Divergent evolution of T cell repertoires: extensive diversity and developmentally regulated expression of the sheep gamma delta T cell receptor.

Sheep gamma delta T cells express an unprecedented repertoire of antigen receptors contributed by increased diversity in both variable and constant region gene segments. Variable region diversity results mainly from the utilization of a large family of duplicated V delta genes that have retained two distinct hypervariable segments comparable with the complementarity determining regions present in other antigen receptor V genes. This implies that sheep V delta chains have been intensely selected during evolution, probably at sites involved in ligand recognition. The sheep gamma delta heterodimer occurs in at least five isotypic variants formed by the association of a single C delta segment with one of five functional C gamma segments, each with distinctive hinge regions. Our analysis also shows that the establishment of a normal peripheral repertoire is both developmentally regulated and dependent on the continual presence of a functional thymus during ontogeny. The existence of an expanded V gene repertoire and multiple receptor isotypes together with the prominence of gamma delta T cells in the sheep immune system argues that this lineage of T cells has a more elaborate functional role in this evolutionary pathway.     

Excision products of the T cell receptor gene support a progressive rearrangement model of the alpha/delta locus.

We have cloned extrachromosomal circular DNAs containing T cell receptor (TCR) delta gene segments in adult mouse thymocytes and splenocytes. We find that the frequency of circular DNA clones carrying germline delta sequences is lower than that of J alpha probe-positive clones, possibly related to increasing 5' distance from the most upstream J alpha segment. This suggests that the TCR alpha/delta locus is successively rearranged from within and that the delta-containing excision products are progressively diluted out by the subsequent cell division which includes further alpha gene rearrangements. In addition, examination of delta gene excision products revealed newly identified V delta subfamilies, the reciprocal joining of two D delta elements, J delta 2 usage in thymocytes and novel sequences homologous to the human delta-gene deleting elements.     

Organization of the human T-cell receptor genes

T lymphocytes recognize antigens through their membrane bound T-cell receptors. Whereas the conventional T-cell receptors are heterodimers of alpha and beta chains, expressed at the surface of CD3+ CD4+ and CD3+ CD8+ T lymphocytes, the gamma delta T-cell receptors are found at the surface of a subset of T-lymphocytes of phenotype CD3+ CD4- CD8-. The synthesis of the T-cell receptor chains results from the junction (or rearrangement) of DNA segments: Variable (V) gene and joining (J) segment for the alpha and gamma chains, V gene, D (diversity) and J segments for the beta and delta chains. In this review, we summarize the recent findings on the genomic organization of the alpha, beta, gamma and delta T-cell receptor loci in human.     

Microanatomic clonality of gamma delta T cells in human leishmaniasis lesions.

T cells bearing gamma delta Ag receptors accumulate in the lesions of patients with localized American cutaneous leishmaniasis (LCL), and are thought to be involved in immunity to the parasite. To obtain clues as to the nature of the Ag recognized by these cells, we analyzed the diversity of the TCR delta-chain in LCL lesions. Using mAb against variable (V) encoded determinants with immunoperoxidase, both V delta 1 and V delta 2 subpopulations were identified in the dermal granulomas. However, within the epidermis of LCL lesions, the majority of the gamma delta T cells were V delta 1 positive. PCR analysis of lesion-derived DNA using oligonucleotide primers for V and junctional (J) gene segments revealed preferential usage of J delta 1 in lesions compared with the peripheral blood of these patients. Nucleotide sequence analysis of the V-J junction indicated limited diversity of gamma delta T cells within specific microanatomic regions. In addition, use of a single diversity (D) gene segment, D delta 3, in V delta 2 cells in lesions was observed, as opposed to multiple D delta gene segment usage in the blood of the same individuals. The distribution, gene segment usage and clonality of gamma delta T cells in lesions of leishmaniasis was remarkably similar to that observed in leprosy. Therefore, gamma delta T cells responding to infection may recognize a limited set of nominal Ag, perhaps common to distinct pathogens and/or those expressed by the host. Our findings are most consistent with a model in which specific gamma delta T cells are clonally selected by these Ag in lesions and undergo oligoclonal expansion within a microanatomic region.     

Molecular characterization of the A/J J558 family of heavy chain variable region gene segments

The nucleotide sequences of the coding as well as the flanking regions of 11 A/J J558 heavy chain variable region (VH) gene segments are presented. Among these J558 VH segments was the unrearranged germline VH gene segment recruited in the predominant A strain-specific anti-arsonate response. Three other VH gene segments that are greater than 92% related to the p-azophenylarsenate (Ars) A VH gene segment were also isolated. Detailed analysis of the nucleotide sequences of these as well as the remaining seven J558 VH gene segments reveal that the J558 VH gene family is composed of distinct, but related, J558 VH subfamilies. Deletion mapping analyses were used to position the Ars A VH gene segment proximally with respect to the DH-JH clusters within the J558 VH gene family and distally with respect to its own J558 subfamily. The documentation of J558 VH subfamilies is discussed in the context of J558 VH family evolution and diversification.     

Repertoire and Organization of Human T-Cell Receptor α Region Variable Genes

A contig of YAC, PAC, and cosmid clones that spanned the human T-cell receptor α variable (AV) gene region on chromosome 14 was assembled. PCR primers corresponding to the members of 32 different subfamilies were used to map AV genes on the genomic clones. Nucleotide sequencing of PCR products derived from different genomic clones was used to discriminate between related AV gene segments that coamplified. The presence of individual AV gene segments on genomic clones was further confirmed by hybridization both to clones and to human genomic DNA from several unrelated individuals. These results suggest that the T-cell receptor α (TCRA) region in humans contains at least 46 distinct AV gene segments that can be grouped into 32 subfamilies based on nucleotide homology. Several subfamilies appear to contain additional members detectable by hybridization that do not map to chromosome 14.     

Thymocyte circular DNA excised from T cell receptor alpha-delta gene complex.

We have characterized thymocyte circular DNA excised from the T cell receptor alpha-delta gene complex. Some delta gene clones contained unusual recombinant structures derived from V-(D)-J joining: (i) a reciprocal joint of direct V to J delta joining, skipping the D delta segment; (ii) a V-D delta coding joint lacking an adjacent D delta-J delta coding joint; (iii) a V- D structure containing two D delta segments. Many of the alpha gen clones contained both coding and reciprocal joints of V alpha-to-J alpha joining on the same structure. Most of these coding joints were out of phase; however, in one clone there was an in-phase V-J alpha structure. Interestingly, some alpha gene clones contained the same V gene sequence as rearranged in the delta gene clone, indicating that the same V gene family, at least in part, could be utilized for both the alpha and delta gene systems.     

Characterization of an avian (Gallus gallus domesticus) TCR alpha delta gene locus.

Mammalian TCR delta genes are located in the midst of the TCR alpha gene locus. In the chicken, one large V delta gene family, two D delta gene segments, two J delta gene segments, and one C delta gene have been identified. The TCR delta genes were deleted on both alleles in alpha beta T cell lines, thereby indicating conservation of the combined TCR alpha delta locus in birds. V alpha and V delta gene segments were found to rearrange with one, both or neither of the D delta segments and either of the two J delta segments. Exonuclease activity, P-addition, and N-addition during VDJ delta rearrangement contributed to TCR delta repertoire diversification in the first embryonic wave of T cells. An unbiased V delta 1 repertoire was observed at all ages, but an acquired J delta 1 usage bias occurred in the TCR delta repertoire. The unrestricted combinatorial diversity of relatively complex TCR gamma and delta loci may contribute to the remarkable abundance of gamma delta T cells in this avian representative.     

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.     

Organization of the V Gene Segments in Mouse T-Cell Antigen Receptor α/δ Locus

The mouse T-cell receptor (TCR) α/δ locus was mapped using 17 Vα and 4 Vδ subfamily-specific probes. Four complementary methods were used: (1) an estimate of the V gene repertoire by Southern blot analysis of genomic DNA with subfamily-specific probes; (2) an analysis of V gene segments deleted by TCR gene rearrangements from a panel of T-cell tumors and hybridomas; (3) an analysis of overlapping clusters of cosmid clones; and (4) an analysis of large DNA fragments separated by field-inversion gel electrophoresis. The α/δ locus spans about 1 Mb. The distance between the 3′-most V gene segment (Vδ1) and the δ constant gene (Cδ) is no more than 150 kb. Sixty-six V gene segments have been mapped physically on cosmids. The members of individual Vα gene segment subfamilies are dispersed throughout the locus. In contrast, the Vδ gene segments Vδ1 to 5 are clustered at the 3′ end of the V gene segment cluster. At least two DNA segment duplications, 45 to 80 kb in length, are present in the locus. These data provide information on the evolution of the α/δ locus and on organizational features that might influence the expression of specific V gene segments in γδ cells.     

Determination of copy number and linkage relationships among five actin gene subfamilies in Petunia hybrida

The actin gene superfamily of Petunia hybrida cv. Mitchell contains greater than 100 gene members which have been divided into several highly divergent subfamilies [1]. Five subfamily-specific probes have been used to compare the actin genes among the Mitchell, Violet 23 (V23) and Red 51 (R51) cultivars of P. hybrida. The sum total of actin genes in these five subfamilies was estimated to be between 10 and 34 members in both V23 and R51. Restriction fragment length polymorphisms (RFLPs) between V23 and R51 were examined with these five probes and eleven different restriction endonucleases. Among the 55 comparisons, 87% exhibited RFLPs. These data indicate extreme divergence between V23 and R51 in DNA sequence and/or the presence of small insertions and deletions surrounding these actin gene subfamilies. This divergence suggests that V23 and R51, which have contrasting phenotypic marker loci on every chromosome, may be useful for the development of a complete RFLP linkage map of the Petunia genome. The segregation of Hind III RFLPs among the progeny of two backcrosses demonstrated that representatives of the five subfamilies of Petunia actin genes exist at four distinct genetic locations and suggested that two of these loci are tightly linked. Apparently, amplification of the numerous members of the Petunia actin gene superfamily occurred via gene dispersal of the original subfamily progenitors and not primarily as a result of amplification of a single chromosomal region.     

Restricted use of T cell receptor V genes in murine autoimmune encephalomyelitis raises possibilities for antibody therapy

Experimental allergic encephalomyelitis (EAE) is a paralytic autoimmune disease induced in susceptible animals by active immunization with myelin basic protein (MBP) or by passive transfer of MBP-specific T helper (TH) lymphocytes. We have analyzed the T cell receptor genes of 33 clonally distinct TH cells specific for a nonapeptide of MBP inducing EAE in B10.PL (H-2u) mice. All 33 TH cells used two alpha variable gene segments (V alpha 2.3, 61%; V alpha 4.2, 39%), the same alpha joining gene segment (J alpha 39), and two V beta and J beta gene segments (V beta 8.2-J beta 2.6, 79%; V beta 13-J beta 2.2, 21%). The anti-V beta 8 monoclonal antibody F23.1 was found to block completely recognition of the nonapeptide by V beta 8 TH cells in vitro and to reduce significantly the susceptibility of B10.PL mice to peptide-induced EAE.     

Genes encoding Xenopus laevis Ig L chains. Implications for the evolution of kappa and lambda chains.

Xenopus laevis Ig contain two distinct types of L chains, designated rho or L1 and sigma or L2. We have analyzed Xenopus genomic DNA by Southern blotting with cDNA probes specific for L1 V and C regions. Many fragments hybridized to the V probe, but only one or two fragments hybridized to the C probe. Corresponding C, J, and V gene segments were identified on clones isolated from a genomic library prepared from the same DNA. One clone contains a C gene segment separated from a J gene segment by an intron of 3.4 kb. The J and C gene segments are nearly identical in sequence to cDNA clones analyzed previously. The C segment is somewhat more similar and the J segment considerably more similar in sequence to the corresponding segments of mammalian kappa chains than to those of mammalian lambda chains. Upstream of the J segment is a typical recombination signal sequence with a spacer of 23 bp, as in J kappa. A second clone from the library contains four V gene segments, separated by 2.1 to 3.6 kb. Two of these, V1 and V3, have the expected structural and regulatory features of V genes, and are very similar in sequence to each other and to mammalian V kappa. A third gene segment, V2, resembles V1 and V3 in its coding region and nearby 5'-flanking region, but diverges in sequence 5' to position -95 with loss of the octamer promoter element. The fourth V-like segment is similar to the others at the 3'-end, but upstream of codon 64 bears no resemblance in sequence to any Ig V region. All four V segments have typical recombination signal sequences with 12-bp spacers at their 3'-ends, as in V kappa. Taken together, the data suggest that Xenopus L1 L chain genes are members of the kappa gene family.