Submolecular Specificity Research Articles

Human 60-kDa Heat Shock Protein Is a Target Autoantigen of T Cells Derived from Atherosclerotic Plaques

Epidemiological studies suggest the potential importance of an inflammatory component in atherosclerosis and support the hypothesis that immune responses to Ags of pathogens cross-react with homologous host proteins due to molecular mimicry. Protein candidates involved may be the stress-induced proteins known as heat shock proteins (HSP). In this study, we report that atherosclerotic plaques harbor in vivo-activated CD4(+) T cells that recognize the human 60-kDa HSP. Such in vivo-activated 60-kDa HSP-specific T cells are not detectable in the peripheral blood. In patients with positive serology and PCR for Chlamydia pneumoniae DNA, but not in patients negative for both, most of plaque-derived T cells specific for human 60-kDa HSP also recognized the C. pneumoniae 60-kDa HSP. We characterized the submolecular specificity of such 60-kDa HSP-specific plaque-derived T cells and identified both the self- and cross-reactive epitopes of that autoantigen. On challenge with human 60-kDa HSP, most of the plaque-derived T cells expressed Th type 1 functions, including cytotoxicity and help for monocyte tissue factor production. We suggest that arterial endothelial cells, undergoing classical atherosclerosis risk factors and conditioned by Th type 1 cytokines, express self 60-kDa HSP, which becomes target for both autoreactive T cells and cross-reactive T cells to microbial 60-kDa HSP via a mechanism of molecular mimicry. This hypothesis is in agreement with the notion that immunization with HSP exacerbates atherosclerosis, whereas immunosuppression and T cell depletion prevent the formation of arteriosclerotic lesions in experimental animals.

Molecular mimicry between Helicobacter pylori antigens and H+, K+ --adenosine triphosphatase in human gastric autoimmunity.

Autoimmune gastritis and Helicobacter pylori–associated gastric atrophy develop through similar mechanisms involving the proton pump H+,K+–adenosine triphosphatase as autoantigen. Here, we report that H. pylori–infected patients with gastric autoimmunity harbor in vivo–activated gastric CD4+ T cells that recognize both H+,K+–adenosine triphosphatase and H. pylori antigens. We characterized the submolecular specificity of such gastric T cells and identified cross-reactive epitopes from nine H. pylori proteins. Cross-reactive H. pylori peptides induced T cell proliferation and expression of T helper type 1 functions. We suggest that in genetically susceptible individuals, H. pylori infection can activate cross-reactive gastric T cells leading to gastric autoimmunity via molecular mimicry.

Antibody and T-cell recognition of α-bungarotoxin and its synthetic loop-peptides

Peptides representing the loops and surface regions of α-bungarotoxin (BgTX) and control peptide analogs in which these sequences were randomized were synthesized and used to map the recognition profiles of the antibodies and T-cells obtained after BgTX immunization. Also, the abilities of anti-peptide antibodies and T-cells to recognize the immunizing peptide and BgTX were determined. Three regions of BgTX were immunodominant by both rabbit and mouse anti-BgTX antibodies. These regions resided within loops L1 (residues 3–16), L2 (residues 26–41) and the C-terminal tail (residues 66–74) of the toxin. The regions recognized by BgTX-primed T-lymphocytes were mapped in five mouse strains: C57BL 6(H-2 b) , Balb c (H-2 d ), CBA (H-2 k ), C3H He (H-2 k ) and SJL (H-2 s ). The H-2 b and H-2 d haplotypes were high responders to BgTX, while the H-2 k and H-2 s were intermediate responders. The T-cell recognition profile of the peptides varied with the haplotype, consistent with Ir gene control of the responses to the individual regions. The submolecular specificities of antibodies and T-cells were compared in three of the mouse strains ( C57BL 6 , Balb c and SJL). In a given mouse strain, there were regions that were strongly recognized by both antibodies and T-cells as well as regions that were predominantly recognized either by antibodies or by T-cells. The peptides were used as immunogens in their free form (i.e. without coupling to any carrier) in two of the mouse strains, Balb c and SJL. In both mouse strains, the peptides gave strong antibody responses. Antibodies against peptide L2 showed the highest binding to intact BgTX. Antibodies against the other peptides exhibited lower binding activity to the intact toxin, and this activity was dependent on the peptide and the mouse strain. The response of peptide-primed T-cells to a given immunizing peptide was not related to whether this region was immunodominant with BgTX-primed T-cells. The ability of peptide-primed T-cells to recognize the intact toxin varied with the peptide and was dependent on the host strain. These results indicate that anti-peptide antibody and T-cell responses are also under genetic control and that their ability to cross-react with the parent toxin is not only dependent on the conformational exposure of the correlate region in intact BgTX.

Antigen Presentation by Non-Immune B-Cell Hybridoma Clones: Presentation of Synthetic Antigenic Sites Reveals Clones that Exhibit no Specificity and Clones that Present Only One Epitope

Recently, we reported the preparation and antigen-presenting properties of hybridoma B-cell clones obtained after fusing non-secreting, non-antigen presenting Balb/c 653-myeloma cells with non-immune SJL spleen cells. It was found that antigen presentation at the clonal level can be specific or non-specific, depending on the particular B-cell clone. In the present work, one specific and one general presenter B-cell clones were tested for their epitope presentation ability to SJL T-cells that were specific to lysozyme or myoglobin. B-cell clone A1G12, a general presenter which presented both lysozyme and myoglobin to their respective T-cell lines, was found to present all five myoglobin epitopes while clone A1L16, a lysozyme specific presenter presented only one of the three epitopes of lysozyme. The latter reveals a hitherto unknown submolecular specificity (to a given epitope within a protein) for antigen presenting cells at the clonal level. Therefore, the specificity of T-cell recognition does not only derive from the T-cell but may also be dependent on the epitope specificity of the antigen-presenting B-cell.

Non-cross-reactive monoclonal antibodies to human chorionic gonadotropin generated after immunization with a synthetic peptide.

Noncross-reactive monoclonal antibodies specific for human chorionic gonadotropin (hCG) were obtained after pre-selection for submolecular specificity with a synthetic peptide immunogen. Mice were immunized with a synthetic peptide representing a segment unique to the beta-subunit of hCG (amino acid residues 109-145), conjugated to diphtheria toxoid. We then derived nine different hybridomas that secreted monoclonal antibodies reactive with both native hCG and isolated C-terminal peptide, after somatic cell hybridization of immune spleen cells with a nonsecretory myeloma cell line. None of the nine monoclonal antibodies, termed beta-hCG-CTPa1----a9, reacted with hLH, hFSH, or hTSH, although these pituitary hormones display extensive amino acid sequence homology with hCG. The noncross-reactive anti-beta-hCG monoclonal antibodies show apparent association constants on the order of 10(9) to 10(10) M-1. A sandwich-type enzyme-linked immunosorbent assay was set up with cut-off values of around 5 mIU/ml. These antibodies might have important implications for: a) improving the diagnosis and clinical management of pregnancy; b) monitoring the course of development of carcinomas which secrete the hormone, through in vitro assays or in vivo radioimmunodetection; c) evaluating the antibodies' therapeutic potential against such carcinomas; d) studying the biologic functions of the C-terminal segment of beta-hCG; and e) addressing the anti-fertility effect of antibodies raised against that segment.

Lens junctional protein: analyzing MP26 with monoclonal antibodies.

Specific antibodies are versatile tools for analyzing cell surface proteins. This study involves the characterization of monoclonal antibodies which are specific for the junctional protein found in the lens fiber cell. This protein can be expected to include regions on the external membrane surface for junction formation, others on the cytoplasmic surface for regulation of junctional properties and, if cell-cell channels are indeed involved, transmembrane domains forming the hydrophilic connection between adjacent cytoplasms. Antibodies to these various regions would provide for an experimental analysis of the junctional protein, e.g., the identification of "active sites" for junction formation. Three monoclonal antibodies specific for the lens junctional protein in the chicken are described here. The first, termed B2, also recognizes the bovine junctional protein, MP26 (5). We have characterized the submolecular specificity of B2 and have found that it binds approximately ten amino acid residues from the C-terminus of MP26. In isolated lens junction preparations, B2 binds to the cytoplasmic surfaces of the lens junctions (both 12 nm and 16 nm thick forms). Thus, we consider MP26 a component of the lens junction. Monoclonal A4, the second antibody considered in detail here, was produced by immunization with lens membranes after treatment with low pH. We have found that lens junctional membranes are separated, or "split," by treatment at pH 2.5-3.0. It appears that A4 binds to the external surface of the junctional membrane; EM studies to confirm this are in progress. In order to map the A4 binding site within the chicken junctional protein and to explore the arrangement of this protein within the membrane, a number of procedures were used to generate fragments of MP26. These included reactions with N-chlorosuccinimide and proteases after acid treatment. Antibody binding to fragments was evaluated with immunotransfer ("Western") procedures. These studies mapped the A4 binding site to the center of the molecule and suggested that MP26 projected externally from the membrane at two different points. These results are consistent with a recent model, based on sequence data (6), for the arrangement of MP26 within the bovine lens membrane.

Preparation of T-lymphocyte lines and clones with specificities to preselected protein sites by [formula omitted] passage with free synthetic peptides: Demonstration with myoglobin sites

Recently, this laboratory has demonstrated that antibodies to preselected regions of a protein can be obtained by immunization with free small synthetic peptides (6–7 residues) without conjugation to a carrier. In the present work, we report the use of free synthetic peptides representing myoglobin (Mb) antigenic sites to prepare T-cell lines and clones of preselected specificities. Lymph node cells from mice primed in vivo with sperm-whale Mb were periodically passaged in vitro with synthetic peptide. After several passages, the peptide-driven long term T-cell cultures responded to the intact protein and exclusively to the peptide that was used to drive the cells. From these cultures, T-cell clones were prepared that responded only to the driving peptide and to the whole protein. The ability to prepare T-cell lines and T-cell clones with preselected submolecular specificities to a protein by driving cultures with desired synthetic peptides affords an important and simple tool for basic immunological investigations and for clinical applications.

The fine specificity of regulatory T cells. IV. Idiotypic complementarity and antigen-bridging interactions in the anti-lysozyme response.

The hen egg-white lysozyme (HEL)-specific B cells which are activated in response to HEL share a cross-reactive idiotype as well as submolecular specificity. Helper T cells of two different specificities are implicated in their optimal activation, one recognizing the idiotype and the other, an epitope on the HEL molecule opposite to the one recognized by B cells. Thus, efficient T-B collaboration involves both idiotypic complementary and antigen bridging.