Relationships among antinuclear antibodies from autoimmune MRL mice reacting with histone H2A-H2B dimers and DNA

A spontaneous, autoreactive autoantibody called SN5–18 (IgG2b, κ) binds to a complex of H2A/H2B/dsDNA in chromatin, but erroneously appears to bind dsDNA when the Ab is used in a form that is not highly purified. Because of this finding, we evaluated the antigenic specificity of a prototypic anti-dsDNA Ab, 3H9/Vκ4, now used widely in transgenic studies of tolerance and autoimmunity. We found that the purified mAb 3H9/Vκ4 binds chromatin and specifically a complex of H2A/H2B/dsDNA, but not dsDNA in solid phase or in solution. When used in the form of culture supernatant or as a standard protein G preparation, mAb 3H9/Vκ4 appears to bind dsDNA, apparently due to nuclear proteins in the preparation that assemble on target DNA. Because of the reported role of VHCDR3 Arg residues in dsDNA binding and the near identity of the SN5–18 sequence to other dsDNA-specific Ab, we tested the contributions of two VHCDR3 Arg residues in SN5–18 to chromatin specificity. We found that both these Arg residues at positions 104 and 106 were required for detectable chromatin binding. These results indicate a role for VHCDR3 Arg residues in chromatin specificity of lupus-derived autoantibodies. Further, they provide an explanation for a possible discrepancy in the form of tolerance observed in different anti-DNA Ig transgene models.

Section: Discussionmentioning

confidence: 99%

Chromatin Specificity of Anti-Double-Stranded DNA Antibodies and a Role for Arg Residues in the Third Complementarity-Determining Region of the Heavy Chain

Guth

Zhang

Smith

et al. 2003

“…However, the SN5-18 Ab binds with high avidity to mouse chromatin, which copurifies with this mAb on protein G columns, thereby necessitating elaborate purification steps (see Materials and Methods). Furthermore, H2A/H2B/DNA is one of the most consistent and early targets of autoantibodies in SLE (62)(63)(64). Finally, a National Center for Biotechnology Information BLAST search (65) with the SN5-18 sequence revealed 22 Abs encoded by the same or closely related V H gene (94 -98% sequence identity).…”

Section: Discussionmentioning

confidence: 99%

A Receptor Presentation Hypothesis for T Cell Help That Recruits Autoreactive B Cells

Zhang

Smith

Guth

et al. 2001

To uncover mechanisms that drive spontaneous expansions of autoreactive B cells in systemic lupus erythematosus, we analyzed somatic mutations in variable region genes expressed by a panel of (NZB × SWR)F1 hybridomas representing a large, spontaneously arising clone with specificity for chromatin. A single mutation within the Jκ intron that was shared by all members of the lineage indicated that the clone emanated from a single mutated precursor cell and led to the prediction that a somatic mutation producing a functionally decisive amino acid change in the coding region would also be universally shared. Upon cloning and sequencing the corresponding germline VH gene, we found that two replacement somatic mutations in FR1 and CDR2 were indeed shared by all seven clone members. Surprisingly, neither mutation influenced Ab binding to chromatin; however, one of them produced a nonconservative amino acid replacement in a mutationally “cold” region of FR1 and created an immunodominant epitope for class II MHC-restricted T cells. The epitope was restricted by IAq (SWR), and the SWR MHC locus is associated with systemic lupus erythematosus in (NZB × SWR)F1 mice. These, and related findings, provoke the hypothesis that autoreactive B cells may be recruited by a “receptor presentation” mechanism involving cognate interactions between T cells and somatically generated V region peptides that are self-presented by B cells.

“…The nucleosome-specific mAbs PR1-3, PL2-6, PL2-3, LG4-1, MRB-4, LG10-1, LG8-1, PL9-7, PL2-8, PL2-7, and MGC 23 were derived from MRL mice and purified by protein G affinity chromatography (31)(32)(33)(34) …”

Section: Serum Samples and Absmentioning

confidence: 99%

“…Nucleosome-containing fractions with nucleosome monomers, dimers, and higher oligomers are shown. B, 33 P-labeled nucleosomes from fractions 4 and 7 above were added to wells precoated with the anti-nucleosome mAbs PL2-3 and PR1-3 or with the anti-NIP mAb 23 or to uncoated wells. The counts retained after washing were determined by measuring ␤ emission in a liquid scintillation counter.…”

Section: Figurementioning

confidence: 99%

Immune Complexes Present in the Sera of Autoimmune Mice Activate Rheumatoid Factor B Cells

Rifkin

Leadbetter²,

Beaudette³

et al. 2000

Self Cite

The fate of an autoreactive B cell is determined in part by the nature of the interaction of the B cell receptor with its autoantigen. In the lpr model of systemic autoimmunity, as well as in certain human diseases, autoreactive B cells expressing rheumatoid factor (RF) binding activity are prominent. A murine B cell transgenic model in which the B cell receptor is a RF that recognizes IgG2a of the j allotype (IgG2aj), but not the b allotype, was used in this study to investigate how the form of the autoantigen influences its ability to activate B cells. We found that sera from autoimmune mice, but not from nonautoimmune mice, were able to induce the proliferation of these RF+ B cells but did not stimulate B cells from RF− littermate controls. The stimulatory factor in serum was found to be IgG2aj, but the IgG2aj was stimulatory only when in the form of immune complexes. Monomeric IgG2aj failed to stimulate. Immune complexes containing lupus-associated nuclear and cytoplasmic autoantigens were particularly potent B cell activators in this system. Appropriate manipulation of such autoantibody/autoantigen complexes may eventually provide a means for therapeutic intervention in patients with certain systemic autoimmune disorders.