The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

The Journal of Immunology, 2008, 180, 3436 -3446
Copyright © 2008 by The American Association of Immunologists, Inc.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bastian, M.
Right arrow Articles by Stenger, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bastian, M.
Right arrow Articles by Stenger, S.
Right arrowPubmed/NCBI databases
*Substance via MeSH
Medline Plus Health Information
*Tuberculosis

Mycobacterial Lipopeptides Elicit CD4+ CTLs in Mycobacterium tuberculosis-Infected Humans1

Max Bastian*, Tobias Braun{dagger}, Heiko Bruns*, Martin Röllinghoff{dagger} and Steffen Stenger2,*

* Institut für Medizinische Mikrobiologie und Hygiene, Universitätsklinik Ulm, Ulm, Germany; and {dagger} Institut für Klinische Mikrobiologie, Immunologie und Hygiene, Friedrich-Alexander Universität, Erlangen-Nürnberg, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In searching for immunogenic molecules with the potential to induce protective immune responses against tuberculosis, we developed an ex vivo model to study frequency, phenotype, and effector functions of human T lymphocytes recognizing hydrophobic Ags of Mycobacterium tuberculosis (M.Tb). To obtain unbiased results, we characterized T lymphocytes responding to a crude cell wall extract (chloroform methanol extract of M.Tb (M.Tb-CME)) containing a broad spectrum of mycobacterial glycolipids and lipopeptides. A significant proportion of T lymphocytes recognized M.Tb-CME (290 IFN-{gamma}+ T cells/105 PBMCs) and developed to effector memory cells as determined by the expression of CD45RO and the chemokine receptors CXCR3 and CCR5. Expanded lymphocytes fulfilled all criteria required for an efficient immune response against tuberculosis: 1) release of macrophage-activating Th1 cytokines and chemokines required for the spatial organization of local immune responses, 2) cytolytic activity against Ag-pulsed macrophages, and 3) recognition of infected macrophages and killing of the intracellular bacteria. Phenotypically, M.Tb-CME-expanded cells were CD4+ and MHC class II restricted, challenging current concepts that cytotoxic and antimicrobial effector cells are restricted to the CD8+ T cell subset. Pretreatment of M.Tb-CME with protease or chemical delipidation abrogated the biological activity, suggesting that responses were directed toward mycobacterial lipopeptides. These findings suggest that lipidated peptides are presented by M.Tb-infected macrophages and elicit CD4+ cytolytic and antimicrobial T lymphocytes. Our data support an emerging concept to include hydrophobic microbial Ags in vaccines against tuberculosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Protection against the intracellular pathogen Mycobacterium tuberculosis (M.Tb)3 requires a fine-tuned interplay between cells of the innate and adaptive immune system. In the ~90% of infected individuals, this results in containment or clearance of the pathogen. Even though this sounds comforting, tuberculosis is a major cause of mortality worldwide because the tubercle bacilli is exceptionally successful and has infected a third of the world population. Despite the introduction of the bacille Calmette Guérin (BCG) vaccination in the first half of the 20th century and the availability of efficient antituberculosis therapy since the 1960s, 2 million deaths occur annually (1). The main reasons for the persistence of this ancient disease are an increasing number of people who are at risk for developing active disease due to coinfection with the human immune deficiency virus, malnutrition, or other factors leading to immunological deficits. In addition, the percentage of drug-resistant bacilli is increasing (2) and essentially untreatable extremely resistant "XDR" strains are emerging (3). Another aggravating issue is the unsatisfying efficacy of BCG that prevents severe disease in childhood, but not the most common pulmonary infection of adults (4, 5). Experts unequivocally agree that new strategies for the prevention of tuberculosis are desperately needed to halt the further spread of this deadly disease. Several candidate vaccines have reached the level of clinical trials. Two vaccines improved classical BCG by inserting the secreted mycobacterial Ag85B (6) or by inserting listeriolysin to facilitate the escape of the bacilli from the phagosome into the cytoplasm (7). Another strategy follows the idea that secreted mycobacterial Ags from the Ag85 complex will induce protective memory T lymphocytes when given as fusion proteins (8, 9, 10) or if expressed in a modified vaccinia virus Ankara (11). Even though the development of these vaccines is far advanced, it will take years until results on the efficacy in field trials will become available. Therefore, the search for additional Ags must continue to keep "the pipeline filled" and have alternative strategies available if required. Current approaches include DNA vaccines, genetically modified viable mycobacteria, and subunit vaccines (12). Traditionally, the search for immunogenic mycobacterial Ags to be included in a subunit vaccine has focused on classical protein Ags stimulating CD4+ T cells (13). More recently, the translation of basic research into vaccine design prompted the search for unconventional Ags that stimulate {gamma}{delta} T cells or CD1-restricted T cells (14, 15, 16). Specifically, lipid Ags such as mycolic acid (17), lipoarabinomannan (18), glucose monomycolate (19), mycoketide (20), mycobactin (21), or diacylated sulfoglycolipid (22) have been shown to induce group I CD1-restricted T cells. Early studies indicated that the majority of T cell clones specific for lipid Ags were negative for CD4 and CD8 (17, 18, 23), but meanwhile the universe of lipid-responsive T cells has expanded to CD4+ (24) and CD8+ lymphocytes (25, 26, 27). Cells responding to nonprotein Ags are likely to be important in protection against tuberculosis because they produce Th1 cytokines, lyse M.Tb-infected cells, and kill the intracellular pathogen (16). Although all these results were obtained by analyzing T cell lines and clones, only one study has demonstrated that there is a reservoir of CD1-restricted lipid responsive T cells in the peripheral blood of M.Tb-primed individuals (28). These studies extended our classical view that potent T cell Ags are restricted to proteins presented by MHC class I or MHC class II molecules. Therefore, we designed the present study to evaluate whether a hydrophobic cell wall extract of M.Tb contains unconventional Ags that induce protective T cell subsets in humans. Specifically, we investigated frequency, phenotype, and function of primary human lymphocytes responding to a chloroform-methanol extract of M.Tb. We identify mycobacterial lipopeptides as potent inducers of a unique CD4+ T cell subset that is equipped with chemotactic, cytolytic, and antimicrobial effector functions.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell culture reagents

Cells were cultured in RPMI 1640 (Biochrom) supplemented with glutamine (2 mM; Sigma-Aldrich), 10 mM HEPES, 13 mM NaHCO3, 100 g/ml streptomycin, 60 g/ml penicillin (all purchased from Biochrom), and 5% heat-inactivated human AB serum (Cambrex) (complete medium (CM)). Experiments involving live M.Tb were performed in the absence of antibiotics.

Ags and Abs

As a source of hydrophobic Ags, we used a chloroform-methanol extract of a virulent M.Tb strain (H37Rv; Dr. J. Belisle, Colorado State University, Ft. Collins, CO) that was originally developed to enrich lysates of ubiquitous mycobacteria for lipopeptides (29, 30) and is now used to purify free lipids of M.Tb (28, 31) such as glucose monomycolate, mycolic acid, phosphatidylcholine, or phosphatidylinositol (32). The lyophilized preparation was dissolved in chloroform:methanol (2:1) and stored at –70°C. Before the experiments, the appropriate amount was dried under vacuum and resuspended in CM by sonication in a preheated water bath. To generate M.Tb extract, mycobacteria were inactivated by gamma irradiation (25 kGy). Subsequently, the bacteria were resuspended in PBS and sonicated three times. The sonicate was centrifuged at 150,000 x g for 1 h and the supernatant was collected. The protein concentration was determined in a BCA protein assay (Pierce) according to the manufacturer’s instructions. Additional Ags were purified protein derivative (PPD), tetanus toxoid (both obtained from Chiron Behring), early secretory antigenic target 6 (ESAT6; Lionex), LPS (Sigma-Aldrich), and a synthetic lipopeptide (Pam3Cys; EMC Microcollections). All Ags were used at 10 µg/ml unless otherwise stated.

The following Abs were used for flow cytometry: anti-TCR-{alpha}β-FITC, anti-CD45RO-PE, anti-CD45RA-allophycocyanin, anti-granzyme B-allophycocyanin, anti-CD83-allophycocyanin, streptavidin-allophycocyanin, biotinylated anti-CD4 (all obtained from Caltag Laboratories), anti-CXCR3-FITC, anti-CCR7-allophycocyanin (R&D Systems), anti-CD1b-FITC, anti-CCR5-PE, anti-CD56-PE, anti-CD3-PerCP, anti-CD8-allophycocyanin (BD Biosciences), biotinylated goat anti-rabbit (The Jackson Laboratory), biotinylated anti-perforin (Alexis), and polyclonal rabbit serum anti-granulysin. A total of 1 µl of Ab was added to a 100-µl cell suspension containing ~5 x 105 cells. Cells were immediately analyzed by flow cytometry (FACSCalibur; BD Biosciences).

Classification of healthy donors and tuberculosis patients

Buffy coats from healthy donors were purchased from the German Red Cross located at the Institute of Transfusion Medicine (Ulm University, Ulm, Germany). Blood donors were classified according to the reactivity to PPD and ESAT6 (33) (Table I). All patients included in this study were undergoing treatment with antituberculosis drugs for 1–8 wk at the time of blood donation. All studies including human subjects were approved by the local ethical review committee.


View this table:
[in this window]
[in a new window]

  		Table I. Classification of blood donorsa

 
Generation of APCs and lymphocytes

PBMCs were isolated using a density gradient centrifugation (Ficoll; Amersham Biosciences). Monocytes were separated by adherence of PBMCs on a plastic surface for 1 h and differentiated with 1000 IU recombinant human GM-CSF (Berlex) and 1000 IU recombinant human IL-4 (Strathmann Biotech) for 2 days. Differentiated monocytes were irradiated using a gamma source (33 Gy). Thirty to 50% of the resulting cell population expressed group I CD1 molecules as determined by flow cytometry. This protocol was used to generate APC for all experiments. For individual experiments, monocytes were incubated for 6 days in the presence of GM-CSF (1000 IU) and IL4 (1000 IU) to obtain immature dendritic cells (DC). Purified T cells were isolated by negative magnetic sorting (Pan-T cell isolation kit; Miltenyi Biotec). CD45RA–/CCR7+ central memory and CD45RA+/CCR7+ naive T cells were purified using a FACS sorter (MoFlo; Nikolaus-Fiebiger Zentrum, Universität Erlangen-Nürnberg, Erlangen, Germany). Purity of the T cell populations was confirmed by flow cytometry and exceeded 98% in all experiments.

Measurement of T cell proliferation

T cell-enriched (nonadherent) PBMC and APCs (5:1 ratio) were resuspended in PBS/2% FCS. CFSE (Alexis) was added (0.5 µM) and cells were incubated at 37°C for 10 min. A total of 2.4 x 106 cells/well were seeded in a 24-well plate in the presence of the chloroform methanol extract of M.Tb (M.Tb-CME) or PPD. After 7–10 days, cells were costained for cell surface markers (CD3, CD8, CD4, CXCR-3, CCR5, CCR7, CD45RO) or intracellular molecules (granulysin, perforin, granzyme B) as previously described (34). CFSElow (proliferated) cells were detected by flow cytometry and further analyzed for the expression of additional parameters. In selected experiments, CFSElowCD3+ cells were purified by flow cytometry and used as Ag-specific responder cells. For selected experiments, central memory and naive T cells (1 x 105) T cells were stimulated with M.Tb-CME in 96-well plates in the presence of 1 x 105 autologous APCs. After 5 days, [3H]thymidine (1 µCi; Amersham Biosciences) was added for the final 18 h of incubation. Incorporation of radioactivity was determined in a beta counter (Inotec).

Measurement of cytokine release

Nonadherent PBMC (1 x 107/well) were stimulated with autologous APCs (2 x 106/well) that had been pulsed with M.Tb-CME or M.Tb extract (both at 10 µg/ml). After overnight incubation, IFN-{gamma} secretion was measured by IFN-{gamma} release assay following the protocol supplied by the manufacturer (Miltenyi Biotec).

For measuring cytokine release by M.Tb-CME-expanded T lymphocytes, CFSElow, CD56-negative cells (5 µl/1 x 107 cells) were sorted. Isolated T cells (2 x 104/well) were stimulated with PPD (10 µg/ml), M.Tb-CME (10 µg/ml), tetanus toxoid (10 µg/ml), LPS (1 ng/ml), or Pam3Cys (10 µg/ml) in the presence of 1 x 104 irradiated, autologous APCs. After 48 h, cell supernatant was collected and concentrations were determined of IFN-{gamma} (Perbio Sciences), TNF-{alpha} (Endogen), CCL5 (R&D Systems), and IL-4 (Endogen) by sandwich ELISA according to the manufacturer’s instruction.

CFSE cytotoxicity assay

A CFSE-based cytotoxicity assay was adapted from Jedema et al. (35). Briefly, nonirradiated APCs were labeled with CFSE (2 µM), seeded in a 96-well round-bottom plate (1 x 104/well) and pulsed with PPD or M.Tb.-CME. Purified proliferated cells (see above) were added at an E:T ratio of 5:1, 2:1, and 1:1. APCs in the absence of T cells served as controls. After overnight incubation, all cells were harvested and incubated with 1 µl of propidium iodide (Sigma-Aldrich) for 5 min. CaliBRITE beads (1 µl; BD Biosciences) were added and the suspension was analyzed by flow cytometry. Acquisition of cells was stopped after 2000 beads had been collected. Live cells were enumerated by counting the number of propidium iodide-negative, CFSEhigh APCs (=N). Specific lysis was calculated according to the following equation: percent lysis = (1 – Ntest/Ncontrol) x 100.

Identification of the Ag-presenting molecules

Cells were plated in 96-well plates (1.2 x 105 cells/well) in the presence of neutralizing Abs (10 µg/ml) to HLA-I (clone W6/32), HLA-II (clone L243; both obtained from BioConcept), or CD1 type 1 molecules (anti-CD1a, clone 10H3; anti-CD1b, clone BCD1b; anti-CD1c; clone F10; all provided by S. Porcelli, Albert Einstein College of Medicine, New York, NY) 30 min before addition of the Ag. After 7 days, CFSE dilution was assessed by flow cytometry and IFN-{gamma} concentration in the supernatants was measured by sandwich-ELISA.

Protease K digestion

M.Tb-CME was treated with 1 mg/ml protease K (Sigma-Aldrich) for 1 h at 50°C (final volume 100 µl) in a water bath. The protease was subsequently denatured for 30 min at 75°C.

SDS-PAGE

SDS-PAGE was performed under denaturing conditions in 15% polyacrylamide gels. Proteins were visualized by silver staining as described elsewhere (36).

Delipidation of M.Tb-CME

Delipidation of M.Tb-CME was achieved by incubation with 1 N sodium hydroxide (Roth) at 50°C for 45 min. pH was subsequently adjusted to 7.4 with 1 N HCl (Roth). PPD was treated identically as a control. For chemical delipidation M.Tb-CME was digested overnight with 0.5 mg/ml lipoprotein-lipase (Sigma-Aldrich) at 37°C. The lipase was subsequently denatured at 75°C for 10 min.

Measurement of intracellular growth of M.Tb

APCs were seeded in 96-well plates at 1 x 104 cells/well and infected with single-cell suspensions of M.Tb at a multiplicity of infection (MOI) of 10. After 4 h of incubation, extracellular bacteria were removed by thorough washing with PBS. Purified, M.Tb-CME-expanded T cells (CFSElow) were added at 2 x 104 cells/well. After 48 h, cell supernatants were collected and filtered through 2-µm syringe filter units (Millipore) and IFN-{gamma} concentration was determined by sandwich ELISA. After further 48 h of incubation, cells were lysed with 0.3% saponin (Sigma-Aldrich) to release intracellular bacteria. Cell lysates were resuspended vigorously, transferred into screw caps, and sonicated in a preheated water bath for 5 min. Aliquots of the sonicate were diluted 10-fold in 7H9 medium. Four dilutions of each sample were plated in duplicate on 7H11 agar plates and incubated at 37°C in 5% CO2 for 14–21 days before determining the number of CFU.

Statistical analysis

The Student t test was used to determine statistical differences between the groups. Differences were considered significant at p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Hydrophobic mycobacterial Ags activate human T cells ex vivo

A critical parameter for the identification of immunogenic Ags is their potential to activate antimicrobial effector mechanisms in primary human lymphocytes. Therefore, we stimulated cells from PPD-reactive healthy donors with M.Tb-CME overnight and measured IFN-{gamma} release and proliferation as correlates of T cell activation (Fig. 1). Stimulated cells and control cultures were stained for IFN-{gamma} and TCR{alpha}β and analyzed by flow cytometry (Fig. 1A). In 25 donors, M.Tb-CME (median: 29 IFN-{gamma}+ T cells/104 PBMCs) and M.Tb extract (median 58 IFN-{gamma}+ T cells/104 PBMCs) elicited robust IFN-{gamma} responses as compared with unstimulated control cultures (median: 11 IFN-{gamma} + T cells/104 PBMCs; p < 0.0002; Fig. 1B). Second, T cell-enriched PBMCs were cultured with M.Tb-CME-treated APCs and CFSE dilution was assessed after 7 days (Fig. 1C). T lymphocytes derived from 14 different donors expanded significantly as compared with control cultures after stimulation with M.Tb-CME (median: 67 CFSElow T cells/103 PBMCs) and M.Tb extract (median: 172 T cells/103 PBMCs) (p < 0.001; Fig. 1D). Proliferated CD3– cells were NK cells presumably responding to IL-2 secreted by Ag-activated T cells (data not shown). These results demonstrate that a considerable proportion of M.Tb-specific T cells circulating in the peripheral blood respond to hydrophobic mycobacterial Ags and may be major players in the establishment of protective cellular immunity in human tuberculosis.


Figure 1
View larger version (38K):
[in this window]
[in a new window]

  		FIGURE 1. Hydrophobic mycobacterial Ags stimulate human T cells ex vivo. A and B, PBMCs of PPD+ donors were stimulated with APCs in the presence or absence of M.Tb-CME. After overnight incubation, an IFN-{gamma} release assay was performed and results analyzed by flow cytometry. Fig. 1 presents a representative donor of 25 (A) and the results of individual donors (B). C and D, CFSE-labeled PBMCs were stimulated with autologous APCs in the presence or absence of M.Tb-CME (10 µg/ml). After 7 days, CD3+ and CFSElow were enumerated by flow cytometry. C, A representative donor of 14; D, the results of all individual donors investigated.

 
M.Tb-CME-expanded T cells are Ag specific

To ascertain that T cell activation was Ag specific and not mediated by an adjuvant effect of hydrophobic Ags on APC (37, 38), we determined whether M.Tb-CME induces maturation of DC. Immature DC were incubated with M.Tb-CME overnight and maturation was determined by CD83 expression, a marker present on mature DC. In contrast to LPS which was included as a positive control, M.Tb-CME did not affect CD83 expression (Fig. 2A). Therefore, T cell activation is not due to activation of DC, even though the investigation of additional markers would be required to definitively confirm this. To ascertain the Ag specificity of M.Tb-CME-expanded T lymphocytes, CFSElow cells were isolated by flow cytometry and restimulated with mycobacterial Ags or irrelevant controls. T cells secreted substantial amounts of IFN-{gamma} in response to M.Tb-CME and PPD (median 1.9 and 3.3 ng/ml), but not to tetanus toxoid or the TLR ligands LPS and Pam3Cys (Fig. 2B). These results demonstrate that T cell activation by M.Tb-CME is Ag specific and independent of the adjuvant activity of the hydrophobic Ags.


Figure 2
View larger version (10K):
[in this window]
[in a new window]

  		FIGURE 2. M.Tb-CME-expanded T cells are Ag specific. A, Immature DC were incubated with M.Tb-CME (10 µg/ml) or LPS (1 ng/ml) overnight. The percentage of CD83+ DC was determined by flow cytometry. {blacksquare}, The average of four independent experiments with different donors. Error bars show the SD. B, M.Tb-CME-expanded T lymphocytes (day 7, CFSElow, CD56 negative) were isolated by flow cytometry and restimulated with PPD, M.Tb-CME, tetanus toxoid (10 µg/ml), or the TLR agonists LPS (1 ng/ml) and Pam3Cys (10 µg/ml) in the presence of autologous APCs. After 48 h, supernatants were harvested and the IFN-{gamma} concentration was determined by ELISA. B depicts the values for 25 individual donors ({circ}, vaccinated donors; •, latently infected donors) and the horizontal lines show the median.

 
M.Tb-CME-specific T cell responses in M.Tb-infected donors

To discriminate whether priming of T cell responses to hydrophobic mycobacterial Ags requires natural infection with M.Tb, we classified 54 donors according to their reactivity to PPD and ESAT6 (Table I) as naive (n = 9), vaccinated (n = 22), or latently infected (n = 23) and measured proliferative responses to M.Tb-CME. Responses in vaccinated donors (median: 52 CFSElow lymphocytes/103 PBMC) were not significantly different from naive donors (median: 26 CFSElow lymphocytes/103 PBMC), whereas T lymphocytes derived from latently infected individuals (242 CFSElow lymphocytes/103 PBMC) readily proliferated (Fig. 3). To confirm that natural tuberculosis infection as opposed to BCG-vaccination primes T cells specific for hydrophobic mycobacterial Ags we recruited 11 patients with active pulmonary tuberculosis that were undergoing the first stage of antituberculosis therapy in our clinic. Strikingly, 10 of 11 patients showed significant proliferation to M.Tb-CME (median: 143 CFSElow lymphocytes/103 PBMC). These results suggest that ex vivo reactivity to hydrophobic mycobacterial Ags reflects recall responses of T lymphocytes that were primed during natural M.Tb infection.


Figure 3
View larger version (13K):
[in this window]
[in a new window]

  		FIGURE 3. M.Tb-CME-specific T cell responses in naive (n = 9), BCG-vaccinated (n = 22), and latently infected donors (n = 23) and tuberculosis patients (n = 11). PBMC were labeled with CFSE and stimulated with M.Tb-CME in the presence of autologous APC. After 7 days, CFSElow lymphocytes were quantified by flow cytometry. Differences between naive donors and latently infected individuals or tuberculosis patients were statistically significant. Horizontal lines show the median.

 
M.Tb-CME-responsive T lymphocytes are CD4+ central memory cells

The major aim of this study was to identify Ags that elicit memory T lymphocytes supporting protection against tuberculosis. Therefore, we analyzed cell surface markers of M.Tb-CME-specific T cells characteristic for functionally defined T cell subsets. PBMC of PPD- and ESAT6-reactive healthy donors (latently infected) were stimulated overnight with M.Tb-CME and IFN-{gamma}-secreting T cells were identified by flow cytometry. Costaining with cell surface markers showed that the majority of IFN-{gamma}-secreting cells expressed CD4 (median 82%), while CD8+ (median 16%) or double-negative (median 3%) cells were underrepresented as compared with total T cells (Fig. 4). A similar distribution was observed when we analyzed cells in a long-term assay (day 7 CFSE dilution; data not shown).


Figure 4
View larger version (13K):
[in this window]
[in a new window]

  		FIGURE 4. The majority of M.Tb-CME-responsive T lymphocytes are CD4+. PBMC were stimulated with M.Tb-CME overnight. Cells were then stained for IFN-{gamma}, CD3, CD4, and CD8. A high-energy allophycoyanin conjugate was used to stain for CD4 and a low-energy allophycoyanin conjugate for CD8 staining. The distribution of CD4 and CD8 is shown for a representative donor (n = 10) for CD3+ T cells (A) and IFN-{gamma}+ cells (B). C, The individual results of the CD4/CD8 distribution of M.Tb-CME-reactive cells of 10 donors is shown. The horizontal line shows the median.

 
Central memory T cells represent the cellular basis for long-lived, immunological memory and are characterized by the dual expression of CD45RO and CCR7 (39). To determine which cellular compartment contains the reactivity to hydrophobic mycobacterial Ag, we stimulated sorted central memory (CD45RA–, CCR7+) and naive (CD45RA+, CCR7+) T cells (Fig. 5A) with M.Tb-CME. In contrast to naive T cells, central memory cells proliferated vigorously (Fig. 5B). Together, this implies that PPD+/ESAT6+ individuals have a pool of circulating CD4+, central memory T lymphocytes specific for hydrophobic mycobacterial Ags.


Figure 5
View larger version (26K):
[in this window]
[in a new window]

  		FIGURE 5. M.Tb-CME-responsive T lymphocytes are central memory T cells. CD3+ cells were purified by magnetic isolation. Central memory T lymphocytes and naive T cells were further isolated by cell sorting according to the expression of CD45RA and CCR7 (A). B, Central memory or naive T cells were stimulated with increasing concentrations of M.Tb-CME in the presence of autologous APCs. After 5 days, [3H]thymidine incorporation was determined. One representative donor of five is shown. The error bars show the SD of the triplicate wells.

 
M.Tb-CME-expanded T cells acquire an effector memory phenotype

Activated central memory T cells proliferate and develop into effector cells that migrate into inflamed or infected peripheral tissues. In Th1 cells, migration is directed by a fine-tuned regulation of chemokine receptors, namely the down-regulation of CCR7 and the up-regulation of CCR5 and CXCR3 (40). To investigate whether this program is initiated in response to M.Tb-CME, we analyzed the chemokine receptor expression profile on proliferated T cells after 7 days of incubation. To obtain a sufficient number of donors, we included vaccinated ({circ}) and latently infected (•) individuals (Fig. 6). The vast majority of CFSElow T lymphocytes expressed CD45RO (median 97%), hardly detectable levels of CCR7 (median 3%; Fig. 6, A and B), and high levels of CXCR3 and CCR5 (median 97 and 63%; Fig. 6, C and D). There were no significant differences between vaccinated and latently infected donors. Therefore, M.Tb-CME induces the expansion of Th1 effector memory T cells that play a major role in protection against M.Tb.


Figure 6
View larger version (29K):
[in this window]
[in a new window]

  		FIGURE 6. M.Tb-CME-expanded T lymphocytes acquire an effector memory phenotype. PBMC from BCG-vaccinated ({circ}) and latently infected donors (•) were labeled with CFSE and stimulated with M.Tb-CME for 7 days. CFSElow cells were further analyzed by flow cytometry. The expression of CD45RO and CCR7 is shown in 1 representative donor (A) and for all 14 independent donors (B). The expression of CXCR3 and CCR5 in 1 representative donor (C) and for all 6 independent donors (D) is shown. The horizontal lines show the median. Differences in CD45R0, CCR7, CXCR3, and CCR5 expression between vaccinated and latently infected donors were not statistically significant.

 
M.Tb-CME induces antimicrobial and CTLs

Although antimicrobial and cytotoxic activity has mainly been attributed to CD8+ T cell subsets (25), we considered the possibility that M.Tb-CME induces a unique subset of cytolytic CD4+ effector cells. To test for this possibility, we determined the intracellular expression of the antimicrobial peptide granulysin and the cytotoxic molecules perforin and granzyme B in cells that had proliferated in response to M.Tb-CME (Fig. 7A). In 14 independent donors (PPD+, ESAT+/–), we detected significant but heterogeneous expression of granulysin (on average 31% of CFSElow T lymphocytes), perforin (75%), and granzyme B (53%) (Fig. 7B). The expression of these molecules was Ag specific, because resting (CFSEhigh) T cells failed to express granular effector molecules (Fig. 7A).


Figure 7
View larger version (14K):
[in this window]
[in a new window]

  		FIGURE 7. M.Tb-CME induces antimicrobial and CTLs. The expression of granulysin, perforin, and granzyme B was determined in M.Tb-CME-expanded (CFSElow) T lymphocytes by intracellular flow cytometry. A, Bold lines indicate the staining of CFSElow T cells, dotted lines the staining of nonproliferated (CFSEhigh) T lymphocytes from the same well. B, Expression of granular effector molecules was determined in CFSElow T cells in 14 individual donors ({circ}: vaccinated donors, n = 6; •: latently infected donors, n = 8). The horizontal line shows the median.

 
Functional analysis of isolated M.Tb-CME-expanded cells

There are at least three critical defense mechanisms of T lymphocytes in the immune response against M.Tb: 1) release of cytokines and chemokines, 2) cytotoxicity, and 3) direct antimicrobial activity. To directly measure the capacity of M.Tb-CME to expand relevant T cell subsets, we sorted cells from vaccinated and latently infected individuals that had proliferated in response to M.Tb-CME and tested them for these functions after in vitro restimulation.

First, we analyzed the secretion of the prototypic Th1 cytokines IFN-{gamma} and TNF-{alpha}. Significant amounts of IFN-{gamma} (median 0.9 ng/ml) and TNF-{alpha} (median 1.0) (Fig. 8, A and B) were secreted in response to M.Tb-CME-pulsed autologous APCs. In contrast, the concentration of the Th2-related cytokines IL-4 and IL-10 was below the detection limit (median <14 pg/ml; Fig. 8, C and D). We recently reported that CCL5 is coordinately expressed with granulysin and perforin in CD8+ T cells (41). Because a substantial portion of M.Tb-CME-expanded cells expressed granulysin and perforin, we investigated whether CD4+ effector cells were also able to secrete CCL5. In response to M.Tb-CME stimulation, significant amounts of CCL5 (median 1.8 ng/ml) and CCL3 (median 2 ng/ml)—another Th1-related chemokine—were released (Fig. 8, E and F). Taken together, isolated M.Tb-CME-reactive T cells release a cytokine and chemokine pattern that is closely associated with protection against tuberculosis.


Figure 8
View larger version (17K):
[in this window]
[in a new window]

  		FIGURE 8. Isolated M.Tb-CME-reactive T cells secrete Th1-related cytokines and chemokines. PBMC from PPD+ donors ({circ}, ESAT6–; •, ESAT6+) were CFSE labeled and stimulated with M.Tb-CME. After 7 days, CFSElow cells were sorted and restimulated with M.Tb-CME for 48 h. Supernatants were harvested and the concentration of IFN-{gamma} (A), TNF-{alpha} (B), IL-4 (C), IL-10 (D), CCL5 (E), and CCL3 (F) was measured by ELISA. Individual results are shown for 25 (A–D) or 12 (E and F) donors. The horizontal line shows the median.

 
To measure cytolytic activity, we adapted a flow cytometry-based assay that directly quantifies the number of living target cells (35). The assay was validated by comparison to the most widely used 51Cr-release assay using PBMC-mediated killing of the NK cell target line K562. Both methods yielded comparable results in all five donors tested, but the flow cytometry-based assay proved to be more sensitive (data not shown) and was therefore used for the following experiments. We incubated sorted, M.Tb-CME-expanded effector cells with CFSE-labeled autologous APC that had been pulsed with PPD (Fig. 9, A–C) or M.Tb-CME (Fig. 9, D and E). M.Tb-CME-expanded T cells significantly reduced the number of viable target cells (CFSE+, propidium iodide negative) (Fig. 9A). The cytotoxic activity was Ag specific, correlated with the E:T ratio (Fig. 9, B and D), and was reproducible in all nine (PPD) and six (M.Tb-CME) donors investigated (Fig. 9, C and E). Cytotoxicity was not inhibited by neutralizing Abs to MHC class I or MHC class II (data not shown). Because IFN-{gamma} release was completely abrogated by MHC class II Abs in the same wells in which lysis was unaffected, we believe that this result is due to the notorious difficulty to neutralize cytotoxicity in short-term assays.


Figure 9
View larger version (27K):
[in this window]
[in a new window]

  		FIGURE 9. M.Tb-CME-expanded T lymphocytes lyse Ag-pulsed APCs. Isolated, M.Tb-CME-expanded T lymphocytes were coincubated with CFSE-labeled, autologous APCs in the presence or absence of PPD (10 µg/ml, A–C) or M.Tb-CME (10 µg/ml, D and E). After overnight incubation, viable APCs were enumerated by flow cytometry. A, A representative histogram for nine experiments is shown. Bold lines show the number of viable (propidium iodide-negative) targets in the presence of T cells and dotted lines show the number of viable targets in the absence of T cells. The numbers give the absolute counts of viable cells. B, CFSE-labeled targets were incubated with PPD-pulsed or control APCs at increasing E:T ratios. One representative assay of nine is shown. C, Specific lysis at an E:T ratio of 1:1 for nine donors is shown. Donors were PPD+, ESAT6– ({circ}) or PPD+, ESAT6+ (•). D, CFSE-labeled targets were incubated with M.Tb-CME-pulsed or control APCs at increasing E:T ratios. One representative assay of six is shown. E, Specific lysis at an E:T ratio of 1:1 for all six donors is shown ({circ}, PPD+, ESAT6–; •, PPD+, ESAT6+).

 
M.Tb-CME-expanded T lymphocytes recognize and kill intracellular M.Tb

A hallmark of protective T cell responses against M.Tb is the recognition of infected cells and optimally killing of the intracellular pathogen (26). We therefore tested M.Tb-CME-expanded T cells for their response to macrophages infected with virulent M.Tb. Coincubation induced the release of significant amounts of IFN-{gamma} (median 200 pg/ml) (Fig. 10A), indicating that hydrophobic Ags are available for T cell recognition on the surface of infected cells. To determine whether recognition of infected is paralleled by killing of the intracellular pathogens, the number of viable bacilli was determined after 96 h of coincubation. In four independent experiments, a 10-fold reduction in CFU numbers was observed in the presence of T lymphocytes as compared with infected APCs in the absence of T cells (p = 0.009) (Fig. 10B). There were no statistically significant differences between vaccinated and latently infected donors in terms of effector functions of isolated M.Tb-CME-amplified T lymphocytes. Together, these results show that M.Tb-CME-expanded CD4+ T cells recognize infected macrophages. Recognition induces a plethora of effector mechanisms, which, in sum, result in killing of the intracellular bacteria.


Figure 10
View larger version (14K):
[in this window]
[in a new window]

  		FIGURE 10. M.Tb-CME-reactive T lymphocytes recognize infected APC and kill intracellular M.Tb. M.Tb-CME-expanded T lymphocytes were sorted and coincubated with M.Tb-infected (MOI 10) APCs. A, After 48 h of incubation of 2 x 104 T cells and 1 x 104 APC, supernatants were harvested and IFN-{gamma} concentration was determined by ELISA. Results of 26 individual donors are shown. B, M.Tb-infected APCs were cultured in the presence (E:T ratio ranging from 1:1 to 3:1) or absence of sorted M.Tb-CME-expanded T cells. After 96 h, lysates of the cells were plated on 7H11 agar plates in four different dilutions and the number of CFUs was counted after 14 days of incubation. Donors were vaccinated ({circ}) or latently infected (•). The horizontal line shows the median of all four experiments performed.

 
HLA-II-restricted lipopeptides are the immunogenic compound in M.Tb-CME

To understand the mechanism of T cell activation, we sought to determine the biochemical nature of the immunogenic compound within M.Tb-CME. First, we aimed at identifying the Ag-presenting molecule by stimulating cultures in the presence of neutralizing Abs to HLA-I, HLA-II, or CD1 type 1 molecules. In nine of nine donors, IFN-{gamma} release was reduced by 80% with HLA-II-blocking Abs, although individual donors showed a minor contribution of the CD1 pathway (Fig. 11A).


Figure 11
View larger version (19K):
[in this window]
[in a new window]

  		FIGURE 11. M.Tb-CME reactivity is due to HLA-II-restricted lipopeptides. A, T cell-enriched PBMCs and autologous APCs of nine PPD+ donors were stimulated with M.Tb-CME in the presence of blocking Abs against HLA-II, HLA-I, or CD1a,b,c (all at 10 µg/ml). Two days after stimulation, the supernatants were collected and IFN-{gamma} concentration was determined by ELISA. Percent inhibition of IFN-{gamma} secretion as compared with M.Tb-CME stimulation is shown for all individual donors. B, M.Tb-CME was treated with protease K before stimulation of PBMC and autologous APC. After 48 h, IFN-{gamma} secretion was compared between cultures stimulated with untreated and treated M.Tb-CME. The results of all 12 donors are shown. The horizontal line shows the median. C, PPD and M.Tb-CME were separated by SDS-PAGE and proteins were detected by subsequent silver staining. D, M.Tb-CME was delipidated by mild sodium hydroxide treatment. IFN-{gamma} secretion of nonadherent PBMC (2 x 104) and autologous APC (1 x 104) in response to nontreated and treated M.Tb-CME was measured after 48 h of incubation by ELISA. The figure presents the individual results of 8 donors. The horizontal line depicts the median. E, M.Tb-CME was treated with lipoprotein lipase. T cell proliferation as measured by CFSE dilution after 7 days of incubation is compared in response to treated vs nontreated M.Tb-CME. Results obtained with six independent donors are shown. Donors were vaccinated ({circ}) or latently infected (•). The horizontal line shows the median.

 
To explore whether an intact peptide backbone was required for the biological activity, we digested M.Tb-CME with protease K before stimulation of the T cells. The treatment completely abolished T cell activation in 11 of 12 donors (p = 0.0009) (Fig. 11B), providing compelling evidence that structurally intact peptides are required for the M.Tb-CME reactivity. To exclude that proteins were present in the M.Tb-CME preparation, we performed SDS-PAGE separation of M.Tb-CME (100 µg) and PPD (10 µg/ml). Silver staining of the SDS gel failed to yield any visible protein bands in the range of 6–170 kDa for M.Tb-CME, confirming the high purity of the preparation.

Because chloroform-methanol extraction strongly enriches the mycobacterial cell wall extract for hydrophobic molecules such as lipids, glycolipids, and lipopeptides, we next examined whether—in addition to a peptide backbone—a lipid moiety was required for M.Tb-CME-induced IFN-{gamma} release. M.Tb-CME was deacylated by mild alkaline hydrolysis which is a common method to deacylate lipopeptides or LPS (42, 43). Deacylation of M.Tb-CME reduced cytokine release in all donors (n = 8; p = 0.0005) (Fig. 11D). Identically treated PPD retained the antigenicity ruling out a toxic effect of the deacylation procedure (data not shown). This suggested that acylated N-terminal fragments of mycobacterial lipoproteins were the immunogenic compounds. To directly test this possibility, we treated M.Tb-CME with lipoprotein lipase, an enzyme that specifically cleaves the diacylglycerol moiety of bacterial lipopeptides (44, 45, 46). Proliferation in response to lipase-treated M.Tb-CME was significantly reduced on average by 70% as compared with the nontreated M.Tb-CME (p = 0.012) (Fig. 11E) strongly suggesting that mycobacterial lipopeptides are capable of eliciting multifunctional CD4+ T lymphocytes.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
This report provides the first comprehensive quantitative and qualitative characterization of primary human T lymphocytes responding to hydrophobic mycobacterial Ags. Lipopeptides are identified as potent immunogens of M.Tb eliciting lymphocytes that are equipped with key functions required for protection against tuberculosis: 1) release of Th1 cytokines, 2) recognition of M.Tb-infected macrophages, and 3) killing of intracellular bacteria. Activated CD4+ T cells are decorated with chemokine receptors that mediate migration to sites of inflammation. We therefore conclude that lipopeptides are attractive candidates to induce effector memory T cells required for protection against M.Tb.

Lipopeptides are a heterogeneous family of hydrophobic molecules present in the outer layer of the bacterial cell wall. They are characterized by a lipobox motif that is crucial for initiation of lipid modifications. The structure responsible for the immunological activity is located in the NH2 terminal triacylated lipopeptide region (47, 48, 49). Lipoproteins have been extensively found in both Gram-positive and -negative bacteria as well as in Treponema pallidum (50), Mycoplasma species (51), and Borrelia burgdorferi (52). The M.Tb genome contains 99 putative lipoproteins (2.5% of the predicted genome) (53) and are major components of the cell envelope. Studies on mycobacterial lipoproteins have focused on defined recombinant or purified molecules, particularly the 19- and 27-kDa molecules. The 19-kDa lipoprotein mediates a complex spectrum of TLR-2 mediated effects on phagocytes including the induction of apoptosis (54), production of IL-12 (55), down-regulation of MHC class II molecules (56, 57, 58), maturation of DC (59), and the activation of neutrophils (60); 19-kDa also induces the vitamin D-dependent up-regulation of cathelicidin that kills intracellular M.Tb (61, 62, 63). A 19-kDa lipoprotein-deficient mutant showed decreased growth in human macrophages and induced lower amounts of proinflammatory cytokines (64). The introduction of the 19-kDa lipoprotein into BCG did not improve (65) or even reduce (66) the efficacy of BCG vaccination in mice. Finally, a 19-kDa-deficient mutant was slightly attenuated in vivo in a high throughput screen of transposon mutants (67). The M.Tb complex-specific 27-kDa lipoprotein is a mitogen for murine splenocytes (68) and drives a Th1-type immune response in vivo, but fails to ameliorate a simultaneous or subsequent infection with virulent M.Tb (69, 70). In vivo, a 27-kDa deficient M.Tb strain was strongly attenuated as compared with the control strains in a mouse model of tuberculosis (71). M.Tb mutants lacking lipoprotein signal peptidase are deficient in the processing of all lipoproteins which results in impaired stimulation of TLR2-reporter cells (72). Lipoprotein signal peptidase mutants are highly attenuated in vitro and in vivo in murine tuberculosis (73). Taken together, these results suggest that mycobacterial lipoproteins are major players in shaping innate immune responses and contribute to the virulence of M.Tb. In contrast to the established effect of lipoproteins on phagocytes, knowledge on their possible role as T cell Ags is scarce. Epitopes of defined mycobacterial lipoproteins (19, 27, 38 kDa) have previously been found to induce MHC-restricted T cell responses (74, 75, 76, 77, 78). Lymphocyte activation was attributed to TCR-mediated recognition of conventional proteins or the facilitated presentation of endogenous ligands by supporting costimulation of APCs (37). In contrast, our open approach using the hydrophobic chloroform methanol extract of M.Tb identified native lipopeptides as potent immunogens for primary human T lymphocytes. We provide several lines of evidence that T cell activation by mycobacterial lipopeptides is Ag specific and not due to an adjuvant effect on APCs. First, M.Tb-CME had no measurable direct effects on the phenotype of APC (Fig. 2A). Second, M.Tb-CME-expanded T cells responded selectively to mycobacterial Ags but not to TLR agonists (Fig. 2B). Most importantly, activation was restricted to M.Tb-primed individuals and was only moderate (BCG-vaccinated) or absent (PPD– donors) in other cohorts. This extends an earlier observation that another class of hydrophobic Ags—mycobacterial lipids—stimulates CD1-restricted CD4+ T cells predominantly in M.Tb-infected individuals (28). In this report, a preparation of mycobacterial lipids induced proliferation and IFN-{gamma} release of CD4+ T lymphocytes. Lymphocyte activation was inhibited to a considerable but variable extent by neutralizing Abs to group 1 CD1 molecules (11–94% for proliferation; 20–78% for IFN-{gamma} release; 2 of 17 donors responded independently of CD1). Our study may offer a partial explanation for this variable inhibition. Even though we also observed CD1-restricted activation, we demonstrate that responses to M.Tb-CME are largely MHC class II restricted (Fig. 11A). It is possible that some of the responses found by Ulrichs et al. (28) may have been MHC class II mediated because MHC class II blocking was not done. Nonetheless, the differences between the studies remain difficult to explain. Possible explanations for differences in the levels of CD1-restricted responses include the distinct compositions of the Ag preparations (e.g., relation of lipids to lipopeptides), different readout systems (ELISPOT vs ELISA, thymidine uptake vs CFSE dilution), or the level of CD1 expression on APCs. In any case, the combination of these two studies is intriguing in terms of defining markers capable of discriminating BCG-vaccinated and M.Tb-infected donors. At this stage, detailed analyses on large and well-defined cohorts are warranted to evaluate whether reactivity to hydrophobic Ags will be a promising correlate of infection with M.Tb.

There are several alternative mechanisms—besides supporting APC costimulation—for how lipidation of proteins could promote Ag-specific T cell responses: 1) improvement of Ag presentation as reported for synthetically palmitoylated peptides (79, 80, 81) by modulating Ag processing (82) and intracellular trafficking (83, 84), as the lipid moiety is required to direct the mycobacterial lipopeptides from the phagosome to the MHC-loading machinery (85). 2) Stabilization of the protein-MHC complex by protrusion of the lipid moiety from the MHC class II-binding groove and attachment to the phospholipid bilayer of the endosomal membrane (86). 3) Unmasking or modification of protein epitopes by posttranslational lipidation thereby increasing the repertoire of peptides fitting into the MHC-binding groove (87). Further knowledge about the contribution of the lipid anchor for the immunogenicity of lipopeptides may open new avenues for the rational design of new vaccines.

M.Tb-CME stimulated a unique CD4+ T cell subset characterized by the expression of chemotactic, cytolytic, and antimicrobial effector molecules. In general, these functions are associated with CD8+ T cells that recognize and lyse infected host cells (25, 26, 41, 88). There have been reports on cytolytic CD4+ T cells specific for M.Tb (89, 90, 91), but the functional relevance of this subset may have been underestimated, because in the peripheral blood CD4+ T cells express only moderate levels of cytolytic effector molecules (41). Here, we demonstrate that lipopeptide-reactive, CD4+ T lymphocytes strongly up-regulate granulysin and perforin upon restimulation indicating that this subset is not only essential in combating viruses (92, 93), but also in antibacterial immunity. The lack of cytolytic activity of CD4+ T cells in CD4 depleted mice may also account for the surprising finding that these mice are particularly susceptible to mycobacterial lung infection although the overall level of IFN-{gamma} in the lung and critical macrophage functions are not affected (94).

T lymphocytes activated by hydrophobic mycobacterial Ags preferentially express a chemokine receptor pattern (CCR5+, CXCR3+, CCR–) that directs them to sites of inflammation (95), e.g., mycobacterial infection (96). In infected tissue, lipopeptide-reactive cells could contribute to elimination of pathogens by the release of Th1 cytokines and chemokines crucial for granuloma formation and containment of M.Tb (97, 98). Notably, M.Tb-expanded T lymphocytes secreted significant levels of the Th1-associated chemokines CCL5 and CCL3 (99, 100) even without Ag-specific restimulation (Fig. 8). We suggest that the constitutive secretion of chemokines represents a "looking for target" signal attracting infected macrophages, while the elevated secretion after Ag encounter may serve as a "found target" signal attracting other effector T cells (41).

In summary, we report the lipopeptide-induced amplification of an MHC class II-restricted, CD4+ T cell subset that unifies all major effector functions required for protection in human tuberculosis. The strong immunogenicity of mycobacterial lipopeptides should be exploited for prophylactic and therapeutic immune intervention strategies against tuberculosis. The next milestone in the course of these studies will be the identification of biochemically defined lipopeptides that elicit this protective T cell subset in humans.


   Acknowledgments
 
We acknowledge the support of our clinical collaborators at the Städtische Klinikum Fürth, the Waldkrankenhaus St. Marien in Erlangen, the Bezirksklinikum Obermain in Kutzenberg, and the Rangauklinik in Ansbach. We are grateful to Kirstin Castiglione and Nives Schwerdtner for their excellent scientific and technical input. We appreciate the gift of unique reagents from Alan Krensky (Stanford University School of Medicine, anti-granulysin serum) and Steve Porcelli (Albert Einstein College of Medicine, New York, CD1-blocking Abs). We thank Markus Schnare and Macarena Bompadre-Beigier for helpful discussions.


   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 German Research Foundation (SFB643), the European Union (VIth Framework, TB-VAC), and the Interdisciplinary Center for Clinical Research in Erlangen. Back

2 Address correspondence and reprint requests to Dr. Steffen Stenger, Institut für Medizinische Mikrobiologie und Hygiene, Universitätsklinik Ulm, Robert Koch-Strasse 8, D-89081 Ulm, Germany. E-mail address: steffen.stenger{at}uniklinik-ulm.de Back

3 Abbreviations used in this paper: M.Tb, Mycobacterium tuberculosis; BCG, bacille Calmette Guérin; CM, complete medium; M.Tb-CME, chloroform methanol extract of M.Tb; PPD, purified protein derivative; ESAT6, early secretory antigenic target 6; DC, dendritic cell; MOI, multiplicity of infection. Back

Received for publication August 10, 2007. Accepted for publication January 2, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Anonymous, . 2007. Global Tuberculosis Control: WHO Report 2007 1-2. World Health Organization, Geneva.
  2. Friedland, G.. 2007. Tuberculosis, drug resistance, and HIV/AIDS: a triple threat. Curr. Infect. Dis. Rep. 9: 252-261. [Medline]
  3. Anonymous, G.. 2006. Emergence of Mycobacterium tuberculosis with extensive resistance to second line drugs, 2000–2004. MMWR Morb. Mortal. Wkly. Rep. 55: 301-305. [Medline]
  4. Brewer, T. F.. 2000. Preventing tuberculosis with bacillus Calmette-Guérin vaccine: a meta-analysis of the literature. Clin. Infect. Dis. 31: (Suppl. 3):64-67.
  5. Trunz, B. B., P. Fine, C. Dye. 2006. Effect of BCG vaccination on childhood tuberculous meningitis and miliary tuberculosis worldwide: a meta-analysis and assessment of cost-effectiveness. Lancet 367: 1173-1180. [Medline]
  6. Horwitz, M. A., G. Harth. 2003. A new vaccine against tuberculosis affords greater survival after challenge than the current vaccine in the guinea pig model of pulmonary tuberculosis. Infect. Immun. 71: 1672-1679. [Abstract/Free Full Text]
  7. Grode, L., P. Seiler, S. Baumann, J. Hess, V. Brinkmann, E. A. Nasser, P. Mann, C. Goosmann, S. Bandermann, D. Smith, et al 2005. Increased vaccine efficacy against tuberculosis of recombinant Mycobacterium bovis bacille Calmette-Guérin mutants that secrete listeriolysin. J. Clin. Invest. 115: 2472-2479. [Medline]
  8. Olsen, A. W., A. Williams, L. M. Okkels, G. Hatch, P. Andersen. 2004. Protective effect of a tuberculosis subunit vaccine based on a fusion of antigen 85B and ESAT-6 in the aerosol guinea pig model. Infect. Immun. 72: 6148-6150. [Abstract/Free Full Text]
  9. Langermans, J. A., T. M. Doherty, R. A. Vervenne, T. van der Laan, K. Lyashchenko, R. Greenwald, E. M. Agger, C. Aagaard, H. Weiler, D. van Soolingen, et al 2005. Protection of macaques against Mycobacterium tuberculosis infection by a subunit vaccine based on a fusion protein of antigen 85B and ESAT-6. Vaccine 23: 2740-2750. [Medline]
  10. Brandt, L., Y. A. Skeiky, M. R. Alderson, Y. Lobet, W. Dalemans, O. C. Turner, R. J. Basaraba, A. A. Izzo, T. M. Lasco, P. L. Chapman, et al 2004. The protective effect of the Mycobacterium bovis BCG vaccine is increased by coadministration with the Mycobacterium tuberculosis 72-kilodalton fusion polyprotein Mtb72F in M. tuberculosis-infected guinea pigs. Infect. Immun. 72: 6622-6632. [Abstract/Free Full Text]
  11. McShane, H., A. A. Pathan, C. R. Sander, S. M. Keating, S. C. Gilbert, K. Huygen, H. A. Fletcher, A. V. Hill. 2004. Recombinant modified vaccinia virus Ankara expressing antigen 85A boosts BCG-primed and naturally acquired antimycobacterial immunity in humans. Nat. Med. 10: 1240-1244. [Medline]
  12. Kaufmann, S. H., A. J. McMichael. 2005. Annulling a dangerous liaison: vaccination strategies against AIDS and tuberculosis. Nat. Med. 11: S33-S44. [Medline]
  13. Boom, W. H., D. H. Canaday, S. A. Fulton, A. J. Gehring, R. E. Rojas, M. Torres. 2003. Human immunity to M. tuberculosis: T cell subsets and antigen processing. Tuberculosis 83: 98-106. [Medline]
  14. Kaufmann, S. H.. 2005. Recent findings in immunology give tuberculosis vaccines a new boost. Trends Immunol. 26: 660-667. [Medline]
  15. Chen, Z. W.. 2005. Immune regulation of {gamma}{delta} T cell responses in mycobacterial infections. Clin. Immunol. 116: 202-207. [Medline]
  16. Behar, S. M., S. A. Porcelli. 2007. CD1-restricted T cells in host defense to infectious diseases. Curr. Top. Microbiol. Immunol. 314: 215-250. [Medline]
  17. Beckman, E. M., S. A. Porcelli, C. T. Morita, S. M. Behar, S. T. Furlong, M. B. Brenner. 1994. Recognition of a lipid antigen by CD1-restricted {alpha}β+ T cells. Nature 372: 691-694. [Medline]
  18. Sieling, P. A., D. Chatterjee, S. A. Porcelli, T. I. Prigozy, R. J. Mazzaccaro, T. Soriano, B. R. Bloom, M. B. Brenner, M. Kronenberg, P. J. Brennan, et al 1995. CD1-restricted T cell recognition of microbial lipoglycan antigens. Science 269: 227-230. [Abstract/Free Full Text]
  19. Moody, D. B., B. B. Reinhold, M. R. Guy, E. M. Beckman, D. E. Frederique, S. T. Furlong, S. Ye, V. N. Reinhold, P. A. Sieling, R. L. Modlin, et al 1997. Structural requirements for glycolipid antigen recognition by CD1b– restricted T cells. Science 278: 283-286. [Abstract/Free Full Text]
  20. Matsunaga, I., A. Bhatt, D. C. Young, T. Y. Cheng, S. J. Eyles, G. S. Besra, V. Briken, S. A. Porcelli, C. E. Costello, W. R. Jacobs, Jr, D. B. Moody. 2004. Mycobacterium tuberculosis pks12 produces a novel polyketide presented by CD1c to T cells. J. Exp. Med. 200: 1559-1569. [Abstract/Free Full Text]
  21. Moody, D. B., D. C. Young, T. Y. Cheng, J. P. Rosat, C. Roura-Mir, P. B. O’Connor, D. M. Zajonc, A. Walz, M. J. Miller, S. B. Levery, et al 2004. T cell activation by lipopeptide antigens. Science 303: 527-531. [Abstract/Free Full Text]
  22. Gilleron, M., S. Stenger, Z. Mazorra, F. Wittke, S. Mariotti, G. Bohmer, J. Prandi, L. Mori, G. Puzo, G. De Libero. 2004. Diacylated sulfoglycolipids are novel mycobacterial antigens stimulating CD1-restricted T cells during infection with Mycobacterium tuberculosis. J. Exp. Med. 199: 649-659. [Abstract/Free Full Text]
  23. Porcelli, S., C. T. Morita, M. B. Brenner. 1992. CD1b restricts the response of human CD4–8– T lymphocytes to a microbial antigen. Nature 360: 593-597. [Medline]
  24. Ochoa, M. T., S. Stenger, P. A. Sieling, S. Thoma-Uszynski, S. Sabet, S. Cho, A. M. Krensky, M. Rollinghoff, E. Nunes Sarno, A. E. Burdick, et al 2001. T-cell release of granulysin contributes to host defense in leprosy. Nat. Med. 7: 174-179. [Medline]
  25. Stenger, S., R. J. Mazzaccaro, K. Uyemura, S. Cho, P. F. Barnes, J. P. Rosat, A. Sette, M. B. Brenner, S. A. Porcelli, B. R. Bloom, R. L. Modlin. 1997. Differential effects of cytolytic T cell subsets on intracellular infection. Science 276: 1684-1687. [Abstract/Free Full Text]
  26. Stenger, S., D. A. Hanson, R. Teitelbaum, P. Dewan, K. R. Niazi, C. J. Froelich, T. Ganz, S. Thoma-Uszynski, A. Melian, C. Bogdan, et al 1998. An antimicrobial activity of cytolytic T cells mediated by granulysin. Science 282: 121-125. [Abstract/Free Full Text]
  27. Rosat, J. P., E. P. Grant, E. M. Beckman, C. C. Dascher, P. A. Sieling, D. Frederique, R. L. Modlin, S. A. Porcelli, S. T. Furlong, M. B. Brenner. 1999. CD1-restricted microbial lipid antigen-specific recognition found in the CD8+ {alpha}β T cell pool. J. Immunol. 162: 366-371. [Abstract/Free Full Text]
  28. Ulrichs, T., D. B. Moody, E. Grant, S. H. Kaufmann, S. A. Porcelli. 2003. T-cell responses to CD1-presented lipid antigens in humans with Mycobacterium tuberculosis infection. Infect. Immun. 71: 3076-3087. [Abstract/Free Full Text]
  29. Brennan, P. J., M. Souhrada, B. Ullom, J. K. McClatchy, M. B. Goren. 1978. Identification of atypical mycobacteria by thin-layer chromatography of their surface antigens. J. Clin. Microbiol. 8: 374-379. [Abstract/Free Full Text]
  30. Eckstein, T. M., S. Chandrasekaran, S. Mahapatra, M. R. McNeil, D. Chatterjee, C. D. Rithner, P. W. Ryan, J. T. Belisle, J. M. Inamine. 2006. A major cell wall lipopeptide of Mycobacterium avium subspecies paratuberculosis. J. Biol. Chem. 281: 5209-5215. [Abstract/Free Full Text]
  31. Hiromatsu, K., C. C. Dascher, K. P. LeClair, M. Sugita, S. T. Furlong, M. B. Brenner, S. A. Porcelli. 2002. Induction of CD1-restricted immune responses in guinea pigs by immunization with mycobacterial lipid antigens. J. Immunol. 169: 330-339. [Abstract/Free Full Text]
  32. Dascher, C. C., K. Hiromatsu, X. Xiong, C. Morehouse, G. Watts, G. Liu, D. N. McMurray, K. P. LeClair, S. A. Porcelli, M. B. Brenner. 2003. Immunization with a mycobacterial lipid vaccine improves pulmonary pathology in the guinea pig model of tuberculosis. Int. Immunol. 15: 915-925. [Abstract/Free Full Text]
  33. Lalvani, A., R. Brookes, R. J. Wilkinson, A. S. Malin, A. A. Pathan, P. Andersen, H. Dockrell, G. Pasvol, A. V. Hill. 1998. Human cytolytic and interferon {gamma}-secreting CD8+ T lymphocytes specific for Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. USA 95: 270-275. [Abstract/Free Full Text]
  34. Gansert, J. L., V. Kiessler, M. Engele, F. Wittke, M. Rollinghoff, A. M. Krensky, S. A. Porcelli, R. L. Modlin, S. Stenger. 2003. Human NKT cells express granulysin and exhibit antimycobacterial activity. J. Immunol. 170: 3154-3161. [Abstract/Free Full Text]
  35. Jedema, I., N. M. van der Werff, R. M. Barge, R. Willemze, J. H. Falkenburg. 2004. New CFSE-based assay to determine susceptibility to lysis by cytotoxic T cells of leukemic precursor cells within a heterogeneous target cell population. Blood 103: 2677-2682. [Abstract/Free Full Text]
  36. Jungblut, P. R., R. Seifert. 1990. Analysis by high-resolution two-dimensional electrophoresis of differentiation-dependent alterations in cytosolic protein pattern of HL-60 leukemic cells. J. Biochem. Biophys. Methods 21: 47-58. [Medline]
  37. Sieling, P. A., W. Chung, B. T. Duong, P. J. Godowski, R. L. Modlin. 2003. Toll-like receptor 2 ligands as adjuvants for human Th1 responses. J. Immunol. 170: 194-200. [Abstract/Free Full Text]
  38. Rosenkrands, I., E. M. Agger, A. W. Olsen, K. S. Korsholm, C. S. Andersen, K. T. Jensen, P. Andersen. 2005. Cationic liposomes containing mycobacterial lipids: a new powerful Th1 adjuvant system. Infect. Immun. 73: 5817-5826. [Abstract/Free Full Text]
  39. Sallusto, F., D. Lenig, R. Forster, M. Lipp, A. Lanzavecchia. 1999. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 401: 708-712. [Medline]
  40. Rivino, L., M. Messi, D. Jarrossay, A. Lanzavecchia, F. Sallusto, J. Geginat. 2004. Chemokine receptor expression identifies pre-T helper (Th)1, pre-Th2, and nonpolarized cells among human CD4+ central memory T cells. J. Exp. Med. 200: 725-735. [Abstract/Free Full Text]
  41. Stegelmann, F., M. Bastian, K. Swoboda, R. Bhat, V. Kiessler, A. M. Krensky, M. Roellinghoff, R. L. Modlin, S. Stenger. 2005. Coordinate expression of CC chemokine ligand 5, granulysin, and perforin in CD8+ T cells provides a host defense mechanism against Mycobacterium tuberculosis. J. Immunol. 175: 7474-7483. [Abstract/Free Full Text]
  42. Brennan, P. J., P. Draper. 1994. Ultrastructure of mycobacteria. B. R. Bloom, Jr, ed. Tuberculosis: Pathogenesis, Protection and Control 271-284. ASM, Washington, D.C.
  43. Seid, R. C., Jr, J. C. Sadoff. 1981. Preparation and characterization of detoxified lipopolysaccharide-protein conjugates. J. Biol. Chem. 256: 7305-7310. [Abstract/Free Full Text]
  44. Shibata, K., A. Hasebe, T. Into, M. Yamada, T. Watanabe. 2000. The N-terminal lipopeptide of a 44-kDa membrane-bound lipoprotein of Mycoplasma salivarium is responsible for the expression of intercellular adhesion molecule-1 on the cell surface of normal human gingival fibroblasts. J. Immunol. 165: 6538-6544. [Abstract/Free Full Text]
  45. Hashimoto, M., Y. Asai, T. Ogawa. 2004. Separation and structural analysis of lipoprotein in a lipopolysaccharide preparation from Porphyromonas gingivalis. Int. Immunol. 16: 1431-1437. [Abstract/Free Full Text]
  46. Shimizu, T., Y. Kida, K. Kuwano. 2005. A dipalmitoylated lipoprotein from Mycoplasma pneumoniae activates NF-{kappa}B through TLR1, TLR2, and TLR6. J. Immunol. 175: 4641-4646. [Abstract/Free Full Text]
  47. Wu, H. C., F. C. Neidhardt, R. Curtiss, III, J. L. Ingraham, E. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, H. E. Umbarger. 1996. Biosynthesis of lipoproteins. C. Neidhardt, III, ed. Escherichia coli and Salmonella typhimurium 1005-1014. ASM, Washington, D.C.
  48. Sutcliffe, I. C., D. J. Harrington. 2004. Lipoproteins of Mycobacterium tuberculosis: an abundant and functionally diverse class of cell envelope components. FEMS Microbiol. Rev. 28: 645-659. [Medline]
  49. Rezwan, M., T. Grau, A. Tschumi, P. Sander. 2007. Lipoprotein synthesis in mycobacteria. Microbiology 153: 652-658. [Abstract/Free Full Text]
  50. Chamberlain, N. R., M. E. Brandt, A. L. Erwin, J. D. Radolf, M. V. Norgard. 1989. Major integral membrane protein immunogens of Treponema pallidum are proteolipids. Infect. Immun. 57: 2872-2877. [Abstract/Free Full Text]
  51. Bricker, T. M., M. J. Boyer, J. Keith, R. Watson-McKown, K. S. Wise. 1988. Association of lipids with integral membrane surface proteins of Mycoplasma hyorhinis. Infect. Immun. 56: 295-301. [Abstract/Free Full Text]
  52. Weis, J. J., Y. Ma, L. F. Erdile. 1994. Biological activities of native and recombinant Borrelia burgdorferi outer surface protein A: dependence on lipid modification. Infect. Immun. 62: 4632-4636. [Abstract/Free Full Text]
  53. Cole, S. T., R. Brosch, J. Parkhill, T. Garnier, C. Churcher, D. Harris, S. V. Gordon, K. Eiglmeier, S. Gas, C. E. Barry, III, et al 1998. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. [Published erratum appears in 1998 Nature 396: 190]. Nature 393: 537-544. [Medline]
  54. Aliprantis, A. O., R. B. Yang, M. R. Mark, S. Suggett, B. Devaux, J. D. Radolf, G. R. Klimpel, P. Godowski, A. Zychlinsky. 1999. Cell activation and apoptosis by bacterial lipoproteins through Toll-like receptor-2. Science 285: 736-739. [Abstract/Free Full Text]
  55. Brightbill, H. D., D. H. Libraty, S. R. Krutzik, R. B. Yang, J. T. Belisle, J. R. Bleharski, M. Maitland, M. V. Norgard, S. E. Plevy, S. T. Smale, et al 1999. Host defense mechanisms triggered by microbial lipoproteins through Toll-like receptors. Science 285: 732-736. [Abstract/Free Full Text]
  56. Noss, E. H., R. K. Pai, T. J. Sellati, J. D. Radolf, J. Belisle, D. T. Golenbock, W. H. Boom, C. V. Harding. 2001. Toll-like receptor 2-dependent inhibition of macrophage class II MHC expression and antigen processing by 19-kDa lipoprotein of Mycobacterium tuberculosis. J. Immunol. 167: 910-918. [Abstract/Free Full Text]
  57. Fulton, S. A., S. M. Reba, R. K. Pai, M. Pennini, M. Torres, C. V. Harding, W. H. Boom. 2004. Inhibition of major histocompatibility complex II expression and antigen processing in murine alveolar macrophages by Mycobacterium bovis BCG and the 19-kilodalton mycobacterial lipoprotein. Infect. Immun. 72: 2101-2110. [Abstract/Free Full Text]
  58. Lopez, M., L. M. Sly, Y. Luu, D. Young, H. Cooper, N. E. Reiner. 2003. The 19-kDa Mycobacterium tuberculosis protein induces macrophage apoptosis through Toll-like receptor-2. J. Immunol. 170: 2409-2416. [Abstract/Free Full Text]
  59. Hertz, C. J., S. M. Kiertscher, P. J. Godowski, D. A. Bouis, M. V. Norgard, M. D. Roth, R. L. Modlin. 2001. Microbial lipopeptides stimulate dendritic cell maturation via Toll-like receptor 2. J. Immunol. 166: 2444-2450. [Abstract/Free Full Text]
  60. Neufert, C., R. K. Pai, E. H. Noss, M. Berger, W. H. Boom, C. V. Harding. 2001. Mycobacterium tuberculosis 19-kDa lipoprotein promotes neutrophil activation. J. Immunol. 167: 1542-1549. [Abstract/Free Full Text]
  61. Thoma-Uszynski, S., S. Stenger, O. Takeuchi, M. T. Ochoa, M. Engele, P. A. Sieling, P. F. Barnes, M. Rollinghoff, P. L. Bolcskei, M. Wagner, et al 2001. Induction of direct antimicrobial activity through mammalian Toll-like receptors. Science 291: 1544-1547. [Abstract/Free Full Text]
  62. Liu, P. T., S. Stenger, H. Li, L. Wenzel, B. H. Tan, S. R. Krutzik, M. T. Ochoa, J. Schauber, K. Wu, C. Meinken, et al 2006. Toll-like receptor triggering of a vitamin D-mediated human antimicrobial response. Science 311: 1770-1773. [Abstract/Free Full Text]
  63. Liu, P. T., S. Stenger, D. H. Tang, R. L. Modlin. 2007. Cutting edge: vitamin D-mediated human antimicrobial activity against Mycobacterium tuberculosis is dependent on the induction of cathelicidin. J. Immunol. 179: 2060-2063. [Abstract/Free Full Text]
  64. Stewart, G. R., K. A. Wilkinson, S. M. Newton, S. M. Sullivan, O. Neyrolles, J. R. Wain, J. Patel, K. L. Pool, D. B. Young, R. J. Wilkinson. 2005. Effect of deletion or overexpression of the 19-kilodalton lipoprotein Rv3763 on the innate response to Mycobacterium tuberculosis. Infect. Immun. 73: 6831-6837. [Abstract/Free Full Text]
  65. Yeremeev, V. V., G. R. Stewart, O. Neyrolles, K. Skrabal, V. G. Avdienko, A. S. Apt, D. B. Young. 2000. Deletion of the 19 kDa antigen does not alter the protective efficacy of BCG. Tuber. Lung Dis. 80: 243-247. [Medline]
  66. Rao, V., N. Dhar, H. Shakila, R. Singh, A. Khera, R. Jain, M. Naseema, C. N. Paramasivan, P. R. Narayanan, V. D. Ramanathan, A. K. Tyagi. 2005. Increased expression of Mycobacterium tuberculosis 19 kDa lipoprotein obliterates the protective efficacy of BCG by polarizing host immune responses to the Th2 subtype. Scand. J. Immunol. 61: 410-417. [Medline]
  67. Sassetti, C. M., E. J. Rubin. 2003. Genetic requirements for mycobacterial survival during infection. Proc. Natl. Acad. Sci. USA 100: 12989-12994. [Abstract/Free Full Text]
  68. Hovav, A. H., L. Davidovitch, G. Nussbaum, J. Mullerad, Y. Fishman, H. Bercovier. 2004. Mitogenicity of the recombinant mycobacterial 27-kilodalton lipoprotein is not connected to its antiprotective effect. Infect. Immun. 72: 3383-3390. [Abstract/Free Full Text]
  69. Hovav, A. H., J. Mullerad, L. Davidovitch, Y. Fishman, F. Bigi, A. Cataldi, H. Bercovier. 2003. The Mycobacterium tuberculosis recombinant 27-kilodalton lipoprotein induces a strong Th1-type immune response deleterious to protection. Infect. Immun. 71: 3146-3154. [Abstract/Free Full Text]
  70. Hovav, A. H., J. Mullerad, A. Maly, L. Davidovitch, Y. Fishman, H. Bercovier. 2006. Aggravated infection in mice co-administered with Mycobacterium tuberculosis and the 27-kDa lipoprotein. Microbes Infect. 8: 1750-1757. [Medline]
  71. Bigi, F., A. Gioffre, L. Klepp, M. P. Santangelo, A. Alito, K. Caimi, V. Meikle, M. Zumarraga, O. Taboga, M. I. Romano, A. Cataldi. 2004. The knockout of the lprG-Rv1410 operon produces strong attenuation of Mycobacterium tuberculosis. Microbes Infect. 6: 182-187. [Medline]
  72. Banaiee, N., E. Z. Kincaid, U. Buchwald, W. R. Jacobs, Jr, J. D. Ernst. 2006. Potent inhibition of macrophage responses to IFN-{gamma} by live virulent Mycobacterium tuberculosis is independent of mature mycobacterial lipoproteins but dependent on TLR2. J. Immunol. 176: 3019-3027. [Abstract/Free Full Text]
  73. Sander, P., M. Rezwan, B. Walker, S. K. Rampini, R. M. Kroppenstedt, S. Ehlers, C. Keller, J. R. Keeble, M. Hagemeier, M. J. Colston, et al 2004. Lipoprotein processing is required for virulence of Mycobacterium tuberculosis. Mol. Microbiol. 52: 1543-1552. [Medline]
  74. Vordemeier, H. M., D. P. Harris, E. Roman, R. Lathigra, C. Moreno, J. Ivanyi. 1991. Identification of T cell stimulatory peptides from the 38-kDa protein of Mycobacterium tuberculosis. J. Immunol. 147: 1023-1029. [Abstract]
  75. Oftung, F., H. G. Wiker, A. Deggerdal, A. S. Mustafa. 1997. A novel mycobacterial antigen relevant to cellular immunity belongs to a family of secreted lipoproteins. Scand. J. Immunol. 46: 445-451. [Medline]
  76. Bigi, F., C. Espitia, A. Alito, M. Zumarraga, M. I. Romano, S. Cravero, A. Cataldi. 1997. A novel 27 kDa lipoprotein antigen from Mycobacterium bovis. Microbiology 143: (Pt. 11):3599-3605. [Abstract/Free Full Text]
  77. da Fonseca, D. P., D. Joosten, R. van der Zee, D. L. Jue, M. Singh, H. M. Vordermeier, H. Snippe, A. F. Verheul. 1998. Identification of new cytotoxic T-cell epitopes on the 38-kilodalton lipoglycoprotein of Mycobacterium tuberculosis by using lipopeptides. Infect. Immun. 66: 3190-3197. [Abstract/Free Full Text]
  78. Mohagheghpour, N., D. Gammon, L. M. Kawamura, A. van Vollenhoven, C. J. Benike, E. G. Engleman. 1998. CTL response to Mycobacterium tuberculosis: identification of an immunogenic epitope in the 19-kDa lipoprotein. J. Immunol. 161: 2400-2406. [Abstract/Free Full Text]
  79. Deres, K., H. Schild, K. H. Wiesmuller, G. Jung, H. G. Rammensee. 1989. In vivo priming of virus-specific cytotoxic T lymphocytes with synthetic lipopeptide vaccine. Nature 342: 561-564. [Medline]
  80. Rees, A. D., A. Faith, E. Roman, J. Ivanyi, K. H. Wiesmuller, C. Moreno. 1993. The effect of lipoylation on CD4 T-cell recognition of the 19,000 MW Mycobacterium tuberculosis antigen. Immunology 80: 407-414. [Medline]
  81. Fonseca, D. P., D. Joosten, H. Snippe, A. F. Verheul. 2000. Evaluation of T-cell responses to peptides and lipopeptides with MHC class I binding motifs derived from the amino acid sequence of the 19-kDa lipoprotein of Mycobacterium tuberculosis. Mol. Immunol. 37: 413-422. [Medline]
  82. Beekman, N. J., W. M. Schaaper, G. I. Tesser, K. Dalsgaard, S. Kamstrup, J. P. Langeveld, R. S. Boshuizen, R. H. Meloen. 1997. Synthetic peptide vaccines: palmitoylation of peptide antigens by a thioester bond increases immunogenicity. J. Pept. Res. 50: 357-364. [Medline]
  83. Loing, E., M. Andrieu, K. Thiam, D. Schorner, K. H. Wiesmuller, A. Hosmalin, G. Jung, H. Gras-Masse. 2000. Extension of HLA-A*0201-restricted minimal epitope by N {epsilon}-palmitoyl-lysine increases the life span of functional presentation to cytotoxic T cells. J. Immunol. 164: 900-907. [Abstract/Free Full Text]
  84. Stittelaar, K. J., P. Hoogerhout, W. Ovaa, R. R. van Binnendijk, M. C. Poelen, P. Roholl, C. A. van Els, A. D. Osterhaus, E. J. Wiertz. 2001. In vitro processing and presentation of a lipidated cytotoxic T-cell epitope derived from measles virus fusion protein. Vaccine 20: 249-261. [Medline]
  85. Neyrolles, O., K. Gould, M. P. Gares, S. Brett, R. Janssen, P. O’Gaora, J. L. Herrmann, M. C. Prevost, E. Perret, J. E. Thole, D. Young. 2001. Lipoprotein access to MHC class I presentation during infection of murine macrophages with live mycobacteria. J. Immunol. 166: 447-457. [Abstract/Free Full Text]
  86. Robinson, J. H., M. C. Case, C. G. Brooks. 1992. Palmitic acid conjugation of a protein antigen enhances major histocompatibility complex class II-restricted presentation to T cells. Immunology 76: 593-598. [Medline]
  87. Engelhard, V. H., M. Altrich-Vanlith, M. Ostankovitch, A. L. Zarling. 2006. Post-translational modifications of naturally processed MHC-binding epitopes. Curr. Opin. Immunol. 18: 92-97. [Medline]
  88. Stenger, S.. 2001. Cytolytic T cells in the immune response to mycobacterium tuberculosis. Scand. J. Infect. Dis. 33: 483-487. [Medline]
  89. Lorgat, F., M. M. Keraan, P. T. Lukey, S. R. Ress. 1992. Evidence for in vivo generation of cytotoxic T cells: PPD-stimulated lymphocytes from tuberculous pleural effusions demonstrate enhanced cytotoxicity with accelerated kinetics of induction. Am. Rev. Respir. Dis. 145: 418-423. [Medline]
  90. Mutis, T., Y. E. Cornelisse, T. H. Ottenhoff. 1993. Mycobacteria induce CD4+ T cells that are cytotoxic and display Th1-like cytokine secretion profile: heterogeneity in cytotoxic activity and cytokine secretion levels. Eur. J. Immunol. 23: 2189-2195. [Medline]
  91. Ottenhoff, T. H., T. Mutis. 1990. Specific killing of cytotoxic T cells and antigen-presenting cells by CD4+ cytotoxic T cell clones: a novel potentially immunoregulatory T-T cell interaction in man. J. Exp. Med. 171: 2011-2024. [Abstract/Free Full Text]
  92. Niiya, H., I. Sakai, J. Lei, T. Azuma, N. Uchida, Y. Yakushijin, T. Hato, S. Fujita, M. Yasukawa. 2005. Differential regulation of perforin expression in human CD4+ and CD8+ cytotoxic T lymphocytes. Exp. Hematol. 33: 811-818. [Medline]
  93. Yasukawa, M., H. Ohminami, Y. Yakushijin, J. Arai, A. Hasegawa, Y. Ishida, S. Fujita. 1999. Fas-independent cytotoxicity mediated by human CD4+ CTL directed against herpes simplex virus-infected cells. J. Immunol. 162: 6100-6106. [Abstract/Free Full Text]
  94. Scanga, C. A., V. P. Mohan, K. Yu, H. Joseph, K. Tanaka, J. Chan, J. L. Flynn. 2000. Depletion of CD4+ T cells causes reactivation of murine persistent tuberculosis despite continued expression of interferon {gamma} and nitric oxide synthase 2. J. Exp. Med. 192: 347-358. [Abstract/Free Full Text]
  95. Iezzi, G., D. Scheidegger, A. Lanzavecchia. 2001. Migration and function of antigen-primed nonpolarized T lymphocytes in vivo. J. Exp. Med. 193: 987-993. [Abstract/Free Full Text]
  96. Mitra, D. K., S. K. Sharma, A. K. Dinda, M. S. Bindra, B. Madan, B. Ghosh. 2005. Polarized helper T cells in tubercular pleural effusion: phenotypic identity and selective recruitment. Eur. J. Immunol. 35: 2367-2375. [Medline]
  97. Lande, R., E. Giacomini, T. Grassi, M. E. Remoli, E. Iona, M. Miettinen, I. Julkunen, E. M. Coccia. 2003. IFN-{alpha}β released by Mycobacterium tuberculosis-infected human dendritic cells induces the expression of CXCL10: selective recruitment of NK and activated T cells. J. Immunol. 170: 1174-1182. [Abstract/Free Full Text]
  98. Seiler, P., P. Aichele, S. Bandermann, A. E. Hauser, B. Lu, N. P. Gerard, C. Gerard, S. Ehlers, H. J. Mollenkopf, S. H. Kaufmann. 2003. Early granuloma formation after aerosol Mycobacterium tuberculosis infection is regulated by neutrophils via CXCR3-signaling chemokines. Eur. J. Immunol. 33: 2676-2686. [Medline]
  99. Schrum, S., P. Probst, B. Fleischer, P. F. Zipfel. 1996. Synthesis of the CC-chemokines MIP-1{alpha}, MIP-1β, and RANTES is associated with a type 1 immune response. J. Immunol. 157: 3598-3604. [Abstract]
  100. Dorner, B. G., A. Scheffold, M. S. Rolph, M. B. Huser, S. H. Kaufmann, A. Radbruch, I. E. Flesch, R. A. Kroczek. 2002. MIP-1{alpha}, MIP-1β, RANTES, and ATAC/lymphotactin function together with IFN-{gamma} as type 1 cytokines. Proc. Natl. Acad. Sci. USA 99: 6181-6186. [Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bastian, M.
Right arrow Articles by Stenger, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bastian, M.
Right arrow Articles by Stenger, S.
Right arrowPubmed/NCBI databases
*Substance via MeSH
Medline Plus Health Information
*Tuberculosis

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS