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The Journal of Immunology, 2002, 168: 2689-2694.
Copyright © 2002 by The American Association of Immunologists

IL-4 and T Cells Are Required for the Generation of IgG1 Isotype Antibodies Against Cardiolipin1

Karsten Fischer*,{dagger}, Helen Collins*, Masaru Taniguchi{ddagger}, Stefan H. E. Kaufmann* and Ulrich E. Schaible2,*

* Department of Immunology, Max-Planck-Institute for Infection Biology, Berlin, Germany; {dagger} Department of Biology, Chemistry, Pharmacy, Free University Berlin, Berlin, Germany; and {ddagger} Division of Molecular Immunology, Center for Biomedical Science, School of Medicine, Chiba University, Chiba, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Infection with Mycobacterium tuberculosis induces Abs against a vast array of mycobacterial lipids and glycolipids. One of the most prominent lipid Ags recognized is cardiolipin (CL). The kinetics of the generation of anti-CL Abs during infection reveals that IgM titers to CL increase over time. Interestingly, at day 30 postinfection CL-specific IgG1 appears, an isotype usually dependent on T cell help. Using an immunization schedule with CL/anti-CL Ab complexes, which induces antiphospholipid syndrome in mice, we show that the generation of IgG1 to CL requires IL-4 and that optimal production is T cell dependent. IgG1 production to CL was impaired in nude (nu/nu) mice devoid in conventional T cells, but was not affected in mice deficient for either {alpha}{beta} TCR+, {gamma}{delta} TCR+, CD4+, CD8+, or NK1.1+ T cells. We conclude that IgG1 production to CL depends on T cell help and IL-4, which can be provided by different T cell populations. This is the first report that IL-4 is indispensable for the induction of IgG1 Abs to lipid Ags.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Mycobacterium tuberculosis is a major human pathogen, which, early in infection, invades host mononuclear phagocytes in the lung to survive in phagosomes. Mycobacteria are characterized by a complex cell wall consisting of unique lipid and glycolipid species (1). A number of these cell wall lipids are released from mycobacteria both in culture and inside macrophages. These released lipids consist of two groups, namely highly glycosylated hydrophilic species, e.g., lipoarabinomannan (LAM)3 and phosphatidylinositol mannosides (PIM), and hydrophobic lipids such as cardiolipin (CL) and trehalose dimycolate (TDM). In infected cells, LAM and PIM are transferred from phagosomes to lysosomes and subsequently to noninfected bystander cells (2, 3). Of the released hydrophobic lipids, lysocardiolipin was detected outside of the phagosomes following cleavage of mycobacterial CL by macrophage lysosomal phospholipase A2 (4). Mycobacterial lipids and glycolipids also represent important Ags for stimulation of the host immune system. Although mycobacteria primarily reside inside phagosomes, their lipids and glycolipids induce specific Abs. Tuberculosis patients develop Abs against both hydrophilic and hydrophobic cell wall constituents (5). The hydrophilic glycolipids PIM and LAM and the hydrophobic lipids TDM and CL were among the Ags predominantly recognized by patient sera (6, 7, 8). Ab isotypes elicited against these lipid Ags are both IgM and IgG. To further characterize the antilipid response elicited upon infection with M. tuberculosis in detail, a mouse model was used. The Ab responses against mycobacterial lipids in mice infected with M. tuberculosis were analyzed, and IgG1 Abs against CL were detected. To elucidate the mechanisms underlying the Ig class switch of anti-CL Abs to IgG1, we used an immunization schedule using CL/anti-CL Ab complexes, which has been described before to induce the antiphospholipid syndrome (APS) in mice (9, 10, 11, 12). We found that the induction of IgG1 Abs to CL was strictly dependent on IL-4, but independent of a single T cell subset, indicating a redundancy in T cell help for the class switch to the IgG1 isotype. This is the first report describing the crucial role of T cells and IL-4 for the induction of IgG1 Abs to lipids.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals

All reagents were purchased from Sigma Aldrich (Deisenhofen, Germany), unless indicated otherwise.

Mice

All mice were bred and housed under specific pathogen-free conditions at our facilities at the Bundesinstitut für gesundheitlichen Verbraucherschutz und Veterinärmedizin (Berlin, Germany). The following mouse strains were used: wild-type C57BL/6 (B6), A{beta}-/-, Kb-/-Db-/- (kindly provided by Dr. F. A. Lemonnier, Institut Pasteur, Paris, France), Fc{gamma}RIII-/-, {delta}-TCR-/-, {beta}-TCR-/-, J{alpha}281-/-, IL-4-/-, IgM-/-, and nude (nu/nu), all back-crossed on a B6 genetic background. Female mice were used at 6–8 wk of age.

Infection

Mice were infected with M. tuberculosis (H37Rv) by aerosol using a Glas-col aerosol generator (Middlebrook, Terre Haute, IN) calibrated to deliver 100–200 bacteria per lung. Inocula were determined at day 1 postinfection.

Immunization

To induce anti-CL Abs, an immunization schedule was used which had been developed to experimentally induce APS in mice (9, 10). Briefly, mice were injected s.c. with 100 µl Allhydrogel (Superfos Biosector, Vedbaek, Denmark) and 100 µl PBS containing 200 µg CL, and either 70 µg anti-CL Ab (IgG1, T1.8 kindly provided by D. G. Russell, Cornell University, Ithaca, NY) or BSA (Sigma Aldrich), or 300 µg affinity-purified human IgG from systemic lupus erythematosus (SLE) patients (kindly provided by Dr. F. Hiepe, Charité, Berlin, Germany).

Qualitative detection of antilipid Abs

The indicated lipids were prepared as described (4, 13). For staining with lipid-specific Abs, 200 µg of total lipid extract of M. tuberculosis, 50 µg CL (bovine, Sigma Aldrich), 50 µg TDM, and 50 µg of a PIM preparation were spotted onto a high performance thin layer chromatography (HPTLC) plate (Merck, Darmstadt, Germany). The HPTLC plate was developed to 8 cm in one dimension using chloroform/methanol/water (65/25/4; v/v/v) as solvent system. Lipids were visualized using 1% {alpha}-Naphtol in 5% H2SO4/ethanol (14). To test sera of mice for the presence of antilipid Abs, sera were taken 50 days postinfection with M. tuberculosis and incubated with lipids separated on HPTLC plates. The HPTLC plates were fixed by dipping them three times in 0.05% Plexigum P28 (Röhm, Darmstadt, Germany) in hexane as described (15). The plates were blocked for 1 h in PBS/10% FCS and subsequently incubated with the sera diluted 1/25 in PBS/10% FCS for 2 h at room temperature. The plates were washed five times with PBS for 10 min, and the peroxidase-labeled secondary Ab goat anti-murine IgM + IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) was added in PBS/10% FCS for 30 min. After five washing steps, the plates were developed using enhanced luminol reagent (NEN, Boston, MA) and hyperfilm ECL (Amersham Pharmacia Biotech, Buckinghamshire, U.K.).

Quantitative detection of antilipid Abs and statistics

High-binding microtiter plates (Polysorb, Nunc, Roskilde, Denmark) were coated with CL at 50 µg/ml in methanol 50 µl/well. The plates were dried and blocked for 1 h with PBS/10% FCS. Sera of mice were added at the appropriate dilutions in PBS/10% FCS and titrated in a series of 2-fold dilutions. After 2 h incubation, the plates were washed five times with PBS. Specific Abs were detected using peroxidase-coupled secondary Abs, goat anti-murine IgM + IgG (Jackson ImmunoResearch Laboratories), rat anti-mouse IgG2a, IgG2b, and IgG3 (1/2000; Southern Biotechnology Associates, Birmingham, AL), and rat anti-mouse IgG1 (1/4000; Serotec, Oxford, U.K.), followed by the addition of the substrate o-phenylenediamine. Absorbance was read at 490 nm. Results were evaluated for statistical significance (p < 0.05) by the nonpaired Mann-Whitney U test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Various lipid species are recognized by sera from M. tuberculosis-infected mice

Mice were infected by aerosol with a low dose of M. tuberculosis H37Rv (200 CFU/lung) and sera were collected after 50 days. Sera were tested for Abs to individual lipids separated by HPTLC. A total lipid preparation from M. tuberculosis H37Rv (total lipids), CL, purified TDM, and a PIM-preparation were separated by HPTLC and detected by {alpha}-Naphtol spray (Fig. 1Goa) or by sera from naive or infected mice (Fig. 1Go, b–d). The PIM fraction contained three different species with varying mannose contents (Fig. 1Goa). Preinfection sera comprised Abs which weakly detected the PIM preparation but none of the other lipids, indicating that in naive animals, Abs to mycobacterial lipids and glycolipids were virtually absent (Fig. 1Gob). In contrast, sera from M. tuberculosis-infected mice recognized various lipids including PIM and CL (Fig. 1Go, c and d). Within the total lipid extract, mycobacterial CL and three additional lipid species, which are as yet unidentified, were detected by Abs from infected animals (Fig. 1Go, c and d, arrows). When tested on bovine CL, these Abs revealed cross-reactivity with mammalian CL. These results demonstrate that CL is one of the major lipids recognized by Abs in M. tuberculosis-infected mice. Therefore, CL was used to further analyze the kinetics of and mechanisms for the induction of antilipid Abs. Sera of mice infected by aerosol with a low dose of M. tuberculosis H37Rv were harvested at days 0, 10, 20, 30, 50, and 200 postinfection, and the titers of anti-CL Abs were determined by isotype-specific ELISA. CL-specific Abs of the IgG2a and IgG2b isotypes could not be detected at any time point in the sera (data not shown). CL-specific IgM could already be detected in low concentrations in sera from uninfected mice. The amount of CL-specific IgM increased during the course of infection (1/30 at day 0 vs 1/1000 at day 200). Sera from naive mice and those infected for 10 days did not contain detectable amounts of CL-specific IgG1. At later time points, i.e., days 50 and 200, the mean titers of anti-CL IgG1 were 1/30 and 1/60, respectively. At day 200 postinfection, anti-CL IgG1 were observed in 80% of the mice, with titers ranging from 1/25 to 1/200.



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  		FIGURE 1. Generation of antilipid Abs in murine tuberculosis. Wild-type B6 mice were infected by aerosol and sera were collected before and 50 days after infection. Lipids were separated on HPTLC plates and either visualized by {alpha}-Naphtol (a) or fixed for immunostaining (b and c). After blocking, HPTLC plates were incubated with sera (diluted 1/25), and specific Abs were detected as described in Materials and Methods. Lane 1, Total M. tuberculosis lipid extract; lane 2, CL; lane 3, TDM; lane 4, PIM preparation (the uppermost band is residual Triton X-114 from the purification process). A total of 50 µg were applied (except for lane 1 in d where 200 µg were used). The result shown is representative of three experiments. e, Sera were collected at the time points postinfection indicated. Titers of anti-CL Abs were measured using ELISA plates coated with CL, and peroxidase-coupled secondary Abs to murine IgM or IgG1. Shown are titers with an OD above background value plus 2 x SD. Each symbol represents an individual animal. The detection limit (1/25) is indicated by a bar.

 
Immunization with CL/anti-CL Ab complexes induces high titers of IgG1 to CL

To further elucidate the mechanism of anti-CL IgG1 induction, an immunization schedule to generate anti-CL Abs was used, which was described before by others to elicit experimental murine APS (12). Mice were immunized s.c. on days 0 and 10 with CL complexed to either purified human anti-CL IgG from SLE patients, a murine monoclonal anti-CL IgG1 Ab, or to BSA, and sera were tested at days 0, 10, and 20 (16). Using this immunization schedule, only low amounts of CL-specific IgM and IgG3, and no IgG2a and IgG2b were detected (data not shown). In contrast, high IgG1 titers to CL were achieved by immunization with CL complexed to either purified IgG from SLE patients which contain high titers of anti-CL Abs or a murine CL-specific mAb, but not with BSA (Ref. 17 ; Fig. 2Go). By day 10 after the first immunization, the average anti-CL IgG1 titer was already 1/350 in both cases, and had increased at day 20 postimmunization to levels of ~1/1000–1/2000.



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  		FIGURE 2. The CL-specific IgG1 Abs are induced by s.c. immunization with CL/anti-CL Ab complexes. Wild-type B6 mice or IgM-/- mice were immunized on days 0 and 10 with 200 µg CL complexed to 300 µg purified human antiphospholipid IgG Abs, the monoclonal T1.8 anti-CL Ab (70 µg), or BSA in Al(OH)3. Sera were collected on days 0, 10, and 20, and the amount of anti-CL IgG1 was measured. Shown are titers with an OD above background value plus 2 x SD. Each symbol represents an individual animal (*, statistical significance, p < 0.05, and **, p < 0.01, Mann-Whitney U test). The detection limit (1/25) is indicated by a bar.

 
The murine anti-CL Ab used for complexing was of IgG1 isotype, therefore, residual Abs used for immunization could contribute to IgG1 titers. This was controlled by using the identical immunization schedule in B cell-deficient IgM-/- mice. At days 10 and 20 postimmunization, sera from these mice contained the administered anti-CL Ab at titers of 1/100, which was significantly lower, and did not increase after the boost, compared with immunized wild-type mice (Fig. 2Go, p < 0.005). This indicates that immunization of mice with CL complexed to a specific Ab elicited a strong anti-CL IgG1 response. This response depended on complexing of CL to an anti-CL Ab, because immunization with either BSA/CL or CL alone did not induce CL-specific IgG1 Abs (Fig. 2Go and data not shown).

IL-4 and T cells are required for anti-CL IgG1 Ab production

IgG isotype switching is usually induced by activation of IgM expressing B cells via IL-4 secreted by T cells (18). To further elucidate the mechanism whereby anti-CL IgG1 are induced, IL-4-/-, nu/nu, and Fc{gamma}RIII-/- mice were immunized with CL/anti-CL Ab complexes. As an internal control, B6 and IgM-/- mice were also immunized. By 10 days after the first immunization, differences between these mouse strains were detected, which were even more pronounced after 20 days (Fig. 3Go). Compared with titers of 1/1600–1/6400 as observed in B6 mice, IL-4-/- mice showed markedly reduced titers. This indicates that IL-4 was critical for promoting the class switch to IgG1 Abs against CL. Furthermore, T cells were required for generation of high titers of anti-CL IgG1, as revealed by low titers in nu/nu mice compared with B6 mice (Fig. 3Go). Notably, anti-CL IgG1 in nu/nu mice with titers of 1/800–1/1600 at 20 days postimmunization were significantly higher than the background titers seen in IgM-/- mice, suggesting IgG1 production independent of conventional T cells. Since only CL complexed to a specific IgG1 Ab induced anti-CL IgG1, the role of FcR was determined. Lack of the major receptor for IgG1, Fc{gamma}RIII, did not influence the generation of high titers of anti-CL IgG1 (Fig. 3Go). Thus, complexing of CL to Abs is not necessary for uptake by APC via the Fc{gamma}RIII for the induction of anti-CL IgG1. This result rather suggests that anti-CL Abs resolve lipid complexes to facilitate better recognition of CL by Ag receptors of B cells or other cellular receptors.



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  		FIGURE 3. Induction of anti-CL IgG1 depends on IL-4, and partially on T cells. Wild-type B6, Fc{gamma}RIII-/-, IL-4-/-, IgM-/-, and nu/nu mice were immunized s.c. on days 0 and 10 with 200 µg CL complexed to 70 µg anti-CL Ab (T1.8) in Al(OH)3. Sera were taken on days 0, 10, and 20, and the amount of anti-CL IgG1 was measured. Shown are titers with an OD above background value plus 2 x SD. Each symbol represents an individual animal. Significant differences at day 20 were observed between B6 and nu/nu mice (p = 0.0079), B6 and IL-4-/- mice (p = 0.0079), and between nu/nu and IgM-/- mice (p = 0.0317). *, statistical significance, p < 0.05 and **, p < 0.01, Mann-Whitney U test.

 
A specific T cell population is not required for the class switch of anti-CL to IgG1

IL-4 is primarily produced by specific CD4+ Th2 cells (18). To determine whether a unique T cell population provided help for optimal IgG1 production to CL, mice deficient in specific T cell populations were used. Mice lacking {gamma}{delta} T cells ({gamma}-TCR-/-), {alpha}{beta} T cells ({beta}-TCR-/-), or V{alpha}14J{alpha}281 NK T cells (J{alpha}281-/-), as well as mice deficient in CD4 or CD8 T cells due to deletion of MHC class I (Kb-/-Db-/-) or MHC class II genes (A{beta}-/-), respectively, were used. All mouse mutants mounted a comparable IgG1 response to CL, which was detectable already at day 10 after the first immunization and further increased by day 20 (Table IGo). Therefore, the induction of the isotype class switch to IgG1 in CL-specific B cells is not dependent on a single T cell subset.


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  		Table I. Induction of anti-CL IgG1 in different knockout mouse strains1

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
During mycobacterial infection, an array of pathogen-derived lipids induce specific Abs. In this study, we show that CL is one of the major B cell Ags of M. tuberculosis in mice eliciting IgM, but also specific IgG1 Abs, and IL-4 is required for induction of class switch to IgG1 in CL-specific B cells. Immunization with CL/anti-CL Ab complexes revealed that complexing of CL to an Ab promoted the IgG1 response to CL, whereas specific uptake of CL/anti-CL complexes via Fc{gamma}RIII was dispensable. Although T cells were critical for the generation of high titers of CL-specific IgG1, the facilitation for class switch was not provided by a single T cell population.

Earlier studies revealed that large quantities of mycobacterial cell wall material, including LAM, PIM, and CL, are released during infection (2, 4). In this study, we show that Abs against such lipids are generated during infection of mice with M. tuberculosis. This is in accordance with studies in tuberculosis patients who develop Abs to LAM, CL, and TDM (6, 8). In mice, we found that anti-CL Abs represent a major part of the B cell response to mycobacterial lipids during M. tuberculosis infection. Low titers of IgM Abs to CL were already detectable in mice before infection, probably representing part of the natural Ab repertoire present in naive mice (19). The titers of CL-specific IgM Abs increased in response to mycobacterial infection, possibly as a result of the continuous release of cell wall lipids from mycobacteria. Later in infection, IgG1 Abs to CL are generated in addition to IgM. The isotype switch of a lipid-specific B cell from IgM to IgG1 can hardly be explained by the help from Ag-specific CD4 T helper cells, due to the fact that lipids are not recognized by conventional CD4 T cells in mice. Furthermore, CL is not a polyvalent T cell-independent Ag, as exemplified by LPS, precluding direct stimulation of B cells via cross-linking of their Ag receptor (20).

Induction of anti-CL IgG1 was dependent on the administration of CL complexed to anti-CL Abs. This finding could explain the delayed appearance of this isotype at 30 days after mycobacterial infection. At this time point, high levels of IgM Abs to CL were detected which could form immune complexes with CL to promote isotype switching. Induction of anti-CL Abs appears to be independent of a single murine isotype used for immunization, i.e., IgG1, because CL complexed to human serum IgG can also induce anti-CL IgG1 in mice. This is in line with reports by Subang et al. (11) that, independent of anti-CL Abs, immunization with CL complexed to serum {beta}2-glycoprotein I can elicit APS in mice.

Anti-CL Ab generation in patients with syphilis or tuberculosis is a well-known phenomenon, and it is intriguing to speculate on a causative relation between APS and an infectious process (21). Recently, APS in humans has been associated with infections by salmonella, helicobacter, and other pathogens (21, 22). Yet clinical symptoms of APS have not been reported in patients with tuberculosis or other mycobacterial infections, despite the presence of anti-CL Abs in their sera. The CL-binding properties of anti-CL Abs elicited upon infection as compared with those produced during autoimmune conditions appear to be different. {beta}2-glycoprotein I has been reported to be required for binding to CL of autoimmune anti-CL Abs, but not for binding of infection-induced Abs (21). However, in leprosy patients, both types of anti-CL Abs have been demonstrated (23). Our ELISA system did not permit the differentiation between these two types of anti-CL Abs due to the presence of serum in the experimental set up.

It has been shown by others that CL/anti-CL Ab complexes and anti-CL Abs alone can induce anti-CL IgG and APS in mice similar to autoimmune conditions in humans (9, 12, 16). We used the APS model using CL/anti-CL Ab complexes to determine whether CL-specific isotype switching to IgG1 depends on IL-4 or a distinct T cell population using various knockout mouse strains. IL-4 was crucial for IgG1-class switch, because IL-4-/- mice had markedly decreased titers of anti-CL IgG1 Abs compared with wild-type mice. Similarly, reduced IgG1 production upon treatment with anti-IL-4 Abs has been reported in the case of autoantibodies to dsDNA in mice prone to the autoimmune disease lupus nephritis (24). The cellular source of IL-4 includes cells of the mast cell/basophil lineage, eosinophils, CD4+ NK T cells, {gamma}{delta} T cells, and conventional CD4+ Th2 cells (25, 26, 27, 28, 29). In the case of anti-CL IgG1, T cells were the dominant cell type providing IL-4 because T cell-deficient nu/nu mice had significantly reduced anti-CL IgG1 titers. Yet nu/nu mice developed small amounts of IgG1 Abs to CL, which increased upon boosting, indicating a T cell-independent mechanism for Ig class switch for lipid-specific Abs. This could be due to IL-4 produced by eosinophils, mast cells, or bone marrow resident pre-B cells (25, 26, 30). The pre-B cells have been shown to rapidly produce IL-4 in response to bacterial products including LAM (30). Preliminary data indicate that CL itself can also stimulate bone marrow cells from mice lacking B and T cells (recombination activator gene-/-) to secrete IL-4 (our unpublished observations). Furthermore, nu/nu mice are not totally devoid of T cells and harbor small numbers of residual T cells, which could also account for the Ig class switch seen in this mouse strain (31). IL-4 could direct the T cell response into the Th2 direction favoring generation of IgG1 to CL and induction of APS. This notion would be in line with a recent report showing that anti-idiotypic treatment ameliorates murine APS by decreasing anti-CL Ab titers, which is paralleled by a switch from a Th2 (IL-4) to a Th1 (IFN-{gamma}, IL-2) response (32).

However, optimal generation of anti-CL IgG1 Abs required T cells. To determine the T cell population responsible for induction of anti-CL IgG1 Abs, mice deficient in defined T cell populations were analyzed. Alterations in IgG1 Ab titers to CL were neither observed in {beta}-TCR -/- mice lacking {alpha}{beta} T cells, nor in A{beta}-/- mice lacking conventional CD4+ T helper cells, arguing against classical T cell help in IgG1 class switch of CL-specific B cells. It has been reported that Abs against GPI-linked parasite proteins depend on nonclassical T cell help by NK T cells which recognize the GPI-anchor via CD1d (26). In the case of anti-CL IgG1 class switch, NK T cells were not required because J{alpha}281-/- mice with a deletion in the predominant TCR repertoire of NK T cells had similar anti-CL IgG1 titers to wild-type mice. Similarly, mice lacking CD8 T cells (Kb-/-Db-/-) or {gamma}{delta}-T cells ({delta}-TCR-/-) did not show reduced titers of anti-CL IgG1 upon immunization. Hence, a unique T cell population cannot be singled out as the sole IL-4 producer inducing class switch of anti-CL Abs to IgG1, and the lack of a single T cell subset can be compensated by another one. This strongly indicates a vast redundancy of the helper function for B cells by different T cell populations.

To date, the way T cells are activated by CL to provide help for B cells remains unknown. It is possible that idiotypic determinants of Ig molecules contained within the CL/anti-CL Ab complexes used for immunization evoke idiotope-specific T cells with helper function (33). However, this notion does not explain the anti-CL IgG1 response seen after M. tuberculosis infection. In this case, bystander T cells could be activated by mycobacterial components directly or via mediators from infected macrophages or dendritic cells, a pathway still to be elucidated.

In conclusion, lipids and glycolipids require complexed Abs and IL-4 to induce Ig class switch to IgG1. The IL-4 is mainly derived from T cells, but its source cannot be pinpointed to a single T cell population. This T cell-dependent mechanism promotes the generation of Abs to lipids and glycolipids in both bacterial infections and autoimmune diseases such as the APS.


   Acknowledgments
 
We thank Dr. David G. Russell and Dr. Falk Hiepe for kindly sharing reagents, Dr. Peter Aichele for reading the manuscript, and Dr. Francois A. Lemonnier for kindly providing the Kb-/-Db-/- mice.


   Footnotes
 
1 This work was funded by grants from the Deutsche Forschungsgesellschaft, Sonderforschungsbereich 421 (to S.H.E.K. and U.E.S.); Bundesministerium für Bildung und Forschung, Stipend for Infectious Diseases (to U.E.S.), and joint project Mycobacterial Infection (to S.H.E.K.). Back

2 Address correspondence and reprint requests to Dr. Ulrich E. Schaible, Department of Immunology, Max-Planck-Institute for Infection Biology, Schumannstrasse 20/21, 10117 Berlin, Germany. E-mail address: schaible{at}mpiib-berlin.mpg.de Back

3 Abbreviations used in this paper: LAM, lipoarabinomannan; CL, cardiolipin; APS, antiphospholipid syndrome; PIM, phosphatidylinositol mannoside; TDM, trehalosedimycolate; SLE, systemic lupus erythematosus; HPTLC, high performance thin layer chromatography; GPI, glycoprotein I. Back

Received for publication October 30, 2001. Accepted for publication January 10, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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K. Fischer, E. Scotet, M. Niemeyer, H. Koebernick, J. Zerrahn, S. Maillet, R. Hurwitz, M. Kursar, M. Bonneville, S. H. E. Kaufmann, et al.
Mycobacterial phosphatidylinositol mannoside is a natural antigen for CD1d-restricted T cells
PNAS, July 20, 2004; 101(29): 10685 - 10690.
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