied in 10 volunteers before and 2 and 6 months after intradermal vaccination with 3 x 106 CFU of Connaught BCG. The mean (± standard error [SE]) levels of inhibition of intracellular BCG growth mediated by live-BCG-expanded PBMC increased from background levels of 34% ± 10% prevaccination to 49% ± 8% at 2 months (P = 0.05) and 68% ± 6% at 6 months (P < 0.02) postvaccination (Fig. 3A). Therefore, mycobacteria-specific memory-immune responses capable of inhibiting BCG growth were induced by intradermal BCG vaccination.



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  		FIG. 3. Detection of antigen-specific memory T cells inhibitory for intracellular BCG. Ten healthy volunteers were recruited into an intradermal BCG vaccine trial. PBMC were harvested prevaccination, 2 months postvaccination, and 6 months postvaccination, and live-BCG-expanded T cells were studied for their ability to inhibit intracellular BCG as described in the legend to Fig. 1. (A) Results for mean (± SE) percent inhibition mediated by live-BCG-expanded T cells compared with that for TT-expanded T cells at the different vaccine trial time points. The data presented were derived from [3H]uridine uptake experiments, but similar results were seen in parallel measurements of CFU. *, P < 0.05; **, P < 0.02 by the Wilcoxon matched-pairs tests comparing postvaccination responses with prevaccination responses. (B) PBMC from three healthy PPD-positive individuals were stimulated with highly purified mycobacterial antigens and intracellular BCG inhibitory activity measured 7 days later by tritiated uridine uptake. Mtb8.4, Mtb40, Mtb9.9, Mtb39, Ag85B, 38kd, and Mtb41 were purified recombinant proteins. Rv LAM and Ra LAM were LAM purified from H37Rv and H37Ra strains of M. tuberculosis, respectively. N-Ag85 was the native complex of Ag85A, -B, -C purified from the Erdman strain of M. tuberculosis. ST-CF was culture filtrate protein harvested from the early-log-phase growth of H37Rv strain M. tuberculosis. Live Connaught strain BCG was prepared as described in Materials and Methods. Shown are mean (± SE) responses for the inhibition of intracellular BCG mediated by PBMC stimulated with the indicated mycobacterial antigen.

T cells expanded with specific mycobacterial antigens inhibit intracellular mycobacterial growth. To further investigate the antigen specificity of mycobacterial growth inhibition detected in our assay, we studied PBMC expanded with purified mycobacterial antigens. Figure 3B presents the results from three independent experiments using PBMC isolated from PPD-positive individuals. The most potent inhibitions of intracellular BCG were seen with PBMC expanded with the complex mixtures of mycobacterial antigens present in ST-CF of M. tuberculosis (74% ± 5%) and expressed by live BCG (77% ± 4%). However, PBMC stimulated with native Ag85 complex purified from the Erdman strain of M. tuberculosis also markedly inhibited intracellular BCG (by 65% ± 10%). In addition, PBMC stimulated with LAM purified from the H37Rv and H37Ra strains of M. tuberculosis and PBMC stimulated with the recombinant proteins Mtb39, Ag85B, 38kd, and Mtb41 inhibited intracellular mycobacterial growth by 20 to 50%. Despite the induction of similar levels of lymphoproliferative responses, PBMC stimulated with recombinant antigens Mtb8.4 and Mtb40 did not inhibit intracellular BCG and served as important negative controls. These results further demonstrate that our assay can measure the antigen-specific inhibitory effects of T cells on intracellular mycobacterial growth.

Cell contact requirements of intracellular BCG-enhancing and inhibitory activities. Regulatory effects on intracellular BCG growth could be mediated by soluble factors or by mechanisms involving direct effector-to-target cell contact. Initial experiments demonstrated that the transfer of supernatants from BCG-enhancing and inhibitory cultures alone did not alter the growth of intracellular mycobacteria (data not shown). To further study contact requirements for BCG-enhancing and inhibitory activities, chambers separated by membrane inserts were used to allow diffusion of soluble mediators but prevent direct contact between effector and target cells. In parallel experiments, effector cells were cultured in the lower chambers in contact with or the upper chambers separated from the infected targets used to quantitate viable BCG 72 h later. Additional BCG-infected targets were added to all upper chambers to activate T cells and ensure that soluble factors induced by antigen stimulation would be produced during the coculture period. Enhancement of BCG growth was detected only when control T cells were cultured in the lower chambers in direct contact with BCG-infected targets (data not shown). Similarly, maximal T-cell inhibition of BCG growth was seen only when antigen-specific T cells were cultured in direct contact with BCG-infected targets (data not shown).

Inhibition of intracellular BCG growth in autologous monocytes by enriched subsets of mycobacteria-specific CD4+ {alpha}ß TCR+, CD8+ {alpha}ß TCR+, and {gamma}{delta} T cells. We next studied the ability of specific T-cell subsets to inhibit intracellular mycobacterial growth. CD4+ {alpha}ß T cells, CD8+ {alpha}ß T cells, and {gamma}{delta} T cells were enriched from total PBMC expanded with live BCG and cocultured with BCG-infected targets. Total rested PBMC were added as in the mixing experiments described above to ensure that any reduction in BCG growth associated with the addition of a purified T-cell subset was due to direct inhibitory effects and not to the absence of BCG-growth-enhancing activity. Table 1 presents the results of multiple experiments assessing the ability of enriched T-cell subsets to inhibit intracellular BCG quantified by tritiated uridinine uptake. CD4+ {alpha}ß T-cell, CD8+ {alpha}ß T-cell, and {gamma}{delta} T-cell fractions were 92% ± 3%, 86% ± 3%, and 87% ± 5% pure, respectively. Significant inhibitory effects were detected with total (n = 7, P < 0.01) and {gamma}{delta} T-cell-enriched (n = 5, P < 0.04) fractions of BCG-expanded PBMC. Inhibitory effects also were detected with enriched CD4+ and CD8+ {alpha}ß T cells but did not achieve statistical significance, perhaps because of the small sample sizes (n = 7, P < 0.4, and n = 4, P < 0.06, respectively). CD4+ {alpha}ß T cells, CD8+ {alpha}ß T cells, and {gamma}{delta} T cells enriched from TT-expanded PBMC did not inhibit BCG growth (data not shown).


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  		TABLE 1. Inhibition of intracellular BCG growth by specific T-cell subsetsa

Differential gene expression associated with inhibition and enhancement of intracellular mycobacterial growth. RNase protection assays were used to study the expression of cytokines, apoptotic markers, mediators of cytolytic activity, and mediators of the direct microbicidal activity predicted to be important for the inhibition or enhancement of BCG growth (Fig. 4A and B; Table 2). Higher levels of IFN-{gamma}, granzyme, perforin, granulysin, FAS antigen, and FAS ligand mRNAs were detected in cultures incubated with inhibitory BCG-expanded T cells compared with cultures incubated with noninhibitory medium-rested T cells. However, similarly increased levels of granzyme, perforin, granulysin, FAS antigen, and FAS ligand mRNAs were expressed in cultures with noninhibitory TT-expanded T cells (Fig. 4B). The comparisons between cultures containing BCG-expanded T cells and medium-rested T cells suggest that Th1 cytokines, apoptosis, perforin and granzyme cytolytic activity, and direct microbicidal activity mediated by granulysin all may be important for the inhibition of intracellular mycobacterial growth. Alternatively, the similar results for granzyme, perforin, granulysin, FAS antigen, and FAS ligand mRNAs detected in cultures with either inhibitory BCG-expanded T cells or noninhibitory TT-expanded T cells pose uncertainty as to whether any of these potential effector mechanisms are important for mycobacterial inhibition. In any case, the results with TT-expanded cells do indicate that the active mechanisms responsible for inhibition of intracellular mycobacteria require the antigen-specific recognition of target cells to be effective. The fact that TNF-{alpha}, TGF-ß, IL-6, and VEGF were increased in cultures with either medium-rested or TT-expanded T cells, compared with that in cultures containing inhibitory BCG-expanded T cells, strongly suggests that these cytokines are involved in the enhancement of intracellular mycobacterial growth mediated by both of these control T-cell preparations.



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  		FIG. 4. Expression of cytokine and cytolytic effector molecule mRNAs associated with intracellular BCG growth inhibition and enhancement. (A) mRNA expression levels for TNF-{alpha}, IFN-{gamma}, TGF-ß, IL-6, and VEGF studied by RNase protection assay. (B) mRNA expression levels of granzymes, FAS, FAS ligand, granulysin, and perforin studied by RNase protection assay. RNAs were prepared from uninfected macrophages, BCG-infected macrophages alone, and cultures of infected macrophages incubated with control T cells or live-BCG-expanded T cells. Total RNA samples isolated after 24, 48, or 72 h of incubation were hybridized with 32P-labeled probes overnight prior to RNase digestion, and then protected mRNA fragments were detected by autoradiography of Quickpoint Novex gels. Representative results from four experiments are presented.


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  		TABLE 2. Induction of immune effector mRNA in cultures of medium-rested control and BCG-expanded T cells incubated with infected macrophagesa

Reciprocal expression of IFN-{gamma} and TNF-{alpha} in cultures associated with intracellular BCG inhibition and enhancement. We measured secreted IFN-{gamma} and TNF-{alpha} by ELISA 72 h after adding T cells to infected macrophages. Significant increases in IFN-{gamma} (P < 0.001) (Fig. 5A) and significant reductions in TNF-{alpha} (P < 0.001) (Fig. 5B) were induced by mycobacteria-specific T cells. TGF-ß and EGF were not detectable above background in these same supernatants (data not shown). Because of these results, we studied the effects of anti-IFN-{gamma} and anti-TNF-{alpha} neutralizing antibodies on intracellular BCG inhibition and enhancement mediated by mycobacteria-specific and control T cells, respectively. Neutralizing-antibody concentrations preventing the detection of free IFN-{gamma} and TNF-{alpha} in culture supernatants did not prevent the inhibition or enhancement of intracellular BCG growth (data not shown). Thus, although IFN-{gamma} responses were associated with the inhibitory activity of mycobacteria-specific T cells, high levels of extracellular IFN-{gamma} were not necessary for these inhibitory effects. In addition, TNF-{alpha} secretion appears to parallel levels of intracellular mycobacterial growth but may not be required for the enhancement of intracellular BCG growth.



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  		FIG. 5. Degrees of reciprocal production of secreted IFN-{gamma} and TNF-{alpha} were associated with BCG inhibitory and enhancing activities, respectively. Levels of IFN-{gamma} (A) and TNF-{alpha} (B) were measured by ELISA in culture supernatants harvested 72 h after incubating rested or expanded T cells with BCG-infected macrophages. The results shown are the means (± SEs) from five experiments. *, P < 0.001 for comparison of supernatants from cultures incubated with antigen-expanded T cells and supernatants from cultures incubated with medium-rested T cells using Wilcoxon matched-pairs tests.


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DISCUSSION
 
Our goal was to develop an assay for measurement of memory-immune T-cell responses inhibitory for intracellular mycobacteria. We stimulated PBMC with mycobacterial antigens to selectively expand antigen-specific, memory-immune T cells. We studied rested and TT-expanded T cells to control for nonspecific innate immunity and bystander effects. T cells expanded with mycobacterial antigens inhibited intracellular BCG (Fig. 1 through 3), while rested or TT-expanded T cells did not, demonstrating the antigen specificity of the inhibitory responses detected. Importantly, BCG vaccination was shown to significantly enhance antigen-specific T-cell inhibition and purified mycobacterial antigens were shown to stimulate inhibitory T cells, both results characteristic of antigen-specific, memory-immune T-cell responses (Fig. 3A and B).

Because M. tuberculosis replicates almost exclusively within macrophages in vivo, we hypothesized that the memory T-cell inhibition of intracellular mycobacteria should correlate with and serve as a surrogate for protective TB immunity. The following results suggest that our assay predominantly measures intracellular inhibitory effects. Infected monocytes were extensively washed to remove extracellular BCG prior to adding effector T cells, <10% of viable mycobacteria were found in assay supernatants prior to saponin lysis, and similar numbers of macrophage nuclei were recovered from all cultures after the coculture period. The apparent requirement for cell contact between inhibitory T cells and infected macrophages suggests that the inhibition of extracellular BCG by secreted effector molecules is not of major importance. Finally, although the addition of uninfected macrophages enhanced BCG growth in cultures incubated with control T cells, this was not the case in cultures incubated with mycobacteria-specific inhibitory T cells (Fig. 2C). These latter results suggest that only minimal release of viable extracellular BCG from the lysis of infected macrophages occurred. Although we cannot be certain that the inhibitory effects detected in our assay are active against intracellular mycobacteria only, our studies to date are consistent with this interpretation.

Extensive previous results indicate that both CD4+and CD8+ {alpha}ß T cells are important for protective TB immunity. Recent data suggest that other T-cell subsets may be important for antimycobacterial immunity as well. Mycobacterial lipids presented by nonpolymorphic CD1 can activate CD4/CD8 double-negative {alpha}ß T cells and CD8+ {alpha}ß T cells in vitro to inhibit intracellular mycobacteria in human macrophages (33, 37, 41, 45), and these may be the type of inhibitory T cells activated by LAM in our experiments (Fig. 3B). In addition, phosphoantigen-responsive human {gamma}{delta} T cells are stimulated by mycobacterial components (29, 34, 40, 46, 47, 50) and increased {gamma}{delta} T-cell responses have been detected in partially resistant, healthy PPD-positive and BCG-vaccinated persons (19, 31). We now present data that {gamma}{delta} T cells expanded with live BCG (Table 1) or M. tuberculosis lysate (data not shown) were the most potent inhibitors of intracellular mycobacteria in our assay system and therefore could be important for vaccine-induced TB immunity. Interestingly, {gamma}{delta} T cells expanded with phosphoantigen (IPP or BrHPP) plus IL-2 did not inhibit intracellular BCG in our assay (Fig. 1B). Inhibitory {gamma}{delta} T cells and phosphoantigen-expanded {gamma}{delta} T cells may have different antigen specificities; phosphoantigen-expanded {gamma}{delta} T cells may not recognize BCG-infected macrophages. Alternatively, phosphoantigen plus IL-2 may be sufficient to stimulate the expansion but not activation of inhibitory effector mechanisms.

{gamma}{delta} T cells can mediate positive and negative immunoregulatory effects (5, 15, 22). The significance of {gamma}{delta} T cells for protective TB immunity has been questioned by studies in {gamma}{delta} T-cell-knockout mice. These mice display only minor increases in susceptibility to mycobacteria (10, 26, 27). However, there is no murine homologue of the phosphoantigen-responsive V{gamma}9/V{delta}2 human T-cell subset (15, 30) and therefore, the murine model has significant limitations with regard to studying the relevance of {gamma}{delta} T cells for human TB immunity. Tsukaguchi et al. (50) demonstrated that human {gamma}{delta} T cells can produce IFN-{gamma} responses and lyse mycobacteria-infected macrophages. Similar to our findings, Dieli et al. reported that V{gamma}9/V{delta}2 T-cell lines could inhibit intracellular mycobacteria (9). Further studies must determine whether molecular vaccines targeting human {gamma}{delta} T cells can lead to enhanced protective-memory immunity.

Our studies suggest that multiple mechanisms are important for the inhibition of intracellular mycobacteria. Increases in IFN-{gamma} mRNA and protein are associated with intracellular growth inhibition (Fig. 4A and 5A). However, transfer of coculture supernatants to BCG-infected macrophages did not inhibit intracellular BCG growth (data not shown) and cell contact was required for inhibitory activity, indicating that high levels of secreted IFN-{gamma} alone were not responsible for the inhibition of intracellular mycobacteria. Inhibitory T cells were associated with increased FAS, FAS ligand, perforin, granzyme, and granulysin mRNAs (Fig. 4B), suggesting that apoptosis, cytolysis, and direct mycobacterial killing may be important for the inhibitory effects observed (44, 45). The fact that comparably high levels of these latter mRNAs were also detected in cultures with noninhibitory TT-expanded cells indicates that the antigen-specific recognition of target cells is necessary for inhibitory effector function.

Mycobacterial survival and replication depend on a complicated interplay between various host and mycobacterial factors. Areas of caseation necrosis and granulomatous inflammation are rich in cytokines and growth factors that may facilitate mycobacterial replication and persistence (11, 49, 54). Increased TGF-ß, VEGF, IL-6, and TNF-{alpha} levels have been detected in patients with active TB disease (48). TGF-ß and EGF can enhance mycobacterial replication in vitro and in vivo (3, 18, 48). IL-6 signaling prevents apoptosis in the mucosal T cells involved in Crohn's disease (1). Both protective and immunopathogenic effects of TNF-{alpha} have been reported (14, 16, 17, 25). TNF-{alpha} production has been shown to correlate with mycobacterial virulence (43), consistent with our findings that TNF-{alpha} may be a marker of ongoing mycobacterial replication. Our results that the enhancement of BCG growth is associated with increased TGF-ß, VEGF, IL-6, and TNF-{alpha} mRNAs further support a role for these cytokines in mycobacterial survival and immunopathology and suggest that nonspecific T cells can either produce these factors or promote their production in BCG-infected macrophages.

In summary, we have developed an in vitro system that can detect human memory immunity protective against intracellular mycobacteria. Multiple T-cell subsets are involved in these protective responses. We have characterized some of the effector molecules potentially involved in intracellular mycobacterial inhibition and growth enhancement. It will be important in future studies to determine the effects on virulent M. tuberculosis of the inhibitory T cells detected in our assay. This assay could be used to screen new TB vaccine candidates for the ability to induce inhibitory T cells. In addition, further study of the growth-enhancing properties of control T cells may provide a better understanding of how mycobacterial pathogens survive in mammalian hosts.


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ACKNOWLEDGMENTS
 
We thank Yasir Skeiky (Corixa Corp.) and Marila Gennaro (NYPHRI) for recombinant mycobacterial proteins, Peter Andersen (Statens Serum Institut) for Mtb culture filtrate, Larry Schlesinger (University of Iowa) for LAM, John Belisle (Colorado State University) for native Ag85 complex, and Jean-Jacques Fournie (INSERM) for BrHPP. We are also grateful to all the volunteers who generously contributed their time and effort for this study and the Saint Louis University Vaccine Center nurses who obtained all clinical blood specimens.

This investigation was supported by National Institutes of Health Vaccine Treatment and Evaluation Unit Contract NO1-AI-45250 and National Institutes of Health AIDS Vaccine Evaluation Unit Contract NO1-AI-45211.


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FOOTNOTES
 
* Corresponding author. Mailing address: Division of Infectious Diseases and Immunology, St. Louis University Health Sciences Center, St. Louis, MO 63110. Phone: (314) 577-8649. Fax: (314) 771-3816. E-mail: hoftdf{at}slu.edu. Back

Editor: S. H. E. Kaufmann


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Infection and Immunity, April 2003, p. 1763-1773, Vol. 71, No. 4
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.4.1763-1773.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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