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The Journal of Immunology, 2006, 176: 2610-2616.
Copyright © 2006 by The American Association of Immunologists

Human NK Cells Positively Regulate {gamma}{delta} T Cells in Response to Mycobacterium tuberculosis1

Ruijun Zhang2,*,{dagger}, Xiaodong Zheng2,*, Baiqing Li*, Haiming Wei* and Zhigang Tian3,*,{dagger}

* Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, China; and {dagger} Institute of Immunopharmacology and Immunotherapy, School of Pharmaceutical Sciences, Shandong University, Jinan, China


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The decrease in NK cell activity and the loss of {gamma}{delta} T cells in active pulmonary tuberculosis patients have been reported. In this study, we observed that the proliferating response of {gamma}{delta} T cells to the heat-treated Ags of Mycobacterium tuberculosis from different individuals was noted to be dependent on the content or function of NK cells in PBMC in a population study. We also found that NK cells were directly rapidly activated by the heat-treated Ags from M. tuberculosis (H37Ra) in vitro; in turn, the activated NK cells improved {gamma}{delta} T cell proliferation both by CD54-mediated cell-cell contact through the forming immune synapse and by soluble factors TNF-{alpha}, GM-CSF, and IL-12, but not IFN-{gamma}. Our results demonstrated that an interaction between NK cells and {gamma}{delta} T cells existed in antituberculosis immunity. Up-regulating the function of NK cells might be beneficial to the prevention and control of pulmonary tuberculosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Tuberculosis remains the worldwide infection disease which is estimated to kill over 2 million people annually. Fortunately, Mycobacterium tuberculosis (M.tb)4 can be controlled by cell-mediated immunity (1), and {gamma}{delta} T cells play an important role in the immune response to M.tb (2, 3, 4). Previous studies demonstrated that {gamma}{delta} T cell activation by live M.tb was dependent on APC in a nonclass I or class II MHC-restricted manner. When mycobacteria were heat-treated, the ability of the bacilli to stimulate {gamma}{delta} T cells was greatly reduced (5, 6). However, the supernatant from the heat-treated mycobacteria induced the proliferation of human {gamma}{delta} T cells. The protein Ags with a molecular mass of ~10–14 kDa in the supernatant were the bioactivators that stimulated the {gamma}{delta} T cells (7). It was also reported that NK cells could directly kill intracellular M.tb (8) and regulate the macrophage cytotoxic activity against M.tb (9). The decrease of NK cell activity and the loss of {gamma}{delta} T cells in an active pulmonary tuberculosis patient have been reported (10, 11), but the relationship between the changes of NK cells and {gamma}{delta} T cells was unclear.

NK cells were not only the key effectors in the innate immune response to various infections and transformed cells (12, 13), but were also the effective regulators in innate and adaptive immune responses (14, 15, 16). NK cells were involved in the maturation of CD8+ CTLs (14) and the activation of B cells (15, 16). Most but not all of the regulation functions of NK cells were mediated by cytokines (15, 16, 17). Recently, it was reported that reciprocal activation interaction occurred between NK cells and dendritic cells by cell-cell contact-dependent and soluble factor-dependent mechanisms (18, 19). Considering the immune regulation function of NK cells, we hypothesized that NK cells might be involved in the immune regulation of {gamma}{delta} T cells in response to M.tb and the loss of {gamma}{delta} T cells might be due to the decrease in NK cell activity. To investigate the role of NK cells in the immune response of {gamma}{delta} T cells to M.tb, we used the heat-treated Ags from M.tb to expand {gamma}{delta} T cells in vitro (20). The results demonstrated that NK cells played a role in the proliferating response of {gamma}{delta} T cells to the heat-treated Ags of M.tb.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Sample collection

Peripheral blood was obtained from 30 healthy tuberculin reactors at Anhui Province Blood Center, under protocols approved by the Institutional Review Boards of the University of Science and Technology of China. All the subjects had been vaccinated with Mycobacterium bovis bacillus Calmette-Guerin during childhood.

Heat-treated Ag collection by fast protein liquid chromatography (FPLC)

The heat-treated Ags from the supernatant of Mycobacterium tuberculosis (H37Ra) were collected according to the protocol as reported (7). The heat-treated Ag preparations were concentrated to 2 mg/ml in double-distilled H2O and injected into Sephacryl S-100 HR. The bioactive fraction with a molecular mass of ~11–13 kDa was collected, and analyzed by SDS-PAGE.

Cell isolation and culture

PBMC were isolated from freshly heparinized venous blood from healthy adult people by Ficoll-Hypaque. NK cell purification and depletion were performed with CD56 magnetic microbeads (Miltenyi Biotec) according to the manual. Simply, after washing twice in washing buffer (PBS supplement with 2 mM EDTA and 0.5% BSA), the freshly isolated PBMC were resuspended in the washing buffer at a cell concentration of 1 x 108/800 µl and added to 200 µl of microbeads/1 x 108, mixed well, and incubated for 20 min at 4°C, before applying the cells on the top of MS column. The labeled (NK cells) and unlabeled cells (NK-depleted PBMC) were collected separately and washed twice in PBS. Purified cells were >95% NK cells, and <0.2% CD56+CD3– cells were remained in NK-depleted PBMC by FACS analysis. Cells were maintained in RPMI 1640 medium (Invitrogen Life Technologies) supplemented with 10% (v/v) FBS (HyClone), 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in 5% CO2. PBMC and NK-depleted PBMC were cultured at 1 x 106/ml, 1.5 ml/well in a 24-well cell culture plate, at 37°C in 5% CO2, in the presence of 20 U/ml recombinant human IL-2 (rhIL-2; PeproTech), 5 µg/ml of the heat-treated Ags, or both, respectively. Cells were cultured for 12 days, with rhIL-2 (10 U/ml) added every 3–4 days. Purified protein derivative (PPD) was used to compare with the heat-treated Ags.

FACS analysis

Freshly isolated PBMC or cultured cells were washed in washing buffer and then were stained with fluorochrome-conjugated mAbs. CD54-FITC, CD56-FITC, CD70-FITC, CD80-FITC, CD86-FITC, CD137-FITC, CD11a-FITC, CD11b-FITC, {gamma}{delta}TCR-FITC, {alpha}betaTCR-PE, CD27-PE, CD56-PE, CD3-PECY5, and CD54-PECY5 were used (all from BD Pharmingen). After incubation for 30 min at 4°C, cells were washed three times with washing buffer, then determined by FACSCalibur (BD Biosciences) and the data were analyzed with WinMDI 2.8 software. An intracellular cytokine assay was applied to detect the expression of IFN-{gamma} in NK cells of PBMC. Simply, cells (5 x 106/ml) were stimulated with PMA (30 ng/ml; Sigma-Aldrich) and ionomycin (1 µg/ml; Sigma-Aldrich). One hour later, monensin (5 µg/ml; Sigma-Aldrich) was added to prevent the secretion of the induced cytokines into the supernatant. After 4 h of culture at 37°C and 5% CO2, the cells were harvested and labeled by appropriate fluorochrome-conjugated Abs for surface molecules as described above for 30 min at 4°C, and fixed with PBS containing 2% (w/v) paraformaldehyde for 20 min at room temperature. After being permeabilized with 100 µl of permeabilization buffer (BD Biosciences) containing mouse serum for 1 h at 4°C, the cells were incubated with anti-IFN-{gamma}-PE mAb in the permeabilization buffer for 1 h at room temperature. The samples were washed with permeabilization buffer twice and analyzed by flow cytometry.

Transwell coculture

NK-depleted PBMC were cultured in 24-well flat-bottom plates at 1 x 106/ml (0.9 ml/well) equipped with a Transwell insert (Costar). NK cells were resuspended at a concentration of 1 x 106/ml and 0.1 ml of cell suspension was added into the upper wells of the Transwell. NK-depleted PBMC were separated from NK cells by 1 mm, but the soluble factors could diffuse freely through a microporous polycarbonate membrane (0.4-mm thick) between the upper and the lower wells. The heat-treated Ags of M.tb or rhIL-2 or both were added to the coculture cells.

ELISA

The supernatants of purified NK cells cultured with the heat-treated Ags of M.tb (5 µg/ml) and rhIL-2 (20 U/ml) were collected at the 18th, 36th, and 72th hour and stored at –70°C for ELISAs. Following the protocol of the kits, the cytokines IFN-{gamma} (detection limit, 5 pg/ml), TNF-{alpha} (detection limit, 10 pg/ml), GM-CSF (detection limit, 10 pg/ml), IL-12 (detection limit, 8 pg/ml), IL-4 (detection limit, 8 pg/ml), and IL-10 (detection limit, 6.35 pg/ml) were examined by ELISA kits (R&D Systems). The data were analyzed with Origin 7.0 software.

Cytotoxicity assay

Cytotoxic activity of NK cells in PBMC was assessed against 51Cr-labeled K562 in a standard 4-h 51Cr-release assay (21). K562 cells (106) were labeled with 200 µCi of sodium chromate for 1 h at 37°C and washed three times. Effector (PBMC) and target (K562) were incubated for 4 h at the E:T of 20:1. Spontaneous release of 51Cr was determined by incubating the target cells with medium alone and was always <10%. Maximum release was determined by adding Triton X-100 to a final concentration of 2%. The percentage of 51Cr release was calculated as follows: 100 x ((experimental release – spontaneous release)/(maximum release –spontaneous release)).

Confocal microscopy

The cell-cell contact between NK cells and {gamma}{delta} T cells in PBMC was investigated using a confocal microscope according to the protocol as reported (22). Cells were gently resuspended and spread onto prewarmed poly-L-lysine (Sigma-Aldrich)-coated slides. Slides were incubated at 37°C for another 15 min to promote cell attachment, fixed in freshly prepared 4% paraformaldehyde/PBS for 20 min, and then finally washed three times in PBS. Then the slides were stained with {gamma}{delta}TCR-FITC, CD56-PE, and CD54-PECY5 for 1 h at room temperature away from light. Slides were washed three times in PBS before being sealed with a 0.17-mm coverslip. The color confocal analysis was performed at x400 magnification on a Zeiss LSM 510 equipped with an argon (488 nm) laser and the pictures were electromagnified 1.5 times. Image collection was performed with Zeiss LSM 510-associated software and the data were analyzed with Zeiss LSM Image Browser software.

Statistical analysis

The Student t test was used to interpret the significance of differences between experimental groups.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
The proliferating response of {gamma}{delta} T cells was correlated to the function of NK cells

To delineate the relationship between NK cells and {gamma}{delta} T cells in response to the heat-treated Ags of M.tb, we randomly selected five blood samples with a lower percentage of CD56+ cells (< 5%) and five with a higher percentage (> 15%) from blood donors, and then examined the proliferating response to the heat-treated Ags of M.tb in the presence of rhIL-2. As shown in Fig. 1A, {gamma}{delta} T cells more rapidly proliferated in a high NK-percentage population than that in a low NK-percentage population. The result was further confirmed by examining NK cell cytotoxicity (Fig. 1B), showing that the higher cytotoxic function was from a higher CD56+ percentage of PBMC, which was correlated to the greater proliferation of {gamma}{delta} T cells in response to the heat-treated Ags of M.tb. In contrast, because the {alpha}beta T cells are not sensitive to the heat-treated Ags of M.tb, the total number of {alpha}beta T cells was only slightly increased, but the percentage of {alpha}beta T cells was relatively decreased. Moreover, the percentages and numbers of CD4+ and CD8+ T cells changed similarly to {alpha}beta T cells (data not shown). The results suggested the numbers and function of NK cells affected the proliferation response of {gamma}{delta} T cells to the heat-treated Ags of M.tb.


Figure 1
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  		FIGURE 1. The proliferation of {gamma}{delta} T cells became lower in individuals with impaired NK cell function. Based on the percent and the lytic activity of NK cells, PBMC samples from healthy individuals were classed into (A) a high percentage group (NK% >15%, n = 5) and a low percentage group (NK% <5%, n = 5), and (B) a high function (lytic activity >20% at E:T = 20:1, n = 5) group and a low function group (lytic activity <10% at E:T = 20:1, n = 5). After 14 days of culture with the heat-treated Ags of M.tb and rhIL-2, the percentages of {gamma}{delta} T cells in each group were analyzed by flow cytometry, respectively. Data are shown as mean ± SD. *, p < 0.05.

 
NK cells activated by the heat-treated Ags of M.tb were involved in proliferation of {gamma}{delta} T cells

To evaluate the contribution of NK cells to the heat-treated Ags of M.tb-induced {gamma}{delta} T cell proliferation, PBMC, which were depleted NK cells or not, from five healthy tuberculin reactors were cultured with the heat-treated Ags of M.tb (5 µg/ml) in the presence of rhIL-2 (20 U/ml) for 12 days, and the number and percentage of {gamma}{delta} T cells were measured by cell calculating and FACS. As reported (20), the frequency of {gamma}{delta} T cells (Fig. 2A) in PBMC was highly increased when cultured with the heat-treated Ags of M.tb and rhIL-2, but not with the heat-treated Ags of M.tb or rhIL-2 alone. Interestingly, if NK cells were depleted, the proliferating response of {gamma}{delta} T cells to the heat-treated Ags of M.tb and rhIL-2 was significantly decreased (Fig. 2A). The cumulative cell amounts of total cells, {alpha}beta T cells, and {gamma}{delta} T cells were then accounted. As shown in Fig. 2B, total cells dramatically declined in response to the Ags of M.tb and rhIL-2 after NK cell-depleted, and the amount of {gamma}{delta} T cells was decreased ~3-fold, but the number of {alpha}beta T cells was altered rarely. These results suggested that NK cells might be involved in the regulation of the proliferation of {gamma}{delta} T cells in response to the heat-treated Ags of M.tb.


Figure 2
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  		FIGURE 2. The depletion of NK cells caused a decline of the proliferation of {gamma}{delta} T cells in response to the heat-treated Ags of M.tb. Freshly isolated PBMC and NK-depleted PBMC were cultured in the presence of either the heat-treated Ags of M.tb, rhIL-2, or both for 12 days in vitro. The proportion (A) and absolute cell number (B) of total T cells, {gamma}{delta} T cells, or {alpha}beta T cells in PBMC were shown. Data are shown as mean ± SD and representative of five experiments. **, p < 0.01.

 
To investigate whether NK cells might be activated after stimulation with the Ags, purified NK cells were cultured in vitro for 48 h in the presence of either rhIL-2, or the heat-treated Ags of M.tb, or both. CD69 expression was highly induced on NK cells when cultured with the Ags and rhIL-2, but not the Ags or rhIL-2 alone (Fig. 3A), and the cytotoxicity of NK cells was also significantly increased if cultured with the Ags and rhIL-2 (Fig. 3B). PPD, the usually used antigenic stimulus for determining recall immune response to bacillus Calmette-Guerin vaccination in vitro, has been reported to induce the proliferation of CD4+ and CD8+ T cells (20, 23). Cultured with PPD (5 µg/ml) plus 20 U/ml rhIL-2 for 48 h, only 21.3% NK cells expressed the surface activation Ag CD69 (Fig. 3A). These results indicated that the heat-treated Ags of M.tb might specifically activate NK cells in the presence of low concentration of rhIL-2, and only the activated NK cells are possibly involved in the regulation of {gamma}{delta} T cell proliferation.


Figure 3
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  		FIGURE 3. Specific activation of NK cells in response to the heat-treated Ags of M.tb. A, Freshly isolated PBMC were cultured in the presence of either the heat-treated Ags of M.tb, rhIL-2, or both, or PPD + rhIL-2 for 48 h. The CD69 expression on CD56+CD3– NK cells was analyzed by flow cytometry. B, The cytotoxicity of NK cells was assessed by a standard 4-h 51Cr-release assay. Target cells were K562 cells. Data are shown as mean ± SD and representative of three experiments.

 
IFN-{gamma} was not required for NK cells to regulate the Ag-specific {gamma}{delta} T cells

It was generally accepted that IFN-{gamma} was the typical cytokine used by NK cells to regulate the innate and adaptive immune responses. We analyzed the expression of intracellular IFN-{gamma} by flow cytometry in NK cells which were activated by the heat-treated Ags of M.tb. The production of IFN-{gamma} by NK cells stimulated with the heat-treated Ags of M.tb and rhIL-2 was higher than that with rhIL-2 alone (Fig. 4A). The neutralizing anti-IFN-{gamma} Ab was used in further studies to determine whether IFN-{gamma} was required in the regulation of the proliferating response of {gamma}{delta} T cells to the heat-treated Ags of M.tb. Our data showed that the percentage of {gamma}{delta} T cells was rarely altered when cultured with or without anti-IFN-{gamma} Ab in PBMC. However, after the depletion of NK cells from PBMC, the percentage of {gamma}{delta} T cells was significantly reduced even when cultured with exogenous IFN-{gamma} (Fig. 4B). Thus, exogenous IFN-{gamma} could not replace NK cells to maintain the proliferation of {gamma}{delta} T cells in NK-depleted PBMC. These results suggested that IFN-{gamma} was not involved in the regulation of {gamma}{delta} T cells by NK cells.


Figure 4
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  		FIGURE 4. IFN-{gamma} was not involved in the regulation of {gamma}{delta} T cells in response to the heat-treated Ags of M.tb by NK cells. A, Intracellular IFN-{gamma} in the purified NK cells (>95%), which cultured with rhIL-2 or the heat-treated Ags of M.tb plus rhIL-2, was detected at the indicated day by flow cytometry. B, Neutralizing anti-IFN-{gamma} Ab was used to determine the requirement of IFN-{gamma} in proliferation of {gamma}{delta} T cells to the heat-treated Ags of M.tb. Depleted-PBMC (NK cells <0.4%) were cultured with the purified NK cells, rhIFN-{gamma}, anti-IFN-{gamma} Ab, or combinations in the presence of the heat-treated Ags of M.tb and rhIL-2. Data are shown as mean ± SD and are representative of three experiments. **, p < 0.01.

 
NK cells regulated the proliferating response of {gamma}{delta} T cells by secreting soluble cytokines

After eliminating the possibility of IFN-{gamma} mediating the regulation of {gamma}{delta} T cells by NK cells, we wondered whether NK cells activated {gamma}{delta} T cells by secreting soluble factors. We cultured NK cells with NK-depleted PBMC by Transwell, a porous membrane through which the soluble cytokines could transmit. After 12 days of culture with the heat-treated Ags of M.tb and rhIL-2, the absolute number of {gamma}{delta} T cells in NK-depleted PBMC plus purified NK cells (1.6 ± 0.16 x 106) was slightly higher than that in NK-depleted PBMC (0.87 ± 0.18 x 106), but markedly lower than that in PBMC (3.1 ± 0.24 x 106) (Fig. 5A). The results suggested that NK cells could partly regulate the proliferation of {gamma}{delta} T cells by secreting soluble factors. We then examined the supernatant of purified NK cells (1 x 106/ml) after coculture with the heat-treated Ags of M.tb and rhIL-2 by ELISAs. At 18, 36, and 72 h, IFN-{gamma}, TNF-{alpha}, GM-CSF, and IL-12 were detectable but IL-4 and IL-10 were rare (Fig. 5B), which suggested that some cytokines secreted by NK cells might play important roles in the regulation of {gamma}{delta} T cells in response to the heat-treated Ags of M.tb. We added the recombinant cytokines in NK-depleted PBMC to confirm whether the exogenous cytokines could replace, at least partly, the function of NK cells in regulating the {gamma}{delta} T cell response to the Ags. As shown in Fig. 5C, the exogenous TNF-{alpha}, GM-CSF, or rhIL-12 alone or in combination were added into the culture system, the proliferating response of {gamma}{delta} T cells to the Ags was significantly enhanced, and the combination of three cytokines was significantly better than any cytokine alone, which strongly suggested that NK cells might regulate the {gamma}{delta} T cell response by secreting soluble cytokines.


Figure 5
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  		FIGURE 5. NK cells regulated the proliferation of {gamma}{delta} T cells by secreting soluble factors. A, NK-depleted PBMC were separately cultured with or without NK cells by a 0.4-µm Transwell insert (NK-depleted PBMC/NK) in the presence of the heat-treated Ags of M.tb (5 µg/ml) and rhIL-2 (20 U/ml) for 12 days in vitro. B, The supernatant of purified NK cells (106/ml) was collected after stimulation with the heat-treated Ags of M.tb (5 µg/ml) and rhIL-2 (20 U/ml) at different time points, and then the concentrations of cytokines were assayed by ELISA. C, TNF-{alpha}, GM-CSF, rhIL-12, alone, or in combination were added to the culture system of NK-depleted PBMC with the heat-treated Ags of M.tb and rhIL-2. Data are shown as mean ± SD. Data are representative of three experiments. **, p < 0.01; *, p < 0.05.

 
CD54 (ICAM-I) played an important role in cell-cell contact between NK cells and {gamma}{delta} T cells

It had been reported that NK cells could activate CTLs and dendritic cells by cell-cell contact (14, 19). As shown in Fig. 5B, the soluble factors secreted by NK cells could not thoroughly replace the function of NK cells, so cell-cell contact was further investigated. We examined the expressions of the costimulating molecules (such as CD40, CD80, CD86, CD70, and CD137) and adhesion molecules (such as CD11a, CD11b, CD54) on NK cells. Unfortunately, most of the surface molecules, except CD54, were negative or rarely altered even after NK cells were stimulated by the heat-treated Ags of M.tb and rhIL-2 (data not shown). CD54 was detectable on the surface of NK cells during cell culture and the expression frequency was increased over the time after stimulation with the Ags (Fig. 6A). Previous reports showed that CD54 was involved in forming the immunological synapse of NK cells (24); we speculated that CD54 was involved in the cell-cell contact of NK cells and {gamma}{delta} T cells. Using confocal microscopy, CD54 was found to polarize at the synapse of NK cells and {gamma}{delta} T cells (Fig. 6B). In the x20 field view, the number of cell-cell contacts was calculated. The number of cell-cell contacts was increased over the culture time (Fig. 6B). To further confirm the role of CD54 molecule, we stimulated the purified NK cells with the Ags for 72 h, and then incubated the activated NK cells with anti-CD54 Ab or isotype Ab, for another 24 h. The Ag-activated CD54 Ab-blocked NK cells were finally added into NK-deleted PBMC in the presence of the heat-treated Ags of M.tb and cultured for 12 days. If the activated NK cells were pretreated by anti-CD54 Ab, the percentage and number of {gamma}{delta} T cells were obviously decreased (Fig. 6C). These results demonstrated that CD54 played an important role in NK-{gamma}{delta} T cross-talk in response to the heat-treated Ags of M.tb stimulation.


Figure 6
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  		FIGURE 6. CD54 (ICAM-1) was involved in the regulation of {gamma}{delta} T cells in response to the heat-treated Ags of M.tb by NK cells. PBMC were stimulated with the heat-treated Ags of M.tb and rhIL-2 and harvested at the indicated days. A, The expression of CD54 on CD56+ NK cells was examined by flow cytometry. B, NK-{gamma}{delta} T contact in PBMC was observed by confocal microscopy after stimulation with the heat-treated Ags of M.tb and rhIL-2. CD54 (red color) were centralized at the interface between NK (orange color) and {gamma}{delta} T cells (green color). The number of cell contacts was calculated in the 20-field view. C, Anti-CD54 mAb-treated NK cells and untreated NK cells were added to the NK-depleted PBMC, then NK-depleted PBMC were stimulated with the heat-treated Ags of M.tb and rhIL-2 for 12 days. The percentage and number of {gamma}{delta} T cells were analyzed by flow cytometry and cell counting. Data were shown as mean ± SD. Data are representative of three experiments. **, p < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
NK cells are innate immune effectors that produce immunoregulatory cytokines which are critical to early host defense against a variety of viral, bacterial, and parasitic pathogens (17, 25). It was reported that IFN-{gamma} and monokines (IL-15 and IL-18) played the crucial roles in regulation of CD8+ T cells against M.tb infection by NK cells (26). However, other cytokines (not IFN-{gamma}) were also involved in cross-talk between the NK cells and {gamma}{delta} T cells from our results (Fig. 5). Different from the publication, adding exogenous IFN-{gamma} could not replace NK cells to improve proliferation of {gamma}{delta} T cells. Furthermore, the anti-IFN-{gamma} Ab failed to block the proliferation of {gamma}{delta} T cells in response to the heat-treated Ags of M.tb (Fig. 4). However, the data from the Transwell insert showed that NK cells regulated the proliferation of {gamma}{delta} T cells by producing TNF-{alpha}, GM-CSF, IL-12 but not IFN-{gamma}, IL-4, and IL-10 (Fig. 5).

Previous studies showed that adhesion molecules and costimulating molecules were involved in the cell-cell contact between or among immune cells, and CD54 was required in the formation of the immunological synapse of NK cells (24). We observed that CD54 was undetectable on the resting NK cells, but almost fully expressed on the surface of NK cells after stimulation with the heat-treated Ags of M.tb (Fig. 6A). It was surprising that CD54 was found to centralize at the interface between NK cells and {gamma}{delta} T cells by confocal microscope, and anti-CD54 Ab markedly reduced the proliferation of {gamma}{delta} T cells (Fig. 6, B and C). Unfortunately, other costimulating molecules (CD80, CD86, CD70, and CD137) and adhesion molecules (CD11a, CD11b) were not detected or were not changed on the surface of NK cells even after stimulation with the heat-treated Ags of M.tb (data not shown), which further suggested the importance of CD54 in cell-cell contact. CD54 is identified as a costimulating factor that binds to LFA-1, thereby promoting the activation of T cells (27). As CD54 is expressed on virtually any cell, it becomes a crucial molecule for the activation of CD8+ T cells in the absence of costimulation provided by CD80/CD86 molecules (28). Our observation demonstrated that NK-{gamma}{delta} T cell interaction by CD54 molecules might play a critical role in regulation of {gamma}{delta} T cells by NK cells.

Recently, it was reported that in C57BL/6 mice infected by aerosol exposure with M.tb, NK cells were increased in the lungs, however, in vivo depletion of NK cells had no influence on bacterial load within the lungs (29). In contrast to the mouse model, Vankayalapati et al. (26) found that depletion of NK cells from PBMC of healthy tuberculin reactors reduced the frequency of M.tb-responsive CD8+IFN-{gamma}+ T cells. The frequency of CD8+IFN-{gamma}+ T cells was restored by soluble factors produced by activated NK cells and was dependent on IFN-{gamma} (26). Interestingly, the capacity of NK cells to prime CD8+ T cells to lyse M.tb-infected target cells required cell-cell contact between NK cells and infected monocytes via CD40-CD40L (26). However, {gamma}{delta} T cells play an important role in the immune response to M.tb (2, 3, 4), and the supernatant from the heat-treated M.tb induced the proliferation of human {gamma}{delta} T cells, which was recognized as a good experimental platform to study anti-tuberculosis {gamma}{delta} T cells (3, 7, 10). Interestingly, different from the stimulation with active M.tb in which CD8+ {alpha}beta T cells rapidly proliferated (20), {gamma}{delta} T cells were the major responding cell population to heat-treated Ag. Human NK cells played the important but different roles in both stimulating systems: NK cells affected {gamma}{delta} T cells in the presence of heat-treated Ag by secreting cytokines, but not IFN-{gamma}, and by directly cross-talking with {gamma}{delta} T cells via CD54, whereas, NK cells affected {alpha}beta T cells in the presence of active bacteria by directly secreting IFN-{gamma} which was dependent on cross-talking between NK-monocyte cells via CD40-CD40L. The observation that human {gamma}{delta} T cells and {alpha}beta T cells differentially respond to the different part of M.tb and via different regulatory mechanisms by NK cells will greatly help to further explore the pathogenesis of pulmonary tuberculosis.

One shortcoming in our study is that the NK cells were from peripheral blood of the tuberculin responder rather than from the pathogen-invading tissue or organs such as lungs and lymph nodes of persons with primary M.tb infection. We know that such experiments would be extremely informative if we use the sample from the pathogen region, but unfortunately, medical ethical limitation makes it difficult to perform these studies in humans. Additional work in mice is being designed to better delineate the contribution of NK cells to the development of the {gamma}{delta} T cell response to stimulation with heat-treated Ag of M.tb in vivo.

In summary, our results demonstrated that an interaction existed between NK cells and {gamma}{delta} T cells. The heat-treated Ags of M.tb specifically activated NK cells, and the activated NK cells then regulated the proliferation of {gamma}{delta} T cells by producing the cytokines and the cell-cell contact in the presence of Ag. CD54 played a crucial role in the cross-talk between the NK cells and {gamma}{delta} T cells. The results suggested that NK cells might participate in the pathogenesis of pulmonary tuberculosis.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Natural Science Foundation of China (Nos. 30125038, 30230340, 30371302, 30371308), the Key Basic Science Program by the Ministry of Science and Technology of China (Nos. 2001CB510009, 2003CB515501), and the Foundation of the Chinese Academy of Science (No. KSCX2-2-08). Back

2 R.Z. and X.Z. equally contributed to this study. Back

3 Address correspondence and reprint requests to Dr. Zhigang Tian, School of Life Sciences, University of Science and Technology of China, 443 Huangshan Road, Hefei City 230027, Anhui, China. E-mail address: tzg{at}ustc.edu.cn Back

4 Abbreviations used in this paper: M.tb, Mycobacterium tuberculosis; rh, recombinant human; PPD, purified protein derivative. Back

Received for publication August 8, 2005. Accepted for publication December 6, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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