Evidence for gene conversion among immunoglobulin heavy chain variable region genes.

We have previously reported that the VH region amino acid sequence of a phosphocholine (PC)-binding hybridoma antibody of CBA/J origin, HP101 6G6 (6G6), differs extensively from the VH regions of other PC-binding antibodies. The sequence of 6G6 VH appears to be derived from a gene homologous to the BALB/c V11 gene, a member of the PC VH (T15 VH) gene family not normally used to encode PC-binding antibodies. The 6G6 VH sequence differs from the translated sequence of V11 by six amino acids, four of which occur at the same position in other members of this gene family. This coincidence led to the proposal that the 6G6 VH gene was derived by gene conversion involving three genes of the PC VH gene family. We report here the nucleic acid sequence of the rearranged VH gene of hybridoma 6G6. This sequence supports our previous suggestion of gene conversion by confirming those differences, relative to the BALB/c V11 gene sequence, that are encoded by other members of this gene family, and extends this correlation to include three silent base pair substitutions as well. In addition, 5' noncoding region sequence and Southern blot analysis using probes derived from the coding and 5' noncoding regions confirm that the 6G6 VH gene is likely to be derived from the V11 homologue in CBA/J mice, and suggest that all three genes believed to be involved in the generation of the 6G6 VH gene are present in the CBA/J genome, a prerequisite for their involvement in gene conversion.

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.

Interaction and sequence diversity among T15 VH genes in CBA/J mice.

Nucleotide sequences of the four genes composing the T15 heavy chain variable region (VH) family of the CBA/J mouse have been determined. Comparison of these sequences with their published BALB/c and C57BL/10 homologues reveals that nucleotide differences found between given alleles of two strains, i.e., CBA/J and BALB/c, are observed in other family members of the same strain. We suggest that these patterns of sequence variation are most readily explained by gene interaction (conversion). Additionally, the sequence of a CBA/J hybridoma, 6G6, proposed to have been generated by gene conversion, is directly encoded by the CBA/J V11 gene indicating that the putative conversion has occurred meiotically in the germline. These results are consistent with the premise that gene correction is occurring frequently among members of this family and that such processes may contribute significantly to the evolution of Ig variable region genes even in the relatively short time frame of inbred strain derivation.

Gene correction in the evolution of the T cell receptor beta chain.

Mutational mechanisms operating at the T cell receptor beta chain locus have been examined by comparison of the CT beta 1 and CT beta 2 gene sequences from Mus pahari, believed to be the oldest living species in the genus Mus, with those of inbred mice. Results indicate that a gene correction event independent of that suggested to have occurred in inbred mice has homogenized the M. pahari CT beta exon 1 sequences, minimizing diversity in this region of the molecule. These observations suggest that correction events such as gene conversion may occur frequently, even in pauci-gene families with as few as two members, and therefore play a significant role in gene diversification or homogenization of small as well as large gene families.

Relationships between B cell and myeloid differentiation. Studies with a B lymphocyte progenitor line, HAFTL-1.

A cell line, HAFTL-1, derived by in vitro transformation of fetal liver cells with v-Ha-ras, was found to have molecular and phenotypic characteristics of pro-B cells recently committed to the Ly-1+ B cell differentiation pathway. Stimulation of these cells with LPS resulted in their differentiation within either the B or myelomonocytic lineages. Thus, lines derived from LPS-stimulated HAFTL-1 cells were shown to be clonally related, as evidenced by common v-ras integrations, but to exhibit characteristics of pre-B cells (ThB expression, continuing DJ heavy chain rearrangements) or mature macrophages (expression of Mac-1 and Mac-2, lysozyme and nonspecific esterase production, phagocytosis) while maintaining their Ly-1+ phenotype. These results suggest that events resulting in the irrevocable commitment to a single lineage occur late in differentiation, at least within the pathway yielding Ly-1+ B cells and a proposed subpopulation of Ly-1+ monocytes and macrophages. Final commitment to these lineages is carefully orchestrated, as evidenced by restricted expression of Ly-5 isoforms and production of IgH transcripts.

Galactan-binding antibodies. Diversity and structure of idiotypes.

A group of eight IgM hybridoma proteins induced with beta(1,6)-D-galactan-containing antigens has been characterized in terms of primary amino acid sequence and idiotype expression. The H chain amino acid sequences reveal very strong homology in the VH segment although several substitutions are seen that suggest the occurrence of somatic mutation in these IgM molecules. Significant sequence variation was observed in CDR-3, the region generated by the D segment, and the two recombination events, VH-D and D-JH. The number of amino acids in this region contributed by the D segment was found to vary from two to six, yet the overall length of CDR-3 was precisely maintained by the addition of amino acids on either side of D during the recombination processes. These additional amino acids are suggested to result from nucleotide addition by repair enzymes. Idiotypic analysis of these proteins, in conjunction with an assessment of the H chain sequences, has permitted an identification of the molecular basis of both cross-reacting and unique idiotypic determinants expressed by these molecules.

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.

Amino acids at the site of V kappa-J kappa recombination not encoded by germline sequences.

Murine V kappa-J kappa recombination is characterized by a maintenance of size at the site of recombination and the use of nucleic acids found only in germline sequences. This is in contrast to heavy chain VH-D-JH assembly where random nucleotides are added at the recombination sites to produce considerable size variation, even though the heptamer/nonomer recombination sequences are identical in both kappa and heavy chain genes. We have examined the origin of an unusual amino acid, Ile, found at the site of V kappa-J kappa recombination in antigalactan antibodies, by sequence analysis of the corresponding rearranged and germline genes. Results indicate that the Ile codon can be generated by use of a single nucleotide 3' of the V kappa segment in combination with the second and third nucleotides of the first codon of J kappa 5 or J kappa 4. However, several antigalactan antibodies express Ile in combination with J kappa 2. An Ile codon cannot be generated by recombination in any reading frame between germline V kappa and J kappa 2 segments. These results suggest that the origin of the Ile codon in lines using J kappa 2 may represent a novel even in murine light chain assembly, possibly similar to the de novo addition of nucleotides observed in heavy chain gene recombination.

Idiotypic self binding of a dominant germline idiotype (T15). Autobody activity is affected by antibody valency.

We have previously described (1-3) an IgM antibody that binds to PC, expresses the T15 idiotype, and binds also to itself or T15 if insolubilized. Because of the simultaneous presence of complementary idiotopes and paratopes this type of antibody has been termed autobody. The self binding involves the antigen-binding site because the F(ab')2 fragment of T15, PC, and no other haptens inhibit the self binding. DNA sequence analysis of 11E7-1 using primer extension cDNA sequencing showed that the variable sequences of H and L chains of 11E7-1 are identical to the germline sequence of the prototype T15 idiotype. Furthermore, monomeric and dimeric T15 IgA were shown to bind to insolubilized T15 and other T15+ antibodies including 11E7-1. Thus, the self-binding activity is an inherent property of the T15 germline sequence. The self binding is highly dependent on the polymeric state of the binding antibody since the IgM pentamer of 11E7-1 is about three fold more effective than the T15 dimer and 50 times more than the T15 monomer. These data suggest that the self-binding activity of a germline-encoded idiotype may play an important role in the biology of its expression, and more specifically, may be responsible for the establishment of its dominant expression.

Genetic basis for expression of the idiotypic network. One unique Ig VH germline gene accounts for the major family of Ab1 and Ab3 (Ab1') antibodies of the GAT system.

Ig germline genes have been isolated from recombinant clones prepared in separate libraries constructed from adult BALB/c liver DNA either in pBR328 plasmid or in EMBL 3 phage. Three clones that gave a very strong positive hybridization signal with a VH anti-GAT-specific probe were completely characterized and sequenced. All three were greater than 95% homologous, with the exception of the 5' noncoding region, which was only 85% homologous but contained characteristic regulatory signals. One of these genes, H10, had a sequence that was completely identical to that of a cDNA derived from a GAT-specific BALB/c hybridoma. Southern blot analysis using Eco RI-digested DNA from rearranged GAT-specific hybridomas revealed that the same gene was used for other GAT-specific VH regions, including one differing from the H10 sequence by 12 nucleotides, which must have been generated by a somatic mechanism. The same H10 germline gene was also used, in most cases without any nucleotide substitution, in hybridomas of the Ab1' set of the GAT idiotypic cascade, suggesting that immunization with Ab2 (antiidiotypic) antibodies preferentially stimulates the direct expression of VH germline genes. Finally, the previous hypothesis that NPa and GAT VH genes were derived from the same germline gene was definitively confirmed, both from sequence data and Southern blot analysis.

Genetics of a new IgVH (T15 idiotype) marker in the mouse regulating natural antibody to phosphorylcholine.

The idiotype present on the Fab of a phosphorylcholine-binding IgA myeloma protein TEPC 15 (T15) of BALB/c origin was found in normal serum of BALB/c mice. Molecules carrying the T15 idiotype in normal serum could be adsorbed with Sepharose phosphorylcholine beads and R36A pneumococci. The T15 idiotype is absent in germ-free BALB/c but appears when the mice are conventionalized. A survey of normal sera of inbred strains for the T15 idiotype showed it to be present in BALB/c, 129, C57L, C58, and ST and absent or in low levels in CBA, C3H, C57BL/6, C57BL/Ka, C57BL/10, SJL, B10.D2, DBA/2, RIII, A, AL, AKR, NZB, and NH inbred strains of mice. The T15 idiotype is associated with some but not all strains carrying the IgC(H) allotypes found in BALB/c. Linkage of genes controlling the T15 idiotype in normal serum to the IgC(H) locus of BALB/c was demonstrated in F(2) progeny of a BALB/c and C57BL cross, Bailey's recombinant inbred strains, C x BD, C x BE, C x BG, C x BH, C x BI, C x BJ, C x BK, and CB20 congenic strains. Among these strains, only those possessing the IgC(H) locus of BALB/c including the F(2) progeny consisting of BALB/c homozygotes and BALB/c/C57BL heterozygotes and C x BG and C x BJ recombinants showed the T15 idiotype.

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.

Nucleic acid and protein sequences of phosphocholine-binding light chains.

An 18-kilobase DNA fragment containing the sequence coding for both the variable and constant regions of the S107 mouse immunoglobulin light chain was cloned from total cellular DNA. The complete nucleotide sequence of the kappa-chain variable-region gene is reported. Determination of the amino acid sequence encoded by the DNA is found to be identical to the protein sequence of the T15 light chain through residue 88. Direct sequence analysis confirmed that the J1 joining segment is used in the recombination event producing the active kappa light chain gene.

Interleukin 6 is essential for in vivo development of B lineage neoplasms.

Interleukin (IL) 6 has been suggested to be the major cytokine responsible for proliferation of neoplastic plasma cells in both human myeloma and mouse plasmacytoma. Much of the evidence supporting this suggestion is derived from in vitro studies in which the survival or proliferation of some plasma cell tumors has been found to be IL-6 dependent. However, it remains unclear whether this dependency is the consequence of in vivo or in vitro selective pressures that preferentially expand IL-6-responsive tumor cells, or whether it reflects a critical in vivo role for IL-6 in plasma cell neoplasia. To address this question, we have attempted to induce plasma cell tumors in normal mice and in IL-6-deficient mice generated by introduction of a germline-encoded null mutation in the IL-6 gene. The results demonstrate that mice homozygous (+/+) or heterozygous (+/-) for the wild-type IL-6 allele yield the expected incidences of plasma cell tumors. In contrast, mice homozygous for the IL-6-null allele (-/-) are completely resistant to plasma cell tumor development. These studies define the essential role of IL-6 in the development of B lineage tumors in vivo and provide experimental support for continued efforts to modulate this cytokine in the treatment of appropriate human B cell malignancies.

Six BALB-c IgA myeloma proteins that bind beta-(1-6)-D-galactan. Partial amino acid sequences and idiotypes.

Six IgA myeloma proteins of BALB/c origin which bind antigens containing beta-(1 --> 6)-D-galactan side chains have been isolated by affinity chromatography on galactoside-BSA-Sepharose columns. Partial amino acid sequences of of the light chains to residue Cys23 and the heavy chains to reside 30 were determined on the automated sequencer. No differences were found among the six V(K) sequences. Among some 50 partial V(K) sequences that have thus far been determined these six chains are the only ones thus far identified in this subgroup; at least 25 V(K) subgroups in the mouse have been identified so far. The heavy chain partial sequences were also very closely related but two differences were found. One protein differed from the other five by having isoleucine instead of leucine at position 5, a second protein differed from the others by having an unidentified amino acid at position 19. Using the highly sensitive inhibition of hemagglutination method it was found that each of the proteins possessed a unique inidividual antigenic determinant.

Immunoglobulin structure: amino terminal sequences of mouse myeloma proteins that bind phosphorylcholine.

The amino terminal sequences of five light and heavy immunoglobulin chains from myeloma proteins of the BALB/c mouse with binding activity to phosphorylcholine are presented. Except for a single substitution in position 4, all five heavy chains have identical amino terminal sequences through the first hypervariable region. Proteins which share unique (idiotypic) antigenic determinants are identical through the first hypervariable region of their light and heavy chains. Proteins with differing idiotypic determinants have light chains of differing amino acid sequence. These observations suggest that the heavy chain plays a more important role than the light chain in determining the phosphorylcholine binding site.

Immune system impairment and hepatic fibrosis in mice lacking the dioxin-binding Ah receptor.

The aryl hydrocarbon (Ah) receptor (AHR) mediates many carcinogenic and teratogenic effects of environmentally toxic chemicals such as dioxin. An AHR-deficient (Ahr-/-) mouse line was constructed by homologous recombination in embryonic stem cells. Almost half of the mice died shortly after birth, whereas survivors reached maturity and were fertile. The Ahr-/- mice showed decreased accumulation of lymphocytes in the spleen and lymph nodes, but not in the thymus. The livers of Ahr-/- mice were reduced in size by 50 percent and showed bile duct fibrosis Ahr-/- mice were also nonresponsive with regard to dioxin-mediated induction of genes encoding enzymes that catalyze the metabolism of foreign compounds. Thus, the AHR plays an important role in the development of the liver and the immune system.

Specific association of repetitive DNA sequences with major histocompatibility genes.

The DNA sequence organization of a 17.8-kilobase segment of porcine DNA, containing a functional major histocompatibility (MHC) gene, has been studied. The DNA flanking the MHC gene contains at least 10 distinct repetitive DNA sequence elements, each of which occurs only once within the 17.8-kilobase DNA segment. Their reiteration frequencies in the genome range from 10(2) to 10(4). The genomic organization of seven of these sequence elements has been examined; all are interspersed with other, unrelated DNA sequences. These seven repeated sequences are not generally associated in the genome. However, they appear to be nonrandomly linked in MHC-associated regions of the genome: at least two additional DNA segments containing MHC-homologous DNA also contain sequences homologous to DNA fragments bearing the seven different repeats. Of the seven sequences, four can be detected in splenic total RNA. These results suggest that these repeated elements are specifically associated with the MHC locus.

Activation of insulin-like growth factor I receptor signaling pathway is critical for mouse plasma cell tumor growth.

Plasma cell neoplasia in humans generally occurs as multiple myeloma, an incurable form of cancer. Tumors with marked similarity can be induced in mice by a variety of agents, including chemicals, silicone, and oncogene-containing retroviruses, suggesting the use of murine tumors as an informative model to study plasma cell disease. Herein, we have focused on the role of insulin-like growth factor I receptor (IGF-IR) signaling in the development of plasma cell disease. The insulin receptor substrate 2/phosphatidylinositol 3'-kinase/p70S6K pathway was found to be either constitutively or IGF-I-dependently activated in all plasma cell tumors. Biological relevance was demonstrated in that plasma cell lines with up-regulated IGF-IR expression levels exhibited mitogenic responses to IGF-I. More importantly, expression of a dominant-negative mutant of IGF-IR in these lines strongly suppressed tumorigenesis in vivo. Taken together, these results demonstrate that up-regulation and activation of IGF-IR and the downstream signaling pathway involving insulin receptor substrate 2, phosphatidylinositol 3'-kinase, and p70S6K may play an important role in the development of a broad spectrum of plasma cell tumors.

Disseminated growth of murine plasmacytoma: similarities to multiple myeloma.

Murine plasma cell tumors share a number of common features with human multiple myeloma, suggesting their possible use as a model for this disease. However, one major difference between the two is the peritoneal localization of murine tumors as opposed to bone marrow residence of malignant plasma cells in early stages of multiple myeloma. We have thus examined the ability of murine plasmacytoma to produce disseminated growth similar to that seen in myeloma or other lymphoid neoplasias. Of four murine cell lines evaluated, all were demonstrated to effect highly metastatic disease involving multiple organs, although variation was observed between lines. A temporal analysis was accordingly performed with the S107 line to assess the pattern of cellular localization. Both light microscopy and PCR analysis revealed that engraftment of plasma cells occurs first in the bone marrow, followed by dissemination to other sites including the spleen, lung, and liver. Cells passaged in vivo through the bone marrow display an entirely different metastatic pattern with no homing preference to bone marrow or any other organ, suggesting the occurrence of a phenotypic change. Microscopic osteolytic lesions were observed adjacent to plasma cell tumor masses in the bone marrow, indicating early stages of bone disease. These findings demonstrate previously unrecognized similarities between the murine and human diseases and suggest the use of this in vivo model for experimental approaches to the treatment of human disease.