Somatic evolution of diversity among anti-phosphocholine antibodies induced with Proteus morganii

The variable region sequences of light and heavy chains (VL and VH) were determined for 11 hybridoma antibodies produced in response to the PC moiety on Proteus morganii. These hybridomas were derived from two separate fusions, one obtained from mice early in a secondary response and the other from late in a secondary response. All of these antibodies possessed a cross-reactive idiotype found on anti-PC antibodies in the M603 family, and exhibited preferential specificity for PC in the context of P. morganii. We found that all of the antibodies were derived from a single VH/VL pair. VH was encoded by V1, DFL16.1 and JH1, and VL was encoded by a consensus VK8 gene and JK5. Antibodies differed from each other by somatic point mutations that occurred at a high rate. The mutations in VL were approximately one-third as abundant as those in VH and were randomly distributed throughout the molecule. Mutations in VH were concentrated in CDR 2 and 3 and had a replacement to silent ratio that was three to six times greater than predicted from random accumulation. Based on the sequence data, a single genealogic tree with multiple branches could accommodate all the hybrids from a fusion. We concluded that in both examples the anti-PC response arose by somatic mutation and stepwise selection from a single precursor. Antigen binding studies with these 11 hybridomas and a 12th that had no mutations revealed that the acquisition of preferential specificity for antigen was dependent on somatic mutation of germline genes. Additional binding studies demonstrated that continued selection during clonal expansion was probably antigen driven. An unexpected finding was five independently selected antibodies from one fusion that had identically mutated VH and VL sequences. We suggest that the hypermutation mechanism is not a continuously active process during clonal expansion and that it is regulated, probably during the mid to late phase of the primary response.     

Molecular basis of an isogeneic anti-idiotypic response.

The nucleotide sequences of the variable region genes expressed in the heavy and light chains of six isogeneic anti-idiotope antibodies recognizing idiotopes on two closely related antibodies with specificity for the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) were determined. In two independently derived anti-idiotope cell lines the same or strongly homologous V kappa, VH and D region genes had originally been rearranged. The two lines express long and partly homologous N sequences (presumed to be not of germ line origin) at the border of D, resulting in CDR3s of unusual length. An unusually long CDR3, partly encoded by N sequences, is also present in the heavy chain of a third anti-idiotope antibody. The VH regions of the three remaining anti-idiotope antibodies originate from a single VH gene which belongs to the same VH group as the VH genes expressed in the other anti-idiotopes. Two of these antibodies, expressing similar V, D and J elements, had been isolated from the same mouse and appear to have diverged from the same B cell precursor by at least two rounds of somatic mutation. Somatic point mutations have occurred in most, if not all anti-idiotope V region sequences. In two instances somatic mutations in J increase the structural homology between anti-idiotopes. The anti-idiotypic response in this system is thus genetically restricted and may depend upon the selection of non-germ line sequences, suggesting an explanation for the low frequency at which anti-idiotope antibodies are expressed in this system.     

NMR evidence for mechanical coupling of phosphate B(I)-B(II) transitions with deoxyribose conformational exchange in DNA

2a2 is the most commonly rearranged gene in the human V(lambda )locus. It has been postulated that certain immunoglobulin genes (including 2a2) are rearranged preferentially because their germline sequences encode structures capable of binding to a range of antigens. Somatic mutation could then increase the specificity and affinity of binding to a particular antigen.We studied the properties of five IgG molecules in which the same heavy chain was paired with different light chains derived from 2a2. The pattern of somatic mutations in 2a2 was shown to be crucial in conferring the ability to bind DNA, but two different patterns of mutation each conferred this ability.Computer-generated models of the three-dimensional structures of these antibodies illustrate the ability of 2a2 to form a DNA binding site in different ways. Somatic mutations at the periphery of the DNA binding site were particularly important. In two different light chains, mutations to arginine at different sites in the complementarity determining regions (CDRs) enhanced binding to DNA. In a third light chain, however, mutation to arginine at a different site blocked binding to DNA.     

Disparity in the kinetics of onset of hypermutation in immunoglobulin heavy and light chains

The present paper describes a comparative analysis of light chains associated with primary and secondary IgM, as well as with secondary IgG antibodies to fluorescein, undertaken in order to explore the relationship between light chain somatic hypermutation and the isotype switch. The data reveal a disparity in the frequency of somatic hypermutation of secondary IgM heavy versus light chains. Among 20 secondary IgM light chains, a mutation frequency of 1/777 nucleotides was defined. In contrast, our previous analysis of the heavy chains of these molecules had identified a mutation frequency of 1/129. Among 17 IgG-derived light chains, obtained from animals killed at the same time point as those from which the secondary IgM antibodies were obtained, we measured a mutation frequency of 1/77. Finally, analysis of 20 light chains derived from primary IgM antibodies revealed a mutation frequency of only 1/1192 nucleotides. These data demonstrate that, prior to the class switch, light chain mutation occurs at a frequency considerably lower than that measured for the associated heavy chain gene. Six additional apparent mutations in the secondary IgM antibody 95B3 were all shared with a set of IgG antifluorescein antibodies belonging to the Vkappa 34 family. It is suggested that these light chains represent the products of a previously uncharacterized germ line gene.     

The specificity properties that distinguish members of a set of homologous anti-digoxin antibodies are controlled by H chain mutations

Five murine A/J strain anti-digoxin mAb (35-20, 40-40, 40-120, 40-140, and 40-160) have highly homologous H and L chain V regions, only differing by somatic mutation, yet differ in affinity and specificity. The availability of the VH and VL genomic clones from one hybridoma, 40-140, has now allowed studies involving in vitro mutagenesis and chain recombination among these five hybridomas. To determine the relative contributions of the mutations found in either VH or VL to the overall binding properties of these antibodies, we recombined the 40-140VH with the VL of each hybridoma. The 40-140VH gene was transfected into hybridoma variants that produce only VL. The recombinant antibodies show that the mutations present in VH, rather than in VL, affect the fine specificity properties of these antibodies, whereas, the mutations among both VH and VL chains are important in determining antigen affinity. From mutations present in VH that affect fine specificity properties, the comparison of the antibody sequences, and from the previously measured binding properties, we predicted and tested selected VH mutations for their ability to alter specificity or affinity by doing site-directed in vitro mutagenesis. The results for the somatic mutations found in this group of antibodies show: 1) VH mutations control the fine specificity properties that distinguish different members of this group; 2) in particular, VH residues 54 and 55 in CDR2 control the distinguishing characteristics of specificities between these antibodies; and 3) by mutagenesis, we had the unusual result of being able to alter Ag specificity without affecting affinity. A computer model of the 40-140 antibody binding site was generated which indicates that VH residues 54 and 55 are highly accessible.     

Rabbit variable kappa light chain regions: subgroups contain polypeptides encoded by multiple genes.

Two identical light chain variable regions were identified in anti-streptococcal Group A-variant antibodies elicited in litter-mate rabbits by hyperimmunization with vaccine. In addition, one rabbit produced two additional clonally restricted antibodies to this polysaccharide antigen. The partial amino acid sequence of the light chain of one of these antibodies was identical with the dominant antibody light chain sequence, while the light chain of the other antibody, also partially established, showed significant variations in the framework-associated regions with identical CDRI and II. Since all of these light chains were from a small subset of rabbit kappa light chain pools (b4 allotype) the data suggest, together with other light chains reported in the literature, that more than one copy of variable region genes are present in the germ-line per subgroup. Furthermore, framework associated amino acid substitutions are not random; this suggests the existence of some "ordered" mechanism for linked amino acid substitutions (presumably recombination). Furthermore one light chain can pair with more than one heavy chain to yield functional antibodies.     

Idiotypic analysis of myeloma proteins: anti-DNA activity of monoclonal immunoglobulins bearing an SLE idiotype is more common in IgG than IgM antibodies

The anti-idiotype 3I which recognizes a determinant on kappa-chains of anti-DNA antibodies in SLE patients also recognizes a determinant on kappa-chains of 82/706 myeloma proteins tested. Twenty-nine of these 82 proteins bind to double-stranded DNA, including two monoclonal IgM, one monoclonal IgA, and 26 monoclonal IgG proteins. DNA binding is much more frequent in the IgG than in the IgM myeloma proteins (p less than 0.001), and is also associated with cationic antibody charge. Two-dimensional gel electrophoresis reveals markedly increased charge heterogeneity of both heavy and light chains of the monoclonal IgG as compared with the monoclonal IgM proteins. We postulate that the increased charge heterogeneity of the IgG-associated 3I-reactive kappa-light chains may reflect somatic mutation, and that DNA specificity within the 3I idiotype system arises by somatic mutation of germ-line genes found in normal individuals. DNA binding may be associated with those mutations that give rise to a cationic immunoglobulin charge.     

Somatic mutation and clonal expansion of B cells in an antigen-driven immune response.

The variable (V) regions of three closely related monoclonal antibodies produced by hybridomas which had been isolated from a single mouse were sequenced at the level of the mRNA. The sequences and the restriction analysis of the immunoglobulin loci carried by the hybridoma cells indicate that the antibodies are derived from cells belonging to a single B cell clone. The sequence data imply a high frequency and stepwise occurrence of somatic point mutations in the expressed V region genes and substantial clonal expansion of B cells in the mouse. The mutations appear to be randomly introduced into heavy and light chain V region genes. Mutations are also seen in the complementarity determining regions which may thus have been involved in the selection of the cells producing the three antibodies.     

Contributions of immunoglobulin heavy and light chains to antibody specificity for lysozyme and two haptens

Chain recombination experiments with a set of structurally and/or functionally related antibodies were performed to assess the role of the heavy (H) and light (L) chains in determining antigen specificity. The results demonstrated that specificity for hen egg white lysozyme vs two haptens (dinitrophenyl or galactan) is H chain determined and for one set of proteins could be attributed specifically to the H3 region. In contrast to hapten vs lysozyme specificity, when reassociated molecules derived from structurally unrelated antibodies that bound nonoverlapping epitopes on lysozyme were tested, localization of binding to a particular epitope on lysozyme could be predominated by either H or L chains. Furthermore, in some cases, unique specificities distinct from those of either parental antibody were formed. Replacement of the native L chain with an isotypically homologous L chain was more likely to restore high affinity protein binding than was replacement of a less related L chain. When isotypically homologous L chains were compared in association with the same H chain, fine specificity profiles were sensitive to substitutions in as few as two residues that could be attributed to somatic mutation. These results demonstrate that both affinity and specificity derive from very subtle interactions between H and L chains and provide examples of how VH assembly, VL-VH pairing, and somatic mutation could contribute to development and maturation of the specificity repertoire.     

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.     

Clonal diversity, somatic mutation, and immune memory to phosphocholine-keyhole limpet hemocyanin

Group II antibodies to phosphocholine (PC)-keyhole limpet hemocyanin in BALB/c mice are genetically diverse and of a defined binding phenotype which recognizes the hapten, phenyl-PC, and PC coupled to protein but not free PC. We sequenced the V regions of 14 kappa and lambda-bearing group II antibodies. Both types show extensive somatic mutations. The pattern of the mutations differs between kappa and lambda antibodies. The nature of the somatic mutation in lambda chains suggests strong Ag selection on the L chain but not the H chain of the lambda-bearing antibodies. The reverse pattern of selection was observed among kappa-containing antibodies wherein the accumulation of replacement mutations in the CDR of the H chain appears to result from selection while changes in the L chain appear unselected. From these findings it appears that somatic mutation plays a major role in anti-PC-keyhole limpet hemocyanin memory development because all 14 antibodies displayed changes from germ-line sequences.     

A molecular and structural analysis of the VH and VK regions of monoclonal antibodies bearing the A48 regulatory idiotype

The results presented in this paper explore the molecular basis for expression of the A48 regulatory Id (RI). A48 RI+ mAb derived from idiotypically manipulated mice molecularly resembled the A48 and UPC 10 prototypes of this system by utilizing a VHX24-Vk10 combination. Id expression by these antibodies was not restricted by a particular D region sequence, JH, or JK segment, but quantitative differences in Id expression were associated with utilization of different members of the VK10 germ-line gene families. The VL sequences of these A48 RI+ mAb has identified amino acid residues lying in four different idiotope-determining regions which may contribute to the structural correlate of this Id. A comparative sequence analysis of the VH regions of these VHX24 utilizing A48 RI+ mAb with several A48 RI+ mAb utilizing VHJ558 or VH7183 VH genes as well as a hybrid transfectoma antibody derived from two A48 RI-, VHJ558 utilizing hybridomas, all suggested that four nonconsecutive positions which lie outside the idiotope-determining regions may contribute structural elements toward expression of this Id. The VH and VL regions of the A48RI+, VHX24-Vk 10+ mAb showed low to moderate levels of somatic mutation which showed different patterns of distribution between the complementary determining region (CDR) and framework regions in the H and L chains. Although the VK sequences contained 50% of the replacement mutations in the CDR, with a replacement/silent mutation ratio of 10, the CDR of the VH sequences contained only 31% of the replacement mutations with a replacement/silent mutation ratio of 0.69.     

Different VL and VH germ-line genes are used to produce similar combining sites with specificity for alpha(1----6)dextrans

Monoclonal antibodies to alpha(1----6)dextrans produced in mice immunized with the T-independent antigens alpha(1----6)dextran or the stearylisomaltosyl oligosaccharides have been characterized immunochemically. To correlate the immunochemical properties of these monoclonal antibodies with their primary structure, we have sequenced the variable (V) regions of the light (L) and heavy (H) chains. Three V kappa germ-line genes belonging to two major gene families were used; differential J usages also contribute to diversity. Five different VH germ-line genes belonging to three major VH families were used. The VH genes were further modified by junctional diversity and differential J usage and possibly by somatic mutations. The effects of these modifications on the fine specificities of anti-alpha(1----6)dextrans are discussed. Thus far, six different combinations of VLJL-VH(D)JH chains that form groove-type combining sites specific for alpha(1----6)dextran have been found. We conclude that entirely different VL and VH can form combining sites specific for the internal linear sequence of alpha(1----6)dextran.     

甲型H1N1流感病毒血凝素单克隆抗体轻链和重链可变区基因的巢式PCR扩增及序列分析

目的：采用巢式PCR对甲型H1N1流感病毒血凝素单克隆抗体的轻链和重链基因进行扩增,对获得的基因进行序列分析,并找出克隆鼠Igκ轻链和重链可变区基因的通用方法。方法：设计22对扩增鼠Igκ轻链可变区和重链可变区基因的引物,对6株鼠抗人甲型H1N1流感病毒血凝素单克隆抗体的轻链和重链可变区基因进行克隆并测序,与NCBI公布的鼠免疫球蛋白序列比对分析。结果：巢式PCR方法可以有效避免单克隆抗体克隆过程的假基因,并且得到的单克隆抗体的氨基酸序列均符合鼠免疫球蛋白可变区特征。结论：建立了克隆鼠免疫球蛋白轻链和重链可变区基因的通用方法,为后期克隆鼠源性单克隆抗体的可变区基因提供了基础,并为研究甲型H1N1流感病毒血凝素与抗体的结合位点提供了实验数据。     

Mutational analysis of arsonate binding by a CRIA+ antibody. VH and VL junctional diversity are essential for binding activity

To assess the impact of various heavy and light chain mutations on p-azophenylarsonate binding, murine antibodies have been produced in insect cells (SF9) utilizing a baculovirus expression system. When expressed in this system, an antibody composed of a canonical CRIA+ heavy and light chain can bind antigen and express idiotype indistinguishably from analogous hybridoma-derived antibodies. Antibodies comprised of either light chains mutant at the V-J junction or heavy chains mutant at the V-D junction were found to be incapable of binding arsonate. In addition, substitutions in the first and second complementarity determining regions of the heavy chain were shown to play a role in arsonate binding, most likely related to affinity maturation targeted at the carrier protein. These results confirm the obligatory role that junctional diversity plays in the generation of arsonate-specific antibodies, as well as extend our understanding of the role of other variable region amino acids in arsonate binding.     

Specificity change of antibody to (4-hydroxy-3-nitrophenyl)acetyl haptens by somatic hypermutation

Change in the specificity of anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibodies (Abs) with time after immunization was studied. The early anti-NP Abs was specific to the ionized (phenolate) form of NP. The specificity changed with time and the late Abs became able to bind to the protonated (phenolic) form as well as the phenolate form of NP. The nucleotide sequences of mRNA coding for variable regions of heavy and light chains suggested that somatic hypermutation contributed to this change of the specificity.     

Immunoglobulin V region variants in hybridoma cells. I. Isolation of a variant with altered idiotypic and antigen binding specificity.

The hybridoma cell line B1-8.delta 1 expresses an IgD antibody with specificity for the hapten 4-hydroxy-3-nitro-phenylacetyl (NP). Two monoclonal antibodies, each recognizing an idiotypic determinant (idiotope) in the variable region of antibody B1-8, and fluorescence-activated cell sorting were used for the isolation of spontaneous somatic cell variants whose antibody product lacks the expression of one of the two idiotopes. The variant cells carry a mutation which is expressed in the variable region of the antibody heavy chains and which reduces the affinity of the antibody for hapten greater than 100-fold. Idiotope loss variants of the selected phenotype are present in the B1-8.delta 1 cell population at low frequency.     

VH genes encoding the immune response to beta-(1,6)-galactan: somatic mutation in IgM molecules

The immune response to beta-(1,6)-galactan in the BALB/c mouse has been well characterized and includes the amino acid sequence determination of 13 monoclonal antibodies. The genetic potential encoding the VH regions of these antibodies has been determined by isolation and sequencing of homologous germline genes. The germline repertoire encoding these proteins was found to consist of two closely related genes. One of these directly encodes the VH segments of seven Gal-binding proteins, and the second directly encodes one additional protein sequence. Sequence variations found in the VH regions of five other Gal-binding proteins can be explained by somatic mutations leading to single base substitutions in the more frequently used gene. Since four of the hybridoma proteins exhibiting somatic mutations are of the IgM class, these results indicate that somatic mutation, in this system, is not associated with class switching and can apparently be initiated early in B-cell development. The two Gal genes are the only members of a very restricted multigene family and probably result from a gene duplication estimated to occur 1.4-2.8 million years ago. Three other genes hybridizing at moderate stringency to a VHGal probe were also sequenced and were found to be members of two additional VHIII families. Studies of the silent to replacement substitution ratios of these and other VH genes indicate that the number of silent substitutions found in immunoglobulin VH genes is lower than expected when compared with proteins such as preproinsulin and globin. Analysis of base composition reflected in these sequences indicates a marked increase of A-T% in the first and second codon positions of complementarity determining regions (CDR) which may be important in facilitating point mutations.