Specificity and molecular characteristics of monoclonal IgM rheumatoid factors from arthritic and non-arthritic mice

Two-hundred twenty-four hybridomas secreting monoclonal IgM rheumatoid factor (hIgMRF) derived from MRL-lpr/lpr, MRL-+/+ and C57BL/6-lpr/lpr autoimmune mice were analyzed with regard to IgG subclass and domain specificity, and some for VH gene expression patterns. Among these mice, only MRL-lpr/lpr develop arthritis. Clonotypes specific for each of the four mouse IgG subclasses and clonotypes reacting with more than one IgG subclass were identified. Although each panel contained several clonotypes, the predominant one differed in each strain (MRL-lpr/lpr, anti-IgG2a; MRL-+/+, combined anti-IgG2a and 2b; C57BL/6-lpr/lpr, anti-IgG1 or combined anti-IgG1, 2a, and 3). The IgG domains recognized by these monoclonals were defined with mutant Ig carrying IgG1 heavy chains that lacked either the CH1 or CH3 domains, variant Ig carrying hybrid IgG2b-2a heavy chains, and IgG fragments. Inhibition of hIgMRF binding to IgG substrates by protein A was also assessed. Most determinants were assigned to the CH3 domain, but determinants in the hinge region, CH2 domain, and in some instances, even in the Fab portion, could also be identified. Hybridization of cytoplasmic RNA from 35 classes of diverse IgG subclass specificity with VH gene probes representing seven of the approximately 10 VH families (7183, S107, Q52, J558, J606, 36-60, X24) indicated that approximately 90% of these clones expressed VH genes belonging to the large J558 gene family. The results indicate that murine IgMRF are extremely heterogeneous in IgG subclass and domain specificities; the genetic background influences RF specificity characteristics that may relate to pathogenicity; and considering the complexity of the J558 VH gene family and reported RF heavy chain assignments to additional VH gene families, it appears that VH genes encoding RF are diverse.     

Molecular characterization of the A/J J558 family of heavy chain variable region gene segments

The nucleotide sequences of the coding as well as the flanking regions of 11 A/J J558 heavy chain variable region (VH) gene segments are presented. Among these J558 VH segments was the unrearranged germline VH gene segment recruited in the predominant A strain-specific anti-arsonate response. Three other VH gene segments that are greater than 92% related to the p-azophenylarsenate (Ars) A VH gene segment were also isolated. Detailed analysis of the nucleotide sequences of these as well as the remaining seven J558 VH gene segments reveal that the J558 VH gene family is composed of distinct, but related, J558 VH subfamilies. Deletion mapping analyses were used to position the Ars A VH gene segment proximally with respect to the DH-JH clusters within the J558 VH gene family and distally with respect to its own J558 subfamily. The documentation of J558 VH subfamilies is discussed in the context of J558 VH family evolution and diversification.     

Structure, map position, and evolution of two newly diverged mouse Ig VH gene families.

We have characterized two novel mouse VH gene families, VH3609N and VHSM7. These VH families have recently diverged from previously defined VH families. The VH3609N family, which may contain only one member in most inbred strains of mice, shares sequence similarity with the VHJ606 family and is located to the 3' side of VHJ606. VHSM7, with at least three members, is related to the VHJ558 family but maps 3' of VHJ558. These findings suggest that physical displacement of VH sequences may facilitate their subsequent divergence. During the early stages of VH gene family evolution that are exemplified by these new families, amino acid replacements have been selected against in frame-work regions and selected for in complementarity-determining regions. This pattern of nucleotide substitution appears to reflect evolutionary pressures to maintain germ-line VH diversity and, possibly, to select for new antibody specificities, as well as to select against mutations resulting in aberrant Ig. The classification of VH sequences with borderline similarity to previously defined VH families is discussed.     

Ig VH gene family repertoire of plasma cells derived from lupus-prone MRL/lpr and MRL/++ mice

VH gene family usage was determined in both spontaneous, in vivo activated plasma cells and LPS-induced plasma cells from individual MRL/lpr mice by using in situ hybridization. It was found that VH gene family expression in spontaneous plasma cells varied from mouse to mouse. Some mice expressed VH families in an apparently random manner similar to that obtained with polyclonal activation. Other mice showed an exaggerated expression of particular VH gene families. VH J558 was overrepresented most frequently, but overrepresentation of VH 7183, Q52, and 36-60 was also observed. Importantly, LPS-induced VH gene family expression in these same mice displaying biased VH family usage in spontaneous plasma cells, appeared normal with no evidence for similar biases in the LPS-induced repertoire. Anti-DNA antibody concentrations and the degree of glomerulonephritis were determined for each mouse to measure the severity of disease. The level of expression of the J558 family was positively correlated with disease severity. The results suggest that the initial autoantibody response is highly diverse but becomes more restricted as the disease progresses.     

Direct quantitative in situ hybridization studies of Ig VH utilization. A comparison between unstimulated B cells from autoimmune and normal mice

This study examines Ig VH utilization in murine lupus with emphasis on the relative contribution of 3' and 5' gene families. We used in situ hybridization with 35S-labeled ssRNA probes to detect VH expression in individual spleen cells. Cells were taken from unmanipulated animals, and were not stimulated in vitro. This approach allows analysis of VH usage among only those B cells which have undergone activation in vivo, while minimizing the potential for skewing in vitro. We compared usage of the 3' 7183 and Q52 families with the more 5' J558 family in adult NZB, MRL-lpr/lpr, and nonautoimmune NIH Swiss mice. VH utilization in the autoimmune strains was proportionate to VH family size, and was not biased toward the 3' families when compared with the Swiss repertoire. Moreover, 3' skewing did not develop in NZB mice with increasing age. Thus, systemic autoimmunity is not associated with impaired normalization of the adult repertoire away from the 3' bias of early ontogeny. Instead, our data support a stochastic model for VH gene usage in the activated B cells and plasma cells of adult lupus mice.     

Northern hybridization analysis of VH gene expression in murine monoclonal antibodies directed to cancer-associated ganglioside antigens having various sialic acid linkages

The expression of the VH genes in 46 murine hybridoma cells that secrete mAb directed to the cancer-associated carbohydrate Ag, especially acidic glycolipids such as gangliosides and sulfated glycoplipids, was analyzed by Northern hybridization of poly(A)+ RNA of hybridoma with cDNA probes for nine VH gene families. Different hybridomas tended to express VH genes of the same family when the cognate Ag had the same or similar carbohydrate structures; i.e., the VH genes of the J558 family (group 1) were preferentially expressed in the mAb directed to various gangliosides that have NeuAc alpha (or NeuGc alpha) 2-3 and/or 2-8 linkage (71%), the most common linkage of sialic acid residues in the gangliosides of higher animals, and the hybridomas directed to sulfated glycolipids also expressed mainly the VH genes of the J558 family (80%). In contrast, the five mAb directed to various gangliosides with NeuAc alpha 2-6 linkage were exclusively encoded by the VH genes of Q52 family (group 2, 100%), and three antibodies directed to gangliosides with a NeuAc alpha 2-9 linkage all expressed genes of J606 family (group 6, 100%). The VH family usage was largely correlated with the linkage of sialic acid residues in the cognate carbohydrate Ag, but was not correlated at all with the difference in the fine specificities toward the core neutral carbohydrate chain, to which the sialic acid residues were attached. These findings suggest that the VH gene family in these anticarbohydrate antibodies is selected, depending primarily on the linkage of the sialic acid residues in carbohydrate Ag; these residues form the immunodominant sugar residue in the respective antigenic determinant.     

VH gene family utilization in colonies derived from B and pre-B cells detected by the RNA colony blot assay.

The immunoglobulin heavy chain variable region (VH) genes of the mouse have been categorized into families based upon sequence homology. Utilizing the RNA colony blot assay we have determined the expression of eight of these families in B cell colonies derived from either surface immunoglobulin positive (sIg+) adult spleen B cells or sIg- fetal liver pre-B cells. We demonstrate, based upon the analysis of greater than 6000 individual colonies, that VH gene usage is a characteristic of the mouse strain studied. C57BL/6 mice most frequently (45%) utilize family VHJ558, the largest VH family, whereas BALB/c mice most frequently (22%) utilize family VH7183, the most JH proximal family in BALB/c mice. Moreover, colonies derived from sIg- fetal liver derived precursors show similar patterns, suggesting that selection based on exogenous antigen is not an important parameter in determining VH gene family usage.     

Strain-dependent restricted VH and VL usage by anti-bacterial levan monoclonal antibodies.

The immune response to polysaccharides is highly regulated and has several distinguishing features, including restricted clonotype and isotype expression. The basis for this highly restricted response is not fully understood. To address these questions in a systematic manner, we have generated a panel of 102 mAb from CBA/CaHN (CBA/Ca) and BALB/cAnN (BALB/c) mice after one and two injections of bacterial levan (BL), a beta(2----6)-linked polyfructosan with beta(2----1)-linked fructose branch points (inulin determinant, In). This panel of mAb was examined for isotype, fine specificity, VH and VL region gene family usage and relationships between these parameters. After one or two injections of BL in both strains, mAb were IgM and IgG3. Fine specificity and VH/VL gene family usage differed markedly, however, between the two strains. Only 4% (2/51) of CBA/Ca mAb recognized the In determinant, whereas 77% (40/51) of BALB/c mAb recognized this epitope. In both strains, VH usage was restricted and certain families were overrepresented. In CBA/Ca mice, the overall response to BL was dominated by VHJ558 (45%, 23/51), the largest VH family, but VH36-60 (27%, 14/51) and VHJ606 (25%, 13/51) were also highly utilized and overrepresented. In BALB/c mice, the overall response to BL was dominated by VHJ606 (79%, 39/49 designated), a relatively small VH family. More importantly, after a single immunization with BL one particular VH/VL pair (VHJ606/ kappa 11) was used by 88% (36/41) of BALB/c mAb and was associated with In reactivity. In summary, BALB/c and CBA/Ca responses to BL differ in fine specificity and VH/VL usage.     

Eleven MRL-lpr/lpr anti-DNA autoantibodies are encoded by genes from four VH gene families: a potentially biased usage of VH genes

The genes encoding 11 independently derived anti-DNA autoantibodies from the lupus-prone mouse strain, MRL-lpr/lpr, were examined with VH, D, and JH gene probes. These autoantibodies do not define new VH gene families, since all of the autoantibodies were encoded by VH genes from four of the nine known gene families. A minimum of nine different VH genes encoded this panel of 11 anti-DNA autoantibodies. These results are consistent with the stochastic use of the VH gene repertoire and the expression of multiple VH genes. However, the data is also consistent with a biased usage of the VH gene repertoire. First, two pairs of autoantibodies, one from the J558 family and one from the 7183 family, appear to express identical or closely related VH genes as determined by the position of two restriction enzyme sites 5' of the expressed VH genes. In addition, three autoantibodies that appear to be sister clones might define a third VH gene that is used repeatedly. Secondly, about 45% of the panel is encoded by the Q52 and 7183 families, which are the 3' most families. These families have been shown to be preferentially rearranged early in B cell ontogeny. This suggests that some anti-DNA autoantibodies might originate from a population of B cells that predominate early in ontogeny. An alternative hypothesis is that the potential bias in VH gene and gene family usage could be due to antigen selection. All four JH genes are expressed, although the JH1 gene appears to be underutilized in both expressed and unexpressed rearrangements. Two members of the panel that bind double-stranded DNA were encoded by two different VH gene families, the S107 family and the J558 family.     

Germ Line Variable Regions That Match Hypermutated Sequences in Genes Encoding Murine anti-Hapten Antibodies

We asked whether there are germ line immunoglobulin variable (V) segments that match sites of hypermutation in V regions encoding murine antibodies. Murine germ line DNA was probed with a panel of short deoxyoligonucleotides identical in sequence to segments of hypermutated V regions from hybridomas generated in the BALB/c response to the hapten 2-phenyloxazolone (Ox). Germ line sequences that match mutations in both heavy and kappa light chain V regions were identified, and clones of some of these germ line V segments were obtained. Comparison of these clones with hypermutated V regions revealed regions of identity ranging in size from 7 to over 50 nucleotides. In an effort to separate the effects of antigen selection from the mutagenic process, we also searched for matches to a panel of silent mutations in VH regions from germinal center B cells. Fourteen silent mutations occur among a collection of 36 hypermutated VH regions from two separate germinal centers of C57BL/6 mice stimulated with the hapten 4-hydroxy-3-nitrophenyl. Matches to nine of these silent mutations can be found among published sequences of C57BL/6 VH regions of the J558 family. Taken together, these data are consistent with the possibility that a template-dependent mutational process, like gene conversion, may contribute to somatic hypermutation.     

VH gene family repertoire of resting B cells. Preferential use of D-proximal families early in development may be due to distinct B cell subsets

The fetal VH gene repertoire was shown previously to be characterized by overrepresentation of D-proximal families, VH 7183 and VH Q52, compared with adult bone marrow B cells in which VH genes were expressed in a more stochastic fashion. To determine the underlying mechanisms of these findings, adult vs fetal progenitors were placed in the same supportive microenvironment and the resulting B lineage cells analyzed for VH gene family expression. The supportive microenvironment was provided by established adult bone marrow stromal cell layers. In this way the relative importance of environmental vs genetic influences could be determined. The fetal B cells and pre-B cells that developed on adult stromal cells maintained a fetal-like VH gene family repertoire with preference for D-proximal families VH 7183 and Q52. In contrast, adult cultured B cells maintained the adult-like repertoire with predominance of the largest family VH J558. Only after long-term incubation was there a change in the expression of particular VH gene families. These findings suggest that the D-proximal VH gene family preference observed early in ontogeny is associated more with the inherent genetic potential of B cell progenitors that predominate during fetal life and less with environmental influences.     

Variable region cDNA sequences and antigen binding specificity of mouse monoclonal antibodies to isomaltosyl oligosaccharides coupled to proteins. T-dependent analogues of alpha(1----6)dextran

Four mouse hybridomas specific for alpha(1----6)dextran, 16.4.12E (IgA kappa, C57BL/6), 28.4.10A (IgM kappa, BALB/c), 35.8.2H (IgG1 kappa, BALB/c), and 36.1.2D (IgM kappa, BALB/c) were obtained by immunization with the T-dependent Ag isomaltohexaose or isomaltotriose coupled to keyhole limpet hemocyanin or to BSA. Immunochemical characterization of the hybridoma antibodies showed that 16.4.12E and 36.1.2D had cavity-type combining sites, recognizing the terminal non-reducing end of alpha(1----6)dextran, whereas 28.4.10A and 35.8.2H had groove-type sites, recognizing internal linear segments of the dextran. The V region cDNA of the H and L chains of the antibodies were cloned and sequenced. VH of 16.4.12E and VH of 36.1.2D belonged to the X24 and Q52 germ-line gene families, respectively. The VH and V kappa sequences of 16.4.12E and V kappa sequence of 36.1.2D were highly homologous to those of W3129, the only anti-alpha(1----6)dextran mAb with a cavity-type site thus far sequenced; 16.4.12E differed from W3129 in the D, JH, and J kappa. VH genes of 28.4.10A and 35.8.2H were homologous to those of several anti-alpha(1----6)dextrans with groove-type sites, but belonged to the J558 germ-line gene family, differed from the other J558 anti-alpha(1----6)dextrans, probably representing a different germ-line subfamily. The L chain sequence of 28.4.10A encoded by V kappa-Ars and J kappa 2 was almost identical to other groove-type anti-alpha(1----6)dextrans obtained by immunizing with the T-independent glycolipid Ag, stearyl-isomaltotetraose. Use of T-dependent Ag such as isomaltosyl oligosaccharide-protein conjugates provides an additional parameter for probing the fine structure of antibody combining sites and evaluating the V-gene repertoire of anti-alpha(1----6)dextrans.     

Variable region sequence analysis of anti-DNA antibodies: evidence for a family of closely related germ-line VH genes encoding lupus autoantibodies.

Cloning and sequencing of the V regions of the anti-DNA monoclonal antibodies (mAbs), H438 and H130, indicate that H438 is encoded by a J558 VH gene, a single D region nucleotide, and unmutated JH1, V kappa-1C and J kappa 1 genes, and the H130 L chain is encoded by a V kappa-21 subgroup gene J kappa 1 gene. Identification of VH438, which shared VH hybridization pattern with 6% of a panel of 352 MRL/lpr hybridomas, suggests that the frequency of J558 use among spontaneously activated B cells in MRL/lpr mice is greater than previously reported. The VHH438 J558 family gene is identical to VHPAR, which encodes the independently derived MRL/lpr autoantibody, MRP-2, and is highly homologous to the previously reported VHH130, which is identical to a BALB/c germ-line VH gene. Comparison of consensus sequences of homologous autoantibodies and previously reported restriction mapping suggest that a minimum of three highly related J558 germ-line genes encode lupus autoantibodies.     

A new murine Ig VH gene family

A novel murine VH gene family, termed VH10, has been found and characterized. Based on RFLP analysis, this family exhibits extensive polymorphism among inbred strains of mice and encompasses two to five members, depending on the Igh haplotype. Analyses of recombinant inbred strains suggest a map position of this family 5' to the 7183 and Q52 VH gene families. A VH10 gene has been found to encode anti-DNA autoantibodies from lupus mice; another one may be a pseudogene.     

Molecular basis of a mouse strain-specific anti-hapten response

The response of C57BL/6 and BALB/c mice to immunization with proteins coupled to (4-hydroxy-3-nitrophenyl)acetyl (NP) is dominated by distinctly different sets of antibodies. The VH gene family previously shown to be involved in the C57BL/6 response has now been shown to have highly homologous counterparts in BALB/c but of five sequenced BALB/c VH regions, none appeared likely to be able to encode an NP-binding protein. The active VH region from a BALB/c hybridoma making a characteristic anti-NP antibody was recovered and sequenced and shown to be quite different from the VH gene family involved in the C57BL/6 response. Comparison of the variation of the closely related VH regions between the two mouse strains showed that there are separate types of evolutionary pressures on the framework and complementarity-determining regions. The molecular basis for strain-specific immune responses appears to be that the structural divergence of VH regions between mouse strains is great enough that different strains use different VH regions for making the predominant class of antibodies to a specific hapten.     

VH and VL gene usage by murine IgG antibodies that bind autologous insulin

To assess the recognition structures of antibodies that bind a self-Ag, we used mRNA analysis to identify the V region genes of IgG antibodies that bind autologous insulin. Four anti-insulin mAb from primary immunization of BALB/c mice use different combinations of H and L chain V region genes. Two VH genes are from the V-gam 3-2 and V-gam 3-8 families that are infrequently expressed in adult BALB/c mice, and two VH genes are members of the J558 family. Each anti-insulin antibody uses a different Vk gene family. Two antibodies express common Vk genes (Ox1 and Vk21C), whereas two other Vk genes are unusual in BALB/c mice. One Vk gene may represent a BALB/c equivalent of the VkOx2 subfamily and another is identical to a Vk used by anti-idiotypic antibodies from C57Bl/6 mice. When compared with known germ-line counterparts, all of the Vk sequences are close to germ-line configuration. In contrast, the germ-line counterparts for the anti-insulin VH genes are not known, however, they differ only in five to seven predicted amino acids from VH of other expressed antibodies. One antibody (mAb 123) differs in one amino acid in complementarity-determining regions 1 and 2 from the VH of the murine tumor BCL1, and another (mAb 126) employs an unmutated DFL16.1 germ-line D segment. These data suggest that antibodies binding autologous insulin use V gene components that are not extensively mutated, even when derived by immunization with heterologous insulin.     

One heavy chain variable region gene segment subfamily in the BALB/c mouse contains 500-1000 or more members

Nucleic acid hybridization studies suggest that 500-1000 or more heavy chain variable (VH) gene segments related to one VH gene segment (J558) are present in the genome of the BALB/c mouse. The implications of this result regarding the overall size, sequence organization, and evolution of the mouse family of VH gene segments are discussed.     

A new cross-reactive idiotype-defined family in the phthalate humoral immune response of mice. I. Linkage of VH-Xmp to IgCH allotype locus and mapping with respect to other known VH genes

A cross-reactive idiotype family was previously identified from a very large library of phthalate-specific hybridoma clones. The prototype of this idiotype family is the hybridoma, 2E9, secreting an IgM antibody with phthalate specificity. A portion of both primary and secondary anti-phthalate antibodies elicited in all BALB/c mice tested expresses the 2E9 cross-reactive idiotype. This idiotype has now been found in the anti-phthalate antibodies of several other inbred strains of mice (A/HeHa, DBA/2, and C3Hf/HeHa) tested but not in C57BL/6 mice. Anti-phthalate antibodies elicited from congenic mice BC.8, which express the same IgCH allotype as BALB/c mice but possess C57BL/6 genetic background, contain the 2E9 cross-reactive idiotype, whereas this idiotype is not expressed on the anti-phthalate antibodies derived from another congenic mouse CB.20, which expresses a C57BL/6 IgCH allotype and a genetic background of the BALB/c strain. These results indicate that the gene controlling the 2E9 idiotype is closely linked to the IgCH allotype locus. The 2E9 cross-reactive idiotype was also found in all of the F1 mice (BALB/c X C57BL/6) tested, and the level of expression of this idiotype in the F1 mice was quantitatively equivalent to the allotype/idiotype homozygous mice. The expression of the 2E9 idiotype in the phthalate repertoire has been followed in 12 different wild mouse populations. As expected, the 2E9 idiotype was observed in a large proportion of the wild mouse strains. Surprisingly, several examples of nonconcordance in the expression of idiotype and allotype were observed in these mice. One likely explanation for the linkage breakdown is a crossing over of the heavy chain constant and variable region gene complexes. In the SM/J inbred strain of mice, where such a crossover has occurred, nonconcordance between allotype and 2E9 idiotype expression was demonstrated. By using the recombinant inbred BXD strains of mice, the VH gene encoding the 2E9 idiotype has been mapped with respect to other known VH gene families. Relative to other VH genes the VH-Xmp is situated very close to the IgCH gene region.