Induction of anti-DNA antibody with DNA-peptide complexes.

Spontaneous anti-DNA antibodies in autoimmune mice have the characteristics of antibodies produced by antigen-specific, clonally selective B cell stimulation. The nature of the somatically derived antibody variable region structures recurrent among spontaneous anti-DNA antibodies suggests that DNA or DNA-protein complexes may provide the antigenic stimulus for autoimmune anti-DNA antibody. Previously we have demonstrated that native mammalian DNA in complexes with an immunogenic DNA-binding peptide Fus1 from Trypanosoma cruzi can induce anti-DNA antibody in mice not genetically prone to autoimmune disease. The induced anti-DNA has similar specificity, structure and immunopathological function as autoimmune anti-DNA. The present experiments were designed to further characterize the immune response to DNA-peptide complexes. There was considerable variation in the antibody responses of mice from different strains to DNA-Fus1 immunizations. The range was from virtually no response in C57BL/6 mice to most robust responses in NZW mice. The full-length 52 amino acid carboxy-extension protein of ubiquitin (CEP) in T. cruzi (TCEP) protein from which Fus1 was derived functions equally well as an immunogenic carrier for DNA. Anti-DNA responses were generally weak even though anti-Fus1 and anti-TCEP responses were very strong. The results are discussed with respect to the contrasting roles of T cell help and peripheral B cell tolerance in controlling immune and autoimmune antibody responses to DNA.

Immunoregulation of Th cells by naturally processed peptide antagonists.

Th cells recognize protein Ags as short peptides bound to MHC class II molecules. Altered peptide ligands can antagonize (inhibit) T cell responses to stimulatory peptides. Peptides generated by APC may contain peptide flanking residues (PFR), which lie outside the minimal binding epitope and can be recognized by the TCR. Our data show that PFR-dependent T cells were found to be potently antagonized by peptides that lack PFR and responded poorly to native protein or the immunogenic epitope delivered by a recombinant influenza virus. These data provide the first evidence that Ag processing generates both stimulatory and antagonist peptides from a single immunogenic epitope, an observation that may have important implications for T cell immunoregulation and autoimmunity.

Expression of an anti-DNA-associated VH gene in immunized and autoimmune mice.

The J558 family BW-16 VH gene is closely associated with the autoimmune response to DNA of lupus-prone mice. We have followed the expression of VHBW-16-encoded heavy chains in cDNA libraries prepared from unmanipulated normal (C3H, AKR) and autoimmune (New Zealand Black/New Zealand White F1) mice, and from mice immunized with a highly immunogenic peptide/DNA complex. The prevalence, clonal heterogeneity, and structural properties (somatic mutation, complementarity-determining region 3 composition) of these H chains were investigated, and the DNA affinity of VHBW-16-encoded hybridoma mAb was measured in solution. We find that H chains encoded by VHBW-16 are very rare in Igs of normal mice, but increase significantly in peptide/DNA-immunized mice, and dramatically in diseased mice. The experimentally induced VHBW-16-encoded H chains are clonally restricted, somatically mutated, partly switched from IgM to IgG, and give rise to anti-DNA Abs with low affinity. In contrast, the VHBW-16-encoded H chains from diseased New Zealand Black/New Zealand White mice are clonally heterogeneous, exclusively of the IgG isotype, and produce high affinity anti-DNA autoantibodies. We conclude that the experimentally induced and spontaneous immune responses to DNA are qualitatively similar, but quantitatively different, and may truly reflect the principles of self immunologic tolerance.

Monoclonal anti-DNA antibodies: structure, specificity, and biology.

Anti-DNA antibodies are a major contributor to the pathogenesis associated with the autoimmune disease systemic lupus erythematosus in mice and human. The accumulation of a large body of structural information on autoimmune anti-DNA antibodies over the past several years, particularly from mice, has provided considerable insight into the structure, function, and biology of this important class of autoantibodies. Even though the germline repertoire of light and heavy chain variable regions that may encode DNA-specific antibodies is very large in mice, there are individual light and heavy chain variable region genes that have been recurrent and preferentially expressed among anti-DNA hybridomas. This has been particularly true for hybridomas producing antibodies that bind duplex, B-form, mammalian DNA (dsDNA). Recurrent somatically derived variable region structures, particularly arginines in the third complementary-determining region of the heavy chain (VH-CDR3), have also been recurrent and preferentially expressed among monoclonal anti-DNA antibodies. In fact specificity for dsDNA can be correlated to the relative amino acid position at which arginines are expressed within VH-CDR3 of anti-DNA. Most important from the results of structural analyses of monoclonal anti-DNA autoantibodies has been the realization that autoimmunity to DNA results from a clonally selective, antigen-specific immune response to DNA. Autoimmune antibodies to DNA have all of the characteristics of secondary immune antibodies. In further support of this hypothesis, we have been able to induce anti-DNA antibodies in normal, nonautoimmune mice by immunization with immunogenic DNA-peptide complexes. The induced antibodies have all of the structural and functional characteristics of autoimmune anti-DNA including the pathogenetic potential to induce glomerulonephritis. This review summarizes the results of research from our laboratory that support the above conclusions.

Immunoglobulin variable-region structures in immunity and autoimmunity to DNA.

Important to the immunopathology associated with the autoimmune disease systemic lupus erythematosus, is the production of autoantibody to DNA. Crucial to understanding the immunological basis for autoimmunity to DNA is knowing whether the anti-DNA autoantibody is the product of clonally-selective, antigen-specific B cell stimulation or non-selective, polyclonal B cell activation. Structural analyses of the immunoglobulin variable-regions of both early, IgM and late, IgG anti-DNA antibodies from lupus-prone (NZB x NZW) F1 mice have indicated that both IgM and IgG anti-DNA autoantibodies are generated by clonally-selective B cell stimulation. Within individual autoimmune mice the later appearing, IgG anti-DNA autoantibodies are structurally similar to the earlier appearing, IgM antibodies, and in some cases both IgM and IgG may be produced by the same B cell clones. The variable-region structural data also suggest that DNA or complexes containing DNA may be the immunogenic stimuli for autoantibody to DNA. In support of this conclusion, normal mice immunized with immunogenic peptide-DNA complexes produce anti-DNA antibodies with structural and serological characteristics similar if not identical to those of autoimmune anti-DNA antibodies. Normal mice immunized with peptide-DNA complexes eventually develop immunopathology that resembles lupus nephritis. These results suggest that autoimmunity to DNA and subsequent autoimmune disease in SLE may result from a specific immune response to DNA containing antigens.

Antigen-specific induction of antibodies against native mammalian DNA in nonautoimmune mice.

Spontaneous anti-DNA antibodies in autoimmune mice have the characteristics of antibody produced by Ag-specific, clonally selective B cell stimulation. The nature of the somatically derived antibody V region structures recurrent among spontaneous anti-DNA antibodies suggests that DNA or DNA-protein complexes may provide the antigenic stimulus for autoimmune anti-DNA antibody. In order to test this hypothesis directly, we have immunized normal, nonautoimmune-predisposed mice with complexes formed with DNA and an immunogenic, DNA-binding peptide. The highly immunogenic peptide, Fus1, forms an internal domain of a 128-amino acid ubiquitin-fusion protein from Trypanosoma cruzi. DNA-Fus1 complexes formed with native calf thymus DNA induced anti-DNA antibody in normal, nonautoimmune-predisposed mice that is similar in isotype and specificity to spontaneous anti-DNA antibody in (NZB x NZW)F1 autoimmune mice. The progressive nature of the development of dsDNA specificity in the immunized mice was also analogous to what is observed in the spontaneous anti-DNA antibody response of autoimmune (NZB X NZW)F1 mice. DNA-Fus1 immunized mice that produced IgG that bound to dsDNA had low to moderate levels of proteinuria and glomerular deposits of IgG. This experimental immunization system may be useful for understanding the immunologic basis for autoimmunity to DNA.