Identification of distinct T cell receptor (TCR)-gamma delta heterodimers using an anti-TCR-gamma variable region serum

T cell hybridomas were generated from CD3+, CD4-, CD8- splenocytes and fetal thymocytes. V gamma 1-expressing proteins present on these murine TCR-gamma delta hybridomas were identified by using an anti-TCR V gamma 1 peptide serum. This antiserum specifically immunoprecipitated 41-kDa TCR V gamma-C gamma 4 chains and 31-kDa TCR V gamma-C gamma 1/2 chains from distinct heterodimers expressed on the TCR-gamma delta T cell hybridomas. The RNA from a hybridoma with a 31-kDa TCR-gamma chain hybridized with a V gamma 1 probe but failed to hybridize with a V gamma 2 probe. In contrast, the RNA from a hybridoma with a 32-kDa TCR-gamma chain hybridized with a V gamma 2 probe. This 32-kDa TCR-gamma chain was not immunoprecipitated by the anti-V gamma 1 serum. These data were consistent with the conclusion that the 31-kDa protein was the product of a V gamma 1 to C gamma 2 rearrangement, whereas the 32-kDa protein was the product of a V gamma 2 to C gamma 1 rearrangement. Furthermore, Southern analyses confirmed that the 32-kDa protein was the product of a V gamma 1.2-J gamma 2 rearrangement, and all three of the 41-kDa TCR-gamma chains were the results of V gamma 1.1-J gamma 4 rearrangements. This was the first demonstration at the clonal level of TCR-gamma proteins which use members of the V gamma 1 gene family, as well as the C gamma 2 constant region. Additional biochemical analyses of the TCR-gamma and -delta proteins from three independently derived C gamma 4-bearing T cell hybridomas suggested that most of the molecular mass diversity observed in the bulk subpopulation of peripheral C gamma 4-containing heterodimers may be contributed by the TCR-delta chains.     

Human T cell gamma-chain gene rearrangements in acute lymphoid and nonlymphoid leukemia: comparison with the T cell receptor beta-chain gene

Rearrangement of germ-line genes coding for T and B cell antigen receptor molecules is an early event in lymphoid development which eventually leads to the generation of clonal diversity in receptor-positive lymphocytes. Three T cell-associated rearranging genes have been described. Two, T alpha and T beta, code for the two polypeptide chains that form the T cell receptor heterodimer. The function of the third gene, the gamma-gene (T gamma), is not known. To learn more about the behavior of T gamma during lymphoid ontogeny, we compared rearrangement of T gamma and T beta genes in leukemic cells arrested at varied stages of lymphoid and myeloid development. We analyzed 38 fresh cell lines and 15 established cell lines from a total of 53 leukemic patients. Cells were immunophenotyped with a panel of monoclonal antibodies recognizing T-, B-, or myeloid-associated surface markers. Sixteen T-lineage cases were studied; 15 displayed both T beta and T gamma rearrangements. The exception (germ-line for T beta and T gamma) was an immature CD2(T11)+, CD3(T3)-, CD7(3A1)+, CD1(T6)+, CD5(T101)+ phenotype. Fourteen non-T non-B leukemias were analyzed; eight were germ-line for both T beta and T gamma, four had rearrangements involving both T beta and T gamma, and two were germ-line for T beta and rearranged to T gamma. Four cases with acute biphenotypic leukemia were studied; two had rearrangements of T beta and T gamma, and two were germ-line for both genes. Cells from nonlymphocytic leukemias were studied in 19 cases. All were found to be germ-line for both T beta and T gamma. Fifty-one of 53 genomic DNA samples were concordant for T gamma and T beta rearrangement. These results indicate that rearrangement of T gamma can occur in leukemic cells of B cell as well as T cell precursor origin, as has been reported previously for T beta.     

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.     

Allelic exclusion at the TCR delta locus and commitment to gamma delta lineage: different modalities apply to distinct human gamma delta subsets

Expression of a beta-chain, as a pre-TCR, in T cell precursors prevents further rearrangements on the alternate beta allele through a strict allelic exclusion process and enables precursors to undergo differentiation. However, whether allelic exclusion applies to the TCR delta locus is unknown and the role of the gamma delta TCR in gamma delta lineage commitment is still unclear. Through the analysis of the rearrangement status of the TCR gamma, delta, and beta loci in human gamma delta T cell clones, expressing either the TCR V delta 1 or V delta 2 variable regions, we show that the rate of partial rearrangements at the delta locus is consistent with an allelic exclusion process. The overrepresentation of clones with two functional TCR gamma chains indicates that a gamma delta TCR selection process is required for the commitment of T cell precursors to the gamma delta lineage. Finally, while complete TCR beta rearrangements were observed in several V delta 2 T cell clones, these were seldom found in V delta 1 cells. This suggests a competitive alpha beta/gamma delta lineage commitment in the former subset and a precommitment to the gamma delta lineage in the latter. We propose that these distinct behaviors are related to the developmental stage at which rearrangements occur, as suggested by the patterns of accessibility to recombination sites that characterize the V delta 1 and V delta 2 subsets.     

Cutting edge: TCR delta gene is frequently rearranged in adult B lymphocytes

TCR gene rearrangement generates diversity of T lymphocytes by V(D)J recombination. Ig genes are rearranged in B cells using the same enzyme machinery. Physiologically, TCR gene is postulated to rearrange exclusively in T lineage, but malignant B precursor lymphoblasts contain rearranged TCR genes in most patients. Several mechanisms by which malignant cells break the regulation of V(D)J recombination have been proposed. In this study we show that incomplete TCR delta rearrangements V2-D3 and D2-D3 occur each in up to 16% alleles in B lymphocytes of all healthy donors studied, but complete VDJ rearrangement was negative at the sensitivity limit of 1%. Data are based on real-time quantitative PCR validated by PAGE and sequencing of the cloned products. Therefore, TCR genes rearrange not exclusively in T lineage. This study opens up further questions regarding the exact extent of the "cross-lineage" TCR or Ig rearrangements in normal lymphocytes, specific subsets in which the cross-lineage rearrangements occur, and the physiological importance of these rearrangements.     

T cell receptor gamma and delta gene rearrangements in scid thymocytes. Similarity to those in normal thymocytes.

The nature of TCR gamma and delta gene rearrangements in 4- to 6-week-old scid thymocytes was examined by using the polymerase chain reaction technique, cloning, and DNA sequencing. Analysis of 78 sequences indicates that TCR gamma and delta gene rearrangements in scid mice generally resemble those in thymocytes from normal young adult mice. V gamma 1, V gamma 2, and V gamma 5 rearrangements are heterogeneous, with extensive N region addition and nucleotide excision from the recombining coding segments. In addition, homogeneous and fetal-like V gamma 3, V gamma 4, and V delta 1 rearrangements are observed. These rearrangements are currently difficult to interpret but may be significant with respect to whether certain homogeneous joints in normal mice are due to cellular selection or to the rearrangement process. scid TCR gamma and delta gene nucleotide sequences also reveal direct V-J delta joining, inter-(V-J-C gamma) cluster joining, and the possibility of inversional rearrangement at the gamma locus. Short sequence homologies may contribute to V(D)J recombination and to the rescue of blocked coding joints.     

Characterization of TCR gene rearrangements during adult murine T cell development

Development of the alphabeta and gammadelta T cell lineages is dependent upon the rearrangement and expression of the TCRalpha and beta or gamma and delta genes, respectively. Although the timing and sequence of rearrangements of the TCRalpha and TCRbeta loci in adult murine thymic precursors has been characterized, no similar information is available for the TCRgamma and TCRdelta loci. In this report, we show that approximately half of the total TCRdelta alleles initiate rearrangements at the CD44highCD25+ stage, whereas the TCRbeta locus is mainly in germline configuration. In the subsequent CD44lowCD25+ stage, most TCRdelta alleles are fully recombined, whereas TCRbeta rearrangements are only complete on 10-30% of alleles. These results indicate that rearrangement at the TCRdelta locus can precede that of TCRbeta locus recombination by one developmental stage. In addition, we find a bias toward productive rearrangements of both TCRdelta and TCRgamma genes among CD44highCD25+ thymocytes, suggesting that functional gammadelta TCR complexes can be formed before the rearrangement of TCRbeta. These data support a model of lineage commitment in which sequential TCR gene rearrangements may influence alphabeta/gammadelta lineage decisions. Further, because TCR gene rearrangements are generally limited to T lineage cells, these analyses provide molecular evidence that irreversible commitment to the T lineage can occur as early as the CD44highCD25+ stage of development.     

Human T cell receptor-gamma and -delta chain pairing analyzed by transfection of a T cell receptor-delta negative mutant cell line

Specific TCR V gamma and V delta segments are found to be coordinately used on subpopulations of gamma delta T lymphocytes. The reasons for this phenomenon are unknown, but may include the inability of particular chains expressing unique V delta and V gamma segments to physically associate. V delta 2 is typically used together with V gamma 2 on human peripheral blood gamma delta T lymphocytes. To examine whether V delta 2 can be used in conjunction with distinct V gamma segments, a TCR- mutant of the human gamma delta T cell line MOLT-13, which expresses parental TCR gamma (V gamma 1.3) but not TCR-delta protein, was transfected with plasmids containing full-length TCR-delta cDNA using either V delta 2 or V delta 3. TCR reconstitution was successful in both transfectants and resulted in TCR protein and RNA levels similar to that of the parental MOLT-13 cell line. These cell lines could be activated through their receptors as assessed by increases in cytoplasmic free calcium. These studies imply that physical constraints cannot explain the observed chain pairing preferences. Other possible explanations are discussed.     

Patterns of T cell receptor gamma gene rearrangements in human CD3+ clones derived from WT31- or Leu7+ cells in relation to non-MHC-restricted cytotoxic activity

Clones were obtained from human peripheral blood WT31-, WT31-CD4-8-, CD4-8- or Leu 7+ cells in the presence of interleukin 2 and phytohaemagglutinin. Almost all clones were CD3+, about 50% were CD4-8- and all clones tested derived from WT31- remained WT31-, indicating that they were expressing a gamma/delta heterodimer in association with CD3. Some clones derived from CD4-8- cells expressing CD3 were WT31- and some were WT31+. All CD3+ clones had T cell receptor (TCR) gamma gene rearrangements; most also had their TCR beta genes rearranged, including all clones derived from Leu 7+ cells. TCR gamma gene rearrangements were noted involving all five known J segments. There was a tendency for V gene segments from the VII and VIII subgroups to be rearranged to J gamma 2 less often than those from the more 5' VI subgroup. Two clones definitely had one rearrangement to C gamma 1 and one to C gamma 2. When clones derived from WT31- cells were considered, the only obvious relationship which emerged was that all clones with both chromosomes rearranged to C gamma 2 had low or negligible cytotoxic activity against natural killer (NK)-sensitive and NK-resistant targets. Several of these clones were expressing CD8 on about 30% of cells. Most clones with rearrangements involving only C gamma 1 had high non-MHC-restricted cytotoxicity while those with at least one C gamma 1 rearrangement had either high or low activity. The only exceptions noted were a clone with a single V9JP rearrangement and a clone with a V9JP and a VI/IIIJP1 rearrangement, which both had low activity. A similar pattern was also found with most clones derived from Leu 7+ cells. The data are consistent with the participation of most types of disulphide-linked (C gamma 1) gamma/delta heterodimers in non-MHC-restricted cytotoxic activity mediated by CD3+ gamma/delta + T cell clones.     

Human V gamma 9-V delta 2 T cell receptor-gamma delta lymphocytes show specificity to Daudi Burkitt's lymphoma cells

Peripheral blood TCR-gamma delta cells with different functional V gamma or V delta gene rearrangements represent two nonoverlapping subsets. The major subset uses the V gamma 9 and the V delta 2 gene segments and the minor subset the V delta 1 gene segments in its functional TCR rearrangement. Upon in vitro activation, these TCR-gamma delta lymphocytes display MHC-unrestricted lytic activity, against a wide variety of tumor cells of distinct histologic origin. Here we show that fresh TCR-gamma delta lymphocytes that express a V gamma 9-V delta 2 encoded TCR display a specific proliferative response to Daudi, Burkitt's lymphoma cells. Moreover, cloned V gamma 9-V delta 2 lymphocytes show the capacity to lyse Daudi cells, whereas none of the cloned V gamma 1 TCR-gamma delta lymphocytes shows such specificity. Nucleotide diversity at the V-D-J junction of the TCR-V delta 2 gene did not contribute to this Daudi cell specificity. Comparison of the MHC-unrestricted cytolytic capacities of the V gamma 9-V delta 2 and the V delta 1 clones using a panel of distinct types of tumor target cells showed that on average, the level of MHC unrestricted lysis of V gamma 9-V delta 2 clones against these tumor cells exceeded that of V delta 1 clones. However, in contrast to all these tumor cell lines, only the Daudi cells showed such an absolute distinction in susceptibility to lysis by V gamma 9-V delta 2 and V delta 1 clones. V gamma 9-V delta 2 clones that were generated with a stimulator cell other than Daudi did not lyse their stimulator cells but nevertheless showed specific cytolysis of Daudi cells. The specific proliferation to and cytolysis of Daudi cells of the entire V gamma 9-V delta 2 subpopulation of TCR-gamma delta lymphocytes is reminiscent of a superantigen response.     

T cell receptor delta gene organization and expression in normal and leukemic natural killer cells

In peripheral blood most NK activity is mediated by CD3- cells with large granular lymphocyte morphology which cannot be assigned to a specific hemopoietic lineage. In accordance with previous studies we have analyzed the organization of the TCR delta gene, which rearranges early in thymic ontogeny, in normal NK cells, and in granular lymphocytes proliferative disorders (GLPD), in an effort to further define their relationship to the T cell differentiation pathway and to identify a possible marker of clonality for CD3- GLPD. The alpha/delta locus was rearranged in five cases of CD3+ GLPD with a biallelic deletion of the C delta region, suggesting V-J alpha rearrangement, whereas CD3- GLPD and normal CD3- NK cells had the delta gene in germ-line configuration, but surprisingly expressed high levels of TCR delta-related mRNA. On the basis of this finding and of the presence of truncated TCR-beta and CD3-epsilon mRNA, we are led to speculate on a possible ontogenic relationship of NK cells to the T cell differentiation pathway at stages preceding TCR gene rearrangement.     

Further evidence for a gamma/delta T cell receptor-mediated TCT.1/CD48 recognition.

We have demonstrated recently that a molecule, termed TCT.1 (Blast-1/CD48), is recognized on the surface of target cells by a series of alloreactive gamma/delta T cell clones generated from PBL of one healthy individual (designated E). Southern blot analyses suggested that these clones express a TCR associating a V3-JP2-C2 gamma-chain and V1-D-J1-C delta-chain. In the present study, we have developed from PBL of a second normal donor (designated G) a novel series of gamma/delta cloned T cell lines with similar functional activity (i.e., specific recognition of TCT.1 protein). The TCR gamma- and delta-chain nucleotide sequences of both the E and G clones were determined. Results show that 1) sequences from all the clones are identical in each individual donor, 2) the delta-chains expressed by the E and the G clones are encoded by distinct gene rearrangements including V1-D-J delta 1 and V1-D-J delta 2, respectively, 3) the gamma-chains expressed by the E and the G clones are encoded by the same genomic variable elements, namely V gamma 3 and JP2, whereas the junctional regions are distinct. Because the latter rearrangement is very infrequent in human peripheral blood, these data support the view that TCT.1/CD48 recognition is likely to be TCR dependent.     

Immature and advanced patterns of T cell receptor gene rearrangement among lymphocytes in splenic culture

Bulk populations and 39 hybridomas from splenic Con A cultures were analyzed for rearrangements among TCR genes: alpha, beta, gamma, and delta. Patterns were categorized to reveal general rules governing gene rearrangement within the activated adult peripheral population. Many patterns of gene rearrangement were consistent with previous studies of T cell lines. Additional points of interest were the following: 1) A large proportion of Con A-stimulated splenic cells bore no TCR gene rearrangements. 2) One splenic hybridoma exhibited an unusual gene pattern, with rearrangements, at alpha and beta, but not J gamma 1 or J gamma 2 loci. 3) Multiple gamma rearrangements were noted other than V1.2-J2 and V2-J1. 4) One hybridoma exhibited TCR gene rearrangements typical of day 14 to 15 fetal thymocytes, as well as rearrangements at immunoglobulin gene loci. 5) Among hybridomas with J alpha rearrangements, homologous chromosomes exhibited rearrangements at similar positions along the J alpha locus.     

Gamma delta T cell receptor analysis supports a role for HSP 70 selection of lymphocytes in multiple sclerosis lesions.

BACKGROUND: Interactions between gamma delta T cells and heat shock proteins (HSP) have been proposed as contributing factors in a number of diseases of possible autoimmune etiology but definitive evidence to support this hypothesis has been lacking. In multiple sclerosis (MS), a chronic inflammatory neurologic disease, HSP and gamma delta T cells are known to colocalize in brain lesions. Analysis of T cell receptor (TCR) gene usage in these lesions has detected evidence of clonality within both the V delta 2-J delta 1 and V delta 2-J delta 3 populations of gamma delta T cells. In our own studies, using direct sequence analysis, a dominant V delta 2-J delta 3 TCR sequence was found in 9 MS brain samples, suggesting a response to a common antigen. In this report, we have examined gamma delta T cell receptor gene usage in MS peripheral blood T cell lines selected for reactivity to HSP 70. MATERIALS AND METHODS: TCR rearrangement patterns for V delta 2-J delta 1 and V delta 2-J delta 3 were studied using the polymerase chain reaction (PCR) and a direct sequencing technique in populations of peripheral blood mononuclear cells (PBMC) cultured with Mycobacterium tuberculosis (M. tuberculosis) purified protein derivative (PPD) and then selected for reactivity to a 70-kD heat shock protein (HSP70). Cells were obtained from health donors, patients with MS, and patients with tuberculosis (TB). PCR products were subjected to direct sequence analysis to look for evidence for clonality within these T cell lines and to define the sequence of the V-D-J (CDR3) region of the TCR. RESULTS: In freshly isolated PBMC, both V delta 2-J delta 1 and V delta 2-J delta 3 gene rearrangement patterns were detected, whereas in HSP70+ T cell lines the predominant delta chain rearrangement pattern was V delta 2-J delta 3. Direct sequence analyses indicated that in cells reactive with HSP70 the V delta 2-J delta 3 sequences were usually oligoclonal and used D delta 3 exclusively. In four of four MS and two of three TB patients, the oligoclonal sequences in the HSP70+ T cell lines were identical to one another and to a dominant sequence previously detected in MS brain lesions. In two of three HSP70+ T cell lines from healthy controls, the oligoclonal sequences differed from those found in both groups of patients but were identical to one another except for a small region of heterogeneity in the second N region. In contrast, in freshly isolated PBMC or in PPD+HSP70- T cell lines, the V delta 2-J delta 3 gene rearrangement patterns were usually polyclonal and dominant sequences were rarely identified. CONCLUSIONS: These results support the conclusion that a subpopulation of gamma delta T cells in MS lesions are responding to HSP 70 and that non-CNS-specific antigens contribute to the pathogenesis of MS.     

Systematic development of distinct T cell receptor-gamma delta T cell subsets during fetal ontogeny

To elucidate the developmental pattern and diversity of murine cluster of differentiation (CD)3-associated TCR-gamma delta heterodimers, adult and fetal thymocytes were examined for cell-surface expression of various gamma- and delta-encoded TCR. Biochemical analysis, using antisera specific for distinct C gamma gene products, revealed the presence of T cells expressing C gamma 1 and/or C gamma 4 heterodimers in adult and fetal CD4- CD8- thymocyte populations. Although CD4-CD8- thymocyte populations express both C gamma 1 and C gamma 4 TCR-gamma delta heterodimers early in fetal thymus development, the relative level of C gamma 4-expressing T cells was significantly lower than previously observed in peripheral lymphoid organs. In addition, biochemical studies revealed the presence of TCR-gamma delta heterodimer(s) expressed during fetal ontogeny which were not detected in adult thymocyte or peripheral lymphoid populations. Studies of N-glycosylation patterns of one of these heterodimers suggested that it contained a rearranged V gamma 3/C gamma 1 gene product. To examine in detail individual TCR-gamma delta heterodimers, a panel of TCR-gamma delta expressing hybridomas was prepared. Biochemical analysis at the clonal level revealed that indeed three distinct TCR-gamma delta heterodimers were present at day 16 of fetal thymus development, with TCR-gamma-chains most likely encoded by V gamma 2/C gamma 1, V gamma 3/C gamma 1, and V gamma/C gamma 4. Together these findings suggest an ordered development of TCR-gamma delta T cells in the thymus and selective expression of distinct TCR-gamma delta subsets in peripheral lymphoid organs such as spleen and lymph nodes.     

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.     

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.     

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.     

A delta T-cell receptor deleting element transgenic reporter construct is rearranged in alpha beta but not gamma delta T-cell lineages.

T cells can be divided into two groups on the basis of the expression of either alpha beta or gamma delta T-cell receptors (TCRs). Because the TCR delta chain locus lies within the larger TCR alpha chain locus, control of the utilization of these two receptors is important in T-cell development, specifically for determination of T-cell type: rearrangement of the alpha locus results in deletion of the delta coding segments and commitment to the alpha beta lineage. In the developing thymus, a relative site-specific recombination occurs by which the TCR delta chain gene segments are deleted. This deletion removes all D delta, J delta, and C delta genes and occurs on both alleles. This delta deletional mechanism is evolutionarily conserved between mice and humans. Transgenic mice which contain the human delta deleting elements and as much internal TCR delta chain coding sequence as possible without allowing the formation of a complete delta chain gene were developed. Several transgenic lines showing recombinations between deleting elements within the transgene were developed. These lines demonstrate that utilization of the delta deleting elements occurs in alpha beta T cells of the spleen and thymus. These recombinations are rare in the gamma delta population, indicating that the machinery for utilization of delta deleting elements is functional in alpha beta T cells but absent in gamma delta T cells. Furthermore, a discrete population of early thymocytes containing delta deleting element recombinations but not V alpha-to-J alpha rearrangements has been identified. These data are consistent with a model in which delta deletion contributes to the implementation of a signal by which the TCR alpha chain locus is rearranged and expressed and thus becomes an alpha beta T cell.