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ler
,
Divisions of
*
Hematology and Oncology and
Dermatology, Department of Medicine, and
Department of Microbiology and Immunology, University of California, Los Angeles, School of Medicine, Los Angeles, CA 90095;
Institut für Klinische Mikrobiologie, Immunologie und Hygiene, Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, Germany;
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Department of Pediatrics, Stanford University, Stanford, CA 94305; and
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Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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24 TCR that recognizes the nonclassical Ag-presenting molecule CD1d. Activation of NKT cells is greatly augmented by the marine sponge-derived glycolipid -galactosylceramide (GalCer). Because human monocyte-derived cells express CD1d and can harbor the intracellular pathogen Mycobacterium tuberculosis, we asked whether the addition of GalCer could be used to induce effector functions of NKT cells against infected monocytes, macrophages, and monocyte-derived dendritic cells. NKT cells secreted IFN-
, proliferated, and exerted lytic activity in response to GalCer-pulsed monocyte-derived cells. Importantly, GalCer-activated NKT cells restricted the growth of intracellular M. tuberculosis in a CD1d-dependent manner. NKT cells that exhibited antimycobacterial activity also expressed granulysin, an antimicrobial peptide shown to mediate an antimycobacterial activity through perturbation of the mycobacterial surface. Degranulation of NKT cells resulted in depletion of granulysin and abrogation of antimycobacterial activity. The detection of CD1d in granulomas of tuberculosis patients supports the potential interaction of NKT cells with CD1d-expressing cells at the site of disease activity. These studies provide evidence that GalCer-activated CD1d-restricted T cells can participate in human host defense against M. tuberculosis infection. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The study of CD1d has been centered on the interesting finding that a marine sponge-derived glycolipid, -galactosylceramide (GalCer),3 is presented by CD1d to NKT cells, a unique subset of T cells found in both humans and mice (7, 8). Human NKT cells express a semi-invariant TCR containing the V24 chain (9, 10) and usually the NK cell marker CD161 (11). Activation of NKT cells by GalCer results in proliferation and release of cytokines (12, 13). Additionally, although NKT cells are primarily CD4+ or double negative (DN), they can be potently cytolytic (7, 14, 15). Indeed, it is the ability of Ag-activated NKT cells to lyse tumor targets that has prompted clinical investigations into the use of GalCer as an antitumor agent (16). By the study of mouse models of infection, CD1d and NKT cells have been implicated in host defense against a number of pathogens including Borrelia burgdorferi, Plasmodium species, and Mycobacterium tuberculosis (17, 18, 19).
Tuberculosis, caused by the intracellular bacterium M. tuberculosis, is a major cause of morbidity and mortality worldwide. Despite available antibiotic therapy, resistant strains are emerging and may reach up to 25% of isolated strains in several regions of the world (20). Therefore, novel therapeutic strategies are desperately needed to protect from uncontrolled spread of multidrug-resistant tuberculosis. In addition to the classic association of M. tuberculosis with infection of alveolar macrophages, intracellular infection of dendritic cells (DC) has also been shown and is important for the initiation of both innate and adaptive immune responses (21, 22).
In the current study, we investigated whether GalCer-activated CD1d-restricted NKT cells could contribute to host defense against M. tuberculosis infection. Monocyte-derived cells, including monocytes, macrophages, and DC were studied for their ability to activate NKT cells by presentation of GalCer in a CD1d-restricted manner. The ability of NKT cells to contribute to antimycobacterial defense was examined according to 1) the production of proinflammatory cytokines, 2) the lysis of infected macrophages, and 3) the delivery of antimicrobial peptides such as granulysin resulting in direct antimycobacterial activity. Although the induction of apoptosis may lead to the death of intracellular bacteria, apoptosis is not a required component of CTL-mediated antibacterial effector function (23). The following studies demonstrate that NKT cells can contribute to each of these three mechanisms and suggest that activation of human NKT cells by GalCer may specifically contribute to host defense against mycobacterial infection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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After informed consent was given, PBMCs from healthy donors were isolated by Ficoll-Hypaque (Pharmacia, Freiburg, Germany) density gradient centrifugation. To generate monocytes, cells were cultured in complete medium (RPMI 1640, 0.1 mM sodium pyruvate, 2 mM penicillin, and 50 µg/ml streptomycin; Life Technologies, Grand Island, NY) supplemented with 1% FCS (Omega Scientific, Tarzana, CA) and were allowed to adhere to culture flasks. After 2 h at 37°C, the nonadherent cells were removed by vigorous washing with PBS. To generate macrophages, adherent cells were cultured in a CO2 incubator at 37°C in complete medium containing 10% human serum for 4 days. To generate immature DCs, adherent cells were cultured for 7 days in complete medium containing 10% FCS, 1000 U/ml GM-CSF (Genetics Institute, Cambridge, MA), and 1000 U/ml IL-4 (Schering-Plough, Madison, NJ), as previously described (24). These cells were nonadherent, displaying typical DC morphology. Purity of the DCs was typically >95%, as determined by flow cytometry.
NKT cell clones
NKT cell clones DN2.B9 and DN2.D5 were established and maintained as previously described (12, 25). Briefly, cells were maintained in the presence of rIL-2 (1 nM; Chiron Diagnostics, Norwood, MA) and stimulated with anti-CD3 (30 ng/ml) and irradiated PBMC approximately every 4 wk. V24+CD4-CD8- phenotype was confirmed by flow cytometry (not shown).
Antibodies
The following Abs were used for flow cytometry: OKT6 (anti-CD1a), OKT3 (anti-CD3), OKT4 (anti-CD4), and OKT8 (anti-CD8) (American Type Culture Collection, Manassas, VA); TUK4 (CD14) and goat anti-mouse-FITC (Caltag, South San Francisco, CA); and C15 (TCR V24; Immunotech, Westbrook, ME) and CD1d 42.1 (BD PharMingen, San Diego, CA). DH4 (anti-granulysin) (26) and tetramethylrhodamine isothiocyanate (TRITC)-conjugated anti-mouse IgG1 (Southern Biotechnology, Birmingham, AL) were used for immunofluorescence labeling of cells. For immunofluorescence labeling of tissue, the following Abs were used: anti-CD1d (BD PharMingen), RPA-M1 (CD14; Zymed, South San Francisco, CA), and DCN46 (DC-specific ICAM-3 grabbing nonintegrin (DC-SIGN); BD PharMingen). The secondary Abs used were isotype-specific goat anti-mouse mouse Abs conjugated to Alexa Fluor 488 or 568 (Molecular Probes, Eugene, OR).
CD1d42.1.1 (anti-CD1d) was used to block CD1d-restricted NKT cell responses and for immunoperoxidase staining (12). IgG isotype controls (Sigma-Aldrich, St. Louis, MO) were used in all experiments.
T cell assays
For measurement of Ag-specific proliferation, NKT cells (5 x 104) were added to irradiated (50 Gy) monocytes or Mo-DCs (104) plated in a 96-well plate to which 100 ng/ml synthetic GalCer (Kirin Brewery, Gunma, Japan) or an equivalent amount of the vehicle DMSO was added. After 2 days, cultures were pulsed with 3[H]thymidine (1 µCi/well; ICN Biomedicals, Costa Mesa, CA) and were harvested 18 h later for evaluation by liquid scintillation counting. To demonstrate the CD1d restriction of the NKT cell lines, 20 µg/ml anti-CD1d blocking Ab (CD1d42.1) was added 30 min before the addition of NKT cells. NKT cell cytokine release was measured by use of a standard sandwich ELISA method (IFN- and IL-4; Endogen, Woburn, MA) using supernatants that were collected after 24 h of culture. The cultures were identical with those described above, with the exception that 25 ng/ml GalCer was used in cultures that were analyzed by ELISA.
Cytotoxicity assays
NKT cell cytotoxicity experiments against CD1d+ target cells were performed as described (27). Target cells were pulsed with GalCer for 16 h, then washed and labeled with 100 µCi of 51Cr (ICN Biomedicals) for 1 h and plated in a 96-well V-bottom plate at a final concentration of 5000 targets/100 µl. T cells were then added at indicated E:T ratios for 4 h. Supernatants were collected and target cell lysis was calculated by quantifying 51Cr release. The spontaneous 51Cr release by monocytes or Mo-DCs in the absence of T cells was <10%. Results are expressed as percent specific lysis and are calculated as follows: 100 x (experimental release - spontaneous release)/(maximum release - spontaneous release).
Growth of M. tuberculosis
M. tuberculosis (virulent strain H37Rv) was grown in suspension with constant, gentle rotation in roller bottles containing Middlebrook 7H9 broth (BD Biosciences, Heidelberg, Germany) supplemented with 1% glycerol (Roth, Karlsruhe, Germany), 0.05% Tween 80 (Sigma-Aldrich), and 10% Middlebrook oleic acid, albumin, dextrose, and catalase enrichment (BD Biosciences). Aliquots from logarithmically growing cultures were frozen in PBS containing 10% glycerol, and representative vials were thawed and enumerated for viable CFU on Middlebrook 7H11 plates. Staining of bacterial suspensions with fluorochromic substrates differentiating between live and dead bacteria (BacLight; Molecular Probes) revealed a viability of the bacteria >90%. Several precautions were taken to minimize clumping of mycobacteria: 1) culture conditions (rotation; Tween) were chosen to support the growth of single-cell suspensions; 2) before in vitro infection, M. tuberculosis bacilli were sonicated to disrupt small aggregates of bacteria; and 3) the multiplicity of infection (MOI) was selected such that there were only two to three bacilli per infected cell.
Infection of monocyte-derived cells
Monocytes, macrophages, or Mo-DCs were infected with single-cell suspensions of M. tuberculosis. After 4 h of incubation at 37°C, adherent monocytes and macrophages were washed three times with antibiotic-free medium. For nonadherent Mo-DCs, cells were harvested and centrifuged at 800 rpm for 8 min. This low-speed centrifugation selectively pellets cells while extracellular bacteria remain in the supernatant. After three cycles of centrifugation, the majority of extracellular bacteria were removed as determined by auramine-rhodamine stain (TB-fluor; Merck, Darmstadt, Germany). Infected cells were then plated at a concentration of 1 x 106 cells/ml in a 24-well plate in complete medium without antibiotics plus 10% human serum. The efficiency of infection, as quantitated by staining of control cultures on Permanox chamber slides (Nunc, Naperville, IL) in every experiment was dependent on the MOI. The microscopic evaluation of infected macrophages under the fluorescence microscope confirmed the absence of any mycobacterial aggregates. Viability of infected cells was determined by trypan blue exclusion and was >99% in all experiments.
Quantification of mycobacterial growth
To measure mycobacterial growth, cells were lysed with 0.3% saponin (Sigma-Aldrich) to release intracellular bacteria. At all time points, an aliquot of unlysed infected cells was harvested and counted. This allowed an exact quantification of cells as well as the determination of cellular viability by trypan blue exclusion. Recovery of cells was >80% in all experiments, with cell viability regularly exceeding 90% of total cells. Lysates of infected cells were resuspended vigorously, transferred into screw-cap tubes, and sonicated in a preheated (37°C) water bath sonicator for 5 min. Aliquots of the sonicate were diluted 10-fold in 7H9 medium. Four dilutions of each sample were plated in duplicates on 7H11 agar plates and incubated at 37°C and 5% CO2 for 21 days.
Flow cytometry
For phenotypic analysis of NKT cells and monocyte-derived cells, 3 x 105 cells were resuspended in 100 µl of staining buffer (2% FCS, 1% NaN3, and PBS without Mg2+/Ca2+) and incubated with unconjugated or conjugated Abs for 30 min on ice. Samples were washed twice in staining buffer and, if necessary, incubated for an additional 30 min on ice with goat anti-mouse-FITC Abs (1:500). Cells were then fixed in 2% paraformaldehyde and stored at 4°C until analysis in a FACScan flow cytometer (BD Biosciences). Intracellular granulysin was detected after fixation (4% paraformaldehyde) and permeabilization (0.3% saponin) of NKT cells and incubated with a polyclonal anti-granulysin rabbit serum (28) or a control serum for 30 min. After washing, an FITC-conjugated donkey anti-rabbit Ab (1/250 final dilution) was added for 30 min in the presence of 0.3% saponin.
Immunoperoxidase and immunofluorescence labeling
Immunoperoxidase labeling was performed as previously described (29) on archived cryostat sections of lymph node specimens obtained from tuberculosis patients (30). Briefly, frozen sections were fixed in acetone for 10 min and then air-dried. Sections were rehydrated in PBS and blocked with 5% horse serum for 30 min and 10 µg/ml CD1d42.1, or the equivalent amount of IgG1 isotype control Ab was added for 2 h. Primary Ab staining was detected using the ABC system (avidin/biotin complex; Vector Laboratories, Burlingame, CA). Slides were counter-stained with hematoxylin. For immunofluorescence staining, tissue sections were blocked in 5% goat serum and labeled overnight with 25 µg/ml anti-CD1d (BD PharMingen). After washing, goat anti-mouse IgG1 conjugated to Alexa Fluor 488 was added at 2 µg/ml for 2 h. After washing, the second primary Abs, either 2.5 µg/ml DCN46 or 1 µg/ml RPA-M1, were added for 1 h. After washing, goat anti-mouse IgG2b conjugated to Alexa Fluor 568 was added at a 1/1000 dilution for 1 h. For immunofluorescence labeling of cultured NKT cells, cells were harvested 1 wk after stimulation with PBMC and anti-CD3 and were adhered to individual wells of glass slides coated with poly-L-lysine (Sigma-Aldrich). Cells were then fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton-X, and 0.01% saponin in PBS with 1% milk and 10% goat serum. After permeabilization, primary Ab, either DH4 (anti-granulysin; 1/50 dilution) or an equivalent amount of isotype control Ab (IgG1), was added for 2 h. After three washes, a TRITC-conjugated anti-mouse IgG1 was added for 1 h (final dilution, 1/1000). To demonstrate the cellular localization of granulysin, nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). Immunofluorescence was examined with a Leica TCS-SP inverted confocal laser-scanning microscope fitted with an argon laser (Leica, Deerfield, IL). Cells were illuminated with 488 or 568 nm of light after filtering through an acoustic optical device. Images were recorded through an optical detector with a 590-nm long-pass filter. Sections stained with DAPI were examined using the multiphoton laser system tuned to 770 to generate UV excitation. Pairs of images were superimposed for colocalization analysis.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The fact that NKT cells are activated in response to the marine sponge-derived glycolipid Ag GalCer, and that Ag presentation occurs through CD1d, is well established (8). However, it has only been recently appreciated that the low levels of CD1d expression on monocytes and Mo-DCs are sufficient to activate NKT cells in vitro (31). To confirm the role of CD1d in NKT cell activation by primary human monocyte-derived cells, we purified fresh human monocytes and generated Mo-DCs to test their ability to activate NKT cell clones in the presence of GalCer. Both cell types express low levels of CD1d that were only detectable after extensive blocking with human serum before the addition of the anti-CD1d Ab (Fig. 1A). Consistent with previous findings (32), culture of adherent PBMCs with GM-CSF and IL-4, a condition which up-regulates group 1 CD1 molecules, did not up-regulate CD1d expression (data not shown).
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-chain V24, and are CD3 positive, but do not express either CD4 or CD8. NKT cell clones were cultured with either monocytes or Mo-DCs that were either pulsed overnight with GalCer or the vehicle DMSO. After 24 h, culture supernatants were tested for IFN- by ELISA. As shown in Fig. 1B, NKT cell responses to Ag-pulsed Mo-DCs resulted in significant IFN- production with an up to 10-fold increase over the control. Pretreatment of GalCer-pulsed Mo-DCs with a blocking Ab to CD1d, but not with an isotype control Ab, abrogated IFN- production. This finding demonstrates the importance of CD1d in the cytokine response by NKT cells. Although the magnitude of IFN- production was similar between the two different NKT cell clones, the ratio of increase was lower for DN2.D5. The higher background cytokine production by DN2.D5 is consistent with the fact that this clone has previously been shown to exhibit autoreactivity to CD1d (13). Lower levels of IFN- were produced in response to Ag-pulsed monocytes (10–30% of levels produced in response to Mo-DCs; data not shown). When cultured with monocytes, NKT cells also produced a modest IL-4 response (not shown). IL-4 production was not measured in the case of Mo-DCs because of the confounding effect of exogenous IL-4 administration. Overall, the expression of this cytokine pattern, Th0 with a notable bias toward Th1 cytokine expression, is consistent with previous studies (31).
To investigate the participation of CD1d in the activation of the NKT cell clones used in these experiments, proliferation assays were performed using GalCer. As previously demonstrated for DN2.B9 (31), the addition of GalCer to monocytes resulted in an up to 5-fold increase in the proliferation of both NKT cell clones as measured by uptake of 3[H]thymidine after 3 days of culture (Fig. 1C). Preincubation of monocytes with the blocking Ab to CD1d reduced the proliferative response, whereas pretreatment with the isotype control Ab did not. Similar experiments were performed using Mo-DCs (not shown) and included effective blocking of proliferation with Ab to CD1d. These results are in agreement with a recent report by Takahashi et al. (33) using other NKT cells. Although proliferation was not completely blocked by the anti-CD1d Ab, these results suggest that administration of GalCer might be sufficient to activate CD1d-reactive NKT cells at sites of disease where CD1d is endogenously expressed.
GalCer enhances the cytotoxicity of NKT cells
Because NKT cell cytotoxicity is greatly enhanced when GalCer is added to tumor targets that express CD1d (15) and because M. tuberculosis resides within cells of the monocyte lineage, we asked whether GalCer can enhance the cytotoxicity of the NKT cells against monocyte-derived cells, thereby facilitating the immune clearance of the mycobacteria. Cytotoxicity assays were performed using a standard chromium release assay with Ag-pulsed monocytes or Mo-DCs as targets. Increasing numbers of NKT cells were added to monocytes or Mo-DCs that were pulsed overnight with GalCer. As opposed to unpulsed controls, the presence of GalCer resulted in efficient lysis (20–25%) of Mo-DCs even at the lowest E:T ratio (Fig. 2). An anti-CD1d Ab inhibited Ag-specific lysis by 
50%. Although at lower levels, lysis of monocytes pulsed with GalCer was also significant with a specific lysis of 25–35% at an E:T ratio of 20:1 (data not shown). These results demonstrate that, in the presence of GalCer, the level of CD1d expressed by monocyte-derived cells is sufficient to direct lysis by NKT cells. Therefore, the administration of GalCer might be therapeutically useful due to its ability to effectively target the cell types that harbor M. tuberculosis for lysis by NKT cells.
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Granulysin is an antimicrobial peptide with activity against many parasites, fungi, and bacteria (including virulent M. tuberculosis) and is expressed in the cytotoxic granules of NK cells, 
T cells, and both CD8+ and CD4+ CTL (34, 35, 36). Based on its ability to perturb mycobacterial membranes, granulysin has been shown to be an important component of the antimycobacterial activity of M. tuberculosis-reactive CTL that are either CD8+ or CD4+ while DN group 1 CD1-restricted CTL failed to express granulysin (36, 37). We sought to clarify whether DN group 2 CD1-restricted NKT cells behaved similar to the group 1 CD1-restricted counterparts in terms of granulysin expression. Intracellular flow cytometry revealed granulysin expression in 34–39% of cells from each clone tested (Fig. 3A). This level of expression is comparable to that seen in M. tuberculosis-reactive cytotoxic CD4+ T cells, and likely reflects that, in an activated T cell clone, only a percentage are activated at a given time (36). Granulysin was detected in a granular pattern in the cytoplasm of NKT cells consistent with their presence within cytotoxic granules (Fig. 3B). The ability of GalCer-activated NKT cells to express granulysin suggested that these cells could mediate an antimicrobial activity.
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As we observed that GalCer is capable of initiating NKT cell lysis of infected host cells and that NKT cells express granulysin, we surmised that these two conditions may combine to achieve efficient killing of intracellular mycobacteria. To test this hypothesis, monocyte-derived cells, either monocytes, macrophages, or Mo-DC, were infected with virulent M. tuberculosis at an MOI of 5:1. After extensive washing to remove extracellular mycobacteria, infected cells were cultured with GalCer before addition of GalCer-naive NKT cells. By 24 h after infection, the addition of NKT cells to infected macrophages treated with GalCer resulted in a >50% growth inhibition of M. tuberculosis when increasing numbers of NKT cells were added (Fig. 4A). This difference was statistically significant at the NKT to macrophage ratios of both 3:1 (p < 0.05) and 10:1 (p < 0.01). Similarly, inhibition of mycobacterial growth was observed in Mo-DC (Fig. 4B; p < 0.01 at a ratio of 10:1). At an E:T ratio of 5:1, neither GalCer alone nor NKT cells alone resulted in significant mycobacterial growth inhibition in either monocytes (Fig. 4C) or Mo-DC (Fig. 4D) while the combination of GalCer and NKT cells resulted in a 2- to 3-fold decrease in mycobacterial growth in both cell types (p < 0.01 for monocytes; p < 0.05 for macrophages). Addition of the blocking Ab to CD1d completely abrogated the growth inhibition in monocytes (Fig. 4C; p < 0.01) and Mo-DCs (Fig. 4D; p < 0.01). These results suggest that the presentation of GalCer through CD1d is responsible for the antibacterial effect of NKT cells.
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GalCer was almost completely diminished by degranulation (MOI of 5:1). These results demonstrate the importance of cytotoxic granules for the antimycobacterial activity of NKT cells. CD1d is present at the site of mycobacterial disease
As CD1d is required to activate GalCer-responsive NKT cells, in order for the GalCer/NKT cell system to be potentially effective as antibacterial therapy, CD1d must be present at the site of disease. To determine whether CD1d-positive cells are available to present GalCer to human NKT cells, we examined lymph nodes from patients with tuberculosis. Fig. 5A shows prominent expression of CD1d by immunoperoxidase staining. CD1d localized to the center of the granulomas, and is detected on the surface of cells that exhibit a distinct dendritic appearance (Fig. 5A). This pattern is reminiscent of the CD1d staining pattern observed in human leprosy lesions (41).
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GalCer, to NKT cells at the site of infection. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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GalCer defend the host from fungal, protozoal, and viral infections (43, 44, 45). Recently, administration of GalCer has also been shown, in a CD1d-dependent manner, to reduce bacterial burden, reduce tissue injury, and prolong the survival of mice inoculated with M. tuberculosis (46). In the current study, we provide evidence that GalCer can activate antimicrobial pathways against M. tuberculosis in a CD1d-restricted manner in humans. The following three mechanisms by which GalCer-activated NKT cells could contribute to host defense against infection were identified: the release of cytokines, cytotoxicity, and direct antimicrobial activity against intracellular M. tuberculosis. These results also suggest that, in contrast to the human group 1 CD1 molecules that are down-regulated by intracellular infection with M. tuberculosis (47), functional expression of human CD1d by infected cells is intact. Therefore, the data provided by the present study, that activation of human NKT cells can mediate antimycobacterial responses, represent a crucial step in the targeting of the NKT cell pathway as a potential treatment modality for human infectious disease.
One important effector mechanism by which NKT cells may contribute to host defense against infection in humans is the production of cytokines. Having originally been recognized for their ability to produce the Th2 cytokine IL-4, NKT cells take on a Th1 phenotype during development and produce significant amounts of IFN- (48). In agreement with previous studies, we found that presentation of GalCer by monocyte-derived cells could activate NKT cells to produce IFN-, and to a lesser degree, IL-4. IFN- can activate phagocytes to kill intracellular organisms through up-regulation of nitrogen-radical production (49), and NKT cell-derived IFN- might be particularly beneficial in the setting of mycobacterial infection because the virulence of strains of M. tuberculosis from human clinical isolates varies inversely with the ability to induce a Th1 response (50). Recent data also indicates that humoral responses can also contribute to host defense because Abs directed against mycobacterial Ags confer protection by directing organisms to granulomas (51). A role for NKT cell-derived IL-4, a mediator of humoral immunity, is suggested by studies demonstrating that NKT cells are necessary for the formation of granuloma-like lesions in mice exposed to mycobacterial cell walls (19). Overall, it appears that GalCer-activated NKT cells can initiate both Th1 and Th2 immune responses that can contribute to host defense against M. tuberculosis infection.
A second important antimicrobial mechanism is cytotoxicity directed against infected cells. The ability of intracellular pathogens, such as M. tuberculosis, to survive within phagocytes represents a major barrier to the eradication of mycobacterial diseases. Extracellular release of the pathogen could allow for reuptake by IFN--activated phagocytes or may expose the bacilli to antimicrobial peptides released by nearby cells (52). In this study, we have demonstrated that NKT cells can lyse phagocytic cells that harbor M. tuberculosis and that this lysis is dependent upon CD1d.
A third mechanism for host defense is direct antimicrobial activity through the release of antimicrobial peptides. In this study, we detected the expression of the antimicrobial peptide granulysin in NKT cells that possess antimicrobial activity against M. tuberculosis residing within human monocytes, macrophages, and Mo-DC. Depletion of cytotoxic granules was associated with the loss of antimycobacterial activity. Granulysin has not yet been identified in the mouse indicating that human and murine NKT may differ in some effector function. However, granulysin is present in human NK cells, CD4+, and CD8+ T cells. Because NKT cells are considered part of the innate immune system, the demonstration that NKT cells can mediate antimicrobial activity, likely through the release of granulysin, further indicates that this host defense pathway has been conserved between the innate and adaptive human T cell systems.
It remains to be determined whether NKT cells recognize natural ligands of the tubercle bacillus and whether such Ags are appropriately presented during the course of in vivo infection. By demonstrating three mechanisms by which human NKT cells can protect against mycobacterial infection—production of protective cytokines, lysis of infected cells, and release of the antimicrobial peptide granulysin—these studies strengthen the argument for further investigations into the potential therapeutic use of GalCer in humans. The plausibility of this approach is supported by the finding that CD1d, the restriction element of the NKT cells, is expressed at the site of disease in tuberculous lymphadenitis.
Effective therapy using GalCer will likely depend upon the access of both NKT cells and GalCer to sites of disease. NKT cells have been detected in the granulomas of mice that were induced by mycobacterial cell walls (19), but whether GalCer will be able to activate these cells in situ is not yet known. Previous studies of pulmonary infection suggest that the effects of GalCer are present at the sites of disease. Systemic administration of GalCer can reduce the CFU of both Pseudomonas aeruginosa and M. tuberculosis in the lungs of infected mice; however, protection against M. tuberculosis is effective only when GalCer is administered early in the course of infection (46, 53). Translation of GalCer therapy into useful treatments of human infection will require investigations into optimal drug delivery and into whether GalCer might be useful in established human infection, perhaps as an adjuvant therapy.
One recently suggested therapeutic strategy is the administration of GalCer-loaded ex vivo-derived DC. This approach has been shown to produce prolonged IFN- responses, as compared with the i.v. administration of GalCer, and reduces the number of melanoma metastases in mice challenged with a melanoma cell line (54). Another alternative to systemic administration of GalCer is direct intrapulmonary administration of aerosolized GalCer. This approach may specifically target alveolar macrophages that may harbor M. tuberculosis while also minimizing the activation of NKT cells by uninfected CD1d-expressing cells at distant sites. Finally, the usefulness of GalCer as adjuvant treatment against intracellular infection has been demonstrated in a murine malaria model when combined with vaccination (55).
The development of novel therapeutic approaches that do not rely on a CD4+ cellular response will be particularly important given the worldwide prevalence of HIV infection and the emergence of multidrug resistance by M. tuberculosis. Future studies are needed to determine whether GalCer will be useful as an adjuvant therapy to vaccination or chemotherapy for tuberculosis. Elucidation of the effector mechanisms of human NKT cells and the pattern of CD1d expression in disease should contribute to the design of studies that will define the utility of GalCer/NKT cell-based therapy for the treatment of infection and malignancy in humans.
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Acknowledgments |
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Footnotes |
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1 This work was supported by a Medical Research Fellowship from the Gianinni Foundation (to J.L.G.), and the German Research Foundation (Sonderforschungsbereich 263), the Erlanger Graduiertenkolleg 592, and the European Union (TB vaccine cluster) (to S.S.), as well as by Grants AI22553 and AR40312 (to R.L.M.) and AI48933 and AI45889 (to S.A.P.) from the National Institutes of Health and a grant from the Irene Diamond Foundation (to S.A.P.). 
2 Address correspondence and reprint requests to Dr. Steffen Stenger, Institut für Klinische Mikrobiologie, Immunologie und Hygiene, Friedrich-Alexander Universität Erlangen-Nürnberg, Wasserturmstrasse 3, D-91054 Erlangen, Germany. E-mail address: steffen.stenger{at}mikrobio.med.uni-erlangen.de
3 Abbreviations used in this paper: GalCer, -galactosylceramide; DN, double negative; DC, dendritic cell; Mo-DC, monocyte-derived DC; DC-SIGN, DC-specific ICAM-3 grabbing nonintegrin; TRITC, tetramethylrhodamine isothiocyanate; DAPI, 4',6-diamidino-2-phenylindole; MOI, multiplicity of infection.
Received for publication July 5, 2002. Accepted for publication January 8, 2003.
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  M. Walch, S. K. Rampini, I. Stoeckli, S. Latinovic-Golic, C. Dumrese, H. Sundstrom, A. Vogetseder, J. Marino, D. L. Glauser, M. van den Broek, et al. Involvement of CD252 (CD134L) and IL-2 in the Expression of Cytotoxic Proteins in Bacterial- or Viral-Activated Human T Cells J. Immunol., June 15, 2009; 182(12): 7569 - 7579. [Abstract] [Full Text] [PDF]
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M. Bastian, T. Braun, H. Bruns, M. Rollinghoff, and S. Stenger Mycobacterial Lipopeptides Elicit CD4+ CTLs in Mycobacterium tuberculosis-Infected Humans J. Immunol., March 1, 2008; 180(5): 3436 - 3446. [Abstract] [Full Text] [PDF]
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 J. Andersson, A. Samarina, J. Fink, S. Rahman, and S. Grundstrom Impaired Expression of Perforin and Granulysin in CD8+ T Cells at the Site of Infection in Human Chronic Pulmonary Tuberculosis Infect. Immun., November 1, 2007; 75(11): 5210 - 5222. [Abstract] [Full Text] [PDF]
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 P. Korosec, K. Osolnik, I. Kern, M. Silar, K. Mohorcic, and M. Kosnik Expansion of Pulmonary CD8+CD56+ Natural Killer T-Cells in Hypersensitivity Pneumonitis Chest, October 1, 2007; 132(4): 1291 - 1297. [Abstract] [Full Text] [PDF]
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L. P. Huang, S.-C. Lyu, C. Clayberger, and A. M. Krensky Granulysin-Mediated Tumor Rejection in Transgenic Mice J. Immunol., January 1, 2007; 178(1): 77 - 84. [Abstract] [Full Text] [PDF]
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J. S. Im, N. Tapinos, G.-T. Chae, P. A. Illarionov, G. S. Besra, G. H. DeVries, R. L. Modlin, P. A. Sieling, A. Rambukkana, and S. A. Porcelli Expression of CD1d Molecules by Human Schwann Cells and Potential Interactions with Immunoregulatory Invariant NK T Cells J. Immunol., October 15, 2006; 177(8): 5226 - 5235. [Abstract] [Full Text] [PDF]
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R. R. Brutkiewicz CD1d Ligands: The Good, the Bad, and the Ugly J. Immunol., July 15, 2006; 177(2): 769 - 775. [Abstract] [Full Text] [PDF]
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F. Stegelmann, M. Bastian, K. Swoboda, R. Bhat, V. Kiessler, A. M. Krensky, M. Roellinghoff, R. L. Modlin, and S. Stenger Coordinate Expression of CC Chemokine Ligand 5, Granulysin, and Perforin in CD8+ T Cells Provides a Host Defense Mechanism against Mycobacterium tuberculosis J. Immunol., December 1, 2005; 175(11): 7474 - 7483. [Abstract] [Full Text] [PDF]
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K. M. Patton, T. C. McGuire, M. T. Hines, R. H. Mealey, and S. A. Hines Rhodococcus equi-Specific Cytotoxic T Lymphocytes in Immune Horses and Development in Asymptomatic Foals Infect. Immun., April 1, 2005; 73(4): 2083 - 2093. [Abstract] [Full Text] [PDF]
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M. Buettner, C. Meinken, M. Bastian, R. Bhat, E. Stossel, G. Faller, G. Cianciolo, J. Ficker, M. Wagner, M. Rollinghoff, et al. Inverse Correlation of Maturity and Antibacterial Activity in Human Dendritic Cells J. Immunol., April 1, 2005; 174(7): 4203 - 4209. [Abstract] [Full Text] [PDF]
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M. Walch, E. Eppler, C. Dumrese, H. Barman, P. Groscurth, and U. Ziegler Uptake of Granulysin via Lipid Rafts Leads to Lysis of Intracellular Listeria innocua J. Immunol., April 1, 2005; 174(7): 4220 - 4227. [Abstract] [Full Text] [PDF]
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K. M. Patton, T. C. McGuire, D. G. Fraser, and S. A. Hines Rhodococcus equi-Infected Macrophages Are Recognized and Killed by CD8+ T Lymphocytes in a Major Histocompatibility Complex Class I-Unrestricted Fashion Infect. Immun., December 1, 2004; 72(12): 7073 - 7083. [Abstract] [Full Text] [PDF]
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J. J. Endsley, J. L. Furrer, M. A. Endsley, M. A. McIntosh, A. C. Maue, W. R. Waters, D. R. Lee, and D. M. Estes Characterization of Bovine Homologues of Granulysin and NK-lysin J. Immunol., August 15, 2004; 173(4): 2607 - 2614. [Abstract] [Full Text] [PDF]
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T. P. Plackett, E. D. Boehmer, D. E. Faunce, and E. J. Kovacs Aging and innate immune cells J. Leukoc. Biol., August 1, 2004; 76(2): 291 - 299. [Abstract] [Full Text] [PDF]
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D. A. Hagge, N. A. Ray, J. L. Krahenbuhl, and L. B. Adams An In Vitro Model for the Lepromatous Leprosy Granuloma: Fate of Mycobacterium leprae from Target Macrophages after Interaction with Normal and Activated Effector Macrophages J. Immunol., June 15, 2004; 172(12): 7771 - 7779. [Abstract] [Full Text] [PDF]
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M. Skold and S. M. Behar Role of CD1d-Restricted NKT Cells in Microbial Immunity Infect. Immun., October 1, 2003; 71(10): 5447 - 5455. [Full Text] [PDF]
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) and DN2. D5 (
), were tested. 
). The results shown were obtained from a single experiment and are representative of three experiments with similar results.
) were cultured for 2 h overnight with 
). The NKT cell clone DN2.B9 was added at an E:T ratio of 5:1. Cells were lysed 18 h after addition of M. tuberculosis. In selected wells, monocytes were preincubated with an anti-CD1d Ab before addition of the NKT cells (