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M Weigert

Publications and source records attributed to M Weigert.

At least 37 records · Page 2Linked to original sources
PubMed1994

Genetic and structural evidence for antigen selection of anti-DNA antibodies.

The primary structure of anti-DNA antibodies is highly diverse, a result of different germline variable (V) gene use, different combinations of immunoglobulin gene segments, peculiar heavy chain complementarity determining region 3 (H-CDR3) segments, and somatic mutations. Nevertheless, tertiary structure predictions reveal common features that yield information about likely contact sites in the anti-DNA combining site. That these contacts are involved with DNA binding is supported by recurrent features of a newly compiled set of homology groups of 13 variable regions of heavy chains (VH) and 11 variable regions of light chains (VL), characteristic pattern of somatic mutations, and the results of site-directed mutagenesis. The role of antigen in the etiology of the autoimmune response is viewed in light of recent data on overlaps between anti-DNA and anti-nucleic acid binding protein specificities.

Amino Acid Sequence↗
PubMed1993

Residues that mediate DNA binding of autoimmune antibodies.

Somatic mutations to arginine (R) are a common feature of a subset of J558 H chain genes that code for the majority of high-affinity, anti-dsDNA antibodies in autoimmune MRL/lpr mice. To examine the consequences of such amino acid substitutions on DNA binding, we reverted three somatic mutations of a prototypic anti-dsDNA H chain gene, VH3H9, and assayed the effect of those reversions by expression in a V lambda 1 L chain-only plasmacytoma line. Reversion of R53 eliminated virtually all dsDNA binding and sharply reduced ssDNA affinity. While the complete germ-line revertant of VH3H9 retained a low level of DNA binding, the substitution of R96, a product of N base addition in the third complementarity determining region (CDR3), with glycine (G) was sufficient to abolish measureable DNA specificity. Antibodies with higher affinity for DNA were generated by introducing arginines into VH3H9 at any one of four positions where somatic mutations to arginine had been identified by sequencing other anti-dsDNA J558 H chain genes. All four arginine mutants showed affinity increments consistent with their direct involvement in DNA binding, although one such mutant, K64R, required the simultaneous reversion of an adjacent aspartic acid (D) to the germ-line glycine. Two variants with three nongerm-line arginines showed further improvements in DNA affinity suggesting that their contributions to DNA binding may be additive. Molecular modeling of antibody and mutant F(ab) structures and calculations of their electrostatic potentials were used as an aid in interpreting the results and in predicting the location and size of possible combining sites.

Amino Acid Sequence↗
PubMed1993

B lymphocytes may escape tolerance by revising their antigen receptors.

To explore mechanisms that prevent autoreactivity in nonautoimmune mice, endogenous immunoglobulin (Ig) light (L) chains that associate with a transgenic anti-DNA heavy chain were analyzed. The antibodies from splenic B cell hybridomas of such mice did not bind double-stranded DNA (dsDNA) and their L chain sequences showed a biased use of V kappa and J kappa gene segments. The 44 L chains in this survey were coded for by just 18 germline genes. Six of the genes, each belonging to a different V kappa group, were used more than once and accounted for three fourths of all sequences. Based on the distribution of V kappa genes, the L chain repertoire in this line of transgenic mice was estimated at 37 V kappa genes. The most frequently observed gene, a member of the V kappa 12/13 group, was identified in 16 hybrids. In addition, the majority of V kappa genes used J kappa 5. We interpret the skewed representation of V kappa and J kappa gene segments to result from negative selection. Based on the data, we suggest that V kappa rearrangements giving rise to anti-dsDNA reactivity are removed from the repertoire by a corrective mechanism capable of editing self-reactive Ig.

Animals↗
PubMed1993

Receptor editing: an approach by autoreactive B cells to escape tolerance.

To determine the fate of anti-DNA antibody-bearing B cells in normal mice, we generated transgenic mice bearing the heavy (H) and light (L) chain genes of a well-characterized anti-double-stranded DNA antibody. This antibody was originally isolated from a diseased MRL/lpr mouse and has characteristics common to spontaneously arising anti-DNA antibodies. Results show that the H/L transgene (tg) immunoglobulin receptor is not expressed by animals bearing both tgs, although single tg animals (H or L) express their transgenes. Young H/L tg animals express few B cells, whereas adult H/L tg animals maintain almost normal B cell numbers. Analysis of the immunoglobulin receptors used by adult B cells shows that all contain the tg H chain in association with endogenous L chains. These B cells transcribe the L tg as well as the rearranged endogenous L chain gene, and loss of endogenous L chain gene transcription results in resurrection of the 3H9 H/L tg product. Examination of the endogenous L chains used by these cells shows that they represent a highly restricted subset of V genes. Taken together, these data suggest that autoreactive transgenic B cells can rearrange endogenous L chain genes to alter surface receptors. Those L chains that compete successfully with the L tg for H chain binding, and that create a nonautoreactive receptor, allow the B cell to escape deletion. We suggest that this receptor editing is a mechanism used by immature autoreactive B cells to escape tolerance.

Animals↗
PubMed1992

Selective pathogenicity of murine rheumatoid factors of the cryoprecipitable IgG3 subclass.

To analyze the involvement of rheumatoid factors (RF) in the generation of cryoglobulins and the development of related tissue injuries, we have established a panel of anti-IgG2a RF mAbs derived from MRL/MpJ-lpr/lpr (MRL-lpr), C3H/HeJ-lpr/lpr, and 129/Sv mice. After injection of hybridoma cells to normal mice, all four IgG3 RF mAbs induced cryoglobulinemia, and various degrees of glomerulonephritis and skin leukocytoclastic vasculitis. In contrast, none of the RF mAbs of the other isotypes generated cryoglobulins or tissue lesions. Since the same observation was obtained with another panel of five clonally related anti-IgG2a RF mAbs of MRL-lpr origin with almost identical heavy and light chain variable (V) regions but five different isotypes, it seems unlikely that the absence of pathogenicity of non-IgG3 RF mAbs was due to differences in fine specificity or V framework regions. In addition, the analysis of serum RF in MRL-lpr mice has demonstrated that a majority of 4 month old MRL-lpr mice produced substantial amounts of IgG3 RF with cryoglobulin activity. Because the cryoglobulin activity is associated with the murine IgG3 heavy chain constant region, RF of this subclass may play a significant role in the development of autoimmune-related tissue injuries, especially in MRL-lpr mice.

Animals↗
PubMed1991

Expression of anti-DNA immunoglobulin transgenes in non-autoimmune mice.

Self-reactive B cells can be regulated by either deletion or inactivation. These manifestations of self-tolerance have been dramatically shown in transgenic mice in which the number of self-reactive cells has been artificially expanded. We have now extended these models to ask if B-cell tolerance as described for non-disease-associated antigens also operates for the targets of autoimmunity. The target we have chosen is DNA. Anti-DNA antibodies are diagnostic of certain autoimmune syndromes in humans and are a characteristic of the murine model of systemic autoimmunity, the MRl/lpr mouse. Antibodies to both single-stranded and double-stranded DNA have been implicated in disease. By generating anti-DNA transgenic mice, we have addressed the question of whether DNA-specific B cells are regulated in normal (non-autoimmune) mice. We indeed found that most transgenic B cells bind DNA, yet we failed to detect secreted anti-DNA. We suggest that as a consequence of their self-reactivity these B cells are developmentally arrested.

Animals↗
PubMed1991

Ig H and L chain contributions to autoimmune specificities.

An Ig H chain expression vector has been constructed by using the V region of 3H9, an antibody that binds ssDNA, dsDNA, and cardiolipin. The H chain construct was transfected into six hybridoma cell lines expressing Ig L chains. All resulting H and L chain combinations had at least some affinity for ssDNA, whereas five also bound dsDNA to a similar degree as 3H9. The loss of dsDNA binding was correlated with a single amino acid difference between two V kappa 8 L chains. A further characteristic of 3H9, its immunofluorescent staining pattern, was shared by four of the recombinant antibodies, whereas its specificity for cardiolipin was shared with five. The transfections reported here show that a V kappa 3 L chain confers specificity for an RNA-associated epitope and that a V kappa 21E L chain prevents cardiolipin binding. These experiments suggest that the 3H9 H chain contributes essential determinants required for binding to DNA as well as cardiolipin but that L chains can modulate or prevent this binding. L chains may also expand the specificity of a recombinant antibody.

Amino Acid Sequence↗
PubMed1990

The BALB/c secondary response to the Sb site of influenza virus hemagglutinin. Nonrandom silent mutation and unequal numbers of VH and Vk mutations.

We have determined the nucleotide sequences of the expressed VH and Vk genes from 13 secondary (2 degrees) hemagglutinin (HA) (Sb) specific hybridomas derived from a single mouse. These antibodies share an Id, H37-68 (68Id) that dominates the 2 degrees HA(Sb) response in this mouse, but is rare or absent from 2 degrees responses of other mice. We find that these antibodies derive from five clones. The H chains of these antibodies are encoded by a single VH gene joined to a variety of DH and JH genes. The length of complementarity-determining region (CDR) 3 and sequence of the D-J junction are restricted, suggesting selection on CDR3 of the H chain. The L chains are more diverse. In the presented examples, they are encoded by the Vk21C and Vk21E genes and a Vk9 gene, and are joined to Jk1, 2, or 4. Each antibody is extensively mutated. The nature and distribution of the mutations suggests that 68Id-producing cells have been selected by Ag, although there are differences regarding the domain (VH, Vk, or both) in which mutations were selected. The implications of these findings on the idiosyncratic nature of the 68Id antibody response to HA(Sb) are discussed. There are two unusual characteristics regarding somatic mutation in these hybridomas. Whereas the expressed VH and Vk21 genes appear to have accumulated mutations at a high rate (1 to 1.5 x 10(-3)/base pairs/division, the expressed Vk9 genes appear to have accumulated mutations at a 5 to 15-fold lower rate than the expressed VH genes in the same cells. There is also a surprisingly high number of parallel silent somatic mutations in the VH genes, of which all but one are clustered to a 28-bp region in framework region 2 and CDR 2-encoding segments. The probability that this could have occurred by a random mutational process is essentially zero.

Amino Acid Sequence↗
PubMed1990

Heavy-chain class switch does not terminate somatic mutation.

We studied the H-chain class switch rearrangement in four groups of clonally related B cell hybridomas, to test the hypothesis that class switch terminates somatic mutation in a B cell. Using switch region-specific probes in Southern blot analysis individual mu-gamma switch rearrangement events can be distinguished. We show that clonally related IgG-producing hybridomas that differ by mutations often share a common switch rearrangement. This indicates that class switch in these cells did not terminate somatic mutation.

Animals↗
PubMed1990

Anti-DNA antibodies from autoimmune mice arise by clonal expansion and somatic mutation.

The proximate cause of autoantibodies characteristic of systemic autoimmune diseases has been controversial. One hypothesis is that autoantibodies are the result of polyclonal nonspecific B cell activation. Alternatively, autoantibodies could be the result of antigen-driven B cell activation, as observed in secondary immune responses. We have approached this question by studying monoclonal anti-DNA autoantibodies derived from unmanipulated spleen cells of the autoimmune MRL/lpr mouse strain. This analysis shows that anti-DNAs, like rheumatoid factors (19), are the result of specific antigen-driven stimulation. In addition, correlation of sequences with fine specificity shows that: (a) somatic mutations can cause specificity for dsDNA and that such mutations are selected for; (b) arginine residues play an important role in determining specificity; and (c) anti-idiotypes that recognize the majority of anti-DNA are probably not specific for any one family of V regions.

Amino Acid Sequence↗
PubMed1988

[Preventive stabilization of the knee joint by a bandage (brace) in an ice hockey player. Sense or nonsense?].

Braces for the knee are used by more and more First League Players to prevent injury to the knee, whether or not the knee has been injured previously. One player of the National Team wearing braces since last season complained of restricted efficiency. Electromyographical examination of his thigh muscles showed severe neurogenic changes. We concluded to refuse that so-called preventive braces.

Adult↗
PubMed1987

How immunoglobulin V kappa genes rearrange.

Recombination at the immunoglobulin kappa light chain locus creates a complete variable region gene and its reciprocal product. The results presented here show that reciprocal products may be substrates for secondary recombination and that at least one V kappa group rearranges by inversion.

Amino Acid Sequence↗
PubMed1987

Variable region sequences of murine IgM anti-IgG monoclonal autoantibodies (rheumatoid factors). II. Comparison of hybridomas derived by lipopolysaccharide stimulation and secondary protein immunization.

We have obtained the complete variable region mRNA sequences of 11 LPS-derived and 14 secondary immunization-derived monoclonal IgM anti-IgG antibodies (rheumatoid factors, RFs). A comparative analysis of these sequences showed that monoclonal RFs derived after polyclonal activation are structurally very similar to RFs derived after secondary protein immunization. This study was undertaken to evaluate the potential relationship between two previously described phenomena: (a) during a secondary response to a protein antigen, RF is produced in quantities that equal or exceed the immunogen-specific antibody; and (b) the frequency of B cells that make RF after polyclonal activation is quite high; 3-10%. It has been unclear whether LPS-stimulated cells that produce IgM anti-IgG that is detected by an in vitro assay are related to the cells that produce RF after in vivo stimulation. The similarity of the antigen receptors found in the two types of RF, however, suggests that most or all of the RF-producing B cells detected after LPS stimulation would also be stimulated during the secondary immune response. Thus, the presence of relatively large number of B cells that can make RF after nonspecific stimulation provides an explanation for the magnitude of RF production accompanying the secondary immune response.

Amino Acid Sequence↗
PubMed1987

A complex translocation at the murine kappa light-chain locus.

We have previously reported that a segment of DNA from a murine plasmacytoma comprises DNA from three chromosomes, the immunoglobulin kappa light-chain locus on chromosome 6, the S mu locus on chromosome 12, and a region on chromosome 15. We now report that the reciprocal product contains DNA from only the kappa locus and chromosome 15 and not from S mu. We conclude that a complex series of events, including both a transposition of DNA and a translocation between chromosomes, generated these imperfect reciprocal products.

Animals↗