New subgroups in the human T cell rearranging V gamma gene locus.

Two new V gamma genes in humans are described from rearrangement in T cell lines, which constitute single members of new V gene subgroups of the T-cell rearranging gamma (TRG gamma) locus. These two genes (herein designated as belonging to V gamma III and V gamma IV subgroups) are located between V gamma I/V gamma II subgroups and the constant (C) gamma genes. The existence of these new genes brings the number of different, potentially useable, human TRG V gamma genes to eight (excluding at least five pseudo V gamma genes) and the number of distinct subgroups to four. Polymorphism in the sequence of the V gamma II subgroup gene is also described and rearranged fragment sizes which make possible an unequivocal assignment of a V gamma rearrangement are given. These results extend previous conclusions of the inherited diversity of the human TRG V gamma locus.     

The human T-cell rearranging gamma (TRG) genes and the gamma T-cell receptor

The human T-cell Rearranging Gamma genes or T-cell Receptor Gamma (TRG) chain genes, like those encoding the T-cell Receptor (TcR) alpha and beta polypeptides, undergo rearrangements specifically in T-cells. The human TRG locus which has been mapped to chromosome 7 (7p15) is composed of 2 constant region genes (TRGC), 5 joining segments (TRGJ) and at least 14 variable gamma genes (TRGV). 8 variable genes are functional and belong to 4 different subgroups. Based on restriction fragments, the TRG rearrangements can be assigned to given V and J segments, in normal T-cells, T leukemias and lymphomas. The product of the rearranged TRG gene is the gamma chain which is expressed at the surface of a subset of CD3+4-8- T lymphocytes lacking the conventional receptor alpha beta. Structural differences exist between the different 'gamma T-cell receptors', the gamma and delta polypeptides being disulfide or non-disulfide linked. Although the TRG+ cells display a cytolytic activity, their precise function remains to be elucidated.     

Genomic organization of the sheep TRG1@ locus and comparative analyses of Bovidae and human variable genes

gammadelta T cells commonly account for 0.5%-5% of human (gammadelta low species) circulating T cells, whereas they are very common in chickens, and they may account for >70% of peripheral cells in ruminants (gammadelta high species). We have previously reported the ovine TRG2@ locus structure, the first complete physical map of any ruminant animal TCR locus. Here we determined the TRG1@ locus organization in sheep, reported all variable (V) gamma gene segments in their germline configuration and included human and cattle sequences in a three species comparison. The TRG1@ locus spans about 140 kb and consists of three clusters named TRG5, TRG3, and TRG1 according to the constant (C) genes. The predicted tertiary structure of cattle and sheep V proteins showed a remarkably high degree of conservation between the experimentally determined human Vgamma9 and the proteins belonging to TRG5 Vgamma subgroup. However systematic comparison of primary and tertiary structure highligthed that in Bovidae the overall conformation of the gammadelta TCR, is more similar to the Fab fragment of an antibody than any TCR heterodimer. Phylogenetic analysis showed that the evolution of cattle and sheep V genes is related to the rearrangement process of V segments with the relevant C, and consequentely to the appartenence of the V genes to a given cluster. The TRG cluster evolution in cattle and sheep pointed out the existence of a TRG5 ancient cluster and the occurrence of duplications of its minimal structural scheme of one V, two joining (J), and one C.     

The human T-cell receptor gamma (TRG) genes

The human T-cell receptor gamma (TRG) chain genes, like those encoding the T-cell receptor alpha- and beta-polypeptides, undergo rearrangements specifically in T cells. The human TRG locus, which has been completely mapped, is composed of two constant region genes (TRGC), five joining segments (TRGJ) and at least 14 variable gamma-genes (TRGV). Eight variable genes are functional and belong to four different subgroups. The product of the rearranged TRG gene is the gamma-chain which is expressed, along with the delta-chain, at the surface of a subset of T lymphocytes. Although some gamma delta + cells display a cytolytic activity, their precise function remains to be elucidated.     

The T cell receptors of human gamma delta T cells reactive to Mycobacterium tuberculosis are encoded by specific V genes but diverse V-J junctions.

The observation that gamma delta T lymphocytes react to mycobacteria has provided an important model for investigation of these cells in the immune response to infection. One important question regarding human gamma delta T cells is the breadth of the T cell repertoire in response to specific pathogens. The present study was undertaken to characterize, in molecular terms, the mycobacterium-specific gamma delta TCR repertoire. Mononuclear cells were isolated from the peripheral blood and pleural fluid of patients with tuberculous pleuritis and stimulated with Mycobacterium tuberculosis in vitro. Cytofluorometric analysis of the expressed gamma delta TCR repertoire of M. tuberculosis expanded cells was performed using anti-V region antibodies. The majority of responding gamma delta T cells express a receptor composed of V delta 2 and V gamma 9 chains. Molecular analysis by PCR amplification confirmed use of the V delta 2 and V gamma 9 gene segments in these cells, and demonstrated predominant usage of J delta 1 and J gamma P gene segments. Analysis of nucleotide sequence at the V-J junctions revealed extensive diversity including nucleotide deletions of V, D, and J gene segments and nucleotide segment additions. The predicted amino acid sequences further indicates diversity in the V-J encoded region of the protein chains. The data indicate that M. tuberculosis-driven expansion of gamma delta T cells in vitro depends on specific pairing of the V delta 2 and V gamma 9 polypeptide chains, without apparent selection of explicit V-J junction regions.     

Rearrangements to the JP1, JP and JP2 segments in the human T-cell rearranging gamma gene (TRG gamma) locus

In the human T-cell rearranging gamma (TRG gamma) locus, five joining (J) segments have been identified: J1, J2 and three additional segments JP, JP1 and JP2. We report the sequence of the germline JP1 segment and compare it with the other human and mouse J gamma segments. We also demonstrate that rearrangements to the three additional J gamma segments can be identified by hybridization of the KpnI digests to the J gamma 1 probe pH60. Since rearrangements to J1 or J2 can be assigned, using the same pH60 probe, to one of the nine variable (V) gamma genes known to rearrange [(1987) EMBO J. 6, 1945-1950], our results show that a unique probe can detect all the TRG gamma rearrangements and be particularly useful for assessing the preferential usage of V gamma and J gamma segments in the TRG gamma-expressing cells.     

Diversity and rearrangement of the human T cell rearranging gamma genes: nine germ-line variable genes belonging to two subgroups

We describe nine T cell gamma variable (V) gene segments isolated from human DNA. These genes, which fall into two subgroups, are mapped in two DNA regions covering 54 kb and probably represent the majority of human V gamma genes. One subgroup (V gamma I) contains eight genes, consisting of four active genes and four pseudogenes. The single V gamma II gene is potentially active. Sequence analysis of the V gamma I genes shows variation clustered in hypervariable regions, but somatic variability is restricted to N-region diversity. Studies on rearrangement in T cell lines and in thymic DNA show that major rearrangements can be observed that are attributable to the five active V gamma genes. In addition, human cells with the phenotype of helper T cells can undergo productive V gamma-J gamma joining.     

Limited diversity and selection of rearranged gamma genes in polyclonal T cells

The role of a T gamma gene product in the immune response is not known. To investigate the participation of the T gamma gene in functional T cells, we estimated its variable (V gamma) gene diversity among mature polyclonal T cells and assayed for in vivo selection of rearranged V gamma genes during the immune response. In this study, we present evidence that functionally mature, normal human T cells have rearranged their T gamma genes but display a limited range of gene rearrangement choices. In contrast to clonal T cell neoplasms, an invariant array of seven T gamma gene rearrangements was found to be proportionately distributed within normal polyclonal T cell populations, as well as in benign polyclonal T cell proliferations incited by a wide variety of pathological conditions. Findings presented here indicate that the likelihood of rearrangement of each human V gamma gene may be fixed. Lack of selection of V gamma genes during the mature T cell immune response implies a limited role of any single V gamma gene at this stage of T cell development.     

Normal T cell development is possible without ''functional'' gamma chain genes.

The T cell-specific gamma gene family is organized into four V, J and C gene segments containing clusters (gamma 1, gamma 2, gamma 3, gamma 4) in germline DNA. We found that the V, J and C elements of gamma 2 are physically linked on a stretch of 6 kb of DNA while those of gamma 3 are found within a 15-kb region. Rearrangements take place only within the clusters, explaining the rigid rearrangement patterns seen in T lymphocytes. New V gamma, J gamma and C gamma gene segments were discovered and characterized allowing the better understanding of the potential germline diversity of the gamma gene family. No correlation with T cell function, i.e. cytolytic or helper, and the type of the productive gamma rearrangement could be established. In contrast we found that functional T cell clones have been able to mature without any functional gamma chain genes.     

Mapping of immunoglobulin variable region genes: relationship to the ''deletion'' model of immunoglobulin gene rearrangement

Five families of variable region genes of mouse kappa chains were analyzed by Southern blot hybridization to determine their relative chromosomal map positions. Map positions were deduced by Vk gene deletion from antibody-producing cells expressing upstream Vk genes and retention in cells expressing downstream genes. The Vk regions expressed in the myelomas M0PC167, MPC11, M0PC21 and ABPC20 are members of Vk families exhibiting one, three, six and six major germline hybridization bands respectively. The gene order of the five families in germline DNA was found to be VM167-VM11-(VM21, VA20)-VABE8-Jk-Ck. As expected in a deletion model of immunoglobulin gene rearrangement, a sequence located just 5' of J1 in germline DNA was found to be absent from some antibody producing cells which had not retained any germline Ck genes. However, other cell lines contained this sequence in rearranged contexts, suggesting that any deletion model of immunoglobulin V-J joining, as well as V gene mapping, must take into account the possibilities of stepwise rearrangements and reintegration of "deleted" DNA.     

Rearrangement and junctional-site sequence analyses of T-cell receptor gamma genes in intestinal intraepithelial lymphocytes from murine athymic chimeras.

The molecular organization of rearranged T-cell receptor (TCR) gamma genes intraepithelial lymphocytes (IEL) was studied in athymic radiation chimeras and was compared with the organization of gamma gene rearrangements in IEL from thymus-bearing animals by polymerase chain reaction and by sequence analyses of DNA spanning the junction of the variable (V) and joining (J) genes. In both thymus-bearing mice and athymic chimeras, IEL V-J gamma-gene rearrangements occurred for V gamma 1.2, V gamma 2, and V gamma 5 but not for V gamma 3 or V gamma 4. Sequence analyses of cloned V-J polymerase chain reaction-amplified products indicated that in both thymus-bearing mice and athymic chimeras, rearrangement of V gamma 1.2 and V gamma 5 resulted in in-frame as well as out-of-frame genes, whereas nearly all V gamma 2 rearrangements were out of frame from either type of animal. V-segment nucleotide removal occurred in most V gamma 1.2, V gamma 2, and V gamma 5 rearrangements; J-segment nucleotide removal was common in V gamma 1.2 but not in V gamma 2 or V gamma 5 rearrangements. N-segment nucleotide insertions were present in V gamma 1.2, V gamma 2, and V gamma 5 IEL rearrangements in both thymus-bearing mice and athymic chimeras, resulting in a predominant in-frame sequence for V gamma 5 and a predominant out-of-frame sequence for V gamma 2 genes. These findings demonstrate that (i) TCR gamma-gene rearrangement occurs extrathymically in IEL, (ii) rearrangements of TCR gamma genes involve the same V gene regardless of thymus influence; and (iii) the thymus does not determine the degree to which functional or nonfunctional rearrangements occur in IEL.     

Diversity, rearrangement, and expression of murine T cell gamma genes

Although the T cell gamma genes are similar in many respects to T cell receptor alpha and beta genes, earlier studies suggested that only a single gamma variable (V gamma) gene is expressed in mature T cells. We report the isolation and characterization of three new rearranged V gamma genes from murine fetal thymocytes. Although each of the new V gamma gene rearrangements is present in fetal thymocytes, two of them are undetectable in mature T cells. The levels of mRNA corresponding to each type of V gamma gene rearrangement in mature T cells are dramatically diminished compared with those in fetal thymocytes, although the abundance of two of the rearranged genes is increased in mature T cells. Our results demonstrate that there is significant expressed variability of gamma genes in immature T cells. Furthermore, the dynamics of gamma gene rearrangement and expression support the idea that gamma genes function in immature T cells.     

L chain isotype regulation in horse. I. Characterization of Ig lambda genes.

Analysis of 10 cDNA encoding lambda L chains of horse Ig indicated that this species may employ a relatively small number of variable region (V lambda) genes in the splenic B cell population. The V lambda sequences of all of the cDNA analyzed were closely related (> 88% identity at the nucleotide level) and were characterized by a deletion of the amino acid residue at position 3 compared with V lambda sequences so far described in other species. The 10 V lambda sequences could be grouped into three groups, V lambda 1 to V lambda 3, on the basis of a number of linked substitutions. Sequences within the groups showed the greatest divergence in the third cdr regions and at the V-J junctions. The junctional variation included amino acid substitutions on both sides of the V-J junction as well as the insertion or deletion of two to four amino acid residues. Four C lambda genes were identified in genomic blots of horse DNA, and three of these were found expressed in splenic cDNA. The fourth C lambda gene may represent a pseudogene, inasmuch as the associated J region possessed a defective heptamer joining sequence. Six of the nine possible V lambda-C lambda combinations were found in the cDNA analyzed, suggesting that genes belonging to groups V lambda 1 through V lambda 3 may rearrange to any one of three J lambda-C lambda genes. One V lambda germline gene was characterized and found to represent a distinct V lambda group (V lambda 4), not represented in the cDNA sequences analyzed. The number of germline V lambda genes was estimated to be 20 to 30, based on analysis of restriction fragments hybridizing with V lambda probes. On the basis of these data, we propose that the V lambda repertoire in horse may consist of relatively limited number of genes, of which only a few may be used at high frequency in the splenic B cell population. The results indicate that predominance of lambda-chains in horse Ig may not simply be due to the presence of a large germline V lambda gene repertoire.     

T cell receptor gamma gene status of human alpha/beta+ and gamma/delta+ T cell clones: absence of V9JP rearrangements in alpha/beta+ clones is not a result of a lack of rearrangements involving more 5' J gamma segments

T cell receptor (TCR) gamma gene rearrangements were examined in panels of human T cell clones expressing TCR alpha/beta or gamma/delta heterodimers. Over half of the alpha/beta+ clones had both chromosomes rearranged to C gamma 2 but this was the case for only 20% of the gamma/delta+ clones. While more than half of the gamma/delta+ clones showed a V9JP rearrangement, this configuration was absent from all 49 alpha/beta+ clones analysed. However, this was not a result of all rearrangements being to the more 3' J gamma genes as 11 alpha/beta+ clones had rearrangement(s) to JP1, the most 5' J gamma gene segment. Both alpha/beta+ and gamma/delta+ clones showed a similar pattern of V gamma gene usage in rearrangements to J gamma 1 or J gamma 2 with a lower proportion of the more 3' genes being rearranged to J gamma 2 than for the more 5' genes. Several alpha/beta+ and several gamma/delta+ clones had noncoordinate patterns of rearrangement involving both C gamma 1 and C gamma 2. Eleven out of fourteen CD8+ clones tested had both chromosomes rearranged to C gamma 2 whereas all clones derived from CD4-8- cells and having unconventional phenotypes (CD4-8- or CD4+8+) had at least one C gamma 1 rearrangement. Twelve out of twenty-seven CD4+ clones also had this pattern, suggesting that CD4-8+ clones had a tendency to utilize more 3' J gamma gene segments than CD4+ clones. There was some evidence for interdonor variation in the proportions of TCR gamma rearrangements to C gamma 1 or C gamma 2 in alpha/beta+ clones as well as gamma/delta+ clones. The results illustrate the unique nature of the V9JP rearrangement in gamma/delta+ clones and the possible use of a sequential mechanism of TCR gamma gene rearrangements during T cell differentiation is discussed.     

Polymorphism of the T-cell receptor gamma variable and constant region genes in a Chinese population

Summary The human T-cell receptor gamma gene region spans 160 kb genomic DNA. Restriction fragment length polymorphisms (RFLPs) have been previously documented for the constant region (TRGC) genes, the joining (TRGJ) segments and the variable (TRGV) genes. We have recently defined the alleles of the T-cell receptor gamma V, J and C genes and we have described seven haplotypes of the V gamma subgroup I genes characterized either by RFLPs or by deletion or insertion of V gamma genes. The number of VI genes may vary from 7 to 10 per haploid genome, the 9-gene haplotype being the most frequent. Allelic fragments can unambiguously characterize the TRGC2 gene with duplication or triplication of the exon 2. These alleles and haplotypes have been analyzed in four different populations (French, Lebanese, Tunisian and Black African). In this paper, we compare these allele and haplotype frequencies with those found in a Chinese population and we describe new TRGV allelic restriction fragments found only in the Chinese samples. These results and the previous data demonstrate the flexibility of the human T cell receptor gamma locus and the importance of unequal crossing-overs in the evolution of that locus. Moreover, they underline the importance of studying these polymorphisms in population genetics.     

Aberrant recombination events in B cell lines derived from a kappa-deficient human.

We have analyzed the structure of Ig kappa chain genes in B cell lines derived from a human individual who cannot synthesize any kappa chains, and whose Igs all contain lambda chains (1). We have characterized secondary DNA recombination events at two kappa alleles which have undergone misaligned V-J recombinations. One such secondary recombination has joined the flanking sequences of a V kappa and a J kappa 2 gene segment as if it were the reciprocal product of a V-J kappa 2 recombination, and resulted in the displacement of the recombined VJ kappa 1 gene segments from the C kappa locus. The non-rearranged form of the V kappa fragment which had recombined with the J kappa 2 flank was cloned. Nucleotide sequencing of this fragment identified a V kappa gene that differed by at least 38% from all previously sequenced human V kappa genes. The other V-J kappa segment analyzed has undergone a secondary recombination at a different site from that described above, at a site within the intervening sequence between the J kappa and C kappa gene segments, similar to the location of secondary recombinations which have occurred in lambda + B cell lines from mice and humans (2,3). These results prove that multiple recombinations can occur at one J kappa-C kappa locus.     

Cloning, sequencing and analyzing of the heavy chain V region genes of human polyreactive antibodies

The heavy chain variable region genes of 5 human polyreactive mAbs generated in our laboratory have been cloned and sequenced using polymerase chain reaction(PCR) technique.We found that 2 and 3 mAbs utilized genes of the VHIV and VHⅢ families,respectively.The former 2 VH segments were in germline configuration.A common VH segment,with the best similarity of 90.1% to the published VHⅢ germline genes,was utilized by 2 different rearranged genes encoding the V regions of other 3 mAbs.This strongly suggests that the common VH segment is a unmutated copy of an unidentified germline VHⅢ gene.All these polyreactive mAbs displayed a large NDN region(VH-D-JH junction).The entire H chain V regions of these polyreactive mAbs are unusually basic.The analysis of the charge properties of these mAbs as well as those of other poly-and mono-reactive mAbs from literatures prompts us to propose that the charged amino acids with a particular distribution along the H chain V region,especially the binding sites(CDRs),may be an important structural feature involved in antibody polyreactivity.     

Immunoglobulin genes of different subgroups are interdigitated within the VK locus

The variable regions of immunoglobulins are encoded by multigene families which are rearranged during B-cell differentiation. These families were classified in groups and subgroups based on their amino acid sequences. Genes belonging to a distinct subgroup are believed to occur in the genome within clusters. We are investigating the organization of human variable region genes of the kappa type (VK genes, ref. 1) in the germline and found now for the first time that VK sequences of three of the four different subgroups are interdigitated within the VK locus. We present evidence for the interspersion of two VKIII genes and a VKII pseudogene within an array of five VKI genes. All eight VK sequences are arranged in the same orientation. An evolutionary model for the generation of this 'mixed cluster' is discussed.