Expression of equivalent clonotypes in BALB/c and A/J mice after immunization with phosphorylcholine.

Analysis of A/J antibody to phosphorylcholine (PC) revealed a striking degree of similarity to PC-binding myeloma proteins of BALB/c origin. By quantitative idiotypic analysis A/J anti-PC antibody was composed to antibodies bearing binding site idiotypic determinants indistinguishable from two different BALB/c myeloma proteins, T15 and M511. Idiotypic determinants of three other PC-binding proteins, W3207, M167, and M603 were not detected. Isoelectric focusing of the light chains verified the presence of antibodies similar to T15 and M511 and indicated the presence of a third antibody whose light chains had a pI identical to that of M603. When the sequence of A/J heavy chains were compared to the heavy chains of T15, M511, and M603, both the framework and first complementarity regions were identical in all cases. Sequences analysis of the light chains through part of the first complementarity region revealed three chains, one similar to each of the myeloma proteins T15, M603, and M167-M511. The latter two sequences differ by only a single amino acid (a single base substitution) in the first 23 residues, suggesting that the two light chains may be very similar if not identical. Thus, BALB/c and A/J mice which differ genetically at multiple loci including the heavy chain allotype complex locus show a remarkable preservation of their anti-PC antibodies. These results indicate that the genes encoding these antibodies are contained in the germ line.

Clonal nature of the immune response to phosphorylcholine (PC). V. Cross-idiotypic specificity among heavy chains of murine anti-PC antibodies and PC-binding myeloma proteins.

Seven mouse myeloma proteins with specificity for phosphorylcholine (PC) were found to share a common antigenic determinant. This group of proteins contained members which differed in genetic origin, heavy chain class, kappa-chain subgroup, individual antigenic determinants and specificity for choline analogues. The cross-idiotypic determinant, VH-PC, was antigenically similar in each of the proteins and was associated with the variable portion of the heavy chain in the region of the antibody combining site. Further studies showed that an indistinguishable determinant was present on IgM anti-PC antibodies isolated from all strains of mice tested regardless of histocompatibility or heavy chain allotype. In view of the finding that this cross-idiotypic determinant was not found on antibodies or myeloma proteins which lacked specificity for PC, the data strongly suggest that a particular heavy chain variable region has been preserved in all mouse antibodies with specificity for PC.

Anti-phosphocholine hybridoma antibodies. I. Direct evidence for three distinct families of antibodies in the murine response.

Biochemical and serological studies were performed on more than 400 anti- phosphocholine (PC) hybridoma proteins (HP) derived from six strains of mice; 26 of these HP were examined in detail. All HP possessed specificity for PC, and all those tested contained an H-chain idiotypic determinant, V(H)-PC, which is shared by PC-binding myeloma proteins (BMP) and anti-PC antibodies. Among the HP, three well-defined and distinct families that correlated well with previous studies on serum anti-PC antibodies were identified. The largest group shared idotypic determinants, an L-chain isoelectric focusing (IEF) pattern, and a binding site specificity with the PC-BMP, T15. Using the same criteria, a second group was found to be strikingly similar to another PC-BMP, M603. The third group possessed an idiotypic determinant and an L-chain IEF profile similar to M511, but differences in binding site specificities were observed among the HP. The latter two groups contained members whose L-chain IEF profiles were not identical to other members of that group. Thus, among strains there is a remarkable degree of conservation among responding anti-PC antibodies, in both the kinds of anti-PC families that exist and the immunochemical and structural characteristics of various members within a family. Differences in at least one parameter were observed in each family, demonstrating that even a relatively restricted response is heterogeneous. However, this diversity seems to operate within certain constraints.

Clonal nature of the immune response to phosphorylcholine. IV. Idiotypic uniformity of binding site-associated antigenic determinants among mouse antiphosphorylcholine antibodies.

A new idiotypic determinant(s) on mouse anti-PC antibodies is described. Antibodies to the determinant(s) were raised in rabbits by immunization with HOPC 8, a PC-binding myeloma protein, and were isolated from HOPC 8 immunoadsorbent by elution with PC. These antibodies react with binding site determinants on anti-PC antibodies raised in all 15 inbred mouse strains tested regardless of histocompatibility or allotype, but fail to react with antibodies of other specificities or with anti-PC antibodies raised in other rodent species. These results correlate closely with other studies which show similar binding specificity of anti-PC antibodies raised in 17 different strains of mice. The site-associated idiotypic determinant(s) is clearly distinct from that detected by mouse anti-HOPC 8 antisera. This latter determinant(s) is present on anti-PC antibodies of only a few strains of mice and may not be in the binding site.

Clonal nature of the immune response to phosphorylcholine. I. Specificity, class, and idiotype of phosphorylcholine-binding receptors on lymphoid cells.

The relationship between receptor molecules on antigen-binding lymphocytes (ABC) and antibody produced by antibody-secreting cells was studied in inbred strains of mice using the immune response to phosphorylcholine (PC) as a model system. Splenic and lymph node lymphocytes of nonimmune mice possess rare lymphocytes which bind (125)I-labeled PC-bovine serum albumin. The frequency of PC-ABC increases after immunization and is paralleled by a rise in the frequency of PC-specific antibody-producing cells. Both of these responses are thymus independent. The receptors on these ABC display specificity for PC and are exclusively of the IgM class. In one of the strains, BALB/c, the receptors possess the same idiotype and fine degree of specificity for PC and two of its analogues, glycerophosphorylcholine and choline, that are characteristic of a PC-binding myeloma, HOPC 8. Furthermore, the idiotype and class of the receptor in these mice do not change during the course of the immune response. These data provide more direct evidence for the immunelogic relevance of receptor-bearing lymphocytes.

Polymorphism in anti-phosphocholine antibodies reflecting evolution of immunoglobulin families.

Complete variable (V) region amino acid sequences were determined for four heavy (H) and one light (L) chain from C57BL phosphocholine (PC)-binding monoclonal antibodies. Additional NH2-terminal sequences were obtained from H and L chains of C57BL and CBA/J origin. When these V regions were compared with previously reported anti-PC sequences, a number of observations could be made regarding the function and evolution of L and H chain segments used in these antibodies. (a) L and H chain V segments are remarkably conserved in these inbred strains, although there has been an accumulation of point mutations identifying apparently allelic forms of VK and VH. (b) Mice of each genotype use the same three VK segments in combination with a single VH segment to produce most anti-PC antibodies. An exception has been noted that indicates the occasional use of a second VH gene segment. (c) Multiple, different DH regions are used by mice of each strain, which suggests that the DH segment sequence plays no critical role in either antigen binding or VH-VL pairing. Furthermore, the DH segments and their corresponding gene families appear to be highly conserved in the inbred strains studied. (d) Most PC-binding antibodies use the JH1 joining segment. All JH1 sequences from C57BL mice differ from the BALB/c JH1 at position 105, which identifies allelic forms of the JH1 region. These studies are a first assessment of the nature of mutational events associated with the evolution of specific multigene immunoglobulin families and indicate that homologous VH, DH, JH, VK, and JK genes are similarly assembled and expressed in PC antibodies from three diverse genotypes.

Anti-phosphorylcholine antibodies of the T15 idiotype are optimally protective against Streptococcus pneumoniae.

In the mouse, most anti-PC antibody is found in one of the three murine anti-PC idiotype families: T15, M603, or M511. The antibodies within each of these idiotypic families have characteristic fine specificities for phosphorylcholine (PC)-analogues. In this paper we compare the ability of hybridoma IgM anti-PC antibodies of the three idiotype families to protect mice from fatal infection with S. pneumoniae. Antibody bearing the T15 idiotype was approximately 8 times as effective as antibody with the M603 idiotype and approximately 30 times as protective as antibody with the M511 idiotype. Reports by others have shown that the heavy chains of virtually all mouse anti-PC antibodies are produced by translocation of a single variable region gene and that the direct translation of this gene (in the absence of somatic mutations) results in heavy chains characteristic of the T15 idiotype. Thus, our findings suggest that the T15 germ line heavy chain variable region gene may have been selected through evolution to code for antibody binding PC-containing pathogens such as S. pneumoniae. Our observations may also explain the existence of regulatory mechanisms that result in maintenance of T15 idiotype expression in murine anti-PC immune responses.

Structural, functional, and idiotypic characteristics of a phosphorylcholine-binding IgA myeloma protein of C57BL/ka allotype.

An IgA phosphorylcholine (PC)-binding myeloma protein with IgCH allotypic determinants different from those of BALB/c mice is characterized. The myeloma, CBPC 2, was induced in the CB-20 strain of mice which is congenic to BALB/c but differs from it by carrying the A15 allotypic determinant of C57BL/ka mice. Sequence analysis of the CBPC 2 light chain through the first hypervariable region, as well as isoelectric point analysis, show that this chain is indistinguishable from that of T15, a PC-binding myeloma protein of BALB/c origin. The heavy chains of CBPC 2 and T15 differ by only two amino acids (positions 14 and 16) through the first hypervariable region. As measured by inhibition of precipitation, both CBPC 2 and T15 have the same specificity for PC, glycerophosphorylcholine, acetylcholine, and choline. In addition, CBPC 2 possesses the binding site-associated idiotypic determinant which is present on T15. However, like normal or induced C57BL/6 anti-PC antibody, it does not possess the nonbinding site idiotypic determinant.

Anti-phosphocholine hybridoma antibodies. II. Functional analysis of binding sites within three antibody families.

The present investigation extends our immunochemical characterization of binding site heterogeneity among a large series of monoclonal anti-phosphocholine (PC) antibodies. Hybridoma proteins (HP) from eight genetically distinct strains are included in this study, yet no strain specific characteristics are observed. These HP, as previously shown (5), are divided into three well-defined families based on public or family-specific Id and L chain isotypes characteristic of three PC-binding myeloma proteins: T15, M603, and M511. All antibodies exhibited some degree of inter- or intra-family heterogeneity, or both. Some of this intra-family diversity was reflected by differential reactivity for PC when attached to three different carriers. In spite of this, the specificity profiles for hapten analogues of PC, as measured by hapten inhibition of binding, were the same for all members of the T15 family. Altering the carrier had no effect, thus suggesting that the binding site pocket for PC is essentially preserved, whereas that for carrier is variable. Similar conclusions were reached for most of the M603 HP, although the binding site is different from the T15 HP. The M511 HP stand in sharp contrast to the HP in the other two families because their binding sites exhibit extensive variability. The independence in reactivity for PC and PC plus carrier offers a rational explanation for idiotypic and/or structural heterogeneity within a family. More importantly it suggests interesting strategies for diversification within one group of antibodies.

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.

The role of a novel VH sequence (V11) in the formation of anti-phosphocholine antibodies.

The immune response to phosphocholine (PC) in mice is highly restricted. Most anti-PC antibodies use heavy-chain variable-region (VH) sequences derived from single VH gene segment, V1. In order to investigate whether a highly homologous VH gene segment, V11, could contribute to the formation of PC-binding antibodies, we carried out chain recombination experiments with M47A, a non-PC binding myeloma protein whose H-chain is encoded by the V11 gene segment, and two PC-binding antibodies, HP101.6G6 (HP6G6) and M511. The H-chains from the non-PC-binding myeloma protein, M47A, formed a functional PC-binding site when paired with L-chains from both PC-binding antibodies. These results suggest that a second VH gene segment, V11, could theoretically be used to form PC-binding antibodies. In addition, these results provide direct evidence that a single H-chain can be used in combinatorial association with different L-chains to form antibodies of differing specificities.

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.

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.

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.

Uniformity in the clonal repertoire for the immune response to phosphorylcholine in mice.

A comparison of the clonal nature of the immune response to phosphorylcholine (PC) was made in nine different inbred mouse strains. Quantitative idiotypic analysis showed that anti-PC antibodies from each strain were composed of antibodies bearing binding-site idiotypic determinants indistinguishable from two different BALB/c myeloma proteins, T15 and M511. Idiotypic determinants of two other PC-binding proteins, M167 and M603, were not detected. Isoelectric focusing of the light (L) chains verified the presence of antibodies similar to T15 and M511 in each strain and indicated the presence of two additional antibodies, one of which has an L chain which cofocuses with M603. Fractionation of anti-PC antibody with anti-idiotypic antibody showed that immunoglobulins bearing T15 and M511 idiotypic determinants are separate and contain L chains that are unifore and resemble those of T15 and M511, respectively. Thus, these mice which differ genetically at multiple loci including the heavy chain allotype complex locus each possess, at least in part, an equivalent set of clonotypes specific for PC. This indicates that the genes encoding these antibodies must be contained in the germ line.

Genetic marker in the variable region of kappa chains of mouse anti-phosphorylcholine antibodies.

A newly discovered genetic marker in the kappa light chains of mouse immunoglobulins is described. This marker, designated kappa-PC8, is located in the L chains of those anti-phosphorylcholine (PC) antibodies which show the same functional and idiotypic characteristics as a PC-binding myeloma protein, HOPC 8 (H8). Analytical isoelectric focusing of these L chains revealed two phenotypes whose strain distribution pattern suggested a genetic association with genes that determine the T lymphocyte surface antigen(s) Ly-2/Ly-3. In four strains , AKR/J, C58/J, RF/J and PL/J (AKR-type, A) the H8-like L chains have a slightly lower isoelectric point than those of C57L/J and 12 other strains (C57L-type, B). Breeding experiments showed that the kappa-PC8-A phenotype is preferentially expressed. The most probable location of the marker is the variable region since other idiotypically related kappa-chains in C57L/J and AKR/J do not show differences in their electrophoretic mobility.

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.

Structural evidence for a polymorphic or allelic form of the heavy chain variable region.

The heavy (H) chains of anti-phosphocholine (PC) antibodies from C57BL/6J and CBA/J were sequenced through the N-terminal 36 residues and compared with previously published sequences of A/J anti-PC antibody and BALB/c PC-binding myeloma proteins T15, M603, and M511. Each of these antibody preparations contained molecules having light (L) chains and idiotypic determinants of T15, M511, and M603 indicating the presence of at least three different anti-PC antibodies in each pool. The structures of the C57BL/6J and CBA/J H chains each revealed a single sequence from positions 1 to 36 (which includes the first complementarity determining region (CDR), and they were identical. The first CDR was identical to that previously found for BALB/c and A/J indicating that this portion of these antibody molecules is highly conserved throughout inbred mice and is probably critical to PC-binding. A surprising finding was that both C57NL/6 and CBA sequence differed from the BALB/c and A/J sequences at two positions, residue 14 and 16. Since each of these strains differs at the allotype locus, the data indicates that the evolution of allotypy in mice occurred after variable region diversity for the particular genes.