Direct measurement of antigen binding properties of CD1 proteins using fluorescent lipid probes.

CD1 proteins are antigen-presenting molecules that bind foreign and self-lipids and stimulate specific T cell responses. In the current study, we investigated ligand binding by CD1 proteins by developing a fluorescent probe binding approach using soluble recombinant human CD1 proteins. To increase stability and yield, soluble group 1 CD1 (CD1b and CD1c) and group 2 CD1 (CD1d) proteins were produced as single chain secreted CD1 proteins in which beta2-microglobulin was fused to the N termini of the CD1 heavy chains by a flexible peptide linker sequence. Analysis of ligand binding properties of single chain secreted CD1 proteins by using fluorescent lipid probes indicated significant differences in ligand preference and in pH dependence of binding by group 1 versus group 2 CD1 proteins. Whereas group 1 CD1 isoforms (CD1b and CD1c) show stronger binding of nitrobenzoxadiazole (NBD)-labeled dialkyl-based ligands (phosphatidylcholine, sphingomyelin, and ceramide), group 2 CD1 (CD1d) proteins were stronger binders of small hydrophobic probes such as 1-anilinonaphthalene-8-sulfonic acid and 4,4'-dianilino-1,1'-naphthyl-5,5'-disulfonic acid. Competition studies indicated that binding of fluorescent lipid probes involved association of the probe with the hydrophobic ligand binding groove of CD1 proteins. Analysis of selected alanine substitution mutants of human CD1b known to inhibit antigen presentation showed that NBD-labeled lipid probe binding could be used to distinguish mutations that interfere with ligand binding from those that affect T cell receptor docking. Our findings provide further evidence for the functional specialization of different CD1 isoforms and demonstrate the value of the fluorescent lipid probe binding method for assisting structure-based studies of CD1 function.

Immunization with a mycobacterial lipid vaccine improves pulmonary pathology in the guinea pig model of tuberculosis.

Lipids and glycolipid molecules derived from Mycobacterium tuberculosis can be presented to T cells by CD1 cell-surface molecules in humans. These lipid-specific T cells are cytolytic, secrete pro-inflammatory cytokines and have bactericidal activity. Here, we describe studies in which lipids from M. tuberculosis were incorporated into liposomes with adjuvant and tested as vaccines in a guinea pig aerosol tuberculosis challenge model. Animals vaccinated with mycobacterial lipids showed reduced bacterial burdens in the lung and spleen at 4 weeks after infection. In addition, the lungs of lipid-vaccinated animals also had significantly less pathology, with granulomatous lesions being smaller and more lymphocytic. In contrast, animals receiving only vehicle control immunizations had granulomatous lesions that were larger and often contained caseous necrotic centers. Quantification of histopathology by morphometric analysis revealed that the overall percentage of lung occupied by diseased tissue was significantly smaller in lipid-vaccinated animals as compared to vehicle control animals. In addition, the mean area of individual granulomatous lesions was found to be significantly smaller in both lipid- and bacillus Calmette-Guerin-vaccinated guinea pigs. These data support an important role for lipid antigens in the immune response to M. tuberculosis infection, potentially through the generation of CD1-restricted T cells. Immunogenic lipids thus represent a novel class of antigens that might be included to enhance the protective effects of subunit vaccine formulations.

Characterization of guinea-pig group 1 CD1 proteins.

CD1 molecules are cell-surface glycoproteins with strong structural similarities to major histocompatibility complex (MHC) class I molecules, and studies in humans and mice have demonstrated that CD1 proteins perform the unique role of presenting lipid antigens to T lymphocytes. Our previous studies have shown that guinea-pigs, unlike the muroid rodents, have an extended family of group 1 CD1 genes. In the current study, we raised monoclonal anibodies (mAbs) against guinea-pig CD1 proteins and generated transfected cell lines expressing individual members of the guinea-pig CD1 family. Our results indicated that multiple members of the guinea-pig CD1 family, including members that are homologous to the human CD1b and CD1c proteins, are expressed at the protein level in transfected cells and in specialized antigen-presenting cells such as monocyte-derived dendritic cells. In addition, CD1 proteins, especially guinea-pig CD1b3, were expressed on a large number of B cells in the guinea-pig, and CD1 expression appeared to be regulated by B-cell maturation or differentiation. Interestingly, three different patterns of intracellular localization were observed for the various guinea-pig CD1 isoforms, a finding that is reminiscent of the distinct patterns of intracellular localization that have been previously demonstrated for human CD1a, CD1b and CD1c. Taken together, these results provide further evidence for substantial similarities between the guinea-pig and human CD1 systems, thus supporting the possibility that the guinea-pig may offer significant advantages as an animal model for the study of the in vivo role of CD1 proteins in infectious and autoimmune diseases.

Induction of CD1-restricted immune responses in guinea pigs by immunization with mycobacterial lipid antigens.

Group 1 CD1 molecules have been shown to present lipid and glycolipid Ags of mycobacteria to human T cells. However, a suitable animal model for the investigation of this component of antimycobacterial immunity has not yet been established. Previously, we found that guinea pigs express multiple isoforms of group 1 CD1 proteins that are homologous to human CD1b and CD1c. In this study, we show that CD1-restricted T cell responses can be generated in guinea pigs following immunization with lipid Ags from Mycobacterium tuberculosis. Splenic T cells from lipid Ag-immunized guinea pigs showed strong proliferative responses to total lipid Ags and partially purified glycolipid fractions from M. tuberculosis. These lipid Ag-reactive T cells were enriched in CD4-negative T cell fractions and showed cytotoxic activity against CD1-expressing guinea pig bone marrow-derived dendritic cells pulsed with M. tuberculosis lipid Ags. Using guinea pig cell lines transfected with individual CD1 isoforms as target cells in cytotoxic T cell assays, we found that guinea pig CD1b and CD1c molecules presented M. tuberculosis glycolipid Ags to T cells raised by mycobacterial lipid immunization. These results were confirmed using a T cell line derived from M. tuberculosis lipid Ag-immunized guinea pigs, which also showed CD1-restricted responses and cytolytic activity. Our results demonstrate that CD1-restricted responses against microbial glycolipid Ags can be generated in vivo by specific immunization and provide support for the use of the guinea pig as a relevant small animal model for the study of CD1-restricted immune responses to mycobacterial pathogens.