Comparison of the Frequencies of Arginines in Heavy Chain CDR3 of Antibodies Expressed in the Primary B-Cell Repertoires of Autoimmune-Prone and Normal Mice

Because the pathogenesis of anti-DNA Ab in SLE is correlated to Ab specificity for native DNA (dsDNA), understanding how such specificity is generated is important. The VH structures of most autoimmune anti-DNA antibodies include at least one arginine in VH–CDR3; moreover, antibody specificity for dsDNA can be correlated to the relative position of arginines in VH–CDR3. The coding sequences for most VH–CDR3 arginines among the anti-DNA MoAbs we have studied to date appeared to have been encoded by sequences generated during V–D–J recombination and would have been expressed in the primary B-cell repertoire. The frequency at which arginine codons are generated during V–D–J recombination therefore could potentially influence the frequency at which DNA-specific B cells are generated in the primary B-cell repertoire. The present study was undertaken to determine whether a higher percentage of B cells in the primary, preautoimmune repertoire of autoimmune-prone (NZB × NZW)F₁ mice have immunoglobulin heavy chains with at least one VH–CDR3 arginine compared to B cells in the primary, preimmune repertoire of non-autoimmune-prone BALB/c mice. The present results indicate that mature B cells in preautoimmune (NZB × NZW)F₁ mice, whether specific for DNA or not, are no more likely to have heavy chains with VH–CDR3 arginines than are B cells in BALB/c mice. The high frequency of recurrence of VH–CDR3 arginines among autoimmune anti-DNA in (NZB × NZW)F₁ mice would appear to derive from the selective oligoclonal expansion of selected B cells that express such structures.     

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.     

The role of somatic mutation in determining the affinity of anti-DNA antibodies

Combinatorial antibody libraries were constructed from the spleen of a patient with concomitant systemic lupus erythematosus and idiopathic thrombocytopenia. Following selection of the libraries with DNA, a panel of 15 anti-DNA Fabs was isolated. Sequence analysis of these antibodies coupled with measurements of their affinities for ss- and dsDNA were used to investigate the role of somatic mutation in affinity maturation of the anti-DNA response. Examination of the germline genes used by these Fabs supports previous studies that suggest there is no restriction of the gene usage in the anti-DNA response. However, data are presented indicating that VH3 genes and the A27 V(kappa) paired with the J(kappa)1 may be over-expressed in the anti-DNA repertoire. Analysis of the role of somatic mutation in increasing affinity for DNA indicates that affinity maturation has occurred and suggests that the CDR1 and CDR2 of the heavy chain are of importance in this process.     

The role of germline gene expression and somatic mutation in the generation of autoantibodies to DNA

Several distinctive features of anti-DNA autoantibodies have been identified by a detailed analysis of the available heavy and light chain sequences. They include unique VH gene segments that are not normally expressed in antibodies to external antigens, somatic mutations which may serve to change the antigenic specificity as well as to increase affinity, a less stringent choice of light chains, and a unique basic peptide in the heavy chain CDR3. It is proposed that in the majority of cases, the regulatory mechanism of self-tolerance in the healthy animal operates via VH gene expression to prevent the synthesis of potentially high affinity anti-DNA autoantibodies.     

Autoimmune NZB/NZW F1 mice utilize B cell receptor editing for generating high-affinity anti-dsDNA autoantibodies from low-affinity precursors

We have previously constructed knock-in (C57BL/6xBALB/c) F1 mice, each expressing an anti-DNA heavy (H) chain (D42), combined with one of three different light (L) chains, namely Vkappa1-Jkappa1, Vkappa4-Jkappa4 or Vkappa8-Jkappa5. All of these H/L chain combinations bind DNA with similar affinity and fine specificity. However, while mice carrying Vkappa1-Jkappa1-transgenic L chain were tolerized almost exclusively by L chain receptor editing, the mice expressing Vkappa8-Jkappa5 L chains utilized clonal anergy as their principal mechanism of B cell tolerance. Vkappa4-Jkappa4 targeted mice exhibited an intermediate phenotype. In the present study, these three H/L chain combinations were backcrossed onto the autoimmune NZB/NZW F1 mice. We find that the mechanism of clonal anergy is abrogated in these mice, but that receptor editing is maintained. Moreover, diseased NZB/NZW mice utilize L chain secondary rearrangements for the generation of high-affinity, anti-dsDNA-producing B cells from low-affinity precursors. The edited B cell clones are not deleted or anergized in the autoimmune animal; rather they are selected for activation, class-switching and affinity maturation by somatic mutation. These results suggest that B cell receptor editing plays an important role not only in tolerance induction, but also in generating high-affinity autoreactive B cells in autoimmune diseases.     

B cell repertoire for anti-DNA antibody in normal and lupus mice: differential expression of precursor cells for high and low affinity anti-DNA antibodies.

The precursor frequency for anti-DNA antibody producing cells and the affinity of antibodies secreted by these cells in both immature prereceptor B cell populations and mature B cell populations were compared between 8-week-old C57BL/6 female mice and 9-month-old B/WF1 female mice by producing a large collection of IgM secreting hybridomas from LPS-stimulated B cells. The data indicate that precursor cells for high affinity anti-DNA antibody are eliminated as they mature in C57BL/6 mice, while a sizable number of such clones are present in mature splenic B cells of aged B/WF1 mice. These results suggest that the emergence of precursors for high affinity anti-DNA producing cells in mature B cell population is an important factor in the pathogenesis of SLE.     

IL-6 contributes to an immune tolerance checkpoint in post germinal center B cells

The generation of a B cell repertoire involves producing and subsequently purging autoreactive B cells. Receptor editing, clonal deletion and anergy are key mechanisms of central B cell tolerance. Somatic mutation of antigen-activated B cells within the germinal center produces a second wave of autoreactivity; but the regulatory mechanisms that operate at this phase of B cell activation are poorly understood. We recently identified a post germinal center tolerance checkpoint, where receptor editing is re-induced to extinguish autoreactivity that is generated by somatic hypermutation. Re-induction of the recombinase genes RAG1 and RAG2 in antigen-activated B cells requires antigen to engage the B cell receptor and IL-7 to signal through the IL-7 receptor. We demonstrate that this process requires IL-6 to upregulate IL-7 receptor expression on post germinal center B cells. Diminishing IL-6 by blocking antibody or haplo-insufficiency leads to reduced expression of the IL-7 receptor and RAG and increased titers of anti-DNA antibodies following immunization with a peptide mimetope of DNA. The dependence on IL-6 to initiate receptor editing is B cell intrinsic. Interestingly, estradiol decreases IL-6 expression thereby increasing the anti-DNA response. Our data reveal a novel regulatory cascade to control post germinal center B cell autoreactivity.     

The regulation and activation of lupus-associated B cells

Summary: Anti‐double‐stranded DNA (anti‐dsDNA) B cells are regulated in non‐autoimmune mice. While some are deleted or undergo receptor editing, a population of anti‐dsDNA (VH3H9/Vλ1) B cells that emigrate into the periphery has also been identified. These cells have an altered phenotype relative to normal B cells in that they have a reduced lifespan, appear developmentally arrested, and localize primarily to the T/B‐cell interface in the spleen. This phenotype may be the consequence of immature B cells encountering antigen in the absence of T‐cell help. When provided with T‐cell help, the anti‐dsDNA B cells differentiate into antibody‐forming cells. In the context of the autoimmune‐prone lpr/lpr or gld/gld mutations, the VH3H9/Vλ1 anti‐dsDNA B cells populate the B‐cell follicle and by 12 weeks of age produce serum autoantibodies. The early event of anti‐dsDNA B‐cell follicular entry, in the absence of autoantibody production, is dependent upon CD4⁺ T cells. We hypothesize that control of autoantibody production in young autoimmune‐prone mice may be regulated by the counterbalancing effect of T‐regulatory (T_reg) cells. Consistent with this model, we have demonstrated that T_reg cells are able to prevent autoantibody production induced by T‐cell help. Additional studies are aimed at investigating the mechanisms of this suppression as well as probing the impact of distinct forms of T‐cell‐dependent and ‐independent activation on anti‐dsDNA B cells.     

Effect of anti-idiotypic antibody on production of anti-DNA antibodies by splenocytes in lupus prone mice

It has been suggested that production of autoantibodies is regulated by idiotype-antiidiotype network. In this study, we examined modulatory effect of the antiidiotypic antibody on the synthesis of anti-DNA antibodies by New Zealand black/New Zealand white F1 mice (B/W F1) splenocytes. The antiidiotypic antibodies were prepared by immunization of a monoclonal anti-DNA antibody derived from B/W F1 to rabbits. The prepared antiidiotypic antibody had specificity to the antigen binding site of anti-DNA antibodies. B/W F1 splenocytes were adjusted to 1 X 10(6) cells/ml and cultured in 1.0 ml aliquots in the presence of varying concentrations of the antiidiotypic antibody for 48 hours. The cells were then washed three times, resuspended in RPMI1640 containing 10% fetal calf serum and cultured again. On days 3 and 7 of the culture, the supernatants were harvested and secretion of anti-DNA antibodies was measured by ELISA. Production of anti-DNA antibodies by B/W F1 splenocytes was suppressed by pretreatment with the antiidiotypic antibody. When the concentration of antiidiotypic antibody was 1 microgram/ml, anti-DNA activity of the supernatants decreased 50%, compared with control on day 3, but the effect was reduced on day 7. The treatment of antiidiotypic antibody did not affect the proliferation and viability of B/W F1 splenocytes. The results indicated that anti-DNA antibodies synthesis were regulated by idiotype-antiidiotype network and could be manipulated by the antiidiotypic antibody.     

Peripheral B cell receptor editing may promote the production of high-affinity autoantibodies in CD22-deficient mice

CD22-deficient mice are characterized by B cell hyperactivity and autoimmunity. We have constructed knock-in CD22-/- mice, expressing an anti-DNA heavy (H) chain (D42), alone or combined with Vkappa1-Jkappa1 or Vkappa8-Jkappa5 light (L) chains. The Ig-targeted mice produced a lupus-like serology that was age- and sex-dependent. High-affinity IgG autoantibodies were largely dependent on the selection of B cells with a particular H/L combination, in which a non-transgenic, endogenous L chain was assembled by secondary rearrangements through the mechanism of receptor editing. Moreover, we present evidence that these secondary rearrangements are very prominent in splenic peripheral B cells. Since CD22 is primarily expressed on the surface of peripheral B cells, we propose a model for the development of a lupus-like autoimmune disease by a combination of peripheral receptor editing and abnormal B cell activation.     

Biased usage of synovial immunoglobulin heavy chain variable region 4 by the anti-glucose-6-phosphate isomerase antibody in patients with rheumatoid arthritis

Rheumatoid arthritis (RA) is the most common inflammatory arthritis, characterized by marked infiltration of mononuclear cells including B cells into the inflamed synovium. Anti-glucose-6-phosphate isomerase (GPI) antibody (Ab) is an arthritogenic Ab in K/BxN T cell receptor transgenic mice, and is also present in some patients with RA. To characterize synovial B cells from anti-GPI Ab-positive RA, synovial immunoglobulin (Ig) heavy chain variable regions (VH) were compared with those of negative individuals. Synovial tissues were obtained from six RA patients (three anti-GPI Ab-positive and three anti-GPI Ab-negative). Ig-VH genes were amplified by PCR using family-specific primers and were subsequently sequenced. In synovial B cells from anti-GPI Ab-positive RA patients, VH4 and JH4 were predominantly expressed (p<0.0001). The immunoglobulin heavy chain complementarity-determining region 3 (IgH-CDR3) length in the synovium of anti-GPI Ab-positive individuals was shorter than that in anti-GPI Ab-negative individuals (p=0.0005). In addition, the IgH-CDR3 of anti-GPI Ab-positive patients was rich in basic-ionized amino acids (arginine, histidine, and lysine) near their central position, suggesting a high affinity. Our results support the notion that Ig-VH4 B cells in RA synovium with anti-GPI Ab are affinity-matured and that anti-GPI Ab might be associated with the skewed IgH-CDR3.     

The in Vitro B-Cell Response to Pneumococcal Polysaccharides in Adults and Neonates

In order to gain insight into the relatively late appearance in ontogeny of responsiveness to T-cell independent (TI) antigens, the in vitro human B-cell response to type 4 pneumococcal polysaccharides (PS4, a human TI-2 antigen) was studied. B cells obtained from adults differentiate into anti-PS4 antibody forming cells upon culturing with PS4, but neonatal B cells obtained from cord blood fail to respond. The culture system used does, however, allow the differentiation of B cells reactive with TD antigens, for example ovalbumin. In order to evaluate the concept that in man the anti-PS4 response is derived from a particular B-cell subset we separated adult B cells on the basis of expression of the determinant recognized by the monoclonal antibody FMC7. The anti-PS4 response is found mainly, but not exclusively, in the FMC7+ subset. The selective unresponsiveness of neonatal B cells to TI-2 antigens is not, however, due to the absence of FMC7+ B cells because, unlike adult blood B cells of which about 50% are FMC7+, 100% of neonatal B cells are FMC7+.     

A second heavy chain permits survival of high affinity autoreactive B cells

Anti-DNA antibody is the serological hallmark of systemic lupus erythematosus (SLE). While antibodies with this specificity may be generated in many individuals, only patients with SLE fail to regulate them effectively. We have demonstrated previously that in non-autoimmune mice transgenic for the heavy chain of the R4A-gamma2b anti-DNA antibody, the existence of high affinity, IgG2b dsDNA binding B cells is tightly correlated with the co-expression of endogenous IgM heavy chain. These cells are anergic. In contrast, low affinity IgG2b dsDNA binding B cells do not express an endogenous heavy chain and represent a population of immunocompetent autoreactive B cells. In order to determine whether the presence of a second heavy chain permits the high affinity autoreactive B cells to escape deletion, the R4A-gamma2b mouse was mated to a strain with a targeted deletion of the transmembrane portion of the mu heavy chain, muMT mice, to produce R4A-gamma2b/muKO mice. Serum titers of anti-DNA antibodies were negligible in both R4A-gamma2b and R4A-gamma2b/muKO mice. In R4A-gamma2b/muKO mice, however, LPS was able to activate a DNA-reactive population although an LPS inducible DNA-reactive population. Light chain gene usage in transgene expressing B cells from R4A-gamma2b/muKO mice was similar to that of the previously defined low affinity anti-DNA B cells that escape tolerance. These data suggest a requirement for a second heavy chain for the survival of this anergic B cell subset.     

Effect of a genetic deficiency of terminal deoxynucleotidyl transferase on autoantibody production by C57BL6 Fas(lpr) mice

Terminal deoxynucleotidyl transferase (TdT) adds nontemplate coded nucleotides (N additions) between the recombining ends of immunoglobulin and T cell receptor genes. These nucleotides add significant diversity to the Ig and TCR repertoires. Amino acids coded for by these nucleotides play a key role in the binding of self antigens by autoantibodies and autoreactive T cells. To determine the effect of a lack of N additions on autoantibody production, we bred the TdT knockout genotype onto the autoimmune C57BL/6-Fas(lpr) background. TdT-deficient mice had significantly lower sera anti-DNA and rheumatoid factor activity than their TdT-producing littermates. C57BL/6-Fas(lpr) TdT-deficient mice had shorter VH CDR3 regions and fewer VH CDR3 arginines [0.6% versus 4. 7%] than their TdT-producing littermates. These data indicate that the absence of TdT limited the production of anti-DNA antibodies and rheumatoid factors in C57BL/6-Fas(lpr) mice, likely due to constraints on Ig diversity secondary to the lack of TdT-derived N additions.     

Cell- and nuclear-penetrating anti-dsDNA autoantibodies have multiple arginines in CDR3 of VH and increase cellular level of pERK and Bcl-2 in mesangial cells

Investigation of characteristics of cell- and nuclear-penetrating anti-double stranded (ds)DNA autoantibodies (autoAbs) is important to understand pathogenesis of lupus nephritis, but has not been clearly explored. The present study reports that three anti-dsDNA monoclonal autoAbs, which contain more than two arginine residues in their CDR3s of variable heavy domain (VH), penetrated into living murine mesangial cells and the cell nuclei. However, an anti-dsDNA monoclonal Ab (mAb) having only one arginine in the CDR3-VH did not penetrate cells. To assess the contribution of antigen-binding sites, especially the VH, in cell- and nuclear-penetration, we evaluated the characteristics of recombinant single chain Fv(scFv), VH, and variable light domain (VL) of a penetrating mAb. The scFv and VH domain, containing arginine in CDR3-VH maintained the ability to penetrate cells and the cell nuclei, whereas the VL domain, having no arginine in CDR3, did not penetrate cells. The penetratingm Abs, scFv, and VH activated ERK and increased cellular protein levels of Bcl-2, whereas the non-penetrating Ab and VL did not. The cell survival was decreased by the penetrating mAbs, scFv and VH, not by the non-penetrating mAb and VL. The data indicate that an antigen-binding site is required for cell-penetration and that positively-charged arginine residues in CDR3-VH contribute to the cell- and nuclear-penetrating ability of a subset of anti-dsDNA autoAbs. Furthermore,the nuclear-penetrating anti-dsDNA autoAbs could possibly function as a pathogenic factor for lupus nephritis by up-regulating ERK activation and Bcl-2 production in mesangial cells. The cell- and nuclear-penetrating VH domain may be exploited as a vehicle for the intra cellular delivery of various useful molecules.     

Follicular exclusion of autoreactive B cells requires FcgammaRIIb

In non-autoimmune mice, the 3H9 transgenic Ig heavy chain can pair with endogenous Iglambda1 light chains to generate B cells with specificity for DNA. These autoreactive cells are actively regulated in vivo, as indicated by the exclusion of lambda1 cells from the splenic B cell follicle and the absence of auto-antibody production. To study the role of Fcgamma receptor IIb (FcgammaRIIb) in peripheral B cell tolerance, FcgammaRIIb(-/-) mice were crossed with C57BL/6 mice bearing a site-directed knock-in of the 3H9 transgene. 3H9FcgammaRIIb(-/-) mice become autoreactive, lose the follicular exclusion of anti-DNA B cells and instead have lambda1 B cells located within splenic germinal centers. They have increased frequencies of splenic auto-antibody-producing cells and elevated titers of IgG anti-DNA auto-antibody. The data implicate an FcgammaRIIb-dependent checkpoint that can exclude autoreactive B cells from splenic follicles. By restricting their participation in germinal center reactions, this putative checkpoint helps attenuate the production of potentially pathogenic auto-antibodies. The data further suggest that this FcgammaRIIb-dependent regulation is B cell autonomous.     

Molecular and serological diversity of anti-DNA autoantibodies from NZB and (NZB X NZW) F1 mice

We have carried out an analysis of the serological and molecular diversity of a panel of monoclonal anti-DNA autoantibodies and serum autoantibodies from NZB and (NZB X NZW) F1 mice, in an attempt to obtain insights into the mechanisms responsible for the development of systemic autoimmune disease. Our data show that the autoantibodies are quite diverse. A dominant, binding-site idiotope on one of our monoclonal autoantibodies is expressed at variable levels in anti-DNA binding antibodies in the sera of both NZB and (NZB X NZW) F1 mice, but on none of the other monoclonal autoantibodies in our panel. We have cloned and sequenced the heavy chain variable region (VH) gene of one anti-DNA hybridoma and by hybridization have determined the VH and V kappa gene segments expressed by 14 others. All of the autoantibodies express members of known V gene subfamilies. A total of four different VH and at least six V kappa subfamilies are expressed by the hybridomas. Thus, a broad spectrum of the total murine Ig repertoire is represented in the anti-DNA autoantibodies present in these strains.     

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.     

Immunoglobulin heavy-chain receptor editing is observed in the NOD/SCID model of human B-cell development

Receptor editing and receptor revision are the two mechanisms of antibody diversity that result in either complete V-gene replacement or the formation of hybrid V genes. We do not yet understand how this process unfolds, because they are rare and difficult to study in vivo. In this study, we describe a family of VH4-34:VH4-61 hybrids isolated from a human B-cell chimeric non-obese diabetic/severe combined immunodeficient mouse. The observation of hybrid immunoglobulin sequences in human B cells that developed in this model system makes it useful for the study of this mechanism of diversification and tolerance.     

Selective strain distribution pattern of a germline VH gene for a pathogenic anti-DNA autoantibody family

Autoimmune NZB mice rarely develop nephritis, but the SNF1, progeny of Crosses between NZB and the normal SWR strain uniformly develop severe lupus nephritis, indicating that the normal SWR strain makes a genetic contribution to the development of nephritis. The SNF1 mice produce a select population of cationic IgG anti-DNA autoantibodies that share a recurrent cross-reactive idiotype called Id564 and these autoantibodies play a prominent role in the development of nephritis. These pathogenic autoantibodies of SNF1 possess the IgCH allotype of the SWR, indicating their origin from the normal parent. The leader-VH sequences of these Id564+ pathogenic anti-DNA autoantibodies are highly homologous and they are also related by 95% homology to a germline gene of normal C57BL/6 mice, called VH-23, that is a member of an anti-NP antibody gene family. Herein, we cloned the flanking and coding regions of the expressed VHDJH genes of the anti-DNA mAb 564, the prototype member of the pathogenic Id564 family. By restriction analysis and partial sequencing, we found that the VH564 gene is related but distinct in its 5' flanking region from all of the known anti-NP VH genes of C57BL/6 and BALB/c mice. Hybridization with four probes complementary to different segments of the flanking and coding regions of the expressed VH564 gene indicated that the germline gene for VH564 is contained in an approximately 5.2 kb EcoRI fragment of SWR genomic DNA. Moreover, high stringency hybridization with oligonucleotide probes complementary to unique CDR2 and 5' flanking sequences of the expressed VH564 gene revealed that the 'approximately 5.2 kb' germline allele for VH564 that is possessed by the normal SWR parental strain is lacking in the NZB parental strain. C57BL/6 mice also lack this allele of the anti-DNA VH564 germline gene, although this strain possesses the highly homologous, anti-NP-related VH-23 germline gene. Thus germline VH genes for certain pathogenic autoantibodies may have a selective strain distribution pattern.