Variable region primary structures of monoclonal anti-DNA autoantibodies from NZB/NZW F1 mice

VH and VL region primary structures of five NZB/NZW F1 derived monoclonal anti-DNA autoantibodies were determined from cloned cDNA. Comparative analysis of VH genes showed that except for two VH genes that shared complete identity the overall VH gene usage was diverse. Comparison of VH genes with those utilized in a variety of antibody responses showed they were generally unique to the autoanti-DNA response although framework homologies allowed assignment of four of five VH genes to existing murine heavy chain gene families. Only one out of five D segments shared homology to existing germline D segments, and all were rearranged to JH3. V kappa genes showed restriction for four of five light chains to the V kappa 1 subgroup. The V kappa 1 subgroup has been shown previously to be utilized in several anti-DNA autoantibodies as well as a variety of antibodies to exogenous antigens. H and L chain amino acid residues associated with the active site of a ssDNA specific autoantibody, 04-01, are discussed based on recently obtained crystallographic data.     

Autoantibodies to anti-DNA with anti-allotypic and anti-idiotypic specificities in (NZB X NZW)F1 mice

The monoclonal A52 (IgG2b, kappa) anti-DNA autoantibody represents a major cross-reactive idiotype in the murine and human autoimmune response to DNA. Examination of sera and purified IgG derived from (NZB X NZW)F1 mice showed that these mice develop an age-dependent binding reactivity with the pure anti-DNA IgG. Three monoclonal antibodies possessing this reactivity were prepared from unprimed female (NZB X NZW)F1 mice. One of these monoclonal antibodies appeared to be directed against allotypic determinants present in the NZB IgG2b; the other two antibodies exhibited a marked preference for idiotypic determinants of the A52 IgG. The IgG anti-allotype and anti-idiotype activities in (NZB X NZW)F1 mice may, therefore, represent the products of a deregulated immune system and/or constitute the normal elements of a functional immune regulation system.     

The VH gene sequences of anti-DNA antibodies in two different strains of lupus-prone mice are highly related

The heavy and light chain V region sequences of an IgG anti-DNA autoantibody (PME77), derived from a lupus-prone (NZB x NZW)F1 mouse have been determined by mRNA sequencing. The V kappa gene segment belongs to the V kappa 1A gene sub-group and is found in several (NZB x NZW)F1 and MRL lpr/lpr anti-DNA antibodies, as well as in other antibodies of unrelated specificities. The VH gene segment appears to represent a unique gene or a subfamily of the large J558 VH gene family of the mouse, and is highly related to a germ-line sequence of a major anti-DNA idiotype (H130, IgM) of MRL mice. This anti-DNA-related VH segment has not been found, so far, to be expressed in antibodies with specificities for external or synthetic antigens; therefore, expression of such specificities may be regulated by powerful mechanisms of self tolerance in the healthy animal. In addition, both the heavy and light chain of the PME77 IgG antibody were found to contain somatic point mutations with a high ratio of replacement to silent mutations in complementarity determining regions. This IgM to IgG sequence relationship suggests an affinity maturation process, which is driven by the autoantigen.     

Differences in Vϰ gene utilization and VH CDR3 sequence among anti-DNA from C3H-lpr mice and lupus mice with nephritis

To investigate the molecular properties of anti-DNA from lpr mice that express high levels of anti-DNA without immune-mediated nephritis, the sequences of VH and Vϰ genes encoding 11 monoclonal anti-DNA antibodies derived from C3H-lpr/lpr (C3H-lpr) mice were studied. All of the C3H-lpr monoclonal anti-DNA bound single-stranded DNA while five also bound double-stranded DNA. Two of the hybridomas were clonally related as determined by Southern analysis and sequencing. Sequence analysis of C3H-lpr anti-DNA revealed the use of VH genes that encode anti-DNA from the MRL-lpr/lpr and (NZB X NZW)F1 mouse models of lupus, although differences occurred in the VH CDR3 amino acid content. In contrast, the Vϰ genes from C3H-lpr mice lacked significant identity with previously reported Vϰ genes for anti-DNA from lupus models. These results indicate that anti-DNA from C3H-lpr mice differ from anti-DNA from lupus mice with nephritis in patterns of V gene expression and suggest a molecular basis for the lack of pathogenicity of anti-DNA in these mice.     

Lack of anti-DNA precursors in lambda chain-bearing B lymphocytes in (NZB x NZW)F1 mice. Evidence for the contribution of V kappa genes to anti-DNA specificity

We have analyzed by the limiting dilution assay on spleen cells from (NZB x NZW)F1 hybrid mice the repertoire of lipopolysaccharide-responsive murine kappa- and lambda 1-secreting B cells committed to the production of anti-DNA autoantibodies to determine the contribution of the heavy and light chains to anti-DNA specificities. Our results demonstrated that anti-DNA precursors were predominantly found in the kappa-secreting B cell population, but not in lambda 1-secreting B cells, while anti-hapten, dinitrophenyl, and anti-tetanus toxoid activities were distributed fairly well in both populations of B cells. This suggests that the V kappa gene segments are critically involved in the generation of anti-DNA specificities, and that at least at the germ-line gene level, the heavy chain V region genes by themselves are not able to confer the anti-DNA autoreactivity.     

The molecular origin of anti-DNA antibodies

The in vitro observation that a single point mutation in the protective anti-phosphorylcholine anti-bacterial antibody, S107, converts it into an autoantibody that reacts with dsDNA has focused our attention on the role of somatic mutation in generating autoantibodies. It has also led us to examine the significance of an individual's prior response to environmental antigens on the subsequent production of autoantibodies. The fact that genes of the S107 heavy chain variable region family could encode autoantibodies made it possible to clone and sequence the relevant germline genes of this small family from autoimmune (NZB x NZW)F1 mice and to compare these to the comparable genes in non-autoimmune mice. The germline genes from the normal and autoimmune mice are quite homologous and the small number of polymorphisms are not likely to predispose the autoimmune mice to the production of autoantibodies. (NZB x NZW)F1 mice respond to immunization with phosphorylcholine with a response that is largely encoded by the VH1 gene of the S107 family. However, when these same mice begin to make autoantibodies, their anti-DNA antibodies which are encoded by this family are in fact derived from the VH11 gene. The VH11 encoded anti-DNA antibodies which have been sequenced are all of the IgG2a subclass, react with dsDNA, and have undergone significant somatic diversification from the germline gene. Analysis of the ratio and location of the replacement and silent mutations suggests that the regulation of the autoantibody response differs from that of the normal response to foreign antigens. Our studies suggest that the utilization of a particular VH germline gene in the immune response to foreign antigens early in life does not lead to the preferential utilization of that same gene in the subsequent production of autoantibodies.     

Somatically mutated IgG anti-DNA antibody clonally related to germ-line encoded IgM anti-DNA antibody.

Among 15 anti-DNA antibody-producing hybridomas derived from a single NZB X NZW F1 mouse, an IgM and an IgG were shown to use the same VH gene of the Q52 family. Using a combination of two primers both consisting of a mixture of oligonucleotides (one complementary to the 5' end of VH segment and one to the 3' end of VH segment of Q52 family) we determined the sequences of several members of germ-line VH genes in the Q52 family derived from NZB and NZW strains. Comparison of the sequences with those of cloned VH cDNA obtained from the hybridomas revealed that the VH sequence of the IgM anti-DNA antibody was identical to that of a cloned NZW germ-line VH gene, except for the priming sites. In contrast, the VH sequence of the IgG counterpart contained somatically mutated nucleotides. Because the IgG anti-DNA antibody showed a higher DNA binding activity than did the IgM antibody, we conclude that these changes in nucleotide sequences were induced and selected through an antigen-driven mechanism as is the case in a normal immune response. It is tempting to speculate that the germ-line encoded, low-affinity IgM autoantibody undergoes somatic mutations and isotype switching, resulting in generation of pathogenic, high-affinity autoantibodies in autoimmune diseases.     

Induction of anti-DNA autoanti-idiotypic antibodies in (NZB X NZW)F1 mice: possible role for specific immune suppression

(NZB X NZW)F1 (B/W) mice spontaneously produce anti-deoxyribonucleic (DNA) acid antibodies. PME77 anti-DNA monoclonal antibody (MoAb) is a syngeneic antibody bearing idiotype present in most B/W sera. In the present investigation the effect of immunization of B/W mice with the PME77 MoAb on the production of PME77 idiotypes and anti-DNA antibodies in B/W mouse sera was investigated. PME77 MoAb immunization regimen induced the production of autoanti-idiotypic antibodies and abrogated the expression of PME77 idiotype in B/W treated mice. In contrast, untreated mice and control B/W mice, receiving NZB polyclonal IgG2b which lacked detectable DNA binding capacity, expressed PME77 idiotopes. These results demonstrate that the expression of idiotype borne by autoantibodies may be modified through the induction of autoanti-idiotypic antibodies.     

Contribution of the gene linked to the T cell receptor beta chain gene complex of NZW mice to the autoimmunity of (NZB x NZW)F1 mice.

We have investigated the contribution to the autoimmune disease of (NZB x NZW)F1 (NZB/W) mice made by the T cell receptor beta (TcR beta) chain gene complex, or genes linked to it, that are derived from the NZW strain. For this we developed the NZW.TcR beta NZB strain, a NZW congenic line carrying the TcR beta of NZB type, and produced NZB x NZW.TcR beta NZB (NZB/W.TcR beta NZB)F1 mice. We compared the amounts of anti-DNA and anti-histone antibodies and also the severity of lupus nephritis in these mice with those in the original NZB/W F1 mice. We obtained evidence for significantly lower serum levels of autoantibodies to double-stranded and single-stranded DNA and histone, and a later onset and a lower incidence of proteinuria in the NZB/W.TcR beta NZB F1 mice than in the original NZB/W F1 mice. These findings clearly indicate that the gene(s) within or closely linked to the TcR beta chain gene complex on chromosome 6 of the NZW strain acts to intensify the feature of systemic lupus erythematosus in the NZB/W F1 strain. The significant relationship of this finding to the strict dependency of NZB/W F1 disease on the H-2d/H-2z heterozygosity is discussed.     

Molecular analysis of the murine lupus-associated anti-self response: involvement of a large number of heavy and light chain variable region genes

The mRNA encoding heavy and light chains of a hybridoma-derived monoclonal IgM, kappa anti-immunoglobulin (rheumatoid factor) and an IgG3, kappa anti-histone autoantibody from systemic lupus erythematosus and arthritis-prone MRL/Mp-lpr/lpr mice have been molecularly cloned, and the nucleotide sequences corresponding to their variable regions have been determined. To investigate whether autoantibodies with specificities frequently observed in lupus disease might share common structural components, the sequences obtained in this study have been compared with those of a monoclonal MRL/Mp-lpr/lpr IgM, kappa anti-DNA autoantibody previously analyzed in our laboratory (J. Exp. Med. 1985. 161: 805). The 3 immunoglobulins employed different heavy chain variable region (VH) genes belonging to the large J588 VH gene family, kappa light chain variable region (V kappa) genes from 3 different V kappa groups, and different diversity and joining segments. Our findings suggest that murine lupus-associated autoantibodies of different specificities do not have genetic components in common to signal their self-reactive nature and are encoded by a large number of immunoglobulin gene elements.     

Enhanced response of autoantibody-secreting B cells from young NZB/NZW mice to T-cell-derived differentiation signals

Spleen cells from young NZB/NZW mice spontaneously produce IgM antihistone and anti-DNA antibodies in culture, and this in vitro autoantibody production is T-cell dependent. In the present studies, we investigated the response of young autoantibody-producing NZB/NZW B cells to various T-cell-derived signals. Stimulation with unprimed allogeneic T cells resulted in a 10- to 20-fold increase in IgM antihistone and anti-DNA antibody production compared with cultures of B cells alone. The responding cells were found in the large B-cell fraction after separation on Percoll gradients. Allo-stimulated B cells from nonautoimmune mice produced much lower absolute amounts of IgM autoantibodies as well as total IgM compared with NZB/NZW cells. Marked IgM antinuclear antibody and total IgM production was also observed when NZB/NZW B cells were cultured with supernatants from TH2 but not TH1 T-helper clones. Although B cells from nonautoimmune mice produced high levels of autoantibodies after stimulation with lipopolysaccharide, only minimal levels were secreted in response to the active supernatants. These results suggest that young NZB/NZW mice have IgM autoantibody-producing B cells that are more sensitive to certain T-cell-derived signals compared with B cells from normal mice. Although these hyperresponsive NZB/NZW cells appear to be in an advanced stage of activation, they require additional T-cell signals to express this abnormality.     

Requirement of H-2 heterozygosity for autoimmunity in (NZB X NZW)F1 hybrid mice

In the F1 hybrid of autoimmune New Zealand Black (NZB) and phenotypically normal New Zealand White (NZW) mice, there occurs a severe systemic lupus erythematosus (SLE)-like autoimmune disease more fulminant than that found in the parental NZB mice. To determine the role of the H-2 complex in the pathogenesis of autoimmune disease of the (NZB X NZW)F1 hybrid, we developed H-2-congenic NZB (NZB.H-2z) and NZW (NZW.H-2d) strains, and compared the degree of autoimmune features between congenic H-2d/H-2d and H-2z/H-2z homozygous F1 hybrids and the original H-2d/H-2z heterozygous (NZB X NZW)F1 hybrid. We found that autoimmune features such as productions of IgG class anti-DNA antibodies and retroviral gp70 immune complexes and the development of renal disease were to a great extent reduced in both H-2 homozygous F1 hybrids, as compared with the H-2 heterozygous (NZB X NZW)F1 hybrid. It would thus appear that the heterozygosity of H-2d haplotype derived from NZB and H-2z from NZW is essential for the autoimmune disease characteristic of the (NZB X NZW)F1 hybrid.     

Effect of the BXSB Y chromosome accelerating gene on autoantibody production

The Y chromosome of the BXSB mouse is able to accelerate and adversely alter the autoimmune disease of inbred BXSB mice and in male F1 hybrids. In order to further study the effects of the BXSB Y chromosome, we developed three inbred congenic strains, each bearing the BXSB Y: CBA/J.BXSB-Y, NZW.BXSB-Y, and NZB.BXSB-Y. The BXSB Y did not induce anti-DNA or anti-red blood cell (RBC) autoantibodies in either CBA/J or NZW congenic strains. Thus, it is an accelerating rather than an inducing factor. NZB.BXSB-Y congenic mice had accelerated anti-RBC but not anti-DNA. Studies of recombinant inbred by BXSB F1 mice indicated that the BXSB Y did not act to promote the activity of the NZB gene underlying anti-DNA. These and studies of (BXSB X NZB.BXSB-Y) F1 mice suggested that BXSB autosomal genes are required for the full anti-DNA accelerating activity of the BXSB Y. These mice provide a basis for future molecular genetic studies of the BXSB Y.     

Variable region sequences of pathogenic anti-mouse red blood cell autoantibodies from autoimmune NZB mice

New Zealand Black (NZB) mice spontaneously develop a severe autoimmune hemolytic anemia due to the production of anti-mouse red blood cell (MRBC) autoantibodies. The contribution of variable region genes and somatic mutations in the pathogenicity of anti-MRBC autoantibodies was investigated by mRNA sequencing of eight NZB anti-MRBC monoclonal autoantibodies, among which five are capable of inducing anemia in BALB/c mice. Here we report that at least three VH gene families (J558, J606 and 3609) and five Vchi subgroups (V chi 8, 9, 19, 21 and 28), in combination with several D, JH and Jchi gene segments, encode anti-MRBC autoantibodies. Thus, the NZB anti-MRBC autoantibodies, whether pathogenic or not, are encoded by a large number of immunoglobulin gene elements and by members of known VH and Vchi gene families with preferential usage of VH gene families most distal to the D regions. The presence of several mutations in the JH gene segments of both IgM and IgG anti-MRBC autoantibodies, whether pathogenic or not, strongly suggests that their VH regions may be highly mutated and that the mechanism of somatic diversification might be important in the generation of anti-MRBC autoantibodies. Our results support the idea that anti-MRBC autoimmune responses are likely to be generated by an antigen-driven mechanism.     

Natural antibodies that react with V-region peptide epitopes of DNA-binding antibodies are made by mice with systemic lupus erythematosus as disease develops.

Cross-reactive idiotypes (CRI) have been detected on anti-DNA autoantibodies associated with lesions typical of systemic lupus erythematosus. In order to analyse the antigenic make up of idiotypes on anti-DNA monoclonal antibodies (mAb) V-88 (IgG1 kappa) and F-423 (IgG3 kappa), derived respectively from an adult (NZB x NZW)F1 and a fetal MRL/Mp-lpr/lpr mouse, a set of overlapping hexapeptides representing the VH and VL regions of mAb V-88 and F-423 were synthesized and reacted with a range of sera in pepscan enzyme-linked immunosorbent assays (ELISA) taken from normal and lupus mouse strains. Serum pools were collected both from normal BALB/c and lupus MRL/Mp-lpr/lpr and (NZB x NZW)F1 mice at 10, 20 and 30 weeks of age and analysed for the presence of spontaneously produced anti-V-region peptide IgM and IgG antibodies. IgM antibodies from both the lupus mice reacted with the same V-region epitopes, and although some epitopes mapped to similar locations in the two mAb, the maps for V-88 and F-423 were not identical. In MRL/Mp-lpr/lpr mice, as lupus disease progressed there was a switch from IgM antibodies to IgG anti-peptide antibodies whose specificity for the peptide antigens coincided with but was better defined than that of the IgM antibodies. The identified idiotopes were located in both complementary determining regions (CDR) and framework region (FR) regions, indicating that some contribute to CRI shared by other related antibodies, while others were unique to either mAb V-88 or F-423. In conclusion, we have dissected and identified a mosaic of antibody V-region idiotopes that contribute to the idiotype of an anti-DNA autoantibody and against which autoantibodies are made naturally in lupus disease.     

Intrastrain recurrent idiotypes among anti-DNA antibodies of (NZB X NZW)F1 hybrid mice

Immunization of NZB and A/J mice against an anti-DNA hybridoma antibody (F227) derived from (NZB x NZW)F₁ (B/W) mice allowed the preparation of two anti-idiotype antisera. These two reagents were shown to recognize different idiotopes of the F227 monoclonal antibody. NZB anti-idiotypic antibodies recognized non-ligand-modifiable idiotypic determinants. These idiotopes were private or present at undetectable level in BW mouse sera since it was found that only two of the 24 B/W mouse sera tested were recognized by these antibodies. Conversely, A/J anti-idiotypic antibodies recognized partially ligand-modifiable idiotopes which were found in all B/W mouse sera tested. These results demonstrate that anti-DNA antibodies share similar idiotypic specificities and suggest that these autoantibodies occur as families of structurally related proteins.     

Does the deletion within T cell receptor β-chain gene of NZW mice contribute to autoimmunity in (NZB × NZW)F1mice?

To determine the transacting genetic factors of NZW contributing to the development of autoimmune disease in (NZB X NZW)F1 (B/W F1) mice, we examined the relationship between the T cell receptor beta chain gene deletion and the severity of autoimmune manifestations in 76 B/W F1 X NZB backcross mice. Very high association between the T cell receptor beta chain gene deletion and the development of autoimmune manifestations including the production of IgG anti-DNA antibodies and circulating retroviral gp70 immune complexes was observed, indicating that a defect in the NZW T cell receptor beta chain gene or a locus closely linked to it contributes to the autoantibody formation in B/W F1.     

Sequential study of C 1 q-binding substances and of anti-DNA antibodies in (NZB X NZW)F1 mice: evidence for an acute phase of the lupus disease in 2-month-old mice.

Longitudinal studies of C 1 q and DNA-binding substances were performed in sera from 38 (NZB X NZW)F1 female mice between days 39 and 150 of life. Results suggest a two-phase evolution of circulating immune complexes and anti-DNA antibodies in young (NZB X NZW)F1 mice and show the existence of an acute phase of the disease during the second month of life of these mice.     

Thymic microenvironment and NZB mice: the abnormal thymic microenvironment of New Zealand mice correlates with immunopathology

There are distinct microenvironmental abnormalities of thymic architecture in several murine models of SLE defined using immunohistochemistry and a panel of mAb dissected at thymic epithelial markers. To address the issue of the relationship between the thymic microenvironment and autoimmunity, we studied backcross (NZB x NZW) F1 x NZW mice in which 50% of offspring develop nephritis associated with proteinuria and anti-DNA antibodies. We reasoned that if thymic abnormalities are associated with development of disease, the correlation of abnormalities with lupus-like disease in individual backcross mice will form the foundation for identification of the mechanisms involved. In parallel, we directed a genetic linkage analysis, using markers previously shown to be linked to nephritis and IgG autoantibody production, to determine if such loci were similarly associated with microenvironmental changes. Our data demonstrate that all (NZB x NZW) F1 x NZW backcross mice with disease have microenvironmental defects. Although the microenvironmental defects are not sufficient for development of autoimmune disease, the severity of thymic abnormalities correlates with titers of IgG autoantibodies to DNA and with proteinuria. Consistent with past studies of (NZB x NZW) F1 x NZW mice, genetic markers on proximal chromosome 17 (near MHC) and distal chromosome 4 showed trends for linkage with nephritis. Although the markers chosen only covered about 10-15% of the genome, the results demonstrated trends for linkage with thymic medullary abnormalities for loci on distal chromosome 4 and distal chromosome 1. We believe it will be important to define the biochemical nature of the molecules recognized by these mAbs to understand the relationships between thymic architecture and immunopathology.     

Successful treatment of autoimmunity in (NZB X NZW)F1 mice with cyclosporin and (Nva2)-cyclosporin: I. Reduction of autoantibodies.

Autoimmune (NZB X NZW)F1 mice were treated with the immunosuppressive agent, cyclosporin, and its new derivative (Nva2)-cyclosporin. Both compounds prevented the development of autoantibodies in young mice, and also reduced the levels of the autoantibodies in old mice. These findings established that autoantibodies, at least in the (NZB X NZW)F1 mice, can be controlled pharmacologically. This study supports the possibility that treatment with cyclosporin and (Nva2)-cyclosporin might be effective in the treatment of certain autoimmune diseases in man.