Subject(s)
Antibodies, Bacterial/immunology , Antigens, Bacterial/immunology , B-Lymphocyte Subsets/immunology , Staphylococcal Protein A/immunology , Staphylococcus aureus/immunology , Superantigens/immunology , Animals , Animals, Newborn , Antibodies, Bacterial/chemistry , Antibodies, Bacterial/metabolism , Antigen-Antibody Reactions , Antigens, Bacterial/chemistry , Antigens, Bacterial/metabolism , Binding Sites , Clonal Deletion , Crystallography, X-Ray , Evolution, Molecular , Humans , Immunoglobulin Fab Fragments/immunology , Immunoglobulin Fab Fragments/metabolism , Immunoglobulin Fc Fragments/metabolism , Immunoglobulin G/metabolism , Immunoglobulin Heavy Chains/metabolism , Immunoglobulin Variable Region/metabolism , Mice , Mice, Transgenic , Protein Structure, Tertiary , Receptors, Antigen, B-Cell/immunology , Staphylococcal Protein A/chemistry , Staphylococcal Protein A/metabolism , Structure-Activity Relationship , Superantigens/chemistry , Superantigens/metabolism , T-Lymphocytes/immunology , Xenopus laevis/immunologyABSTRACT
The bacterial toxin protein A from Staphylococcus aureus (SpA) interacts with B cell antigen receptors encoded by variable region heavy chain (V(H)) clan III genes via a V region framework surface that has been highly conserved during the evolution of the adaptive immune system. We have investigated the consequences of exposure to this prototypic B cell superantigen, and found that treatment of neonates or adults induces a T cell-independent deletion of a large supraclonal set of susceptible B cells that includes clan III/V(H) S107 family-expressing lymphocytes. In studies of different SpA forms, the magnitude of the induced deletion directly correlated with the V(H)-specific binding affinity/avidity. Upon cessation of SpA exposure, the representation of conventional splenic (B-2 subset) lymphocytes normalized; however, we found that the V(H) family-restricted deficit of peritoneal B-1 cells persisted. SpA treatment also induced a persistent loss of splenic S107-mu transcripts, with a loss of certain natural antibodies and specific tolerance to phosphorylcholine immunogens that normally recruit protective antimicrobial responses dominated by the S107-expressing B-1 clone, T15. These studies illustrate how a B cell superantigen can exploit a primordial Achilles heel in the immune system, for which B-1 cells, an important source of natural antibodies and host immune responses, have special susceptibility.
Subject(s)
B-Lymphocytes/immunology , Genes, Immunoglobulin , Receptors, Antigen, B-Cell/immunology , Staphylococcal Protein A/immunology , Superantigens/immunology , Adult , Animals , Antibody Affinity , Flow Cytometry , Humans , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Variable Region/genetics , Infant, Newborn , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Receptors, Antigen, B-Cell/genetics , T-Lymphocytes/immunologyABSTRACT
Evolution of the Ab system has yielded three clans of VH region genes that are represented in almost every known higher species with an adaptive immune system. These clans are defined by sequence homologies primarily in highly conserved framework (FR) subdomains, which serve a scaffolding function maintaining the conformation of loops responsible for Ag binding. Structural analyses indicate that the VH FR1 and FR3 form a conserved composite exposed surface, which has been implicated in interactions with B cell superantigens. To directly investigate the expression of clan-defined supraclonal sets, we exploited the evolutionary distance of the chicken immune system and the selection power of phage display, to derive Abs diagnostic for clan III Ig. Using a specially tailored immunization and selection strategy, we created recombinant avian single chain Fv Abs specific for the clan III products, including those from the human VH3 family, and the analogous murine 7183, S107, J606, X24, and DNA4 families, and binding was competitive with natural B cell superantigens. The archetype, LJ-26, was demonstrated to recognize a clan-specific surface expressed in diverse mammalian, and also the Xenopus and chicken, immune systems. In flow-cytometric studies with LJ-26, we found that treatment of heterozygous T15i transgenic mice with a model B cell superantigen induced a clan III-restricted clonal deletion. These studies demonstrate the utility of a novel recombinant serologic reagent to study the composition of the B cell compartment and also the consequences of B cell superantigen exposure.
Subject(s)
Antibodies, Monoclonal/chemistry , Antibody Specificity , Clonal Deletion , Immunoglobulin Heavy Chains/chemistry , Immunoglobulin Variable Region/chemistry , Superantigens/pharmacology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/metabolism , Antigens, Bacterial/metabolism , B-Lymphocytes/immunology , Binding Sites, Antibody , Binding, Competitive/immunology , Chickens , Conserved Sequence , Epitopes, B-Lymphocyte/metabolism , Evolution, Molecular , Flow Cytometry , Humans , Immunoglobulin Fab Fragments/metabolism , Immunoglobulin Heavy Chains/metabolism , Immunoglobulin Variable Region/metabolism , Immunoglobulins/metabolism , Mice , Mice, Transgenic , Models, Immunological , Molecular Sequence Data , Protein Conformation , Recombinant Proteins/immunology , Recombinant Proteins/metabolismABSTRACT
ProtoClear is a proprietary technique for clearing albumin and immunoglobulin G (IgG) from human serum samples. Albumin constitutes 57-71% of total serum protein and IgG ranges from 8-26%. Removal of these two proteins alone clears approximately 75% of the total protein present in serum and allows the detection of the remaining proteins that are present in far lower concentrations. ProtoClear effectively removed >95% of human serum albumin (HSA) and >97% of human IgG as measured by an anti-HSA competitive immunoassay and a radial immunodiffusion assay, respectively. ProtoClear was far more specific at removing albumin and IgG than Cibracon Blue Dye chromatography (Cibracon Blue), the typically utilized alternative. Comparing two-dimensional (2-D) gels of serum cleared by either Cibracon Blue or by ProtoClear, it was apparent that Cibracon Blue removed a number of proteins in addition to albumin. Following removal of albumin and IgG from serum, we found a significant improvement in the resolution of polypeptide spots detected on two-dimensional gels.
