Antibodies that bind complex glycosaminoglycans accumulate in the Golgi.
ABSTRACT: Light (L) chains that edit anti-DNA heavy (H) chains rescue B-cell development by suppressing DNA binding. However, exceptional editor L chains allow B cells to reach splenic compartments even though their B-cell receptors remain autoreactive. Such incompletely edited B cells express multireactive antibodies that accumulate in the Golgi and are released as insoluble, amyloid-like immune complexes. Here, we examine examples of incomplete editing from the analysis of variable to joining (VJ) gene junction of the variable (V?x) editor L chain. When paired with the anti-DNA heavy chain, VH56R, the V?x variants yield antibodies with differing specificities, including glycosaminoglycan reactivity. Our results implicate these specificities in the evasion of receptor editing through intracellular sequestration of IgM and the release of insoluble IgM complexes. Our findings can be extrapolated to human L chains and have implications for understanding a latent component of the Ig repertoire that could exert pathogenic and protective functions.
Project description:Pathogenic anti-DNA antibodies expressed in systemic lupus erythematosis bind DNA mainly through electrostatic interactions between the positively charged Arg residues of the antibody complementarity determining region (CDR) and the negatively charged phosphate groups of DNA. The importance of Arg in CDR3 for DNA binding has been shown in mice with transgenes coding for anti-DNA V(H) regions; there is also a close correlation between arginines in CDR3 of antibodies and DNA binding. Codons for Arg can readily be formed by V(D)J rearrangement; thereby, antibodies that bind DNA are part of the preimmune repertoire. Anti-DNAs in healthy mice are regulated by receptor editing, a mechanism that replaces κ light (L) chains compatible with DNA binding with κ L chains that harbor aspartic residues. This negatively charged amino acid is thought to neutralize Arg sites in the V(H). Editing by replacement is allowed at the κ locus, because the rearranged VJ is nested between unrearranged Vs and Js. However, neither λ nor heavy (H) chain loci are organized so as to allow such second rearrangements. In this study, we analyze regulation of anti-DNA H chains in mice that lack the κ locus, κ-/κ- mice. These mice show that the endogenous preimmune repertoire does indeed include a high frequency of antibodies with Arg in their CDR3s (putative anti-DNAs) and they are associated mainly with the editor L chain λx. The editing mechanisms in the case of λ-expressing B cells include L chain allelic inclusion and V(H) replacement.
Project description:To investigate the manner in which B cells with lambda light (L) chains undergo receptor editing, we have studied hybridoma panels from 56R/kappa-deleted (kdel) mice. 56R/kdel mice only produce four L chains (lambda1, lambda2, lambda3, and lambdaX). They also have a simplified heavy (H) chain repertoire: All B cells start out with a 56R anti-DNA H chain. A few frankly autoreactive 56R lambda1 cells appear to escape into the periphery, but the majority of the peripheral B cell repertoire in 56R/kdel is made up of B cells expressing the 56R H chain with the lambdaX L chain. Surprisingly, 56R lambdaX B cells are multireactive, binding to a variety of self and nonself antigens, including dsDNA (albeit at reduced affinity compared with the other lambda L chains). Another significant population in the 56R/kdel mouse consists of allelically included B cells that express lambdaX along with another L chain. The multireactivity of both 56R lambdaX and 56R lambdaX/lambda1 receptors could contribute to autoimmunity if these B cells were to become activated. Also found among 56R/kdel hybridomas are clones that have inactivated the H chain and secrete only L chains. These clones may represent products of exhaustive rearrangement. Multireactivity, allelic inclusion, and L chain secretion are three consequences of editing at the lambda locus that may predispose toward the development of autoimmunity.
Project description:To determine the molecular and functional properties of human rheumatoid factors (RF), we established stable hybridomas and Epstein-Barr virus-transformed B cell lines from the synovial fluid or peripheral blood of three patients with rheumatoid arthritis and one patient with systemic lupus erythematosus. 17 cell lines were obtained that produced high-titer immunoglobulin M (IgM) RF that reacted exclusively with rabbit but not human IgG or IgG of other mammalian species. Certain anti-rabbit IgG RF also had specificity for other mammalian antigens (Ag), including cytoskeletal proteins and intracellular proteins found in HeLa cells, as well as for Ag present in an extract prepared from the cell wall of group A streptococci. 13 of the 17 RF contained lambda-type light (L) chains, of which 12 were classified serologically as members of the lambda-L chain variable region (V lambda) subgroup, designated V lambda III. The heavy chain V region (VH) and V lambda sequences of nine of these IgM lambda RF were determined at the cDNA level. Five VH genes in three VH families were used by these antibodies (Ab), including VH1 (dp21/1-4b and dp10 [51p1]/hv1051), VH3 (dp38/3-15 and dp77/13-21), and VH4 (dp70/4-4b). The deduced V gene-encoded amino acid sequences of the lambda chains of these IgM lambda RF confirmed their serological classification as lambda III, and they were further classified as members of the relatively uncommon V lambda III subgroup, designated V lambda IIIb. Based on cDNA analyses, nine were the product of three different V lambda III b germline genes. Two such genes, designated hsiggll150 and hsiggll295, were cloned and sequenced from genomic DNA. Unique combinations of these VH and V lambda III b genes could be related to distinctive patterns of reactivity among the IgM lambda RF. Although the VH and V lambda regions of these Abs were expressed primarily as germline-encoded sequences, four of nine multireactive Abs had extensive V region mutation, indicative of an Ag-driven process. The finding that lambda IIIb L chains are preferentially found among anti-rabbit IgG RF, and that some of these Ab have specificity for other protein, cellular, and bacterial Ag, provides new insight into the pathogenesis of RA and related diseases.
Project description:Tolerance to dsDNA is achieved through editing of Ig receptors that react with dsDNA. Nevertheless, some B cells with anti-dsDNA receptors escape editing and migrate to the spleen. Certain anti-dsDNA B cells that are recovered as hybridomas from the spleens of anti-dsDNA H chain transgenic mice also bind an additional, Golgi-associated antigen. B cells that bind this antigen accumulate intracellular IgM. The intracellular accumulation of IgM is incomplete, because IgM clusters are observed at the cell surface. In the spleen, B cells that express the heavy and light chains encoding this IgM are surface IgM-bright and acquire the CD21-high/CD23-low phenotype of marginal zone B cells. Our data imply that expression of an Ig that binds dsDNA and an additional antigen expressed in the secretory compartment renders B cells resistant to central tolerance. In the periphery, these B cells may be sequestered in the splenic marginal zone.
Project description:The processes that govern the generation of pathogenic anti-DNA autoantibodies in human systemic lupus erythematosus (SLE) are largely unknown. Autoantibodies may arise as a consequence of polyclonal B cell activation and/or antigen-driven B cell activation and selection. The role of these processes in humoral autoimmunity may be studied by molecular genetic analysis of immunoglobulin (Ig) variable (V) regions of antibodies that are characteristic of SLE. We have analyzed the gene elements that encode a high affinity, IgG anti-double-stranded DNA autoantibody secreted by a monoclonal Epstein-Barr virus (EBV)-transformed cell line derived from a patient with active SLE. In addition, we have identified, cloned, and sequenced the germline counterparts of the VH and VL genes expressed in this autoantibody. The comparison of both sets of gene elements shows that the autoantibody VH and VL regions harbor numerous somatic mutations characteristic of an antigen-driven immune response. The light chain expressed in this autoantibody is a somatically mutated variant of the kv325 germline gene that is frequently associated with paraproteins having autoantibody activity and with Ig molecules produced by malignant B cells that express the CD5 antigen. Furthermore, the utilized DH segment has been repeatedly found in multireactive, low affinity IgM anti-DNA autoantibodies from SLE patients and healthy individuals. These results suggest that pathogenic IgG anti-DNA autoantibodies in human SLE may arise through antigen-driven selection of somatic mutations in the gene elements that frequently encode multireactive IgM autoantibodies.
Project description:We developed a DNA barcoding method to enable high-throughput sequencing of the cognate heavy- and light-chain pairs of the antibodies expressed by individual B cells. We used this approach to elucidate the plasmablast antibody response to influenza vaccination. We show that >75% of the rationally selected plasmablast antibodies bind and neutralize influenza, and that antibodies from clonal families, defined by sharing both heavy-chain VJ and light-chain VJ sequence usage, do so most effectively. Vaccine-induced heavy-chain VJ regions contained on average >20 nucleotide mutations as compared to their predicted germline gene sequences, and some vaccine-induced antibodies exhibited higher binding affinities for hemagglutinins derived from prior years' seasonal influenza as compared to their affinities for the immunization strains. Our results show that influenza vaccination induces the recall of memory B cells that express antibodies that previously underwent affinity maturation against prior years' seasonal influenza, suggesting that 'original antigenic sin' shapes the antibody response to influenza vaccination.
Project description:We recently developed base editing, a genome-editing approach that enables the programmable conversion of one base pair into another without double-stranded DNA cleavage, excess stochastic insertions and deletions, or dependence on homology-directed repair. The application of base editing is limited by off-target activity and reliance on intracellular DNA delivery. Here we describe two advances that address these limitations. First, we greatly reduce off-target base editing by installing mutations into our third-generation base editor (BE3) to generate a high-fidelity base editor (HF-BE3). Next, we purify and deliver BE3 and HF-BE3 as ribonucleoprotein (RNP) complexes into mammalian cells, establishing DNA-free base editing. RNP delivery of BE3 confers higher specificity even than plasmid transfection of HF-BE3, while maintaining comparable on-target editing levels. Finally, we apply these advances to deliver BE3 RNPs into both zebrafish embryos and the inner ear of live mice to achieve specific, DNA-free base editing in vivo.
Project description:Caveolin-1 (Cav1) regulates the nanoscale organization and compartmentalization of the plasma membrane. Here we found that Cav1 controlled the distribution of nanoclusters of isotype-specific B cell antigen receptors (BCRs) on the surface of B cells. In mature B cells stimulated with antigen, the immunoglobulin M BCR (IgM-BCR) gained access to lipid domains enriched for GM1 glycolipids, by a process that was dependent on the phosphorylation of Cav1 by the Src family of kinases. Antigen-induced reorganization of nanoclusters of IgM-BCRs and IgD-BCRs regulated BCR signaling in vivo. In immature Cav1-deficient B cells, altered nanoscale organization of IgM-BCRs resulted in a failure of receptor editing and a skewed repertoire of B cells expressing immunoglobulin-? heavy chains with hallmarks of poly- and auto-reactivity, which ultimately led to autoimmunity in mice. Thus, Cav1 emerges as a cell-intrinsic regulator that prevents B cell-induced autoimmunity by means of its role in plasma-membrane organization.
Project description:Antibody variable regions are composed of a heavy and a light chain, and in humans, there are two light chain isotypes: kappa and lambda. Despite their importance in receptor editing, the light chain is often overlooked in the antibody literature, with the focus being on the heavy chain complementarity-determining region (CDR)-H3 region. In this paper, we set out to investigate the physicochemical and structural differences between human kappa and lambda light chain CDR regions. We constructed a dataset containing over 29,000 light chain variable region sequences from IgM-transcribing, newly formed B cells isolated from human bone marrow and peripheral blood. We also used a published human naïve dataset to investigate the CDR-H3 properties of heavy chains paired with kappa and lambda light chains and probed the Protein Data Bank to investigate the structural differences between kappa and lambda antibody CDR regions. We found that kappa and lambda light chains have very different CDR physicochemical and structural properties, whereas the heavy chains with which they are paired do not differ significantly. We also observed that the mean CDR3 N nucleotide addition in the kappa, lambda, and heavy chain gene rearrangements are correlated within donors but can differ between donors. This indicates that terminal deoxynucleotidyl transferase may work with differing efficiencies between different people but the same efficiency in the different classes of immunoglobulin chain within one person. We have observed large differences in the physicochemical and structural properties of kappa and lambda light chain CDR regions. This may reflect different roles in the humoral immune response.
Project description:In contrast to traditional CRISPR-Cas9 homology-directed repair, base editing can correct point mutations without supplying a DNA-repair template. Here we show in a mouse model of tyrosinaemia that hydrodynamic tail-vein injection of plasmid DNA encoding the adenine base editor (ABE) and a single-guide RNA (sgRNA) can correct an A>G splice-site mutation. ABE treatment partially restored splicing, generated fumarylacetoacetate hydrolase (FAH)-positive hepatocytes in the liver, and rescued weight loss in mice. We also generated FAH+ hepatocytes in the liver via lipid-nanoparticle-mediated delivery of a chemically modified sgRNA and an mRNA of a codon-optimized base editor that displayed higher base-editing efficiency than the standard ABEs. Our findings suggest that adenine base editing can be used for the correction of genetic diseases in adult animals.