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.     

Altered selection processes of B lymphocytes in autoimmune NZB/W mice, despite intact central tolerance against DNA

Anti-DNA autoantibodies are the hallmark of systemic lupus erythematosus and the (NZBxNZW)F1 (NZB/W) murine model. To investigate potential defects in B cell tolerance, we followed the development of anti-DNA-specific B cells in 2-5-month-old mice transgenic for an unmutated muH chain in the normal C57BL/6 and in the NZB/W background. When the transgenic H chain was combined with a random kappa L chain repertoire about 60% of the antibodies bound to DNA. The analysis of the B cell repertoire in the spleen showed extensive receptor editing and a deletion of DNA reactivity in the C57BL/6 as well as in the autoimmune NZB/W background. NZB/W compared to C57BL/6 transgenic mice had a higher frequency of anti-DNA B cells among follicular B cells that were not censored by central tolerance mechanisms. Furthermore, positive selection of B cells with a recurrent rearrangement into the marginal zone compartment was more pronounced in NZB/W mice. Serum levels of transgenic IgM and of anti-DNA autoantibodies indicate a polyclonal activation of hyperactive B cells in the transgenic NZB/W mice. We propose different B cell receptor signaling thresholds for the NZB/W compared to C57BL/6 B cells. This could explain the quantitative differences in the B cell repertoire as well as the hyperactivity of B cells from NZB/W mice.     

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.     

Positive and negative selection of antigen-specific B cells in transgenic mice expressing variant forms of the V(H)1 (T15) heavy chain

Four variant forms of the V1 (T15-H chain) gene are synthesized in mice. Each V1 variant pairs with a distinct L chain to produce a binding site having specificity for phosphocholine (PC). Transgenic mice expressing variant forms of the V1 gene were analyzed to elucidate the factors driving B cell selection into the peripheral repertoire. In all four lines of H chain transgenic mice analyzed, transgene expression caused complete allelic exclusion of endogenous H chains in the bone marrow (BM), whereas most splenic B cells expressed endogenous H chains. The number of sIgM(+) BM B cells and their sIg receptor number was reduced compared to that of normal transgene-negative controls, suggesting that B cells expressing transgene-encoded H chains were being negatively selected in the BM. Mice expressing autoreactive forms of the V1 transgene with lower affinity for PC (M603H and M167H) exhibit positive selection of PC-specific B cells into the spleen, whereas mice expressing the higher affinity T15H variant exhibited elevated PC-specific B cells in the peritoneal cavity but few V(H)1(+) splenic B cells. These data suggest that the higher affinity T15-id(+) B cells preferentially survive in the peritoneal cavity. When these H chain transgenes were crossed into the mu MT knockout mouse in which surface expression of endogenous H chains is blocked, the percent of splenic V(H)1(+) PC-specific B cells increased up to 5-fold and T15-id(+) B cells were detectable in the spleen of T15H mice. This implies that T15-id(+) PC-specific B cells can be selected into the periphery, but they compete poorly with follicular B cells expressing endogenous Ig.     

Insights into the regulation of immunoglobulin light chain gene rearrangements via analysis of the kappa light chain locus in lambda myeloma

Accumulating evidence indicates that B cells may undergo sequential rearrangements at the light chain loci, despite already expressing light chain receptors. This phenomenon may occur in the bone marrow and, perhaps, in germinal centers. As immunoglobulin (Ig)kappa light chains usually rearrange before Iglambda light chains, we analysed, by polymerase chain reaction, the Igkappa locus of bone marrow mononuclear cells from 29 patients with Iglambda myeloma to identify earlier recombinations in marrow plasma cells. The results demonstrated that Igkappa alleles were inactivated via the kappa-deleting element, presumably prior to V(kappa)-J(kappa) rearrangement, in many cases. Eighteen alleles (16 myeloma clones, 55%) showed V(kappa)-J(kappa) rearrangements, with increased utilization of 5' distant V(kappa) and 3' distant Jkappa gene segments (Jkappa4, 56%), an indication of multiple sequential rearrangements. In-frame, potentially functional V(kappa)-J(kappa) rearrangements were found in approximately one-third of available rearrangements (as expected by chance), each one in different myeloma clones: three were germline encoded, while one had several nucleotide substitutions, suggesting inactivation after the onset of somatic hypermutation. Three of four potentially functional V(kappa)-J(kappa)rearrangements involved V(kappa)4-1, a segment considered to be associated with autoimmunity. These findings provide insights into the regulation of light chain rearrangements and support the view that B cells may occasionally undergo sequential light chain rearrangements after the onset of somatic hypermutation.     

Stochastic pairing of Ig heavy and light chains frequently generates B cell antigen receptors that are subject to editing in vivo

We examined the generation and selection of the B cell antibody repertoire through crossing of mice bearing distinct Ig heavy (H) and light (L) chain rearranged variable region transgenes. Ig gene knock-in and transgenic mice whose H and L chains pair to form a non-autoreactive, functional B cell antigen receptor (BCR) have significantly reduced pre-B cells in the bone marrow as their B cell progenitors rapidly differentiate into surface IgM(+) B cells. The presence of a pre-B cell compartment in these Ig transgenic mice, however, indicates the induction of receptor editing. Here, 18 distinct combinations of H and L chains were generated that we showed could pair in vitro to form BCRs of unknown specificities. Of these, nine induced receptor editing in vivo as evidenced by the presence of pre-B cells and endogenous L chain rearrangements in mice bearing these H and L chain transgenes. These data thus suggest that about half of the emerging antibody repertoire is negatively selected during B lymphopoiesis due to the likely encoding of autoreactive or non-functional BCRs.     

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.     

Negative regulation of autoreactive B cells in transgenic mice expressing a human pathogenic cold agglutinin

Cold agglutinins (CA) are autoantibodies that bind to erythrocyte carbohydrates at low temperatures and induce complement-mediated cell lysis, thus causing hemolytic anemia. Tolerance mechanisms towards CA-expressing B cells and the factors inducing pathogenic CA production are unknown. In order to develop an animal model for CA disease, we have produced transgenic mice expressing the heavy or the light chain of a human CA, previously shown to be pathogenic to the mouse. Expression of the human H chain alone resulted in a B cell maturation block at the pro-B stage, and did not induce allelic exclusion. In double-transgenic mice, co-expression of the human H and L chains restored B cell development but the majority of bone marrow cells expressing the human IgM were eliminated by deletion. In the periphery, B cells were depleted, and a large proportion of the remaining cells co-expressed a human and a murine H chain, secreting "mixed" IgM. A few autoreactive cells, predominating in the peritoneal cavity, escaped tolerance mechanisms and secreted transgenic IgM. The autoreactive B cells are amenable to polyclonal stimulation, making these transgenic mice a suitable model for a human autoimmune disease.     

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.     

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.     

Combinatorial immunoglobulin light chain variability creates sufficient B cell diversity to mount protective antibody responses against pathogen infections

To analyze how combinatorial light (L) chain diversity influences the B cell repertoire, we studied mice with a homozygous immunoglobulin-heavy-chain null mutation (mu MT), in which the B cell developmental block was overridden by the expression of a transgenic immunoglobulin mu heavy (H) chain derived from a vesicular stomatitis virus Indiana serotype (VSV-IND)-neutralizing Ab (T11 mu MT mice). The randomly integrated transgene could not undergo secondary rearrangements and was expressed in combination with endogenous kappa or lambda chains. T11 mu MT mice had a skewed B cell repertoire as evidenced by 30-60% VSV-IND-specific peripheral B cells and spontaneous VSV-IND-neutralizing serum titers. Upon immunization, T11 mu MT mice mounted specific IgM antibody responses against VSV-IND but, interestingly, they also responded against VSV New Jersey serotype (VSV-NJ), lymphocytic choriomeningitis virus, poliovirus and Salmonella typhi porins. Variable-region sequence analysis revealed that VSV-NJ-specific antibodies expressed numerous L chains in combination with the transgenic H chain, which was devoid of hypermutations. Thus, in T11 mu MT mice combinatorial L chain variability alone is able to build up a sufficiently complex B cell repertoire to mount protective immunoglobulin responses against a variety of pathogens.     

Relative importance of different human aPL derived heavy and light chains in the binding of aPL to cardiolipin

INTRODUCTION: Previous studies have suggested the importance of somatic mutations and arginine, asparagine and lysine residues in the complementarity determining regions (CDRs) of antiphospholipid antibodies (aPL) implicated in the pathogenesis of the antiphospholipid antibody syndrome. The relative contributions of the heavy and light chains of aPL in binding to cardiolipin (CL) were assessed by pairing the heavy and light chains of two IgG, beta(2)GPI dependent aPL (IS4 and CL24) with different partner chains from other IgG, beta(2)GPI independent aPL (UK4) and anti-DNA antibodies (B3 and 33H11). METHODS: Four heavy (V(H)) and five light (V(L)) chain variable sequences from three aPL and two anti-DNA antibodies were cloned into expression vectors containing appropriate gamma(1), lambda or kappa constant region cDNA. Paired combinations of heavy and light chain expression plasmids were transfected into COS-7 cells allowing transient expression of whole IgG molecules, which were harvested and tested for the ability to bind CL and DNA by enzyme-linked immunosorbant assay (ELISA). RESULTS: Whole IgG was produced from 19 heavy/light chain combinations. IS4V(H) was dominant in conferring the ability to bind CL with four of the five V(L) tested. The identity of the V(L) region paired with IS4V(H) was important in determining the strength of binding to CL. IS4V(H) contains multiple arginine residues in CDR3, which may have accumulated due to antigen driven selection. It is likely that these arginine residues may interact with CL. The combination B3V(H)/B3V(L) also bound CL, but none of the other 14 combinations showed any binding in this assay. CONCLUSION: Whole IgG molecules capable of binding CL were produced by in vitro expression in COS-7 cells. Arginine residues play important roles in binding to CL and double-stranded DNA. However, different patterns of mutation to arginine are associated with binding to each of these antigens.     

Effect of different Ig transgenes on B cell differentiation in scid mice

In this study we show that the ability of bone marrow pre-B cells to differentiate into B cells in H/L chain transgenic scid mice correlates with the ability of the transgenes to inhibit initiation of endogenous kappa gene rearrangement. Initiation of rearrangement was scored by assaying for DNA double-strand breaks (DSB) at the recombination signal/coding borders of J kappa 1 and J kappa 2. In H/L chain transgenic scid mice that develop B cells, we found little or no DSB; whereas in H chain only transgenic scid mice, in which pre-B cells are unable to give rise to B cells, we found a normal level of DSB but no VJ kappa coding joints. As scid mice are deficient in the repair of DSB, we suggest that initiation of kappa gene rearrangement in H chain transgenic scid mice causes B lineage cells to die at the late pre-B stage. In one transgenic scid line (Y-Sp6), which falls to generate B cells despite containing a H and L chain transgene, we found evidence for loss of B lineage cells at two stages of development: the pro-B to pre-B transitional stage and the late pre-B stage.      

Germinal center checkpoints in B cell tolerance in 3H9 transgenic mice

Regulation throughout B cell maturation and activation prevents autoreactive B cells from entering germinal center (GC) reactions. This study shows that a subset of autoreactive B cells in V(H)3H9 micro IgH transgenic mice escapes these serial checkpoints and proceeds into splenic GC. GC B cells isolated from these mice all express the transgenic V(H)3H9 micro heavy chain, some co-express light chains that yield an anti-dsDNA specificity and some have somatic mutations, consistent with their GC origin. Nonetheless, B cell tolerance is ultimately preserved as serum titers of anti-dsDNA antibodies are not elevated. These observations suggest that those autoreactive GC B cells that escaped earlier checkpoints and possibly also those cells that acquire autoreactivity de novo by mutating their antigen receptor are arrested within the splenic GC before differentiating further into antibody-secreting plasma cells.     

An antibody VH gene that promotes marginal zone B cell development and heavy chain allelic inclusion

The Ig heavy (H) chain plays a pivotal role in the regulation of primary B cell development through its association with a variety of other proteins including Igalpha and Igbeta, the surrogate light chain components and bona fide L chains, to form transmembrane signaling complexes. Little is known about how alterations in the structure of the H chain variable region influence association with these proteins, or the signaling capacity of the complexes that form. Here we describe a line of VH 'knockin' mice in which the transgene-encoded VH region differs by eight amino acid residues from the VH region in a VH knockin line we previously constructed and characterized. The transgenic H chain locus in the line of mice we characterized earlier efficiently promotes H chain allelic exclusion and all phases of primary B cell development, resulting in the generation of mature B1, marginal zone (MZ) and follicular (FO) B cell compartments. In contrast, the transgenic H chain locus in the new line fails to enforce allelic exclusion, as evidenced by the majority of peripheral B cells expressing two H chains on their surfaces. Moreover, this locus inefficiently drives bone marrow B lymphopoiesis and FO B cell development. However, this H chain locus does promote MZ B cell development, from precursors that appear to be generated during fetal and neonatal life. We discuss these data in the context of previous findings on the influence of Ig H chain structure on primary B cell development.     

Abnormal kappa:lambda light chain ratio in circulating immune complexes as a marker for B cell activity in juvenile idiopathic arthritis

Patients with juvenile idiopathic arthritis (JIA) have been shown to have elevated levels of circulating immune complexes (CICs) which correlated with disease activity. Our aim was to assess B cell activity by measuring the amount of and the kappa:lambda chain immunoglobulin light (L) chain ratio in CICs from JIA patients and to determine potential evidence for either an antigen-driven response or B-cell receptor editing. We used an enzyme-linked immunosorbent assay to measure kappa and lambda chains present in the CICs from the sera of patients with JIA. Statistical analysis was performed using Pearson's correlation, one-way ANOVA and Bonferroni post hoc analysis. Sera from 44 JIA patients were examined for the concentration of L chains in CICs. Healthy controls had a kappa:lambda chain ratio of 1.2:1, whereas this ratio was reversed among JIA subgroups with RF-positive polyarthritis (1:1.2), RF-negative polyarthritis (1:1.3), oligoarthritis (1:2.3) and systemic-onset arthritis (1:2.5). In addition, overall lambda chain selection was not significantly associated with a particular immunoglobulin heavy (H) chain and occurred with all immunoglobulin isotypes. We showed preferential selection of lambda chains contributing to the formation of potentially pathogenic CICs from JIA patients, of all onset types compared to healthy controls, in an H chain-independent manner. The reversal of kappa:lambda chain ratio within the JIA CICs and association with all immunoglobulin isotypes demonstrated the potential for L chain editing. Furthermore, we conclude that a reversal of the normal kappa:lambda chain ratio in JIA CICs may be used as a marker for increased B-cell activity.     

Evidence for selection of a population of multi-reactive B cells into the splenic marginal zone

Antibody reactivity to self-antigens is a normal component of the immune system. To study the mechanism by which self-reactive B cells are generated and maintained, we analyzed B cell development in transgenic mice that express a rearranged VH81X heavy chain from the pre-immune repertoire. In these mice, > 95% of B cells express the transgene in association with a variety of kappa light chains but V kappa 1 C being the dominant light chain. These transgenic B cells with identical V kappa 1C-J kappa 5 joins do not normally secrete IgM in vivo, but antibodies derived from these B cells, through LPS activation in vitro or after hybridoma immortalization, are self-reactive and recognize an ubiquitous epitope(s) on intracytoplasmic proteins from different tissues. They have the phenotype and localization pattern of long-lived marginal zone B cells and their development in vivo is blocked by injection of soluble VH81X-V kappa 1CJ kappa 5 IgM antibody. The observations in this transgenic mouse provide evidence for positive selection of a population of self-reactive B cells. These B cells enter the peripheral pool of B cells where they localize in the marginal zone of the spleen and, in contrast to other transgene-expressing B cells, do not secrete IgM antibody.