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Somatic mutation occurs at a low rate in the rearranged antibody V region genes of the hybridoma line B1‐8. delta 1 which expresses an antibody with specificity for the hapten 4‐hydroxy‐3‐nitro‐5‐iodo‐phenylacetyl (NIP). A mutant was selected which had lost a binding site‐related idiotope but retained most of its other idiotypic determinants. The mutant had concomitantly lost NIP binding and acquired specificity for dinitrophenylated bovine serum albumin. It carried a single point mutation in position 50 of the heavy chain, resulting in the replacement of an arginine by a glycine.

Idiotope Ac38, a V region determinant of the lambda 1 chain‐bearing, germ line encoded antibody B1‐8, is expressed at high frequency (approximately 1/40) in lambda 1 chain‐bearing B cells. Here, we describe the isolation of lambda‐positive hybridomas from C57BL/6 mice which had been immunized with antibody Ac38, the antibody recognizing idiotope Ac38. In Northern blot analysis, mRNA isolated from 10 such hybridomas hybridizes with a cDNA probe from the VH gene expressed in the cell line B1‐8. Amino acid sequence analysis of the VH regions of four of the hybridoma proteins reveals that they are all derived from related, though distinct, germ line VH genes. In one case the sequence data suggest that extensive somatic mutation has taken place. Only one of the four sequences derives from the same VH gene that is expressed in the cell line B1‐8. Together with earlier evidence, the present data demonstrate that the Ac38 idiotope is a marker for at least five VH and three D region genes in the C57BL/6 germ line. This explains the high frequency at which this idiotope is expressed in the B cell population. In addition, our sequence determinations identify two VH genes in the C57BL/6 strain which are closely related (and possibly allelic) to two known BALB/c VH genes. One of these genes is the gene expressed in the BALB/c myeloma MOPC 104E.

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.

Plasmacytoma cells, transfected with a vector encoding a membrane‐bound IgM molecule, do not show cell surface IgM expression, although complete IgM molecules are assembled intracellularly. The isolation of a surface IgM‐positive variant allowed us to analyse molecular requirements of surface IgM expression. Only in surface IgM‐positive cells, a 34‐kd protein (B34) was found to be associated with IgM. B34 is a glycoprotein which forms a disulphide‐linked homodimer. The surface IgM‐positive variant cell line expressing B34 also contains transcripts of the pre‐B and B cell specific mb‐1 gene. The data are discussed in the context of a possible IgM‐antigen receptor complex.

The mouse hybridoma line B1‐8.delta 1 secretes a monoclonal IgD, lambda 1 anti‐(4‐hydroxy‐3‐nitrophenyl)acetyl (NP) antibody with defined idiotypic determinants. Two spontaneous V‐region variants (B1‐8.V1/V2) with altered idiotope pattern were selected and the structural variation was located to the variable region of the heavy chain. The amino acid sequences of the B1‐8. delta 1 and variant heavy chain V regions were determined. The variant VH regions are identical. Wild‐type and variant VH regions differ in 10 positions. Single amino acid exchanges are found in the first and second framework at positions 20 and 43. The majority of replacements (eight substitutions) is clustered in the second complementary‐determining region (CDR 2). There are no differences in CDR 1 and CRD 3 and the JH region. The variant, which at first glance appears to have undergone a series of point mutations, arose by recombination, possibly gene conversion, between the rearranged VDJ gene of the wild‐type (B1‐8.delta 1) and a neighbouring germ line VH gene encoding all of the substitutions.

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.

In the heterozygous T lymphoma line LDHB, variants which have lost the expression of individual H‐2 class I genes are spontaneously generated in vitro at a frequency of 10(‐1)‐10(‐2). A cell line (HK13) in which the class I gene Kk is stably expressed (frequency of loss variants less than 10(‐4) was selected from LDHB cells by fluorescence activated cell sorting. Further selection of HK13 cells for high Kk expression led to the isolation of the HK22 line which expresses twice as much Kk as HK13. From HK13 and HK22 cells, spontaneous structural variants of Kk having lost individual serological determinants of the Kk wild‐type molecule, were isolated by fluorescence activated cell sorting. Such variants occur at a frequency of 10(‐6)‐10(‐7) per cell per generation. The analysis of these variants indicates that they carry mutations in the Kk structural gene and that HK13 cells express a single Kk gene which appears to be duplicated in HK22 cells. We did not find evidence for the generation of variants expressing ‘alien’ class I products in the LDHB cell line. The instability of class I gene expression in LDHB cells and the transition to stable expression may represent steps of T‐cell differentiation in the thymus.

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.

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.

In cultures of murine bone marrow cells colonies of 10(3)‐10(4) cells were identified which consisted to a large part of pre‐B and B cells. Cell mixing experiments with genetically marked cells indicated that each colony is derived from a single progenitor cell not yet committed to the expression of either IgH locus. A concanavalin‐A‐mediated electrofusion method allowed us to rescue and amplify individual cells from a given colony by hybridization with X63.Ag8.653 cells. The molecular analysis of 12 such hybridomas revealed that all IgH loci were rearranged into DJH or productive or nonproductive VHDJH complexes. Most kappa and all lambda light chain loci were in germline configuration. Kappa chain expression was only seen in heavy (mu) chain expressing hybridomas. Hybridomas from a given colony were heterogeneous in terms of DJH and VHDJH rearrangements and in no cell was more than one productive VHDJH complex detected. None of the productive VHDJH complexes contained a VH gene of group 1 (J558), the largest VH gene family with about half of the VH genes. This is in marked contrast to VH gene usage in splenic B cells.

The influence of structural variation, previously observed in a panel of V186.2 VH/V lambda 1‐expressing anti‐NP antibodies from the secondary response, on the affinity of these antibodies was examined by site‐specific mutagenesis and recombinant antibody construction. A tryptophan––leucine exchange at position 33 in the VH segment of all but one of the high‐affinity antibodies is the most frequently observed somatic mutation and by itself leads to a 10‐fold higher affinity; all other somatic exchanges are irrelevant for affinity selection. In the single case of a high‐affinity antibody without this common exchange, high affinity is mediated by a combination of mutations (including a one‐codon deletion) in VH and the particular D‐JH rearrangement carried by this antibody. The data indicate that the pattern of somatic diversification through hypermutation is shaped by affinity selection, but that only a single point mutation is available in the VH and the VL gene of lambda 1 chain‐bearing anti‐NP antibodies which by itself leads to an increase of hapten‐binding affinity. Based on the analysis of two secondary response antibodies from which somatic mutations in VH and VL have been eliminated, it is also concluded that the recruitment of B cell clones into the pathway of hypermutation involves a mechanism which is not based upon affinity differences towards the antigen.

The nucleotide sequences of the variable regions of lambda 1 chain bearing anti‐NP antibodies from the secondary response of C57BL/6 mice were determined. The data indicate that the V186.2 VH gene which dominates the primary anti‐NP response is expressed in nine out of 10 secondary response antibodies and is extensively mutated. In the V lambda 1 regions somatic mutations are less frequent. While point mutations predominate, there is suggestive evidence for two conversion events, one involving a one‐codon deletion. Most, but not all, secondary response antibodies have a higher affinity (up to 10‐fold) for the hapten than is seen in the primary response. The increase in affinity correlates with ‘parallel’ mutations in CDRs of H and L chains, likely to play a role in hapten binding. The analysis of VDJH rearrangements demonstrates that the secondary response lambda 1 chain‐bearing antibodies are produced by a diverse set of B cell clones, which are only rarely expressed in primary responses. These clones are characterized by N‐sequence‐mediated heterogeneity in the 3′ half of CDR3, where the germ line sequence of the D element DFl16.1 predominates in primary response antibodies. The antibodies analyzed in this and in previous work were isolated from idiotypically suppressed mice in order to evaluate whether, intraclonally, idiotype suppression selects antibody mutants into the memory pool, through suppression of the wild‐type. A selection of this type was not detectable. However, idiotype suppression may control the pattern of clonotypes expressed in the primary versus the secondary response.