Structure and rearrangement of the T cell receptor J alpha locus in T cells and leukemic T cell lines

The vast majority of T cells express an antigen receptor (TcR) composed of an alpha/beta heterodimer. The alpha and beta chains are encoded for by a set of variable (V), joining (J) and constant (C) region genes. Unlike the J genes of the beta chain which are limited in number and are clustered close to the constant region, the J alpha genes are spread over an 85-kilobase DNA region, upstream of the C alpha gene. We have isolated the complete J alpha locus, bounded on the 5' side by the C sequence of the delta gene and on the 3' side by the C sequence of the alpha gene. The experiments described here demonstrate that the J gene segments extend 75 kb 5' of C alpha and participate equally in generating the diversity of the alpha chain in peripheral T cells. Similarly, in leukemic T cell lines, rearrangements occurred over the entire locus and involved both alleles. Densitometry data suggest that in most peripheral T cells both alleles also are rearranged; thus, allelic exclusion in the alpha locus does not occur at the level of rearrangement. In three cell lines, an identical rearrangement has occurred on one allele in a region located 10 kb from the 5' end of the locus.     

Molecular determination of T-cell receptor alpha and beta chain genotypes in human families

Polymorphism in genes encoding the alpha and beta chain of the human T cell receptor has been detected by Southern blot analysis. Genomic DNA samples were isolated from B lymphoblastoid cell lines derived from members of families, each family including at least one individual with a recombinant HLA haplotype. T cell receptor alpha and beta chain haplotypes could be assigned in the families on the basis of observed restriction fragment length polymorphism (RFLP). Polymorphism in the alpha chain gene was detected in BglII digests using an alpha chain probe that included the V, J, C, and 3' untranslated sequences. A probe consisting of only the constant region (C alpha) revealed no polymorphism indicating that the polymorphic fragment hybridized to V, J, or 3' untranslated sequences of the alpha chain. Polymorphism in beta chain genes was observed in BglII digested DNA samples using a probe that corresponds to the constant region (C beta). Polymorphic C beta restriction fragments of 10.0 and 9.2 kilobase segregated in six of the eight families studied. Recent structural data for the C beta region suggest that the polymorphic BglII site lies in the region 5' to the C beta 2 gene. These polymorphisms should serve as markers for alpha and beta chain complexes allowing genetic studies of these immunologically important gene families.     

Non-random features of the repertoire expressed by the members of one V kappa gene family and of the V-J recombination.

The 5' and 3' flanking sequences of 14 members of the V kappa Ox (VK 4/5) gene family of BALB/c mice have been established. The family was unusual in the number of bases between the codon for Pro 95 and the heptamer sequence; most members contained four but there were also examples of none. A conserved leader sequence was used to amplify the genomic DNA of rearranged genes in order to analyze the spleen B cell repertoire of non-immunized animals. The library contained many members with virtually identical sequences to one or other of the already known members of the family. In addition, there were repeats of other sequences, allowing the definition of 12 hitherto undefined members of the family. Only 3 out of 96 could have originated by gene conversion, or as artefacts of the amplification procedure, and only 2 were putative somatic mutants. The frequency of expression of different members of the V kappa Ox gene family was not random, and some germ-line genes were unrepresented in the library. The high frequency of V kappa Ox1-J kappa 5 is in line with the dominance of this combination in the oxazolone response. An analysis of the junctional segment showed that although in most cases the diversity was due to trimming, there were exceptions indicating de novo additions (N or P bases). The average number of bases trimmed from the V kappa and the J kappa segments was not the same. There was no correlation in the number of bases trimmed from V kappa or J kappa in each recombination. The implications of asymmetric trimming in terms of the mechanism of recombination are discussed.     

Conserved nucleotide sequences at the 5' end of T cell receptor variable genes facilitate polymerase chain reaction amplification

Alignment of all available nucleotide sequences of mouse and rat alpha/beta T cell receptor (TcR) variable (V) regions revealed the presence of relatively conserved sequences at the 5' end of the V gene segments. Based on these conserved sequences, degenerate primers were developed for use in the polymerase chain reaction (PCR). The degenerate primers developed on the basis of the conserved sequences at the 5' end of rat and mouse V gene segments are expected to enable the amplification of all mouse and rat TcR alpha/beta chain V regions. To test their applicability, the primers were used for the amplification of the V region of the TcR alpha/beta expressed by rat T cell lines. After amplification, the TcR V regions expressed were cloned and sequenced. The Z1a T cell line was shown to use the same TcR V gene segments (V alpha 2 and V beta 8.2), as most other experimental allergic encephalomyelitis associated T cell lines, but had different D and J segments. In spite of these differences at the nucleotide level, a remarkable conservation of the amino acid sequence at the V beta D beta J beta junction was found. Alignment of a large number of human V alpha and V beta gene segments revealed the presence of similarly conserved sequences. Degenerate primers based on these conserved sequences enabled the amplification of TcR V regions of human T cell lines.     

Restricted alpha/beta receptor gene usage of idiotype-specific major histocompatibility complex-restricted T cells: selection for CDR3-related sequences.

We have sequenced the T cell receptor (TcR) V alpha and V beta genes of seven independent BALB/c CD4+ T cell clones specific for the immunoglobulin lambda 2 light chain produced by the MOPC 315 myeloma (lambda 2(315)). All the clones recognize a peptide of residues 91-101 of lambda 2(315) and are restricted by the major histocompatibility complex (MHC) molecule I-E(d). The results indicate that in BALB/c mice, this anti-idiotypic response uses a very limited number of TcR. The four clones which cross-react between Phe94 and Tyr94 peptide analogues use very similar receptors (V alpha 3, J alpha 1, V beta 6, J beta 1.1). The V alpha 3 gene used by all of these clones is identical and has not been previously described. Although the four clones differ in nucleotide sequence in the V/J borders, two had identical receptors at the amino acid level. One of the cross-reactive clones exhibits a heteroclitic response to the Tyr94 peptide variant resulting from a single amino acid exchange in the V/J junction of the alpha chain. The three remaining clones which recognize only the Phe94 and not the Tyr94 peptide have somewhat more diverse TcR, however, two of these three clones use V beta 6. One of these non-crossreacting clones is alloreactive, the specificity of which can be attributed to differences in the N-D-J sequences. Taken together these data indicate that this T cell response to an immunoglobulin idiotope is very restricted in terms of the TcR used. These data in conjunction with recently published results indicate that, although there can be strong preference for individual V alpha or V beta gene segments, certain V alpha/V beta combinations are preferentially selected for interacting with a given peptide/MHC combination, and that the CDR3-related regions are crucial for antigen fine specificity and alloreactivity.     

The human T cell receptor alpha-delta locus: a physical map of the variable, joining and constant region genes

In this study a physical macro-restriction map of the entire human alpha locus that spans about 1000 kilobase pairs and includes the V alpha, J alpha and C alpha genes is presented. Evidence is provided that gene duplications were involved in the increase of genomic diversity of V alpha genes. In addition, we show a detailed map of a 40-kb region located approximately 100 kg upstream of the human C alpha gene. Direct evidence is provided to support that the human alpha chain locus, like the murine, also contains another T cell constant region gene in the alpha chain locus, the human delta chain gene. In addition, two J segments and one D segment have been identified. Using these genomic probes, we show that several T cell lines, including those known to express the surface gamma/delta heterodimer, have rearranged this region. The design of two separate centers of rearrangement within one locus that are involved in rearrangement events at different times, and the presence of high number of J segments in this region, may render the locus highly vulnerable to chromosomal translocation during T cell development.     

Frequency and genetic characterization of V(DD)J recombinants in the human peripheral blood antibody repertoire

Antibody heavy-chain recombination that results in the incorporation of multiple diversity (D) genes, although uncommon, contributes substantially to the diversity of the human antibody repertoire. Such recombination allows the generation of heavy chain complementarity determining region 3 (HCDR3) regions of extreme length and enables junctional regions that, because of the nucleotide bias of N-addition regions, are difficult to produce through normal V(D)J recombination. Although this non-classical recombination process has been observed infrequently, comprehensive analysis of the frequency and genetic characteristics of such events in the human peripheral blood antibody repertoire has not been possible because of the rarity of such recombinants and the limitations of traditional sequencing technologies. Here, through the use of high-throughput sequencing of the normal human peripheral blood antibody repertoire, we analysed the frequency and genetic characteristics of V(DD)J recombinants. We found that these recombinations were present in approximately 1 in 800 circulating B cells, and that the frequency was severely reduced in memory cell subsets. We also found that V(DD)J recombination can occur across the spectrum of diversity genes, indicating that virtually all recombination signal sequences that flank diversity genes are amenable to V(DD)J recombination. Finally, we observed a repertoire bias in the diversity gene repertoire at the upstream (5') position, and discovered that this bias was primarily attributable to the order of diversity genes in the genomic locus.     

Predominant use of a particular alpha-chain in suppressor T cell hybridomas specific for keyhole limpet hemocyanin

We isolated and sequenced the rearranged genomic variable (V) and joining (J) gene segments of T cell receptor alpha-chain gene from two independent keyhole limpet hemocyanin (KLH)-specific suppressor T cell (Ts) hybridomas (BW5147 x C57BL/6 KLH-Ts). These nucleotide sequences were compared with those of germline DNA from kidney and also with cDNA of alpha-chain (VJ alpha 281) previously isolated from Ts hybridoma (34S-281) with KLH/H-2b Ts activity. The entire V alpha and J alpha sequences of all three Ts hybridomas were exactly identical and were encoded by the germline V alpha and J alpha gene segments without any mutations, except for 2-nucleotide deletions from both the 3' end of V alpha and 5' end of J alpha gene segments, respectively, and a 1-nucleotide (guanine) insertion in the junctional (N) region which was not encoded by the germline gene. Six additional KLH-Ts hybridomas, further analyzed, also possessed the same alpha-chain, indicating the preferential usage of the particular alpha-chain in these hybridomas. As chromosome analysis demonstrated a different pattern in each clone, these hybridomas appear to be independent. More surprisingly, 0.5-1.5% of the total functional T cell alpha-chain mRNA in the thymus and spleen of unprimed C57BL/6 mice was found to be of this particular alpha-chain. These results suggest that the repertoire of KLH-Ts is strictly limited.     

Antibody repertoires generated by VH replacement and direct VH to JH joining

The immunoglobulin heavy chain repertoire is generated by somatic rearrangement of variable (V(H)), diversity (D(H)), and joining (J(H)) elements. It can be further diversified by V(H) replacement, where nonrearranged V(H) genes invade preexisting V(H)D(H)J(H) joints. To study the impact and mechanism of V(H) replacement, we generated mice in which antibody production depends on the replacement of a nonproductive V(H)D(H)J(H) rearrangement inserted into its physiological position in the immunoglobulin heavy chain locus. In these mice a highly diverse heavy chain repertoire resulted from V(H) replacement and a second process of noncanonical V(D)J recombination, direct V(H) to J(H) joining. V(H) replacement rarely generated detectable sequence duplications but often proceeded through recombination between the conserved homologous sequences at the 3' end of V(H). Thus, V(H) replacement is an efficient mechanism of antibody diversification, and its impact on the overall antibody repertoire could be greater than anticipated because it frequently leaves no molecular footprint.     

Expression and demethylation of germinally-transmitted BALB/c mouse mammary tumor virus DNA in Abelson MuLV B-lymphoid cell lines

Murine mammary tumor virus (MMTV) RNA expression, DNA organization and DNA demethylation were examined in BALB/c B-lymphoid cell lines produced by transformation with the Abelson murine leukemia virus (AbMuLV). The MMTV DNA sequences in AbMuLV B cell lines, based on restriction mapping with EcoRI, PstI, BglII, BamHI and SacI and molecular hybridization with cloned probes of the MMTV LTR, gag-pol or env gene regions, were identical to the germinally-transmitted MMTV DNA complement of BALB/c mice. Several AbMuLV B cell lines expressed MMTV poly(A+)-RNA at detectable levels. MMTV poly(A+)-RNA for the env gene, 3.8 kb, and the long terminally redundant (LTR) region, 1.7 kb, were detected in some AbMuLV B cell lines. MMTV DNA sequences in the AbMuLV B cell lines were at least partially sensitive to digestion by the methylation-sensitive restriction endonucleases HhaI and HpaII. HhaI-sensitive sites were present in Units I, II and III of the germinally-transmitted MMTV DNA and were localized specifically near the 5' end of the 5' and 3' LTRs of both Units II and III. HpaII-sensitive sites were localized near the 3' end of the 3' LTRs of Units II and III, and at a cellular site 2.1 kbp 5' to the 5' LTR. These observations demonstrate that the germ line MMTV DNA sequences of BALB/c mice are expressed in cells of B lymphocyte origin, and suggest a correlation between MMTV RNA expression and selective demethylation in the LTR regions of germinally-transmitted MMTV DNA sequences.     

Formation of multimers of linear satellite RNAs.

A 22-base region of turnip crinkle virus satellite-RNA C (sat-RNA C) is involved in the accumulation of monomeric and dimeric forms. Deletions within the region inhibited the accumulation of sat-RNA C monomers. However, normal ratios of dimers to monomers occurred if the 22 bases were replaced by 22 unrelated bases or if the location of this region was altered. Therefore, these specific 22 bases are not involved in the accumulation of sat-RNA C monomers. Examination of the sequences at the junctions of multimers of all three turnip crinkle virus sat-RNAs revealed the deletion of bases corresponding to the 3' and 5' ends of monomeric units as well as the addition of nucleotides not present in monomers. Based on these results, we present a model to explain the formation of multimers of linear subviral RNAs associated with turnip crinkle virus. Our model suggests that multimers are formed by the reinitiation of replication by the replicase before release of the nascent strand. We have previously proposed the same mechanism for the formation of defective interfering RNAs, chimeric sat-RNAs, and sat-RNA recombinants in the turnip crinkle virus system (Cascone, Carpenter, Li, and Simon. (1990). EMBO J. 9, 1709-1715).     

Lambda light chain constant and variable gene complements in wild-derived inbred mouse strains

Previous investigations of lambda light (L) chains in wild-derived inbred (WDI) mouse strains revealed large variations in serum levels of this L chain type as well as differences in antibody responses in which lambda chains predominate. In the present study a diverse group of WDI strains was analyzed by Southern blot analysis using probes for V lambda, C lambda 1 and C lambda 2 genes in an attempt to correlate the complement of lambda genes present in these strains with their lambda expression and with their taxonomic classification. All strains studied had two or three DNA sequences that strongly hybridized with a V lambda probe derived from BALB/c; most strains had V lambda sequences on restriction fragments similar in size to V lambda 1 and V lambda 2 sequences from BALB/c. Comparison of C lambda 1 and C lambda 2 genes in WDI mice to those of BALB/c revealed extensive variation both in number of hybridizing fragments and in their sizes. There were no obvious correlations observed between C lambda and V lambda patterns obtained for mouse strains of any phylogenetic group suggesting that constant and variable regions of lambda have evolved independently. In contrast to variations found for C lambda, a single kappa constant region gene appears to be conserved throughout the various mouse species.