Positive and negative selection of antigen-specific B cells in transgenic mice expressing variant forms of the V(H)1 (T15) heavy chain.

Four variant forms of the V1 (T15-H chain) gene are synthesized in mice. Each V1 variant pairs with a distinct L chain to produce a binding site having specificity for phosphocholine (PC). Transgenic mice expressing variant forms of the V1 gene were analyzed to elucidate the factors driving B cell selection into the peripheral repertoire. In all four lines of H chain transgenic mice analyzed, transgene expression caused complete allelic exclusion of endogenous H chains in the bone marrow (BM), whereas most splenic B cells expressed endogenous H chains. The number of sIgM(+) BM B cells and their sIg receptor number was reduced compared to that of normal transgene-negative controls, suggesting that B cells expressing transgene-encoded H chains were being negatively selected in the BM. Mice expressing autoreactive forms of the V1 transgene with lower affinity for PC (M603H and M167H) exhibit positive selection of PC-specific B cells into the spleen, whereas mice expressing the higher affinity T15H variant exhibited elevated PC-specific B cells in the peritoneal cavity but few V(H)1(+) splenic B cells. These data suggest that the higher affinity T15-id(+) B cells preferentially survive in the peritoneal cavity. When these H chain transgenes were crossed into the mu MT knockout mouse in which surface expression of endogenous H chains is blocked, the percent of splenic V(H)1(+) PC-specific B cells increased up to 5-fold and T15-id(+) B cells were detectable in the spleen of T15H mice. This implies that T15-id(+) PC-specific B cells can be selected into the periphery, but they compete poorly with follicular B cells expressing endogenous Ig.

A method for monoclonal antibody isotype switching: anti-CD60 VH expression in a heavy chain-deficient hybridoma variant.

Alteration of monoclonal antibody isotype is desirable for a variety of purposes, including obtaining an improved reagent for investigative or therapeutic use. A variety of approaches for isotype switching, particularly from IgM to various IgG subclasses, have been described. Antibodies that recognize carbohydrate determinants on glycoproteins, glycolipids, or polysaccharides are generally of the IgM isotype. This includes all available antibodies to the human CD60 antigen, a determinant with cell coactivating properties described on a subset of T lymphocytes and on other cell types. In this report a new method for monoclonal antibody isotype switching is presented. A plasmid containing the VH regions of anti-CD60 linked to C gamma 1 was transfected into a spontaneously arising variant of the CD60 hybridoma that produced kappa light chain but no heavy chain. This transfected hybridoma line maintains stable production of useful quantities of IgG1 monoclonal anti-CD60 in vitro and in vivo.

Mutations in Ig V(D)J genes are distributed asymmetrically and independently of the position of V(D)J.

To study the distribution of somatic mutations in the DNA flanking the 5' side of rearranged Ig V genes, the 5' region of 14 to 21 mutant forms of three different V(D)J were sequenced. These were compared with a fourth V(D)J for which the flanking sequences of 10 mutant forms are known. The leader intron of these four V genes varied from 82 to 365 nucleotides in length. Analysis of the data showed that: 1) The mutation frequency is distributed asymmetrically with respect to the V(D)J exon with skewing in the 3' direction. 2) The distribution of mutations 5' of V(D)J seems to be related to the size of the leader intron. The significance of these findings for models of hypermutation is discussed.

Influence of membrane Ig receptor density and affinity on B cell signaling by antigen. Implications for affinity maturation.

We have initiated a series of experiments to explore the effect of changes in density of the surface Ig receptor (mIg) on Ag:mIg interactions. We transfected into the surface Ig-negative B cell line M12.4 H and L chain constructs known to effect a 10-fold change in antibody binding affinity for the naturally occurring hapten phosphocholine (PC). Two sets of stable transfectants were generated and those expressing levels of mIg comparable to the range normally seen on splenic B cells were studied. One set expressed an unmutated VH and an unmutated VL. The second set expressed the same pair of V regions except for a single somatic change in CDR3 of VH; this substitution increases the affinity of antibody for PC from 3 x 10(4) M-1 to 3 x 10(5) M-1. Ag:mIg interactions were assessed in the transfected cell lines by measuring calcium mobilization induced by stimulation with soluble PC Ag. As expected, the mutation that increased affinity for PC increased the sensitivity of transfectants to PC Ag. Relatively small changes in receptor number had a dramatic effect in the quantity and quality of a calcium response. Significantly, we found that Ag-specific signaling could occur with only a few thousand receptors per cell. Signaling differences were most noticeable with PC protein Ag (T-dependent form) compared with PC polysaccharide Ag (T-independent form). These results suggest that the down-regulation of mIg that follows B cell activation may have evolved to assist in the selection of B cell clones with higher affinity for Ag. Furthermore, the results also provide an explanation for why selection of higher affinity clones can occur with protein Ag but only poorly so with polymeric Ag.

Junctional diversification in the generation of the precursor of a discrete immune response.

Phosphocholine (PC)-specific antibodies that arise in the mouse in response to Proteus morganii (PM) and use V1-DFL16.1-JH1 are characterized by a number of recurring mutations. Most striking is an invariant A for G substitution in codon 95 of VH which results in an asparagine instead of aspartate at that position. Because of the apparent importance of this substitution in an anti-PC(PM) response, we wanted to determine the molecular basis for this base change. A cDNA library derived from pre-immune splenic B cells was examined for the frequency of VDJ containing the A substitution at 95 and the presence of additional point mutations in these sequences. Six different cDNA were isolated which contained an A substitution at the VD junction (frequency 0.00009); a seventh positive cDNA could not be examined. The V segments of four of these cDNA matched known germline genes and were, therefore, unmutated. Two others closely matched V in families whose members have not all been characterized, hence, it is not known whether the mutations observed are somatic or germline in origin. Sequences of 35 cDNA clones, containing the same V segment but differing in D, J and junctional nucleotides, revealed no mutations. These results indicate that the A substitution generated at codon 95 is most likely a product of V-DJ joining.

Antigen binding and idiotype analysis of antibodies obtained after electroporation of heavy and light chain genes encoding phosphocholine-specific antibodies: a model for T15-idiotype dominance.

Antibodies bearing the T15 idiotype dominate the murine primary immune response to phosphocholine (PC). Analysis of antigen binding of antibodies derived from V1:DFL16.1:JH1 (VH1) germline and N region-derived variant heavy (H) chains and kappa 22, kappa 24, and kappa 8 light (L) chains demonstrates that the T15H:kappa 22L (T15) antibody binds PC at least 20-40 times better than other antibodies derived from alternate germline forms of the VH1 H chain and kappa 22, kappa 24, or kappa 8 L chains. To achieve affinities in the same range as the T15 antibody, kappa 24 and kappa 8 L chain-containing antibodies must have H chains derived from variant N region or somatically mutated VH1 genes. Single amino acid differences at the VD junction of the various germline and N region variant VH1 H chains dictate the L chain that can associate with the H chain to produce a PC-specific antibody. Several H:L combinations give rise to T15 or M167 idiotype-positive antibodies that lack specificity for PC, and single amino acid substitutions or insertions at the VH1:D junction result in the loss of T15 or M167 idiotopes. Based on these observations, our data support a molecular model involving both preferential gene rearrangement and antigen-driven B cell selection to explain T15 idiotype dominance in the immune response to PC. In the absence of N region diversification, large numbers of neonatal B cells bearing the T15H:kappa 22L surface immunoglobulin M (sIgM) receptors would be selected and expanded by autologous or environmental PC antigen into the long-lived peripheral B cell pool.

Position of the rearranged V kappa and its 5' flanking sequences determines the location of somatic mutations in the J kappa locus.

Somatic hypermutation is known to occur in the VJ kappa exon and its flanking sequences, yet little is known about the hypermutation mechanism or its exact target within the rearranged locus. Mutations may occur at the same frequency, spanning a region from the leader intron to 3' of J kappa 5, regardless of which J is chosen for VJ rearrangement. Another possibility is that mutations may be limited to the rearranged VJ kappa and its immediate flanking sequences. To distinguish between these possibilities, the JC introns of 21 alleles with V kappa rearranged to J kappa 1 were sequenced, and mutations were located. The frequency of mutations was determined for different sections of the intron and compared with the frequencies of mutations found in the JC intron of a set of VJ kappa 5 alleles. The results showed that mutations were concentrated in and around the rearranged VJ, regardless of whether J kappa 1 or J kappa 5 was used. These data imply that the hypermutational mechanism focuses on rearranged V genes.

Somatic hypermutation of the JC intron is markedly reduced in unrearranged kappa and H alleles and is unevenly distributed in rearranged alleles.

Somatic hypermutation of the Ig genes occurs in rearranged V(D)J and its flanking sequences after Ag stimulation. Even though C regions and unrearranged V segments have been found to lack mutations, it is not known whether the mutational mechanism can be active in unrearranged J segments and their flanking regions. By polymerase chain reaction and direct sequencing of the 500 bp at the 5' end of the JC intron of hybridoma DNA derived from splenic B cells, we show that the frequency of mutations in unrearranged J regions of kappa and H chain genes is 0/7849 bp (upper 95% confidence interval, less than 0.00038) and 1/3209 bp (upper 95% confidence interval, less than 0.0015), respectively. The frequency (f) for the same region of rearranged kappa and H chain genes was 29/9380 bp (95% confidence, 0.0021 less than f less than 0.0044) and 16/2750 bp (95% confidence, 0.0033 less than f less than 0.0094), respectively. The significantly higher frequency of mutations in the rearranged alleles indicates that rearrangement is needed to effect full activation of the mutational mechanism. The data also show that mutations occur predominantly in the 5'-most 250 bp of the JC kappa intron. Statistical analysis of the distribution of mutations within the 5'-most 521 bp of the JC kappa intron reveals significant deviation (p = 0.000085) from a theoretically determined uniform distribution, indicating that mutations are not evenly distributed within this region.

Patterns and extent of isotype-specificity in the murine H chain switch DNA rearrangement.

We have analyzed the configuration of the H chain locus of 41 hybridomas by Southern blot analysis. Each H chain switch region was determined to be germ line, rearranged, or deleted. Including 13 previously analyzed hybridomas, 60% of those with rearrangements on both alleles showed a correlation of the two alleles, i.e., both the expressed and the nonexpressed alleles have rearranged to the same H chain constant region gene segment. When the two H chain alleles did not rearrange to the same gene, they often rearranged to neighboring H chain genes. These results support a role for isotype-specific factors in H chain switch recombination. The action of these isotype-specific factors may be propagated to some extent along the chromosome, which would lead to rearrangements to neighboring genes.

Patterns of mutations and selection in antibodies to the phosphocholine-specific determinant in Proteus morganii.

The contribution of somatic mutation to the generation of an antibody response was investigated by using the phosphocholine (PC) determinant in the bacterium Proteus morganii as the model Ag. The response to this determinant is restricted to a single VH/VL pair and apparently is derived from only one or two precursors per mouse. In this study we examined hybridoma antibodies from nine individual mice which produced representatives of 12 different clones. We found that all antibodies reactive with the PC Ag of P. morganii contained somatic mutations; the number ranged from 2 to 20. Two clusters of mutations were observed, one in complementarity-determining residue (CDR) 2 and the other in CDR 3 of VH. Examination of a three-dimensional model of M603, an antibody with the same V region composition as the anti-PC antibodies under study, showed that these clusters occupied an area of the binding site which presumably interacts with carrier elements of the PC epitope in P. morganii. A high incidence of recurring mutations were found in both clusters, and one of these was invariant, leading to an Asn for Asp substitution at 95. Ag binding studies with these antibodies and an additional one, which was unmutated except for the invariant substitution at 95, showed that: 1) antibodies having only the 95Asn mutation failed to bind the PC Ag of P. morganii, 2) addition of a second recurring mutation, at 52a (CDR 2), was sufficient to create strong binding to the P. morganii Ag, and 3) accumulation of mutations was directly correlated with increased binding activity for Ag. These results show that somatic mutations play a critical, if not essential, role in generating specificity for this PC Ag, and that Ag, and most likely a carrier element of the epitope, is a primary force in the continued selection and expansion of Ag-reactive B cells.

Molecular analysis of clonal stability and longevity in B cell memory.

We used the antiphosphocholine response induced by Proteus morganii and an adoptive transfer protocol to study the contribution of individual clones to B cell memory. Spleen cells from donor mice immunized with P. morganii were injected into irradiated hosts. These recipients were then immunized and their spleen cells fused 12 to 14 wk thereafter. The sequences of hybridoma VH and VL were obtained and DNA rearrangements at both V region loci were studied to ascertain clonal relationships. In all three adoptive transfer experiments, each mouse of a pair receiving cells from the same donor contained hybridomas which were clonally related to each other. In two of these experiments paired recipients possessed cells that had identically mutated V genes. These results lead us to conclude that once a B cell clone(s) dominates a response, progeny of that clone form the memory cell population for many months. Moreover, stability appears to be generated in some memory B cells through inactivation of the hypermutation mechanism.

Genetics of the phosphocholine-specific antibody response to Streptococcus pneumoniae. Germ-line but not mutated T15 antibodies are dominantly selected.

The role that somatic mutations play in the phosphocholine-specific, antibody response to Streptococcus pneumoniae was examined by studying sets of hybridomas from different individual mice. As expected most of the cell lines were from the T15 anti-phosphocholine family and were not encoded by the v1 gene of the T15 VH family and V kappa 22. A minority of antibodies were from the M603 (v1/V kappa 8) and M511 (v1/V kappa 24) families. Three additional antibodies were encoded by the v11 gene of the T15 family; two were paired with a V lambda and the other with a V kappa 1 gene. In vitro binding studies showed that T15- and M603-like antibodies had the highest affinity for S. pneumoniae. Complete sequencing of the VH and VL mRNA from 25 of the hybridomas revealed somatic mutations in 11 of the antibodies. A total of 17 independently derived T15 positive cell lines were studied in detail, six of these were mutated. These mutations were scattered throughout the V regions and the replacement to silent ratio was typical of that for framework regions. Statistical evaluation of the placement of mutations showed that there was a slight but significantly decreased frequency of mutations in complementarity determining regions. Comparisons of mutated and unmutated T15-related antibodies showed that mutations caused a decrease in binding to S. pneumoniae in every case. These results argue that the optimal specificity for this molecular form of phosphocholine is encoded in the germline and that Ag-driven events favor selection of B cells expressing these germ-line encoded antibodies.

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.

IgA isotype-restricted idiotypes associated with T15 Id+ PC antibodies.

Idiotypes are believed to be due to the structural conformation of the variable region of immunoglobulins (Ig). We have found an idiotype (C3-24) that requires both variable and constant regions of the heavy chain to be expressed. C3-24 Id is associated with both the T15 variable region from anti-phosphorylcholine (PC) antibodies and the constant region for the alpha-heavy chain. High titer anti-PC serum from a variety of inbred strains of different Ig haplotypes failed to express C3-24 Id. However, when IgA but not IgG or IgM fractions were isolated from a pool of anti-PC serum from BALB/c mice, more than 70% of the molecules expressed C3-24 Id. The high frequency of the expression of C3-24 Id in IgA anti-PC hybridoma proteins from mice of different Ig haplotypes and in the IgA fraction of normal anti-PC antibodies from BALB/c and presumably other strains of mice suggests that idiotypic determinants produced by the three-dimensional product of VH and CH regions may not be unusual.

Antigen-specific anti-phosphocholine antibodies: binding site studies.

The present investigation extends our initial evaluation of the evolution of antigen selection mechanisms for antibodies of a "single" specificity. The binding sites of 11 mouse anti-PC antibodies produced in response to the bacterium P. morganii or the nematode A. suum were characterized for both hapten and hapten plus carrier specificity. All of the anti-P. morganii HP belonged to the M603 anti-PC antibody family, whereas all the A. suum HP belonged to the M511 family. Of the eight anti-P. morganii HP, six exhibited a fine specificity profile for PC and choline analogues only slightly different from M603 Id+ HP induced by S. pneumoniae and PC-protein. These six and a seventh HP, whose hapten binding profile was unique, were also unusual in showing strong reactivity for a soluble PC containing extract from P. morganii. All three anti-A. suum-specific HP studied in detail had hapten-binding profiles remarkably similar to each other, a finding that is in contrast to M511 Id+ HP to S. pneumoniae and PC-protein. All three HP also showed evidence for preferential binding activity for A. suum, although this was not as dramatic as that seen with the anti-P. morganii HP. These data support our hypothesis that antigen selection of anti-PC antibodies occurs not so much for PC itself as it does for the carrier (microbial) determinants to which PC is attached.

The circular dichroism of phosphocholine-specific mouse hybridoma and myeloma proteins: unusual properties of the hybridoma protein 101.6G6.

The circular dichroism (CD) spectra of five myeloma and six hybridoma proteins specific for phosphocholine were measured in the 250-310-nm range. The effect on the CD spectra of adding phosphocholine was also examined. The five myeloma proteins all had distinctive native spectra and, except for M603 and W3207, unique changes occurred on ligand binding. The hybridomas were chosen as pairs from each of the three known families of phosphocholine-specific immunoglobulins. Those from the T15 or M603 families resembled the appropriate prototype. However, the proteins from the M167 family were all distinctively different in their CD properties. In particular, the hybridoma protein 101.6G6 showed large CD changes on hapten binding and values for the association constant for phosphocholine of 1.1 X 10(5) M-1 and of 5.8 X 10(2) M-1 for acetylcholine were obtained by CD spectrophotometric titration. The CD properties of the proteins are interpreted in the light of the sequence data so far available, including the possible role of the D-segment.

T15 D region germ line amino acid sequences distinguished by monoclonal anti-idiotope antibody.

Monoclonal antibody NL16, prepared with phosphorylcholine (PC)-binding myeloma protein C.BBPC3 (C3), identified an idiotope (C3-16 Id) that was present on T15 IdX+ myeloma proteins (MP) C3, T15, and H8, but not the T15 IdX- MP M167 and M603. The binding of C3 to NL16 is PC inhibitable, indicating that C3-16 Id is site associated. Inhibition studies with PC-specific hybridoma proteins (HP) demonstrated that the T15-type L chain VK22 and elements of the H chain were required for C3-16 Id expression. Studies of amino acid sequences of these PC-binding HP and MP showed that VK22+, T15 IdX+ HP, and MP that use the T15 D region (YYGSS) sequences were always C3-16 Id+. However, the reverse was not true, because all but one VK22+, T15 IdX+ HP with D region sequence changes were C3-16 Id-. This suggested that NL16 defined a specificity mainly determined by the D region of the H chain. A direct test of this hypothesis with heterologous heavy/light chain recombinant molecules obtained from C3-16 Id+ and C3-16 Id- HP of known sequence, showed that the D region was critical to idiotope expression. Additionally, an examination of the amino acid sequences of VK22+, T15 IdX- HP, HPCG14, and HPCM6 suggest that profound changes in the D region may also alter the expression of T15 IdX (an Id defined by a multispecific antiserum from A/He mice). The C3-16 Id+ was found in anti-PC serum of most Ig haplotype-inbred strains except for CBA/J, C3H, and PL, which are all of the Igh-Cj haplotype. Amino acid sequences of PC-binding CBA and PL HP showed marked changes in the D region from the T15 type, and this may account for the C3-16 Id- character of Igh-Cj strains.

The effects of idiotype on the ability of IgG1 anti-phosphorylcholine antibodies to protect mice from fatal infection with Streptococcus pneumoniae.

Anti-phosphorylcholine (PC) antibodies of the mouse are found in three different idiotype families: T15, M603 and M511. These subgroups exhibit different specificities for PC analogs and utilize light chains of different VL subgroups. In this study we have found that IgG1 antibodies of the T15 idiotype are much more protective against pneumococcal infection than IgG1 antibodies of the M511 or M603 idiotypes. This finding provides additional evidence that the T15 VH and VL genes may have evolved to protect mice from infection with PC-bearing pathogens.