Novel ganglioside antigen identified by B cells in human medullary breast carcinomas: the proof of principle concerning the tumor-infiltrating B lymphocytes.

The potential tumor-recognizing capacity of B cells infiltrating human breast carcinoma is an important aspect of breast cancer biology. As an experimental system, we used human medullary breast carcinoma because of its heavy B lymphocytic infiltration paralleled to a relatively better prognosis. Ig-rearranged V region V(H)-J(H), Vkappa-Jkappa, and Vlambda-Jlambda genes, amplified by RT-PCR of the infiltrating B cells, were cloned, sequenced, and subjected to a comparative DNA analysis. A combinatorial single-chain variable fragment Ab minilibrary was constructed out of randomly selected V(H) and Vkappa clones and tested for binding activity. Our data analysis revealed that some of the V(H)-J(H), Vkappa-Jkappa, and Vlambda-Jlambda region sequences were being assigned to clusters with oligoclonal predominance, while other characteristics of the Ab repertoire were defined also. A tumor-restricted binder clone could be selected out of the single-chain variable fragment kappa minilibrary tested against membrane fractions of primary breast tumor cells and tumor cell lines, the V(H) of which proved to be the overexpressed V(H)3-1 cluster. The specific binding was confirmed by FACS analysis with primary breast carcinoma cells and MDA-MB 231 cell line. ELISA and thin layer chromatography dot-blot experiments showed this target Ag to be a ganglioside D3 (GD3). Our results are a proof of principle about the capacity of B cells infiltrating breast carcinomas to reveal key cancer-related Ags, such as the GD3. GD3-specific Abs may influence tumor cell progression and could be used for further development of diagnostic and/or therapeutic purposes.

J chain in the nurse shark: implications for function in a lower vertebrate.

J chain is a small polypeptide covalently attached to polymeric IgA and IgM. In humans and mice, it plays a role in binding Ig to the polymeric Ig receptor for transport into secretions. The putative orthologue of mammalian J chain has been identified in the nurse shark by sequence analysis of cDNA and the polypeptide isolated from IgM. Conservation with J chains from other species is relatively poor, especially in the carboxyl-terminal portion, and, unlike other J chains, the shark protein is not acidic. The only highly conserved segment in all known J chains is a block of residues surrounding an N-linked glycosylation site. Of the eight half-cystine residues that are conserved in mammalian J chains, three are lacking in the nurse shark, including two in the carboxyl-terminal segment that have been reported to be required for binding of human J chain-containing IgA to secretory component. Taken together with these data, the relative abundance of J chain transcripts in the spleen and their absence in the spiral valve (intestine) suggest that J chain in nurse sharks may not have a role in Ig secretion. Analysis of J chain sequences in diverse species is in agreement with accepted phylogenetic relationships, with the exception of the earthworm, suggesting that the reported presence of J chain in invertebrates should be reassessed.

Use of a single VH family and long CDR3s in the variable region of cattle Ig heavy chains.

We have analyzed transcripts encoding the variable regions of Ig heavy chains from adult and fetal bovine splenocytes and bovine x mouse heterohybridomas. The 13 adult, seven fetal and two heterohybridomas transcripts as well as the six genes that were sequenced had > 83% identity to each other in the VH-encoded regions (FRs 1-3 and CDRs 1 and 2). By this criterion, all the bovine sequences were assigned to one family, which corresponds to the bovine homolog of the murine Q52 family. Southern blot analysis of genomic DNA demonstrated that homologs of other murine VH families such as 7183, S107 and 36-60 were present in the genome, but transcripts from these families were not detected in rapid amplification of cDNA ends (RACE)-PCR amplified products or in individual clones. The sequences of the adult transcripts using the mu isotype showed extensive somatic mutation indicating that the process of somatic hypermutation begins earlier in development of the bovine B cell. The length of CDR3 from V(D)J rearrangements averaged 21 amino acids, which is larger than other mammalian CDR3s. Analysis of CDR3s from 23 fetal transcripts revealed a preference for a reading frame in the putative D genes which is rich in glycine and tyrosine, and is also extensively mutated in adults. The bovine immune system appears to utilize Ig VH genes of a single family, but generates antibody diversity by extensive somatic mutation and long CDR3s which are subsequently hypermutated.

A second immunoglobulin light chain isotype in the rainbow trout.

A novel immunoglobulin (Ig) light chain isotype, termed IgL2, has been isolated from trout lymphoid tissues both by reverse transcription - polymerase chain reaction (PCR) and screening of cDNA libraries. The CL domain of the new isotype shares only 29% residues with a recently cloned trout IgL isotype, termed IgL1, which has some similarities to Ckappa and Clambda isotype domains of several vertebrate species. Using anchored PCR, a VL element rearranged to CL2 was isolated. It is a member of a new VL family (VL2) of which four members were sequenced. These differ in the sequence of CDR1 and CDR2 but are remarkably similar in CDR3, i. e., at the junction between VL and JL segments. VL elements are rearranged to novel JL elements which differ from those described for VL1-CL1 rearrangements. Two cDNA clones contained JL-CL2 segments but no VL segments. The JL segments were preceded by typical rearrangements signal sequences [RSS, nonamer-23 base pair (bp) spacer-heptamer]. Further upstream of RSS were located two to three near identical 53 bp repeats, each of which included a 16 bp sequence similar to KI and KII sequences located at similar places in human and mouse Jk1 genes. These sequences are believed to act as binding sites for the protein KLP, which could be a transcriptional factor involved in the synthesis of germline Jk transcripts. Their phylogenic conservation in vertebrates suggests that they have an important role in B-cell differentiation. Remarkably, an RNA species of about 0.7 kilobase is the predominant IgL mRNA in trout spleen and coincides in size with JLCL2 transcripts. Genomic DNA blot analysis indicates that the trout L2 locus has a cluster-like organization similar to the trout L1 locus and the IgL locus of several teleost fish. A phylogenic analysis of VL2 and CL2 corroborates their low similarity to other vertebrate IgL chains and suggests an ancient diversification of the IgL locus.

The VDJ repertoire expressed in human preB cells reflects the selection of bona fide heavy chains.

In early steps of B cell differentiation, mu chains are transiently expressed in association with a surrogate light chain (psi L) composed of the lambda-like and VpreB monomorphic polypeptides, thus forming a putative preB receptor. Using a monoclonal anti-VpreB antibody, preB cells were isolated from two adult human bone marrow samples and their VDJ repertoire analyzed at the transcription level. All VH families were identified and further analysis focused on VH3 sequence analysis of 37 distinct VDJ cDNA clones. The VH3 genes expressed in the two bone marrow samples were also encountered in fetal liver and adult peripheral blood lymphocytes with a roughly similar contribution of 3.30, 3.23, 3.9 and 3.53. The characteristic features of the preB repertoire as compared to the activation B repertoire include the quasi absence of somatic mutations, limited N diversity and a shorter third complementarity-determining region (CDR3). It also significantly differs from the fetal repertoire, which makes higher usage of DQ52 and has CDR3 of even shorter lengths. The almost constant presence of glycine residues in the CDR3 and predominance of JH4 with a low level of DQ52 DH usage, suggest that preB cell clones are submitted to an initial selective pressure which should be antigen independent. The bona fide heavy chains would be merely selected for their ability to interact with the surrogate light chains, thus shaping the repertoire that will be co-expressed with immunoglobulin light chains in IgM molecules.

Structure and diversity of rat T cell receptor alpha-chain genes.

We have sequenced 22 TCR-alpha clones isolated from two cDNA libraries derived from rat thymocytes and heart allograft-infiltrating lymphocytes. Sixteen different V alpha and 17 J alpha genes were characterized in these clones. The V alpha genes could be divided into nine subfamilies, each containing from one to three members. Nucleotide sequence comparisons to mouse alpha genes revealed the existence of mouse homologues for all the J alpha s and 8 of 9 V alpha subfamilies. One of the rat V alpha subfamilies appeared to have no known mouse counterpart. Southern blot analysis of germ-line DNA with cDNA probes specific for six different subfamilies demonstrated the existence of two to nine V alpha genes per subfamily. A minimum number of 35 V alpha genes was estimated for the nine rat subfamilies characterized. These analyses also detected two different RFLP patterns among five different inbred rat strains, including strain-specific loss of hybridization bands for some V alpha, indicating that deletion events may have occurred in the rat alpha gene locus. The characterization of rat TCR V alpha genes should facilitate a better understanding of T cell role in pathologic conditions such as autoimmune diseases and graft rejection.

Clonal characterization of the human IgG antibody repertoire to Haemophilus influenzae type b polysaccharide. III. A single VKII gene and one of several JK genes are joined by an invariant arginine to form the most common L chain V region.

To characterize the L chain V region repertoire of IgG anti-Haemophilus influenzae type b capsular polysaccharide (Hib-PS) antibodies, clonal antibodies were purified from immune serum and internal amino acid sequences of VKII anti-Hib-PS L chains obtained. We examined VKII L chains because it is the most common VL family expressed in the anti-Hib-PS response. Comparison of VKII amino acid sequences, including the entire CDR2 and CDR3 regions, of five anti-Hib-PS clonal antibodies from four unrelated individuals revealed complete identity with the exception of a single CDR3 residue from one antibody. When the sequence of these antibodies was compared with known VKII genes and myeloma proteins, it was found to be identical to the human VKII gene, A2, whose genomic sequence is presented here. In addition, all five of the VKII anti-Hib-PS antibodies examined contain an arginine inserted at the V-J junction. Finally, in contrast to the extraordinary homology of the VKII-encoded residues, there is variability in the JK gene utilization by these antibodies. These results demonstrate that the most common L chain V region in IgG anti-Hib-PS antibodies is the product of a single germ-line gene. The invariant arginine insertion suggests that this residue has an important role in Ag binding.

Clonal diversity, somatic mutation, and immune memory to phosphocholine-keyhole limpet hemocyanin.

Group II antibodies to phosphocholine (PC)-keyhole limpet hemocyanin in BALB/c mice are genetically diverse and of a defined binding phenotype which recognizes the hapten, phenyl-PC, and PC coupled to protein but not free PC. We sequenced the V regions of 14 kappa and lambda-bearing group II antibodies. Both types show extensive somatic mutations. The pattern of the mutations differs between kappa and lambda antibodies. The nature of the somatic mutation in lambda chains suggests strong Ag selection on the L chain but not the H chain of the lambda-bearing antibodies. The reverse pattern of selection was observed among kappa-containing antibodies wherein the accumulation of replacement mutations in the CDR of the H chain appears to result from selection while changes in the L chain appear unselected. From these findings it appears that somatic mutation plays a major role in anti-PC-keyhole limpet hemocyanin memory development because all 14 antibodies displayed changes from germ-line sequences.

Analysis of variable region genes encoding a human anti-DNA antibody of normal origin. Implications for the molecular basis of human autoimmune responses.

In order to investigate the genetic basis for natural anti-DNA immune responses, we isolated and sequenced the variable gene elements (VH and VL) encoding an anti-DNA antibody expressed by a human hybridoma of normal origin (Kim4.6) and compared these sequences with those reported for four other human anti-DNA antibodies. The Kim4.6 antibody leader and VH segments were identical in nucleotide sequence with the VH1.9III germ-line VH3 gene, and the Kim4.6VL segment showed 98% nucleotide sequence identity with a V lambda I subgroup gene expressed in a Burkitt's lymphoma. Comparative analysis of Kim4.6 and other human hybridoma anti-DNA antibodies indicated that anti-DNA immune responses are diverse in terms of VH and VL gene utilization but may exhibit a bias toward rearrangement of VH genes that are over-represented in the fetal pre-B cell repertoire. Moreover, Kim4.6 and three of four other sequenced human anti-DNA antibodies appear to use a germ-line diversity gene, DXP'1, which may represent a counterpart of the DFL16.1 segment utilized in murine responses to the hapten nitrophenyl. Taken together, our findings indicate that anti-DNA immune responses can be encoded by nonmutated VH genes and that the elements and molecular mechanisms which engender this response are essentially the same among natural and lupus-associated anti-DNA antibodies. Our data also suggest that natural autoimmune responses originate early in B cell ontogeny as is consistent with the hypothesis that autoreactivity plays a major role in shaping the normal immune repertoire.

Nucleotide sequences of eight human natural autoantibody VH regions reveals apparent restricted use of VH families.

Eight full length cDNA were isolated from EBV transformed human PBL derived from different normal individuals. Five were derived from antibodies with the characteristics of natural polyreactive antibodies. Three were either monoreactive or bireactive. The most striking feature of the structure of these molecules was their utilization of VH families. Although three used the large VHIII family and one used the large VHI family, the other four used genes derived from two of the recently defined small human VH families VHIV and VHV. Three of the molecules represent VHIV expressed sequences and one is the first example of a VHV gene used in an antibody of defined specificity. The nucleotide sequences of some of the molecules were remarkably similar in their VH gene segments to previously described VH genes. The data suggest that natural autoantibodies may use a restricted portion of the VH repertoire, and, in addition, that some polyreactive antibodies may be germ line encoded. The implication of these findings for the origin and diversity of the human B cell repertoire is discussed.

T cell receptor gene segment usage in a panel of hen-egg white lysozyme specific, I-Ak-restricted T helper hybridomas.

We have studied the relationship between MHC-restricted, Ag-specific recognition and TCR structure in a panel of seven Th-hybridomas specific for the foreign protein Ag, hen egg-white lysozyme, and the I-Ak class II MHC molecule. The fine specificity of these Th cells had been determined previously by their reactivity to a panel of APC lines bearing mutant I-Ak molecules and to proteolytic fragments of HEL. TCR gene segment composition was determined by cDNA cloning and DNA sequencing. A heterogeneous, yet repetitive usage of gene segments was observed within the panel. The same V alpha C10-J alpha MD13 rearrangement was used in three of the hybridomas, two with identical Ag and MHC-restriction fine specificities. The prevalent usage of the V beta 14 gene segment and members of J beta 2 cluster was noted. Inasmuch as gene segment usage did not correlate with MHC-restriction or Ag fine specificity alone, these results favor an interactive Ag model of T-cell recognition, in which Ag and MHC are recognized as a bimolecular complex.

The DJH complex remains active in recombination to VH segments after the loss of mu-chain expression in mu-positive pre-B cells.

AT11-2 is an Abelson virus-transformed B precursor cell line which is capable of differentiating Ig- from mu+ cells via functional recombination of VH segments to preexisting DJH complexes. We describe here that after a mu+ subclone (VDJ+/DJ) generated from Ig- AT11-2 (DJ/DJ) cells by in vitro functional VH to DJH recombination subsequently lost mu-chain expression either by the recombination of a pseudo VH segment to the VHDJH+ allele or by the deletion of VHDJH+ allele, a novel productive joining of VH segments to the preexisting DJH complex occurred. These results indicated that VH to VHDJH rearrangement was not suppressed in mu-chain producing cells and that the DJH complexes still remained active in the recombination to VH segments after the loss of mu-chain expression. Our results may also suggest that VH to DJH rearrangement, but not VH to VHDJH rearrangement, is suppressed in mu-chain producing cells to maintain allelic exclusion. Our cell differentiation system should continue to be valuable for elucidating the mechanism of suppression and associated implications regarding allelic exclusion.

Ig VH, DH, and JH germ-line gene segments linked by overlapping cosmid clones of rabbit DNA.

Overlapping cosmid clones of rabbit germ-line DNA containing VH, DH and JH gene segments were isolated. The map of this cluster of cosmid clones indicated that the rabbit VH and JH regions were separated by 63 kb. Hybridization of Southern blots of these cosmid clones with two different DH segment probes identified a total of six DH segments within the region between the VH and JH regions. The nucleotide sequences of the JH region and one of the DH segments have been determined. The DH segment has conserved heptamer and nonamer sequences separated by 12 and 11 bp at the 3' and 5' sides, respectively, of the coding region and hence, appears to be a functional gene. The nucleotide sequence of the JH region revealed four functional JH gene segments and one JH pseudogene. Inasmuch as the JH region had previously been linked by contiguous overlapping clones with C mu, C gamma, C epsilon, and one C alpha gene, this VH-DH-JH cluster and the clones containing the Ig H chain C region genes represent 190 kb of contiguous germ-line DNA of the Ig H chain locus.

Structural and evolutionary comparisons of four alleles of the mouse Igk-J locus which encodes immunoglobulin kappa light chain joining (J kappa) segments.

The Igk-J locus of the mouse encodes the immunoglobulin kappa light chain joining (J) segments. Four Igk-J alleles have been described on the basis of restriction enzyme length polymorphisms. The nucleotide sequences of the Igk-Ja allele (type strain, C.C58), Igk-Jc allele (type strain, SJL/J), and Igk-Jd allele (type strain, SK/CamRk) have been determined and are compared with the previously reported Igk-Jb allele sequence (type strain, BALB/c). The mouse sequences are also compared with published sequences for rat and human J kappa sequences. Far more differences were found between the Igk-Ja allele and the other mouse alleles than between any two of the latter. These result in two amino acid substitutions which distinguish the J2 and J3' segments of the Igk-Ja allele from the other three alleles. Use of the Phylogenetic Analysis Using Parsimony program to generate a phylogenetic tree strongly indicates that after divergence from the rat ancestor, there appears to have been an early split between the Igk-Ja allele and the evolutionary precursor of the other mouse alleles. There also appears to have been far less divergence from the ancestral condition in the Igk-Ja allele than in the other alleles. Also, the presence of only one convergent mutation among the four mouse alleles provides strong evidence against any crossing over within the Igk-J locus during the history of these alleles. Finally, the differences in rates of evolution of the Igk-J alleles are in marked contrast to the relatively uniform rates of divergence of four alleles of a mouse V kappa gene, Igk-VSer.

The structure of V alpha and J alpha segments in the mouse.

Antigen receptors on most T-cells are heterodimeric glycoproteins, comprised of an alpha chain and a beta chain. These chains are encoded by discontiguous variable (V), diversity (D) and joining (J) gene segments that rearrange to produce a contiguous and functional alpha or beta chain gene. To investigate the size and diversity of the germline repertoire of alpha-chain gene segments, we have characterized and sequenced 20 alpha chain cDNAs. Among these cDNA clones, we have found 4 J alpha and 4 V alpha sequences that have not yet been described. The relationship of these "new" gene segments to those already characterized is discussed.

Comparison of latent and nominal rabbit Ig VHa1 allotype cDNA sequences.

The genetic basis for the expression of a latent VH allotype in the rabbit was investigated. VH region cDNA libraries were produced from spleen mRNA derived from a homozygous a2a2 rabbit expressing an induced latent VHa1 allotype and, for comparison, from a normal homozygus a1a1 rabbit expressing nominal VHa1 allotype. The deduced amino acid sequences of the nominal VHa1 cDNA were concordant with previously published VHa1 protein sequences. A comparison of two complete VH-DH-JH and six partial VHa1 sequences reveals highly conserved sequence within VH framework regions (FR) and considerable diversity in complementarity-determining regions and D region sequences. Two functional JH genes or alleles are evident. Amino acid sequencing of the N-terminal 15 residues of pooled affinity-purified latent VHa1 H chain showed complete sequence identity with the nominal VHa1 sequences. Possible latent VHa1-encoding cDNA clones, derived from the a2a2 rabbit, were selected by hybridization with oligonucleotide probes corresponding to the VHa1 allotype-associated segments of the first and third framework regions (FR1 and FR3). cDNA sequence analysis reveals that the 5' untranslated regions of nominal and latent VHa1 cDNA were virtually identical to each other and to previously reported sequences associated with VHa2 and VHa-negative genes. Moreover, some latent VHa1 genes encode FR1 segments that are essentially homologous to the corresponding segment of a nominal VHa1 allotype. In contrast, other putative latent genes display blocks of VHa1 sequence in either FR1 or FR3 that are flanked by blocks of sequence identical to other rabbit VH genes (i.e., VHa2 or VHa-negative). These composite sequences may be directly encoded by composite germ-line VH genes or may be the products of somatically generated recombination or gene conversion between genes encoding latent and nominal allotypes. The data do not support the hypothesis that latent genes are the result of extensive modification by somatic point mutation.

Specific H chain junctional diversity may be required for non-T15 antibodies to bind phosphorylcholine.

The secondary antibody response of mice to phosphorylcholine (PC) shows a markedly different clonal profile than the primary response. In particular, the T15 antibodies that dominate the primary response are a minor part of secondary IgG antibodies, whereas 511 and 603 antibodies become a more prominent part of the PC-specific secondary response. These three anti-PC families differ only in L chain usage. We partially sequenced the IgH chain mRNA of a series of secondary T15 and 511 hybridomas to determine the role of somatic mutation and affinity maturation in these changes in clonal profile. None of the sequenced T15 antibodies showed somatic mutations or affinity increases. In contrast, all of the 511 antibodies had extensive somatic mutation and most had significantly increased affinity for nitrophenyl-PC. The failure of T15-expressing B cells to contribute to the secondary IgG response thus is likely to be explained by their inability to undergo (or tolerate) substantial somatic mutation and affinity maturation. We also noted that all 511 antibodies sequenced by us or others had an extra amino acid encoded at the VH-D junction by either N region addition or diversity of VH-D joining. Published sequences also show a 603 family-specific change at the VH-D junction. The frequency with which these changes, which appear obligate for PC binding, occur may determine the under-representation of these clonotypes in the primary anti-PC response. The affinity maturation in 511 antibodies after somatic mutation appears to account for their expansion in the secondary response.