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.     

Monoclonal autoantibodies to subnucleosomes from a MRL/Mp(-)+/+ mouse. Oligoclonality of the antibody response and recognition of a determinant composed of histones H2A, H2B, and DNA.

MRL/Mp(-)+/+ mice produce antinuclear antibodies and develop a spontaneous autoimmune syndrome with lupus-like nephritis. We obtained a panel of seven histone-reactive IgG mAb from a single MRL/Mp(-)+/+ mouse. These antibodies do not react significantly with DNA or individual histones, but bind strongly to the histone H2A-H2B dimer and even more strongly to the H2A-H2B-DNA complex. These antibodies also bind to whole nuclei when tested by immunofluorescence, indicating that they recognize an epitope accessible in chromatin. The V region sequences of these antibodies have been determined. The H chain third complementarity-determining regions of these antibodies are similar to those found in anti-DNA antibodies even though the antibodies in our panel do not react with DNA in the absence of histones, suggesting that DNA is part of the subnucleosome epitope. Several of these antibodies are clonally related, supporting the hypothesis that the activation of these clones is Ag-driven. Analysis of the sequences of these antibodies indicates that they derive from autoreactive B cells that were clonally expanded and whose V region genes have undergone numerous somatic mutations.     

A human and a mouse anti-idiotypic antibody specific for human T14(+) anti-DNA antibodies reconstructed by phage display

Little is known about human anti-idiotypic antibodies. Phage display methodology was used to reconstruct these antibodies from lupus patients, which recognize a subset (T14(+)) of anti-DNA antibodies. Antigen-specific B cells were isolated from the blood using a peptide based on a complementarity determining region (V(H)CDR3) of the prototypic T14(+) antibody. cDNA fragments of the V(H) and V(L) genes prepared from the cells were expressed as phage displayed single chain Fv (scFv) fragments using the pCANTAB-5E phagemid vector. From a reactive clone obtained, the Ig genes used were identified to be V(H)3, D5-D3, J(H)4b, V(kappa)I and J(kappa)2. The heavy chain was highly mutated, especially in CDR3, which bears mutations mostly of the replacement type; this region is also unusual in being extremely long due to a D-D fusion. In contrast, a mouse hybridoma antibody, made to the same T14(+) peptide and transformed as a scFv fragment, uses a short V(H)CDR3 comprising five amino acids, three of which are tyrosines. Tyrosines may be important for antigen binding because two of these also exist in the human V(H)CDR3. The light chains of both antibodies may also contribute to the specificity of the protein, because their V(L) segments, including the CDRs, are highly homologous to each other.     

Burkitt's lymphoma is a malignancy of mature B cells expressing somatically mutated V region genes.

BACKGROUND: The developmental stage from which stems the malignant B cell population in Burkitt's lymphoma (BL) is unclear. An approach to answering this question is provided by the sequence analysis of rear-ranged immunoglobulin (Ig) variable region (V) genes from BL for evidence of somatic mutations, together with a phenotypic characterization. As somatic hypermutation of Ig V region genes occurs in germinal center B cells, somatically mutated Ig genes are found in germinal center B cells and their descendents. MATERIALS AND METHODS: Rearranged V kappa region genes from 10 kappa-expressing sporadic and endemic BL-derived cell lines (9 IgM and 1 IgG positive) and three kappa-expressing endemic BL biopsy specimens were amplified by polymerase chain reaction and sequenced. In addition, VH region gene sequences from these cell lines were determined. RESULTS: All BL cell lines and the three biopsy specimens carried somatically mutated V region genes. The average mutation frequency of rearranged V kappa genes from eight BL cell lines established from sporadic BL was 1.8%. A higher frequency (6%) was found in five endemic cases (three biopsy specimens and two BL cell lines). CONCLUSIONS: The detection of somatic mutations in the rearranged V region genes suggests that both sporadic and endemic BL represent a B-cell malignancy originating from germinal center B cells or their descendants. Interestingly, the mutation frequency detected in sporadic BL is in a range similar to that characteristic for IgM-expressing B cells in the human peripheral blood and for mu chain-expressing germinal center B cells, whereas the mutation frequency found in endemic BL is significantly higher.     

Germline V genes encode viable motheaten mouse autoantibodies against thymocytes and red blood cells

Autoantibodies against thymocytes and RBC may contribute to the pathophysiology of homozygous viable motheaten (mev) autoimmune disease. Whether the production of these autoantibodies in mev mouse results from polyclonal nonspecific B cell activation or specific Ag-driven stimulation is not known. To understand the mechanisms involved in the induction of antithymocyte autoantibody response in mev mouse, we have studied the fine antigenic specificity, structure, and origin of three antithymocyte autoantibodies derived from mev splenic B cell hybridomas. Western blot analysis showed that these mAb bind to polypeptides of 33 and 105 kDa present in RBC and thymocytes, respectively. Additional specificities for the epitopes present in other polypeptides distinguished these three autoantibodies. Northern hybridization and flow microfluorimetry analysis indicated that these hybridomas are derived from the Ly1+ B cell subset. These autoreactive Ly-1 B cell hybridomas, chosen on the basis of their specificity, expressed L chain V genes from a single VK family (VK9) and VH genes from J606 and S107 families. Hybridomas UN34.11 and UN42.5 expressed the VK9 gene identical to that used by peritoneal Ly1+ B cells from various mouse strains and malignant B lymphoma cells secreting anti-mouse RBC treated with proteolytic enzyme bromelin and anti-SRBC antibodies. The third hybridoma, S2-14.2, used a VK9 gene identical to that expressed by MOPC41. None of the VK genes encoding these autoantibodies showed any somatic mutations. In the case of VH genes, the two hybridomas UN42.5 and S2-14.2 derived from two separate fusions, used identical VH genes from the J606 family. The third hybridoma UN34.11 used unmutated V11 germline VH gene, a member of the S107 family. Southern hybridizations, using oligonucleotide probes specific for CDR1 and CDR2, showed that the VH genes encoding the J606 autoantibodies were derived from a germline gene found in the 6.7-kb fragment of EcoRI-digested germline DNA. This germline VH gene is distinct from VH22.1 germline gene that codes for antigalactan antibodies. Sequence analysis of this gene showed perfect homology with the rearranged VH genes confirming the lack of somatic mutations. Thus, our data demonstrate that antithymocyte antibody response occurring in mev mouse is polyclonal and it involves Ly-1 B cells expressing unmutated germline VH and VK genes. These results indicate that antigen driven stimulation may not play an important role in the induction of anti-thymocyte antibody response in mev mouse.     

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.     

Germline antibody V regions as determinants of clonal persistence and malignant growth in the B cell compartment.

Antibody V gene expression was studied in a subpopulation of murine B cells (Ly1 B) which was enriched by cell transfer and had earlier been shown to persist in the immune system over long periods of time. Among 17 hybridomas derived from Ly1 B cells of two different mice, eight were progeny of only three different B cell precursors which apparently had expanded to clones of large size, in the absence of detectable somatic mutation of their antibody V regions. Furthermore, several clonally independent cells expressed identical, unmutated V genes. These data define a novel pathway of B cell development in which cells expressing a selected set of germline antibodies are continuously propagated in the organism. A Ly1 B cell leukemia derived from a similar transfer experiment expressed a VH gene that had been isolated in three independent Ly1 B cell hybridomas, suggesting that the leukemic cells had been equally selected in this pathway.     

Probing the human antibody repertoire to exogenous antigens. Characterization of the H and L chain V region gene segments from anti-hepatitis B virus antibodies.

Structural studies of human antibody V regions have been largely limited to those involving the fetal repertoire, autoantibodies, and malignant cell rearrangements, leaving the "normal" repertoire relatively unexplored. In this study we describe the nucleotide sequences of the H and L chain V regions of four antibodies specific for the surface Ag of the hepatitis B virus. Monoclonal cell lines were derived from healthy individuals who received standard immunizations with the serum-derived or recombinant hepatitis B virus vaccines by fusion of PBL to a heterohybridoma cell line, SPAZ-4. We utilized the polymerase chain reaction to amplify the H and L chain V regions for cloning and sequencing. The four antibodies express the following V region combinations: VHIII/V lambda V, VHIII/V kappa II, VHIV/V kappa I, VHV/V lambda III. When compared to germline genes with the closest sequence homology, all of the V regions appear to have undergone somatic mutation, ranging from 3.4 to 11.3% for the H chain, and 5.1 to 9.2% for the L chain. Analysis of the mutations shows them to be typical for an Ag-driven immune response.     

Clustered H chain somatic mutations shared by anti-p-azophenylarsonate antibodies confer enhanced affinity and ablate the cross-reactive idiotype

A cluster of four consecutive CDR2 somatic mutations are shared by the VH regions of two independently isolated hybridoma antibodies with specificity for p-azophenylarsonate (Ars). The mutations appear to be derived by a series of independent events. To assess the influence of these shared somatic mutations on antibody affinity for Ars and on idiotypy, we introduced them, via site-directed mutagenesis, into the V region of an anti-Ars antibody that was otherwise unmutated and we eliminated them from the mutated context of one of the two antibodies in which they were originally found. Results of affinity measurements by fluorescence quenching and of idiotypic binding assays performed on these engineered mutants demonstrated that the shared mutations increased affinity for Ars and eliminated the predominant Id associated with strain A anti-Ars antibodies and four of five idiotypes defined by anti-idiotypic mAb. These results support the interpretation that a strong affinity-based selection pressure has favored the clonal expansion of B cells with receptors containing these mutations despite the loss of a predominant Id. Thus, in producing antibodies containing V regions conferring high affinity for Ag, the combined processes of somatic mutation and clonal selection have generated a common structural solution through parallel repeats.     

Structural and functional implications of a restricted antibody response to a defined antigenic region on the influenza virus hemagglutinin.

A group of hybridoma antibodies that recognize structurally overlapping epitopes on the influenza virus hemagglutinin have been analyzed for the sequence of their immunoglobulin heavy and light chain variable regions. All VH regions derive from the same gene family, and only two Vk genes, from different families, are involved. The repetitive and restricted use of these variable region genes indicates that considerable structural requirements influence the generation of antibodies specific for this region of the hemagglutinin. The degree of amino acid variability which is permissive for interaction with this region suggests that two thirds of the possible replacement mutations may abolish either antibody function or specificity. Analysis of the somatic mutation which occurred in the individual antibodies indicates that the light chains acquired replacement mutations at the rate predicted for random mutation. The heavy chains, however, accumulated a 3-fold excess of replacement mutations over that predicted for random accumulation, correlating with the dominant role they apparently play in determining fine differences in the specificity of these antibodies. The effect of somatic mutation on the clonal amplification and diversification of these B cell lineages is discussed.     

Tracing B cell development in human germinal centres by molecular analysis of single cells picked from histological sections.

Germinal centres are areas of intense B lymphocyte proliferation inside primary B cell follicles in spleen and lymph nodes. Rearranged V genes from single human B cells, isolated from histological sections of two such structures by micromanipulation, were amplified and sequenced. Cells from the follicular mantle were clonally diverse and largely expressed germline V genes. Germinal centres were dominated by a few large B cell clones dispersed throughout these structures and exhibiting intraclonal diversity by ongoing somatic hypermutation. Pronounced counterselection of replacement mutations seen in one of the germinal centres may indicate a late phase of the germinal centre reaction. A polyclonal population of activated B cells expressing unmutated antibodies in the dark zone of the other germinal centre may represent the initial founder cells.     

Convenient plasmid vectors for construction of chimeric mouse/human antibodies

Chimeric antibodies composed of mouse-derived variable regions and human-derived constant regions have been developed for clinical use. However, construction of chimeric mouse/human genes in expression vectors is time-consuming work. In this study, we developed convenient vectors for construction of chimeric mouse/human antibodies. The protocols are as follows: In mouse hybridomas and B cells, most active VH and V kappa genes can be identified as rearranged bands by Southern hybridization of EcoRI- and HindIII-digested DNAs with JH and J kappa probes, respectively, and such fragments can be isolated in lambda-EcoRI and lambda-HindIII vectors, respectively. We constructed two plasmids: pSV2-HG 1 gpt contains human C gamma 1 and Ecogpt genes, and only one EcoRI site upstream of the C gamma 1 gene; pSV2-HC kappa neo contains human C kappa and neo genes, and only one HindIII site upstream of the C kappa gene. An isolated EcoRI fragment containing a VHDHJH gene and a HindIII fragment containing a V kappa J kappa gene are inserted into pSV2-HC kappa neo, respectively. Both resulting plasmid DNAs are co-transfected into SP2/0 cell, a non-Ig-secreting mouse myeloma. Transformants are selected by both mycophenolic acid and G418. With this procedure, it takes only 2 months to obtain chimeric antibodies.     

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.     

Novel secondary Ig VH gene rearrangement and in-frame Ig heavy chain complementarity-determining region III insertion/deletion variants in de novo follicular lymphoma

Human germinal center B cell tumors retain the ability of their nontransformed counterparts to somatically hypermutate Ig V genes by nucleotide substitution. Among a survey of 60 primary previously untreated, clonal, follicular lymphomas we have identified a rare V(H) rearrangement variant and two other in-frame nucleotide insertion/deletion variants within complementarity-determining region III of the Ig heavy chain. The neoplastic origin of the V(H) rearrangement variant was directly demonstrated in cells isolated by microdissection from malignant follicles. In all three cases a common clonal origin for the variants was demonstrated by complementarity-determining region III nucleotide sequence homology and shared somatic mutations in germline encoded positions in framework region IV. The monoclonal nature of the tumors was independently confirmed by demonstrating a single t(14;18) translocation breakpoint in the two cases with a detectable translocation. All the variants occurred in functional V(H) rearrangements, which in two cases were directly shown to encode functional Ab molecules. Both recombination-activating genes 1 and 2 were expressed in lymph node tumor cells containing the V(H) rearrangement variant, although recombination-activating gene expression among a panel of lymphomas was not limited to this variant.     

Absence of Ig V region gene somatic hypermutation in advanced Burkitt's lymphoma

Somatic hypermutation of rearranged Ig V region gene plays a major role in generating antibody diversity. Recently, V mutation has been established as a major mechanism of tumor escape from anti-Id immunotherapy. We cloned and sequenced the expressed Ig H and L chain V regions from a case of B acute lymphoblastic leukemia in order to evaluate B cell stages associated with V region mutation, and to determine which tumors would be better suited to Id directed immunotherapy. A consensus VH and V lambda sequence representing tumor at diagnosis was obtained by conventional cDNA cloning in lambda gt10 from a heterohybridoma. Primers which flanked both V regions were used in a modified polymerase chain reaction to generate multiple independent sequences from tumor cells harvested at relapse. In order to exclude mutations due to infidelity of the amplification procedure, single cDNA templates of known sequence were also amplified. The polymerase chain reaction proved to be an effective procedure to obtain multiple clones, but replication in M13 was associated with a low rate of base misincorporation. The results indicate that there is no evidence for biologically significant ongoing mutation in this t(8;14) B cell tumor when comparing sequences at diagnosis and relapse. Thus, V somatic mutation may be restricted to a discrete B cell stage whose malignant counterpart is follicular lymphoma.     

Clonal characterization of the human IgG antibody repertoire to Haemophilus influenzae type b polysaccharide. IV. The less frequently expressed VL are heterogeneous

We previously demonstrated that the human anti-Haemophilus influenzae type b polysaccharide (Hib-PS) VL repertoire is dominated by a product of the V kappa II gene, A2, and that V kappa II-A2 anti-Hib-PS antibodies have little or no somatic mutation in VL. To further study this VL repertoire, we studied non-A2 anti-Hib-PS antibodies that were identified either serologically or by amino-terminal amino acid sequence analysis. Of 15 non-A2 anti-Hib-PS antibodies from 12 vaccinated adults, we found four V lambda, five V kappa I, one non-A2 V kappa II, four V kappa III, and one V kappa IV antibodies. As expected, all but two of these subjects also produced V kappa II-A2 antibodies. Interestingly, one of these subjects lacks the A2 gene in the germ line. However, both subjects who did not produce detectable V kappa II antibody did produce normal amounts of total anti-Hib-PS antibody after vaccination. Candidate V kappa genes for the non-A2 antibodies were identified by comparison of up to 60 VL amino acid residues, including CDR1 and CDR2, with all sequenced V kappa genes. V kappa I antibodies appear to be products of three newly sequenced V kappa I genes, O8, O18, and L11, that are reported here. The O8 and O18 genes encode identical amino acid sequences. The non-A2 V kappa II antibody is a likely product of the A1 or A17 genes, the V kappa III antibodies are likely products of the A27 gene, and the V kappa IV antibody is a product of the single V kappa IV gene, B3. Unlike V kappa II-A2 antibodies, the V kappa I, V kappa III, and V kappa IV antibodies differed by one to five CDR residues from the germ line product of the candidate genes, suggesting the presence of somatic mutations. Thus, anti-Hib-PS antibodies can be divided into two types, the most frequently observed A2 antibodies with little or no somatic mutation and non-A2 antibodies that likely contain somatic mutations.     

Variable regions of antibodies to synthetic polypeptides. II. Analysis of variable region genes encoding antibodies specific for (T,G)-A--L

Monoclonal antibodies specific for the synthetic polypeptide antigen (T,G)-A--L have been produced in two strains of mice, C57BL/10 and C3H.SW. The genes encoding the variable (V) regions of these antibodies have been studied by using the DNA hybridization technique of Southern, as well as by gene cloning and sequencing. Hybridization of DNA from 14 different cell lines with a kappa-chain probe revealed that the different cell lines used one of two different gene rearrangements to encode the recombined V region gene. There was a perfect correlation between light chain rearrangement, idiotype expression, and fine specificity. Hybridization analyses of the heavy chain revealed a more complex pattern. Seven hybridomas had the rearranged heavy chain V region genes on a 4.4 kb EcoRI restriction fragment. Others were found on restriction fragments that differed in length by several hundred base pairs. The recombined heavy chain V region genes were cloned from three different hybridoma cell lines secreting anti-(T,G)-A--L antibodies, all of which express the same idiotype and fine specificity pattern. Restriction mapping and sequencing indicate that all three utilize the same V gene, identified as the 186-2 germline gene. However, different D and J genes are used to encode each of the antibodies. In contrast to the results seen in other antigen systems, heavy chain D and J genes do not have a major influence on idiotype expression and fine specificity of antibodies to the synthetic polypeptide (T,G)-A--L.     

Nucleotide sequence of messenger RNA encoding VHDJH and VKJK of a highly conserved idiotype-defined primary response anti-hapten antibody.

The primary humoral immune response of mice to the hapten phthalate (Xmp) is focused upon two adjacent immunodominant negatively charged carboxyl groups on a benzene ring that are in positions meta and para to the azolinkage (i.e., Xmp) to the protein carrier keyhole limpet hemocyanin. A significant fraction of the anti-Xmp antibodies raised in several different inbred mouse strains (BALB/c, DBA/2, A/HeHa; C3H, and SM/J), and many wild mouse populations express a cross-reactive Id, CRIXmp-1. This CRIXmp-1 is conspicuously absent in C57BL/6 mice. In order to obtain a better understanding of the events and parameters that influence the selection and regulation of the primary response B cell repertoire, and to explore the structural basis of Ag binding, we have determined the nucleotide sequence of the entire V region gene complexes, which encode the H and L chains of these highly conserved and dominant CRIXmp-1+ antibodies. Our data establish that the H chain gene complex consists of a single VH germ-line gene that is identical to VH Oxazolone-1, encoding the H chain of another highly conserved and dominant cross-reactive Id family associated with the primary response to Oxazolone. In CRIXmp-1+ Xmp-specific hybridomas this gene is joined to a limited set of D region sequences that express a conserved amino acid motif-GLR. At least three of the five D regions examined are coded for by DFL16.2. This VHD complex can be utilized with one of three different JH region genes (JH1, JH2, and JH4) without any significant effect upon antibody fine specificity or Id. In spite of this lack of JH fidelity all of the CRIXmp-1+ hybridomas have precisely maintained the same length in the H chain CDR3 and FRW4 by altering either the length of the D segment or the length of JH. Nucleotide sequence analysis of the VL gene complex of CRIXmp-1+ anti-Xmp antibodies indicates that the L chain V region is also encoded by a single germ-line gene. The amino acid sequence predicted from the nucleotide sequence of the VKJK from Xmp-specific CRIXmp-1+ hybridomas is identical to the sequence of the anti-arsonate antibody 1210.7, which is the prototype of another Id family (CRI) that is conserved and dominant in BALB/c mice.