B D Stollar

Polyspecificity of Monoclonal Lupus Autoantibodies Produced by Human-Human Hybridomas

We studied the serologic properties of monoclonal autoantibodies that were produced by hybridomas derived from lymphocytes of patients with systemic lupus erythematosus. The hybridomas were made by fusion of a human lymphoblastoid cell line, GM 4672 (derived from a patient with multiple myeloma), with peripheral-blood or splenic lymphocytes from six patients with lupus. Thirty monoclonal autoantibodies, selected for their ability to react with denatured DNA, were analyzed. Eighteen of them reacted with three or more additional polynucleotides, including native DNA, left-handed double-helical DNA (Z-DNA), poly(l), and poly(dT). Ten reacted both with nucleic acids and the phospholipid cardiolipin. The multiple binding reactions of the monoclonal autoantibodies may be explained by the presence of appropriately spaced phosphodiester groups in both the polynucleotides and the phospholipid. The sharing of antigenic groups by polymers of different natures may contribute to the apparent diversity of serologic reactions in systemic lupus erythematosus. These findings suggest that DNA itself need not be the immunogenic stimulus for autoantibody formation in this disease.

Production of autoantibodies by human-human hybridomas.

Peripheral blood lymphocytes and splenocytes of patients with autoimmune disease were used to prepare human-human hybridomas that produce autoantibodies. Because exogenous immunization was not used, the hybridoma antibodies were derived from B cells that spontaneously produced autoantibodies. 108 hybrids grew from 4,254 wells (2.5%). Optimal conditions for obtaining hybridomas with the GM 4672 myeloma line included initial growth in 2-ml wells, the use of 44% polyethylene glycol, a mononuclear cell/GM 4672 cell ratio 5:1, and prior stimulation of the B lymphocytes with pokeweed mitogen. Hybridoma supernatants had activity against ssDNA, platelets, and erythrocytes. The results demonstrate the feasibility of producing human-human hybridomas from lymphocytes of patients with various autoimmune diseases.

High resolution detection of DNA–RNA hybrids in situ by indirect immunofluorescence

WE describe here a new method for the detection of RNA–DNA hybrids in cytological preparations with which we have revealed the locations of hybrid molecules on polytene chromosomes. The critical reagent is an antiserum raised in rabbits against poly(rA)·poly(dT) complexed with methylated bovine serum albumin, originally described by Stollar1. The specificity and resolving power of the indirect immunofluorescence procedure are demonstrated using in situ hybridisation of 5S rRNA (ribosomal RNA) to polytene chromosomes of Drosophila melanogaster as a model system. The method has significant advantages over the autoradiographic procedures2–5 used so far.

The structural basis for DNA binding by an anti-DNA autoantibody.

We have used single and multiple site-directed mutagenesis, and molecular modeling, to identify critical residues in the DNA binding site of MAb 2C10, an IgG anti-dsDNA autoantibody from an MRL/lpr lupus mouse. Simultaneous replacement of four Arg residues in the CDR3H abolished binding activity. With one exception, replacement of any one of these Arg residues reduced the activity to 20-50% of the unmutated scFv activity. Arg to Asp replacements had a slightly greater effect than Arg to Ala replacements. In the one exceptional case, replacement of Arg99 with Ala actually increased DNA binding five-fold and replacement by Asp had little effect. Mutation of Phe32 and Asn35 to A1a in CDRIH decreased DNA binding, whereas replacement of Arg31 with A1a had negligible effect. Ala substitution of any one of a cluster of Asp residues in CDR1L increased DNA binding three to six-fold, confirming previous findings that the L-chain of MAb 2C10 is not favorable for DNA binding. The L-chain does participate in shaping the selectivity of antigen binding, and mutation of CDR3L residue Asp92 or Asn93 caused a decrease in DNA binding activity. Directed mutagenesis, consistent with a molecular model, indicates that: several CDR amino acids contribute to DNA binding, without one residue dominating; both VH and VL CDR3 domains contribute to specificity of binding whereas the CDR1L hinders DNA binding. The results suggest a significant role for electrostatics in the interaction of DNA with MAb 2C10.

Immunoglobulin heavy chain gene expression in peripheral blood B lymphocytes.

cDNA libraries for IgM heavy chain variable regions were prepared from unmanipulated peripheral blood lymphocytes of two healthy people. Partial sequencing of 103 clones revealed VH gene family use and complete CDR3 and JH sequences. The libraries differed in the two subjects. In one persons cDNA the VH5 family was overexpressed and the VH3 family underexpressed relative to genomic complexity. In the second persons cDNA, VH3 was most frequently expressed. In both libraries, JH4 was most frequent. VH segments of several clones were closely related to those in fetal repertoires. However, there was also evidence of mutation in many cDNAs. Three clones differed from the single nonpolymorphic VH6 germline gene by 7-13 bases. Clones with several differences from VH5 germline gene VH251 were identified. CDR3 segments were highly diverse. JH portions of several CDR3s differed from germline JH sequences. 44% of the clones had DH genes related to the DLR and DXP families, most with differences from germline sequences. In 11 DLR2-related sequences, several base substitutions could not be accounted for by polymorphism. Thus, circulating IgM-producing B cell populations include selected clones, some of which are encoded by variable region gene segments that have mutated from the germline form.

Characterization of genomic poly(dT-dG).poly(dC-dA) sequences: structure, organization, and conformation.

Hybridization studies suggest the abundant presence of poly(dT-dG).poly(dC-dA) (TG-element), a potential Z-DNA sequence, in eucaryotic genomes. We have isolated and characterized TG-elements from different locations in the human genome: from randomly isolated clones, associated with the actin gene family, and linked to another repeated element. The results indicate that the following features are typical of these TG-elements: the elements consist of 20 to 60 base pairs of (dT-dG)n.(dC-dA)n, the sequences characterized in our study were not flanked by direct or inverted repeats, the sequences are interspersed rather than in satellite blocks, the elements are not usually associated with other repeated elements, and some of the elements are found near coding sequences or in introns. Studies on the conformation of a genomic TG-element in a supercoiled plasmid indicate several distinct properties of the TG-element: it is in the Z-form only at low ionic strength, S1 nuclease recognizes its Z-form with a marked preference for one of the B-Z junctions, and the sensitive region extends for 20 base pairs near the B-Z junction. In contrast to the result with the supercoiled plasmid, S1 nuclease failed to recognize the TG-element in minichromosomes.

Reaction of systemic lupus erythematosus antinative DNA antibodies with native DNA fragments from 20 to 1,200 base pairs.

Double-stranded DNA fragments of varying sizes were isolated and tested for binding to systemic lupus erythematosus (SLE) antinative DNA antibodies. Fragments of 20-25, 40-50, 90-110, and 160-180 base pairs (bp), along with intermediate-size pieces were isolated by preparative gel electrophoresis of a limited micrococcal nuclease digest of calf thymus DNA. Larger helical polynucleotides of 160-200, 380, 600-1,000, and 1,200 bp were isolated by preparative gel electrophoresis of DNA from chicken erythrocyte nucleosomes and oligonucleosomes. The fragments behaved as base-paired structures as tested by thermal denaturation, resistance to S1 nuclease, and serological assays with antibodies to native or denatured DNA. At a concentration of 0.27 muM, fragments of 20-25 bp were able to react with two SLE sera in competition with native DNA. With these and two other sera, DNA of 40-50 bp was a much more effective competitor. One serum required DNA greater than 180 bp for competition in the concentration range tested. Denatured fragments were much less effective than native fragments. The results emphasize the heterogeneity of SLE antinative DNA antibodies, confirm that secondary structure of the antigen is important for specific binding to these antibodies, and support the suggestion that bivalent binding to one molecule may be important for high functional affinity.

Anti-DNA antibodies: aspects of structure and pathogenicity

Abstract. Anti-DNA antibodies contribute to the pathology of systemic lupus erythematosus. Their depositon in tissue lesions could result from localization of preformed immune complexes of antibodies with DNA or nucleosomes, or from cross-reaction of anti-DNA antibodies directly with tissue proteins. Structural analyses contribute to understanding their pathogenic potential. Primary structures of lupus immunoglobulin G double-stranded DNA-binding autoantibodies are determined by immunoglobulin genes with mutated variable region segments, indicative of selection by immunizing antigen. Arginine, lysine and asparagine residues in complementarity-determining region favor DNA binding. Heavy-chain variable regions make major contributions to DNA binding; affinity and specificity of binding are modulated or can be abrogated by the light-chain variable domain. Crytallographic structure is known for a few antibody-DNA complexes and several ligand-free Fab fragments. Computer modeling supplements this limited information. Structural information of lupus antibody interactions with both DNA and cross-reacting molecules will support use of ligands to inhibit tissue deposition of the antibodies and prevent lesion formation in lupus.

Z-DNA-specific antibodies in human systemic lupus erythematosus.

Naturally occurring antibodies to left-handed Z-DNA have been shown to be present in the sera of human patients with systemic lupus erythematosus (SLE). These antibodies are of two types. One type reacts with both denatured DNA and Z-DNA. The other type is specific for Z-DNA and remained in the serum after removal of the cross-reactive antibody by extensive absorption on a denatured DNA affinity column. The antibodies appear to be specific for SLE and do not appear frequently in other rheumatic diseases. The presence of the antibody in SLE is correlated with the clinical manifestations of the disease, in parallel with antibodies to native and denatured DNA.

Repertoire cloning of lupus anti-DNA autoantibodies.

To investigate the autoantibody repertoire associated with SLE, we have created phage display IgG Fab libraries from two clinically active SLE patients and from the healthy identical twin of one of these patients. The libraries from the lupus discordant twins were found to both include unusually large representations of the V(H)5 gene family. By panning with DNA, the SLE libraries each yielded IgG anti-double-stranded (ds) DNA autoantibodies, which are characteristic of lupus disease. These included a V(H)5 autoantibody from the affected twin, that has a targeted cluster of mutations that potentially improves binding affinity. The recovered IgG anti-dsDNA autoantibodies expressed the same idiotypes associated with the in vivo IgG anti-dsDNA response of the respective SLE donor. Heavy-light chain shuffling experiments demonstrated a case in which the in vitro creation of anti-dsDNA binding activity required restrictive pairing of a heavy chain with Vlambda light chains similar to those in circulating anti-dsDNA autoantibodies. By contrast, IgG anti-ds autoantibodies could not be recovered from the library from the healthy twin, or from shuffled libraries with heavy chains from the healthy twin. These repertoire analyses illustrate how inheritance and somatic processes interplay to produce lupus-associated IgG autoantibodies.