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(Journal of Leukocyte Biology. 2002;72:115-124.)
© 2002 by Society for Leukocyte Biology

Selective expansion of perforin-positive CD8+ T cells by immature dendritic cells infected with live Bacillus Calmette-Guérin mycobacteria

Yasuko Tsunetsugu-Yokota*, Hideto Tamura{dagger}, Mikiko Tachibana{dagger}, Kiyoyuki Ogata{dagger}, Mitsuo Honda{ddagger} and Toshitada Takemori*

* Department of Immunology and
{ddagger} The First Group of AIDS Research Center, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, Japan; and
{dagger} The Third Department of Internal Medicine, Nippon Medical School, Bunkyo-ku, Sendagi, Tokyo, Japan

Correspondence: Yasuko Tsunetsugu-Yokota, M.D., Ph.D., Department of Immunology, National Institute of Infectious Diseases, 1-23-1, Toyama-cho, Shinjuku-ku, Tokyo 162-8640, Japan. E-mail: yyokota{at}nih.go.jp


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ABSTRACT
 
Live, but not dead Bacillus Calmette-Guérin (BCG) is partially protective against infection by Mycobacterium tuberculosis, which causes a disease with high mortality in immune compromised individuals. We have shown that uptake of BCG induces maturation of immature dendritic cells (DCs) regardless of the viability of the bacteria. Importantly, when T cells are cocultured with live BCG-infected DCs, the proportion of CD45RA- perforin+ CD8+ T cells is markedly expanded markedly; however, little expansion is seen when T cells are cocultured with DCs harboring heat-killed BCG. The direct contact of T cells with live BCG-infected DCs was required for the expansion of perforin+ CD8+ T cells. These CD8+ T cells demonstrated a high level of killing activity against BCG-infected macrophages. There was little contribution of cytokines, including IFN-{gamma}, TNF-{alpha}, and IL-12, to the expansion of CD8+ T cells by live BCG-infected DCs. We found that the interaction between BCG-infected DCs and CD8+ T cells through CD40/CD40L was crucial for the expansion and maturation of CD8+ T cells, the process of which was CD4-independent. In contrast, blocking the CD58/CD2 but not the CD40/CD40L interaction reduced production of IFN-{gamma} without affecting the maturation of CD8+ T cells. This indicates that the production of IFN-{gamma} and perforin by CD8+ T cells is mediated by distinct signals delivered from BCG-infected DCs. Thus, BCG-specific CD8+ CTL memory cells may be maintained for a long period of time in BCG-vaccinated hosts, and these cells could mature rapidly into effectors through the potent antigen-presenting function of DCs upon mycobacterial infection.

Key Words: live and dead BCG • maturation of DCs • IFN-{gamma} • CD40/CD40L • CTL


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INTRODUCTION
 
Bacillus Calmette-Guérin (BCG) is used worldwide as a live, attenuated vaccine against Mycobacterium tuberculosis (MTb) infection. Although the protective efficacy of BCG against MTb is quite variable (from 80% to 0%) depending on the regions where trials are carried out, BCG is considered to be partly effective against childhood and disseminated tuberculosis (reviewed in ref [1 ]). It has since been found that in different vaccine preparations, there is an enormous variation (from 5% to 45%) in the proportion of viable bacilli. Studies using animal models have shown that only live BCG is protective against challenge with virulent MTb [2 ], and dead BCG is ineffective; therefore, variation in the viability of bacillus present in BCG vaccine preparations is probably responsible for the inconsistent efficacy of BCG against MTb infection.

Live mycobacteria taken up by macrophages can replicate in a specialized intracellular compartment (phagosome) by inhibiting fusion with acidic lysosome (reviewed in ref [3 ]). Using the mouse model of BCG vaccination, Daugelat et al. [4 ] demonstrated that secreted proteins from live BCG induced maximum T cell responses, and T cells of mice vaccinated with heat-killed BCG responded only to several somatic antigens. Recently, a phagosome coat protein, TACO, was identified. This molecule is retained by live mycobacteria and allows them to survive within macrophages [5 ] by inhibiting fusion of the phagosome with lysosomes. Furthermore, using confocal microscopy, it has been revealed that phagosomal membrane permeability is increased in macrophages infected with live but not formalin-killed BCG [6 ]. Thus, part of the protective effect of live BCG may be due to secreted proteins produced by live mycobacteria residing in phagosomes for long periods of time. However, the basic mechanism of how vaccination with the live organism leads to protective immunity is still not fully understood.

Macrophages, T cells, interferon-{gamma} (IFN-{gamma}), and tumor necrosis factor {alpha} (TNF-{alpha}) are four components of the immune response against tuberculosis, which can control disease progression. Activated macrophages can kill intracellular mycobacteria to some extent, but the effect may not be enough to eliminate them completely. From studies using major histocompatibility complex (MHC) classes I- and II-deficient knockout mice, it was suggested that CD4+ and CD8+ T cells play an important role in the control of mycobacterial infection [7 ]. When CD4+ helper T cells (Th) recognize mycobacterial antigen presented by antigen-presenting cells (APCs), an immune response against the mycobacteria is generated. Some of CD4+ T cells become memory cells and mediate a delayed-type hypersensitivity (DTH) reaction, which is thought to be a distinct mechanism from protection [8 ]. CD8+ T cells also recognize infected macrophages and become cytotoxic T cells (CTLs) [9 ]. Activated CD4+ and CD8+ T cells produce Th1-type cytokines such as IFN-{gamma} and TNF-{alpha}, and infected macrophages produce interleukin (IL)-12. These cytokines are considered to regulate the immune response to mycobacterial infection, based on studies of mice and humans with genetic defects in cytokines and cytokine receptors [8 ].

Turner and Dockrell [10 ] stimulated peripheral blood mononuclear cells (PBMC) from vaccinated individuals with BCG and found that live BCG activated more CD8+ T cells than dead BCG following irradiation. The level of activation was measured by the expression of the IL-2 receptor, and they showed the killing activity of CD8+ T cells stimulated with live BCG only. Because they stimulated whole PBMC with BCG, the actual cells presenting BCG antigen were not identified. In a similar study, Smith et al. [11 ] showed that BCG-specific CD8+ T cells were capable of producing IFN-{gamma}, TNF-{alpha}, and perforin and that these cells exhibited CTL activity against target cells expressing a variety of mycobacterial antigens. These studies demonstrated that BCG-specific memory CD8+ T cells did exist in vaccinated individuals and that these cells could be activated in vitro and develop into CTLs. Although the role of CD8+ T cells in immunity to tuberculosis is less well understood, their role in controlling disease is believed to be more important in the chronic phase than in the acute phase [8 ].

Monocyte-derived dendritic cells (DCs) generated by stimulation with IL-4 and granulocyte macrophage-colony stimulating factor (GM-CSF) are immature and able to take up macromolecules by macropinocytosis or by mannose receptor-mediated endocytosis [12 ]. Upon maturation following TNF-{alpha}, lipopolysaccharide (LPS), IL-1ß, or CD40 signaling, they exhibit potent antigen-presenting activity. Recently, it was shown that DCs were susceptible to mycobacterial infection and that the infection induced maturation of these cells [13 , 14 ]. Therefore, infected DCs could serve as potent APCs for the activation of CD4+ as well as CD8+ T cells in the initiation of the immune response against BCG or MTb. To clarify the mechanism of the distinct effect of live versus dead BCG on immunity to tuberculosis, we analyzed the interaction of BCG-infected DCs with T cells in vitro. Here, we demonstrate that the difference between live and dead BCG is that DCs harboring live BCG induce a more marked expansion of perforin+ CD8+ T cells and a higher level of killing of BCG-infected macrophages compared with DCs harboring heat-killed BCG. This effect was not mediated by cytokines or other soluble factors, but by the direct contact between DCs harboring live BCG and T cells. Furthermore, activation of CD8+ T cells by BCG-infected DCs did not require any help by CD4+ T cells. Thus, the interaction between BCG-infected DCs and memory CD8+ T cells may play a critical role in the maturation of BCG-specific CTLs.


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MATERIALS AND METHODS
 
Reagents
Anti-mannose receptor, fluorescein isothiocyanate (FITC)-conjugated anti-mouse immunoglobulin G (IgG), FITC-conjugated CD40, and phycoerythrin (PE)-conjugated CD45RA were purchased from Coulter Corp. (Miami, FL). Hybridomas producing monoclonal antibodies (mAb), anti-CD1a (OKT6, IgG1), anti-CD3 (OKT3, IgG2a), anti-CD8 (OKT8, IgG2a), anti-CD14 (3C10, IgG2b), anti-CD20 (1F54, IgG2a), anti-CD16 (3G8, IgG1), anti-CD11b (OKM-1, IgG2b), anti-CD58 (Ts2/9.1.4.3, IgG1), anti-CD40L (5C8, IgG2a), anti-human leukocyte antigen (HLA)-DR (L231, IgG2a), anti-HLA-ABC (W6/32, IgG2a), and mouse IgG2a used as a control (NK1.1) were obtained from the American Tissue Culture Collection (Manassas, VA), and the IgG fractions were purified from the culture supernatants. APC-streptavidin, FITC-perforin, and FITC-IgG2b were purchased from Pharmingen (BD Bioscience, San Jose, CA). Anti-CD16, anti-CD1b mAb, anti-CD80, and anti-CD86 mAb were kindly provided by Dr. H. Yagita (Dept. of Immunology, Juntendo University, School of Medicine, Tokyo, Japan) and Dr. M. Azuma (Dept. of Allergy and Immunology, National Children’s Medical Research Center, Tokyo, Japan). Monensin and saponin were purchased from Sigma Chemical Co. (St. Louis, MO), and ethidium monoazide acetate (EMA) was obtained from Molecular Probes Inc. (Eugene, OR).

Bacterial cells
Mycobacterium bovis BCG Tokyo strain was obtained through the courtesy of Dr. S. Haga [Dept. of Bacteriology, National Institute of Infectious Diseases (NIID), Tokyo, Japan]. The bacteria were maintained in 10 ml 7H9 Middle Brook medium (Difco Lab., Detroit, MI) containing 10% acid citrate dextrose enrichment (BBL, Cockeysville, MD). Before the experiment, the aggregated bacteria were sedimented at 1 g for 30 min, and then the supernatant was passed through a nylon mesh. The bacterial solution was sonicated with three consecutive, 5-s pulses (30W), and the optical density (OD) at 550 nm was measured. They were serially diluted, inoculated onto 1% Ogawa medium (Kyokuto Pharmacy Co. Ltd., Tokyo, Japan), and the number of colony-forming units (CFU) at 1.0 OD550 was determined to be approximately 2 x 107 CFU/ml. The viability of the bacteria was examined using fluorescent microscopy after staining bacteria smeared on a slide glass with a mixture of 25 µl ethidium bomide (0.04 mg/ml) and 50 µl fluorescein diacetate (0.5 mg/ml). It was always more than 80%. The bacteria were washed and resuspended in RPMI-1640 medium at a concentration of 5–10 x 105 CFU per ml. DCs were infected with BCG at multiplicity of infection (MOI) 10 (except in Fig. 1 at MOI 40) overnight at 37°C. In some experiments, half of the live BCG solution was heat killed at 95°C for 20 min. Phagocytosis of live and killed BCG by DCs was confirmed using the modified Ziehl-Neelsen stain.



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  		Figure 1. Maturation of immature DCs by live or heat-killed BCG. Monocyte-derived DCs were incubated with medium alone (solid line) or infected with live (dotted line) or heat-killed (broken line) BCGs at MOI 40 overnight. Infected cells were washed and cultured for a further 2 days. The expression of surface antigens of DCs was analyzed by FACS using mAb against (a) CD83 or (b) CD1a, HLA-DR, CD40, CD80, CD86, CD58 (LFA-3), the mannose receptor (mannose R), CXCR4, and CCR5. A representative result of three donors is shown. The shadowed histogram in each panel represents the background staining.

DC, macrophage, and lymphocyte preparation
The buffy coat was obtained from 200 ml venous blood of healthy volunteers, and PBMC were isolated by Ficoll-Hypaque density gradient (Lymphosepal, IBL, Gunma, Japan). Monocytes were isolated by direct binding to anti-CD14-conjugated magnetic beads using membrane attach complexes (MACS; Miltenyi Biotec, Cologne, Germany), and the samples were then incubated for 30 min at 37°C to allow the attachment of adherent cells to the plate. These adherent cells were cultured with RPMI 1640 supplemented with 2 mM L-glutamine, 10% fetal bovine serum (FBS), and antibiotics at a cell density of 1 x 106/ml. To obtain immature DCs, 5 ng/ml IL-4 (PeproTech EC Ltd., London, England) and 500 U/ml GM-CSF (a kind gift from Dr. M. Tatsumi, Dept. of Veterinary Science, NIID) were added to the medium. To obtain macrophages, RPMI-1640 medium containing 5% FBS, 10% human AB serum, and 500 U/ml GM-CSF was used. After 7 days of culture, DCs expressed a high level of CD1a and a marginal level of CD83.

CD14-negative cells were incubated with antibodies against CD11b, CD20, CD14, and CD16 for 15 min on ice and washed with MACS buffer [0.5% bovine serum albumin, 1 mM ethylenediaminetetraacetate in phosphate-buffered saline (PBS)]. The cells then were incubated with goat anti-mouse-IgG-conjugated magnetic beads for 15 min at 10°C. The purified T cells were kept frozen at -135°C until use. To obtain CD8+ and CD4+ T cells, T cells were incubated with anti-CD4- or anti-CD8-conjugated microbeads, respectively, for negative selection. For the purification of CD8+ T cells, two consecutive MACS column (LS) purifications were required. The purities of CD8+ and CD4+ T cells were >90% and 95–98%, respectively, with varying levels of contamination with double negative T cells.

Cocultures and fluorescein-activated cell sorter (FACS) analysis of T cells activated by BCG-infected DCs
DCs cultured overnight with live or heat-killed BCG were washed three times by centrifugation at 1000 rpm for 10 min to remove extracellular bacteria. After washing, DCs (1–2.5x105/ml) were cocultured with T cells (1x2.5x106/ml) in RPMI-1640 medium with 10% FBS in the absence of exogenous cytokines. At day 3, half of the medium was replaced with a fresh medium containing 20 U/ml IL-2, which was a baculovirus product kindly provided by Dr. M. Tatsumi (NIID). On days 7 to 8, cultured T cells were harvested and analyzed. To determine whether the distinct effect between live and heat-killed BCG taken up by DCs is mediated by soluble factors, the coculture of T cells with live, BCG-infected DCs was carried out, separated from the coculture of T cells with heat-killed, BCG-infected DCs by a polycarbonate membrane in a trans-well chamber (Transwell; Corning Costar Corp., Cambridge, MA). These cultured T cells were analyzed separately on days 7–8 as described above. In experiments with blocking mAb to inhibit interaction between DCs and T cells, BCG-infected DCs were preincubated with mAb (at final concentration of 20 µg/ml) for 20 min at 37°C before T cells were added to the culture.

Flow cytometric detection of intracellular perforin was performed as described previously with some modifications [15 ]. Briefly, secretion of perforin was blocked by incubating activated T cells in the presence of 2 µg/ml monensin at 37°C for 6 h. The cells were collected, washed, and resuspended in cold PBS containing 2% FBS and 0.05% NaN3 (staining buffer). They were stained first with biotinylated CD8 and EMA (5 µg/ml) for 20 min on ice under room light, washed, and then incubated on ice for an additional 20 min with PE-conjugated CD45RA and APC-streptavidin for surface staining. Then, cells were washed and fixed with 4% formaldehyde in PBS for 20 min at room temperature, followed by washing with permeabilization buffer containing 0.5% saponin in the staining buffer. The fixed and permeabilized cells were stained with FITC-perforin or FITC-IgG2b (control) for 30 min on ice, washed with staining buffer, and analyzed by FACScalibur (BD Bioscience) using the Cell Quest program. In some experiments, the data were reanalyzed using Flow Jo software (Tree Star Inc., San Carlos, CA). All the data of intracellular staining were shown by gating lymphocyte forward scatter/side scatter (FSC/SSC low) and live cells (EMA low) with 50,000 events.

CTL assay
Cytotoxic T cell activity was measured by the chromium release assay as described previously [16 ]. Briefly, macrophages infected with live BCG 1 day before the CTL assay were labeled with Chromium-51 (ICN Biomedicals Inc., Costa Mesa, CA) at 37°C for 1 h. Labeled macrophages (2.5x103 cells in 100 µl) were plated into a 96-well round-bottom plate, and serially diluted effector T cells were added to each well (final 200 µl in total). After 4.5 h incubation at 37°C, 30 µl supernatant was transferred to the Lumaplate (Packard BioScience Company, Meriden, CT), dried, and counted with Topcount (Packard BioScience). The percentage of specific lysis (% killing) was calculated as 100 x [(cpm released with effectors)-(spontaneous cpm released)]/[(cpm released by detergent)-(spontaneous cpm released)].

For the blocking experiment, mAb (at a final concentration of 10 µg/ml) were added to labeled macrophages and incubated at 37°C for 20 min before addition of effector cells.

Cytokine enzyme-linked immunosorbent assay (ELISA)
Supernatants from T cells cocultured with uninfected, killed or live BCG-infected DCs were harvested on day 3 and stored at -30°C. Cytokine levels in supernatants were determined by ELISA. Commercial ELISA kits were used to measure levels of human TNF-{alpha}, IL-10 (both from Boehringer Roche, Basel, Switzerland), IFN-{gamma}, and IL-12 (both from Coulter Corp.).


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RESULTS
 
Induction of maturation of monocyte-derived, immature DCs by the uptake of live or heat-killed BCGs
We first investigated the phenotypic alteration of DCs that resulted from the uptake of live or heat-killed BCG. The bacteria were added to the culture of DCs at MOI 40 and were maintained for 2 days. BCG was efficiently taken up by DCs irrespective of the viability of bacteria, and no apparent difference was observed with respect to the number and localization of bacteria in the cytoplasm. We then compared the expression of surface molecules on DCs harboring live or heat-killed BCG (designated as live BCG+ or heat-killed BCG+ DCs) with FACS (Fig. 1 ). Uptake of live and killed BCG caused the phenotypic change in immature DCs from CD83low to CD83high, which is a marker for maturation of DCs [17 ] (Fig. 1a) . In addition, the uptake of live or heat-killed BCG resulted in the increased expression of maturation-associated molecules, HLA-DR, CD40, CD58 [lymphocyte function-associated antigen-3 (LFA-3)], CD80, CD86, and CXCR4, whereas it caused the down-regulation of the mannose receptor and CCR5 (Fig. 1b) . These results suggest that immature DCs become phenotypically mature following the uptake of BCG, irrespective of the viability of the bacteria.

Selective expansion of perforin-positive CD8+ T cells from T cells cocultured with live BCG+ DCs
DCs are potent APCs and efficiently activate naive and memory T cells [18 ]. To characterize the activation of T cells by live and heat-killed BCG+ DCs, DCs were infected overnight with live or heat-killed BCGs, washed to remove extracellular bacteria, and then cocultured with autologous T cells. Three days later, half of the medium was removed and frozen for cytokine analysis, and IL-2 was added to the culture to maintain the activated T cells. One week later, cells were harvested, and the expression of perforin was examined. As shown in Figure 2 , the proportion of perforin+-activated (CD45RA-) T cells was increased markedly at day 8 after cultivation with live BCG+ DCs (46%), whereas the proportion was lower following cultivation with heat-killed BCG+ DCs (16%; middle panel). The perforin+-activated T cells consisted largely of CD8+ cells (96%). We observed that CD8+ T cells, but not CD4+ T cells increased threefold in number after cultivation with live BCG+ DCs (data not shown), of which 75% expressed perforin. In contrast, 18% of CD8+ T cells expressed perforin after cultivation with heat-killed BCG+ DCs (right panel). The selective expansion of perforin+-activated CD8+ T cells by cultivation with live BCG+ DCs was observed in five out of eight donors examined, and the proportion of perforin+-activated CD8+ T cells ranged from 31.5% to 75%. The induction of perforin+ CD8+ T cell expansion was less or minimal when cells were cocultured with heat-killed BCG+ DCs (3.8–18%) or with uninfected DCs (0.4–8.2%). Therefore, although DCs were activated similarly by the uptake of live or heat-killed BCGs, the level of perforin+ CD8+ T cell expansion that resulted from coculture of T cells with live or heat-killed BCG+ DCs was significantly different (P=0.0061).



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  		Figure 2. A higher level of CD45RA- perforin+ T-cell expansion was induced in vitro by live BCG+ DCs compared with that by heat-killed BCG+ DCs. Monocyte-derived DCs were uninfected (a) or infected with heat-killed (b) or live (c) BCGs at MOI 10 overnight, washed, and then cocultured with autologous T cells (>98% CD3+). Half of cells were harvested at day 3, and the culture was continued until day 8 in the presence of IL-2. Cells were fixed with 4% formalin/PBS after surface staining of CD8 and CD45RA with EMA. Fixed cells were then permeabilized, and the intracellular staining of perforin was carried out. A representative result of five donors is shown in the left and middle panel for the expression of CD45RA and perforin in T cells at days 3 and 8, respectively. In the right panel, the expression of CD45RA and perforin in gated CD8+ T cells at day 8 is depicted. The proportion of CD8+ T cells from the total T-cell population at day 8 consisted of 17.1% (a), 17.5% (b), and 45.6% (c).

Selective expansion of perforin+ CD8+ T cells induced by live BCG-infected DCs is not mediated by soluble factors
A variety of cytokines regulate the immune responses during mycobacterial infection [8 ]. Therefore, it is possible that a distinct profile of cytokines might contribute to the different levels of expansion of perforin+ CD8+ T cells induced by live BCG+ or heat-killed BCG+ DCs. To determine whether this was occurring, we measured the levels of IFN-{gamma}, TNF-{alpha}, IL-10, and IL-12 in the culture supernatant of T cells cocultured with live or heat-killed BCG+ DCs at day 3. DCs infected with BCG produced only a low level of these cytokines per se (data not shown). As shown in Figure 3 , the level of these cytokines was significantly increased in the coculture of T cells with live or heat-killed BCG+ DCs, but not with uninfected DCs (P<0.05). In some donors, the level of TNF-{alpha} and IL-12 produced during coculture of T cells with live BCG+ DCs was higher than that of T cells with heat-killed BCG+ DCs. However, the level of production of these cytokines did not correlate with the level of perforin+ CD8+ T-cell expansion. Therefore, these cytokines may not contribute to the expansion of perforin+ CD8+ T cells induced by live BCG+ DCs.



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  		Figure 3. Cytokine production in the coculture of T cells with live or heat-killed BCG+ DCs. The culture supernatant of purified T cells cocultured with uninfected DCs (no BCG), heat-killed BCG+ DCs (killed), or live BCG+ DCs (live) for 3 days was collected, and the level of IFN-{gamma}, TNF-{alpha}, IL-10, and IL-12 was measured by ELISA. Circles represent the results of five donors, and bars indicate the average value. The level of cytokines produced by DCs alone was below the detection limit.

To examine whether any other cytokine(s) induced by live BCG+ DCs were responsible for the selective expansion of perforin+ CD8+ T cells, T cells mixed with live BCG+ DCs and those with heat-killed BCG+ DCs were cultured in a trans-well culture plate separated by a semipermeable membrane. As shown in Figure 4 , the proportion of perforin+ CD8+ T cells increased markedly during coculture with live BCG+ DCs; however, there was little change in the T cells cocultured with heat-killed BCG+ DCs, despite the free traffic of soluble factors through the membrane. This result suggests that the direct contact of CD8+ T cells with DCs infected with live BCG is required for the expansion and maturation of CD8+ T cells.



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  		Figure 4. The higher level of expansion of CD45RA- perforin+ T cells by live BCG+ DCs is not mediated by soluble factors but by direct contact. A live BCG+ DC (1x105) and T cell (1x106) mixture (upper chamber, solid line) was cultured in a trans-well plate from a mixture of heat-killed BCG+ DCs and T cells (lower chamber, dotted line) by a semipermeable membrane for 7 days. After cultivation, cells were harvested separately, and the level of intracellular perforin in CD8+ T cells was analyzed by FACS as described in the legend to Figure 2 . The isotype-matched (IgG2b) control staining of CD8+ T cells is shown as a shadowed histogram. This result is representative of two experiments performed.

BCG-infected macrophages are killed by perforin-positive CD8+ T cells
We examined the killing activity of perforin+ T cells that had expanded during coculture with BCG+ DCs. We selected a donor who developed a high proportion of perforin+ CD45RA- CD8+ T cells (70–80%) after cultivation with live BCG+ DCs. Targets used for measurement of CTL activity were autologous macrophages that were infected with live BCG overnight. A representative result of CTL activity with various ratios of effector-to-target is shown in Figure 5a . As expected, cells recovered from the coculture with live BCG+ DCs showed a high level of CTL activity compared with those recovered from the coculture with heat-killed BCG+ DCs. No CTL activity was detected when uninfected autologous macrophages were used as targets. Similarly, in the other five donors, who developed a lower proportion of perforin+ CD45RA- CD8+ T cells (30–50% of total CD8+ T cells), the level of CTL activity was always higher in the cultured cells with live BCG (20–30%) than in those with heat-killed BCG (11–19%). Thus, it appears that coculture with live BCG+ DCs causes an expansion of BCG-specific CTLs in association with the expression of perforin.



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  		Figure 5. A higher level of CTL activity in T cells cocultured with live BCG+ DCs is induced compared with that from T cells cocultured with heat-killed BCG+ DCs, both restricted by MHC class I or CD1b. (a) Purified T cells (1x106 per well) were cocultured with uninfected DCs ({circ}), heat-killed ({blacktriangleup}), or live (•) BCG+ DCs (1x105 per well) in a 24-well culture plate for 7 days and were used as effector cells. Autologous macrophages infected with live BCGs and labeled with chromium were used as target cells. Effector and target cells were mixed at various E:T ratios and cultured for 4 h. CTL activity was calculated as 100 x [(cpm released with effectors)-(spontaneous cpm released)]/[(cpm released by detergent)-(spontaneous cpm released)]. A representative result of two independent experiments is shown. These effector cells did not kill uninfected macrophages. (b) The BCG-infected macrophages were chromium-labeled, washed, placed into a 96-well, U-bottom plate, and then precultured for 20 min at 37°C in the presence of mAb against CD1b, HLA-ABC, CD1a, or isotype-matched, control IgG (G1 and G2a) at a final concentration of 20 µg/ml. The effector cells were prepared as in (a), added to the preincubated macrophages at an E:T ratio of 100:1, and incubated for 4 h at 37°C. The percent killing was calculated as aforementioned in (a). This result is representative of two experiments performed.

Both MHC class I-restricted and CD1-restricted CTL clones were isolated from patients infected with MTb [9 ]. We then determined whether there was a qualitative difference, with respect to CTL restriction, between T cells activated with heat-killed BCG+ DCs and those activated with live BCG+ DCs using the same effector cells shown in Figure 5a . Target (T) macrophages were labeled and preincubated for 20 min in the presence or absence of various mAb, and then effector (E) cells were added at an E:T ratio of 100:1. We observed that the killing activity of T cells cocultured with live BCG+ DCs was blocked by anti-HLA-ABC (52% inhibition) and anti-CD1b mAb (35% inhibition); however, anti-CD1a mAb or isotype-matched control IgG (Fig. 5b) had little effect. The effect of these blocking mAb was comparable between T cells activated with heat-killed BCG+ DCs and those activated with live BCG+ DCs, although the level of CTL activity was proportionally higher in the latter. The blocking effect of anti-HLA-ABC and anti-CD1b mAb was consistently observed in cells from two other donors, despite the fact that the CTL activity of these donors was low (20–25% at E:T ratio of 100:1). Taken together, the results suggest that MHC class I- and CD1b-restricted CTL effectors can be generated during in vitro coculture of T cells with DCs harboring BCG, irrespective of the viability of BCG.

Activation and maturation of BCG-specific CTLs are CD4+ T-cell-independent
We next examined whether the selective expansion of CD8+ T cells and maturation to effector cells required the presence of CD4 Th cells in this BCG+ DC-T cell coculture system. CD8+ and CD4+ T cells were purified by negative selection and mixed (1:1) or separately cocultured with uninfected or live BCG+ DCs for 7 days. As shown in Figure 6 , coculture of CD8+ T cells with BCG+ DCs in the absence of CD4+ T cells resulted in a substantial expansion of CD8+ T cells with expression of intracellular perforin (Fig. 6b , right panel, 70.6%). Coculture of the same number of CD4+ and CD8+ T cells with BCG+ DCs resulted in the dominant growth of CD8+ T cells expressing perforin at a frequency similar to that observed in the absence of CD4+ T cells (Fig. 6c , right panel, 73.8%). Uninfected DCs did not induce the expansion of activated perforin+ CD8+ T cells (Fig. 6a , right panel, 0.68%). Taken together, the results support the view that the direct contact of T cells with live BCG+ DCs causes preferential activation of BCG-specific, memory CD8+ T cells and that CD4+ T cells may not play an essential role in this process.



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  		Figure 6. CD4 help is not required for the activation of CD45RA- perforin+ CD8+ T cells. CD4+ (left panel) and CD8+ (right panel) T cells were negatively selected by MACS and cocultured separately (a and b) or mixed at 1:1 (c) with uninfected (a) or live-BCG+ DCs (b and c) for 7 days. Fresh medium containing IL-2 (20 U/ml) was supplied at day 3 of coculture. After cultivation, cells were stained with anti-CD45RA mAb and with anti-CD4 (left panel) or anti-CD8 (right panel) mAb followed by intracellular perforin staining as described in the legend to Figure 2 . For FACS analysis, CD4+ (left panel) or CD8+ (right panel) cells were gated, and the level of CD45RA and perforin was analyzed. A representative result of three donors is shown. The percent of CD45RA- and perforin+ cells is listed in each panel.

We noticed that a substantial number of perforin+ CD4+ T cells were activated (29.2%), especially in the presence of CD8+ T cells (Fig. 6c , left panel). These cells may also contribute to the population of effector CTLs as described previously [19 ].

The crucial role of the CD40/CD40L interaction between CD8+ T cells and BCG-infected DCs
It is known that CD4+ T cells activate DCs through the CD40/CD40L interaction (reviewed in [17 ]). These activated DCs [20 21 22 ] can prime naive CD8+ T cells or activate memory CD8+ T cells without CD4+ T cell help. CD40L is predominantly expressed on activated CD4+ T cells [23 ], but weak expression can also be detected in a small population of activated CD8+ T cells [23 24 25 ]. In contrast to CD4+ T cells, however, the biological significance of CD40L expression on CD8+ T cells is less known. Therefore, to investigate the role of the CD40/CD40L interaction in the selective expansion of perforin+ CD8+ T cells through the direct contact with live BCG-infected DCs, we cocultured BCG-infected DCs and purified CD8+ T cells in the presence of mAb against CD40L (CD154). For comparison, the effect of mAb against LFA-3 (CD58), which acts as an immunological synapse to strengthen the T cell receptor signaling [26 ], was also analyzed. Because the production of IFN-{gamma} by CD8+ T cells is considered a hallmark of CTL activation, the level of IFN-{gamma} in the culture supernatant at day 3 was measured. The results of five donors were expressed as the percentage of IFN-{gamma} production relative to the control culture in the presence of isotype-matched, irrelevant mouse mAb. As shown in Figure 7a , the level of IFN-{gamma} was not reduced in the presence of anti-CD40L, and anti-LFA-3 mAb suppressed IFN-{gamma} production significantly to less than 20%.



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  		Figure 7. The CD40/CD40L interaction is involved in the activation and maturation of CD8+ T cells in coculture with BCG+ DCs. CD8+ T cells (1x106) and live BCG-infected DCs (1x105) were cocultured in the presence of mAb against CD40L, LFA-3, or control IgG (cont; 20 µg/ml at final concentration). Three days later, half of the culture supernatant was collected and replaced with fresh medium containing IL-2 and the same concentration of mAb. The amount of IFN-{gamma} in the culture supernatant at day 3 was measured by ELISA (a). Circles represent the result of four donors, which are expressed as the percentage of the level of IFN-{gamma} produced in the control antibody-treated culture. On day 8, cells were harvested, and CD45RA and perforin expression was analyzed by FACS. The representative results of three donors in five are depicted (b). The percentages of CD45RA- and perforin+ cells are listed in each panel.

At day 8, the perforin content in CD8+ T cells treated with isotype-matched control, anti-CD40L, or anti-LFA-3 mAb was examined by FACS. The number of cells recovered did not differ among these cultures. It is interesting that the development of CD45RA- perforin+ CD8+ T cells was strongly inhibited by anti-CD40L mAb, irrespective of the level of CD45RA- perforin+ CD8+ T cells. The representative results of three among five donors were shown in Figure 7b . Thus, despite a similar level of IFN-{gamma} (Fig. 7a) , the activation and maturation of CD8+ T cells were suppressed variably in the presence of the anti-CD40L mAb. In contrast, the expansion of perforin+ CD8+ T cells was not affected by anti-LFA-3. Taken together, it appears that BCG-infected DCs deliver a signal to induce the activation and maturation of memory CD8+ T cells through the CD40/CD40L interaction, which may be distinct from a signal for IFN-{gamma} production.


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DISCUSSION
 
BCG stimulation of immature DCs is known to induce their maturation [13 ]. The present study demonstrated that immature DCs matured following the uptake of BCG, irrespective of the viability of the bacteria. However, stimulation of T cells in vitro with live BCG-infected DCs caused expansion of perforin-containing CD8+ T cells, which had killing activity against BCG-infected macrophages to a greater extent than did stimulation with DCs harboring heat-killed BCG. The efficient expansion and maturation of CD8+ T cells required direct contact between live BCG-infected DCs and CD8+ T cells; soluble factors including IFN-{gamma}, TNF-{alpha}, and IL-12 did not appear to contribute to this reaction.

In our in vitro system, the administration of mAb against HLA-ABC or CD1b into a coculture of T cells with live BCG+ DCs reduced CTL activity against BCG-infected cells, which is compatible with the notion that MHC class I- and CD1b-restricted antigenic presentation may activate BCG-specific CTLs. It has been shown that phagosome-membrane permeability increases in macrophages infected with BCG, thereby allowing large cytoplasmic or mycobacterial proteins to access the MHC class I presentation pathway [6 ]. Thus, the classical TAP-dependent MHC class I pathway could be used for antigen presentation of proteins secreted from live BCG in the phagosomal compartment. Thus, it is conceivable that the antigens delivered continuously by live BCG, but not by heat-killed BCG, into antigen-presenting compartments of DCs may affect the level of CD8+ T cell expansion and maturation. MHC class I- and CD1-restricted CTLs are known to be present in patients infected with MTb [9 ], although the role of CD1-restricted CTLs in protection against human MTb infection remains unknown. In our in vitro system, most of the expanded T cells (70–80%) were CD8; however, it remains to be elucidated whether these cells with CD1-restricted CTL activity are {gamma}{delta} T cells with the CD4- CD8- phenotype, as has been described by Canaday et al. [27 ].

Activation of T cells requires signaling through the T-cell receptor/CD3 complex and CD28. In addition, LFA-1 and LFA-3 have a role in the formation of a T cell synapse, through interaction with CD54 (intercellular adhesion molecule-1) and CD2, respectively [26 ]. Furthermore, it has been suggested that the interaction of CD40L with its receptor CD40 plays an important role in cell-mediated immunity (reviewed in ref [28 ]). Results from studies using CD40L knockout mice infected with lymphocytic choriomenigitis virus (LCMV) suggest that CD40L is not required for primary CTL responses [29 , 30 ] but is essential for the maintenance of long-term control of virus replication by CTL [29 , 31 ]. CD4+ T cells activate DCs through the CD40/CD40L interaction, which is important for the activation of CD8+ T cells (reviewed in ref [32 ]). Therefore, it is not clear from these in vivo studies whether the impaired CTL function in the chronic phase of LCMV infection is a result of the lack of CD40/CD40L interactions between CD4+ T cells and DCs, between CD8+ T cells and DCs, or both. Regarding mycobacterial infection, Campos-Neto et al. [33 ] demonstrated that CD40L knockout mice infected with MTb were equally resistant as wild-type C57BL/6 mice. The resistance to MTb in these mice was ascribed mainly to a high level of IFN-{gamma} and TNF production by CD4+ T cells, which can occur independently of the CD40/CD40L pathway. Thus, activated macrophages may be potent enough to control MTb infection, without progression to the chronic disease. In this model, the role of CD40L in CD8+ CTL would be very little.

CD40-mediated activation of DCs is accompanied by the up-regulation of accessory molecules, such as CD86, CD80, CD58 (LFA-3), and MHC class II [17 ]. Such activated DCs following CD40 signaling [20 21 22 ] or stimulation with LPS [34 ] can prime naive CD8+ T cells or activate memory CD8+ T cells without CD4+ T cell help. In this context, the present study has shown for the first time that BCG infection also causes maturation of DCs, which are responsible for the expansion and maturation of CD8+ CTLs through the CD40/CD40L interaction, without help from the CD4+ T cell. The potent APC function of BCG-infected DCs could be associated with the increased expression of CD80/86, CD58, and other accessory molecules. However, our results support the possibility that the stimulation of CD40L, through CD40 on activated DCs, delivers a signal directly to the memory CD8+ T cells to induce maturation of CTLs, as has been proposed by Shepherd and Kerkvliet [35 ] in their allograft model. Furthermore, blocking CD2/CD58, but not the CD40/CD40L interaction, significantly reduced the production of IFN-{gamma} without affecting the expansion of perforin+ CD8+ T cells, which indicated that IFN-{gamma} produced by activated CD8+ T cells was not essential for the maturation of CD8+ T cells into CTL effectors. As a result of this, it was demonstrated that the antigen-specific CTL response was not impaired in IFN-{gamma} knockout mice infected with Listeria monocytogenes [36 ] or with influenza virus [37 ].

Perforin is a granule protein and one of the essential mediators for killing infected macrophages by forming pores in the plasma membrane. The granules of CTL also contain a collection of serine proteases, granzymes, and another important molecule, granulysin, which kills mycobacteria directly [38 ]. Because granulysin is exocytosed along with perforin, CTLs with perforin-dependent killing activity are considered to be more important for protection against MTb infection than CTLs that use the Fas/FasL-mediated killing pathway [39 ]. In a mouse model of MTb infection, perforin knockout mice showed only modest susceptibility and did not differ from wild-type mice during the early phase of the infection [40 , 41 ]. Presumably, perforin-dependent CTLs are required later during the chronic phase of infection to attack the remaining macrophages that harbor live bacteria, following the decline of the initial host response. Thus, the immune response against a mycobacterial infection would consist of the combination of activated macrophages and helper and cytotoxic T cells and their cytokines in the early phase and CTLs in the chronic phase of infection [8 ].

The present results support the view that CD8+ CTL memory cells specific for BCG are generated and maintained for long periods of time in BCG-vaccinated hosts and that these cells mature into effectors upon restimulation through interaction with potent APCs. In line with this notion, Lewinsohn et al. [42 ] demonstrated that PPD-negative, BCG-nonvaccinated donors had no demonstratable CD8+ T cell responses to MTb. Most Japanese received BCG vaccination early in life and were at least once PPD-positive, although PPD reactivity of adult donors was not known at the time when we performed this study. Of note, in some adults with recent positive reactivity to the PPD skin test, T cells did not develop into perforin+ CD8+ T cells in response to live BCG-infected DCs. This observation has led us to speculate that there is variability in the maintenance of functional memory T cells responsible for DTH and CTL in BCG-vaccinated individuals. It would be worthwhile analyzing whether protection against MTb could be achieved by the induction of a long-term memory CTL response through vaccination with DCs infected with live BCGs. Furthermore, such DC-based methods might represent an advantage over conventional lymphoproliferation assays and IFN-{gamma} responses in ex vivo studies of the correlates of protective immunity.


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ACKNOWLEDGEMENTS
 
This work was supported by the Program for Promotion of Fundamental Studies in Health Sciences of the Organization for Pharmaceutical Safety and Research of Japan. We thank Dr. H. Yagita (Juntendo University, School of Medicine, Tokyo) and Dr. K. Kato (National Cancer Center) for providing mAb and hybridomas. We also thank Dr. N. Waganabe (University of Tokyo, Institute of Medical Science) for his help and suggestions to analyze intracellular perforin by FACS.

Received August 7, 2001; revised January 8, 2002; accepted February 11, 2002.


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