Analysis of autoimmune bone marrow by antibody‐phage display: Somatic mutations and third complementarity‐determining region arginines in anti‐DNA γ and κ V genes

Abstract

Objective. To examine anti-double-stranded DNA (anti-dsDNA) IgG autoantibodies from the bone marrow of individuals with systemic lupus erythematosus (SLE).Methods. A library of single-chain variable fragments (scFv) was constructed from SLE bone marrow complementary DNA of ␥, , and isotype by cloning into the pHENIX phagemid vector. The library was screened with dsDNA in solution, and 2 anti-DNA phage, DNA1 and DNA4, were isolated and their Ig V genes sequenced. Soluble scFv corresponding to DNA1 and DNA4, and their heavy (H)-and light (L)-chain recombinants, were prepared, purified, and analyzed for binding to DNA by enzyme-linked immunosorbent assay.Results. DNA1 and DNA4 used different Ig H-chain (3-30 and 5-51, respectively) and L-chain (DPK15 and DPK22, respectively) V genes. The ratios of replacement mutations to silent mutations in DNA1 and DNA4 suggest that their V genes were selected for improved antigen binding in vivo. The recombinant between DNA4VH and DNA1VL showed the highest relative affinity for both single-stranded DNA and dsDNA. These 2 Ig subunits contained third complementarity-determining region arginines and had acquired the majority of replacement mutations.Conclusion. Anti-dsDNA IgG autoantibodies from the bone marrow of SLE patients exploit diverse V genes and cationic V-D-J and V-J junctions for DNA binding, and accumulate replacement mutations that enhance binding.Anti-double-stranded DNA (anti-dsDNA) antibodies represent an important specificity among the diverse autoantibodies that are found in systemic lupus erythematosus (SLE), yet their molecular characterization remains incomplete (for review, see ref. 1). AntidsDNA IgG autoantibodies use a variety of recurrent heavy (H)-and light (L)-chain V-region (V H and V L ) genes, and anti-dsDNA-combining sites often acquire somatic replacement mutations during the process of clonal expansion (2,3). Frequently, these replacement mutations generate arginine, asparagine, or lysine side chains whose functional groups are suitable for contacts with DNA. These replacement mutations, and the arginine residues found in the third complementaritydetermining region (CDR3) of many anti-DNA H and L chains, are consistent with positive selection of antidsDNA autoantibodies for improved binding to DNA. Selection for DNA binding was demonstrated in murine anti-dsDNA autoantibodies by reversion of somatic replacement mutations and by in vitro mutagenesis of arginines at the V-D-J junction (4,5).The detailed characterization of anti-dsDNA V genes has been further advanced by the expression of antibody fragments on the surface of bacteriophage (3,6). This heterologous expression system affords the opportunity to screen a large, highly diverse crosssection of the V-gene repertoire.

Blebs and Apoptotic Bodies Are B Cell Autoantigens

“…We used recombinant Ab fusion proteins: D56R/S76R, derived by in vitro mutagenesis and Ab engineering from 3H9, a murine autoantibody that exhibits dual specificity for DNA and anionic phospholipids (34), and DNA4/1, a human anti-DNA scFv, isolated by phage display of Igs encoded in SLE bone marrow mRNA (36). The binding of D56R/S76R was specific for cells in the execution phase of apoptosis, as inhibition of caspase activity largely blocked the interaction between the scFv and cells (Fig.…”

“…To explore the mechanism of Ab binding to apoptotic cells, we used D56R/S76R and compared its binding to DNA4/1, a human anti-DNA autoantibody (36). Confocal microscopy revealed that only D56R/S76R preferentially interacts with blebs on the apoptotic cell surface.…”

B cells in autoimmune diseases: Insights from analyses of immunoglobulin variable (Ig V) gene usage

“…Occasionally, clonally related B cells expressing V genes that have also been reported in disease-related autoAbs have been isolated from target tissues of patients with autoimmune diseases, such as SLE [1,4,75] or SS [53,76]. However, the number of patients whose Ig V genes have been analyzed in detail is far too small and the individual patterns of V gene usage are too divergent as to allow any correlations to be established with gene utilization of diseasespecific antibodies.…”

Anti–double‐stranded DNA antibodies, nucleosomes, and systemic lupus erythematosus: A time for new paradigms?

“…Experimental and descriptive observations collectively provide evidence that anti‐ssDNA and anti‐dsDNA antibodies arise by molecular and cellular processes common to classic T cell–dependent immune responses (14, 19, 20, 23). This generalized conclusion is based indirectly on analysis of the variable‐region structures of monoclonal anti‐DNA antibodies (14, 15, 20, 24–29), and directly on data from experiments in which immunologically normal mice were immunized with DNA–peptide complexes (21, 30–34). Furthermore, these studies demonstrated selection of positively charged structures in the heavy chain variable (V H ) regions of anti‐dsDNA antibodies, indicating that low‐avidity antibodies to DNA converge toward the negatively charged dsDNA with increasing avidity as the immune response progresses.…”