The Armadillo (Dasypus novemcinctus): A Witness but Not a Functional Example for the Emergence of the Butyrophilin 3/Vγ9Vδ2 System in Placental Mammals.

1-5% of human blood T cells are Vγ9Vδ2 T cells whose T cell receptor (TCR) contain a TRGV9/TRGJP rearrangement and a TRDV2 comprising Vδ2-chain. They respond to phosphoantigens (PAgs) like isopentenyl pyrophosphate or (E)-4-hydroxy-3-methyl-but-2-enyl-pyrophosphate (HMBPP) in a butyrophilin 3 (BTN3)-dependent manner and may contribute to the control of mycobacterial infections. These cells were thought to be restricted to primates, but we demonstrated by analysis of genomic databases that TRGV9, TRDV2, and BTN3 genes coevolved and emerged together with placental mammals. Furthermore, we identified alpaca (Vicugna pacos) as species with typical Vγ9Vδ2 TCR rearrangements and currently aim to directly identify Vγ9Vδ2 T cells and BTN3. Other candidates to study this coevolution are the bottlenose dolphin (Tursiops truncatus) and the nine-banded armadillo (Dasypus novemcinctus) with genomic sequences encoding open reading frames for TRGV9, TRDV2, and the extracellular part of BTN3. Dolphins have been shown to express Vγ9- and Vδ2-like TCR chains and possess a predicted BTN3-like gene homologous to human BTN3A3. The other candidate, the armadillo, is of medical interest since it serves as a natural reservoir for Mycobacterium leprae. In this study, we analyzed the armadillo genome and found evidence for multiple non-functional BTN3 genes including genomic context which closely resembles the organization of the human, alpaca, and dolphin BTN3A3 loci. However, no BTN3 transcript could be detected in armadillo cDNA. Additionally, attempts to identify a functional TRGV9/TRGJP rearrangement via PCR failed. In contrast, complete TRDV2 gene segments preferentially rearranged with a TRDJ4 homolog were cloned and co-expressed with a human Vγ9-chain in murine hybridoma cells. These cells could be stimulated by immobilized anti-mouse CD3 antibody but not with human RAJI-RT1Bl cells and HMBPP. So far, the lack of expression of TRGV9 rearrangements and BTN3 renders the armadillo an unlikely candidate species for PAg-reactive Vγ9Vδ2 T cells. This is in line with the postulated coevolution of the three genes, where occurrence of Vγ9Vδ2 TCRs coincides with a functional BTN3 molecule.

T-cell receptor excision circles in primary immunodeficiencies and other T-cell immune disorders.

PURPOSE OF REVIEW: The thymus gland provides an environment for the production of rearranged diversified populations of T-cell receptors (TCRs) expressed on peripheral T cells. These receptors respond to nonself-antigens and are tolerant to self-antigens. During TCR rearrangement processes, unused excised DNA fragments create byproducts termed TCR excision circles (TRECs). Although these byproducts have no function, their detection in the peripheral blood stream is a clear indication that a rearrangement process has occurred. Their enumeration in the latest circle created during TCR delta deletion and the final TCR alpha rearrangement can determine thymus output. RECENT FINDINGS: Since the first description of its monitoring changes in the frequency of recent thymic emigrants with age and in human immunodeficiency virus (HIV)1 infection, TREC assessments have been used in many clinical settings in which T-cell immunity is involved, including diagnosing, understanding and monitoring T-cell immunodeficiencies, HIV infection, aging, autoimmune diseases and immune reconstitution after bone marrow transplantation. SUMMARY: Confounding factors, such as cell division, cell death, longevity of the naïve T cells, and intracellular degradation, are known to affect TREC levels, yet measurement of TREC content is still considered the most reliable tool for tracking recent thymic emigrants. Its recent implantation for neonatal screening to diagnose severe combined immunodeficiency by using dry blood spots from Guthrie papers makes TREC the most accurate noninvasive tool to detect T-cell immune disorders. Together with determination of the TCR repertoire, TREC contents can give a clear insight into peripheral T-cell homeostasis.

Reversible contraction by looping of the Tcra and Tcrb loci in rearranging thymocytes.

Reversible contraction of immunoglobulin loci juxtaposes the variable (V) genes next to the (diversity)-joining-constant ((D)JC) gene domain, thus facilitating V-(D)J recombination. Here we show that the T cell receptor beta (Tcrb) and T cell receptor alphadelta (Tcra-Tcrd) loci also underwent long-range interactions by looping in double-negative and double-positive thymocytes, respectively. Contraction of the Tcrb and Tcra loci occurred in rearranging thymocytes and was reversed at the next developmental stage. Decontraction of the Tcrb locus probably prevented further V(beta)-DJ(beta) rearrangements in double-positive thymocytes by separating the V(beta) genes from the DJC(beta) domain. In most double-negative cells, one Tcrb allele was recruited to pericentromeric heterochromatin. Such allelic positioning may facilitate asynchronous V(beta)-DJ(beta) recombination. Hence, pericentromeric recruitment and locus 'decontraction' seem to contribute to the initiation and maintenance of allelic exclusion at the Tcrb locus.

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.

Regulation of TCR delta and alpha repertoires by local and long-distance control of variable gene segment chromatin structure.

Murine Tcrd and Tcra gene segments reside in a single genetic locus and undergo recombination in CD4- CD8- (double negative [DN]) and CD4+ CD8+ (double positive [DP]) thymocytes, respectively. TcraTcrd locus variable gene segments are subject to complex regulation. Only a small subset of approximately 100 variable gene segments contributes substantially to the adult TCRdelta repertoire. Moreover, although most contribute to the TCRalpha repertoire, variable gene segments that are Jalpha proximal are preferentially used during primary Tcra recombination. We investigate the role of local chromatin accessibility in determining the developmental pattern of TcraTcrd locus variable gene segment recombination. We find variable gene segments to be heterogeneous with respect to acetylation of histones H3 and H4. Those that dominate the adult TCRdelta repertoire are hyperacetylated in DN thymocytes, independent of their position in the locus. Moreover, proximal variable gene segments show dramatic increases in histone acetylation and germline transcription in DP thymocytes, a result of super long-distance regulation by the Tcra enhancer. Our results imply that differences in chromatin accessibility contribute to biases in TcraTcrd locus variable gene segment recombination in DN and DP thymocytes and extend the distance over which the Tcra enhancer can regulate chromatin structure to a remarkable 525 kb.

Clonal T-cell receptor gamma and delta gene rearrangements in T-cell acute lymphoblastic leukemia at diagnosis: predictor of prognosis and response to chemotherapy.

Risk-based treatment assignment requires the availability of prognostic factors that reliably predict clinical outcome. Junctional regions of T-cell receptor (TCR) genes provide the best tool to study clonality, lineage association and minimal residual disease (MRD) in T-ALL. In this study, we have analyzed the suitability of clonal TCR gamma and delta junctional gene rearrangement status of T-ALL patients at diagnosis as a prognostic marker for T-ALL. We studied peripheral blood samples of 50 newly diagnosed patients with T-ALL in India for incidence of clonal TCR gamma and delta junctional region gene rearrangements by PCR-coupled heteroduplex analysis. Of these, 17 T-ALL patients uniformly treated on MCP 841 protocol were followed for more than 40 months (range: 41.26-55.82 months; mean: 49.26) and their clonal TCRgammadelta genotype was correlated with clinical outcome with respect to duration of complete remission, disease-free survival (DFS) and event-free survival. We also compared the clinical and biological features of TCRgammadelta + T-ALL and TCRalphabeta + T-ALL for their relative order of significance. Thirty per cent (15 of 50) of Indian T-ALL patients exhibited clonal rearrangements of both TCR gamma and delta genes. A significant proportion of these patients (73.3%, 11 of 15 P < 0.005) showed predominant usage of VgammaI-Jgamma1.3/2.3 with Vdelta1-Jdelta1 genes. A statistically significant association of L2 and L1 FAB blast morphology with TCRgammadelta + T-ALL and TCRalphabeta + T-ALL, respectively was observed (P = 0.001 by Fisher's Exact Test). The survival rate in DFS group was higher for TCRgammadelta + T-ALL compared to TCRalphabeta + T-ALL (P = 0.1378 by Log rank test). Thus we have identified clonal TCR gamma and delta junctional gene rearrangement status of T-ALL patients at diagnosis as a prognostic marker and predictor of response to chemotherapy. In future, this may help in designing tailored and risk-adjusted (less aggressive and less toxic) therapies for subset of T-ALL patients.

Distinct requirements for IL-7 in development of TCR gamma delta cells during fetal and adult life.

TCRgammadelta-transgenic IL-7(-/-) mice were generated to determine whether T cells containing productively rearranged TCRgammadelta genes have additional requirements for IL-7 within the thymus or peripheral lymphoid tissues. Differences in developmental requirements for IL-7 by TCRgammadelta cells were noted and were linked to derivation from fetal- vs adult-type precursors in the thymus. Although TCRgammadelta cells are absent from IL-7(-/-) mice, TCRgammadelta cells were restored to the thymus and periphery by expression of TCRgammadelta transgenes. Endogenous TCRgamma chains were expressed by IL-7(+/-) but not IL-7(-/-) TCRgammadelta-transgenic mice, providing direct support for findings that IL-7 is necessary for rearrangement and expression of TCRgamma genes. The number of TCRgammadelta thymocytes was 10-fold reduced in TCRgammadelta-transgenic IL-7(-/-) embryos; however, adult TCRgammadelta-transgenic IL-7(-/-) or IL-7(+/-) mice had similar numbers of fetal thymus-derived TCRgammadelta cells in their skin. Thus, fetal TCRgammadelta cells required IL-7 for TCR rearrangement, but not for proliferation or survival in the periphery. In contrast, the numbers of TCRgammadelta cells in other tissues of TCRgammadelta-transgenic IL-7(-/-) mice were not completely restored. Moreover, coincident with the transition from the first to second wave of T cell precursors maturing in neonatal thymus, thymus cellularity of TCRgammadelta-transgenic IL-7(-/-) mice dropped significantly. These data indicated that in addition to TCRVgamma gene rearrangement, TCRgammadelta cells differentiating from late fetal liver or adult bone marrow precursors have additional requirements for IL-7. BrdU incorporation studies indicated that although IL-7 was not required for TCRgammadelta cell proliferation, it was required to prolong the life span of mature TCRgammadelta cells.

Homeostatic regulation of intestinal villous epithelia by B lymphocytes.

The epithelial cell of the small intestine is one of the most rapidly regenerating cells in the body. However, the cellular mechanism and biological significance underlying this rapid regeneration remain elusive. In this study we examined the intestinal epithelia of mutant mice that lack B and/or T cells and those of normal littermates. The absence of B cells in Ig mu-chain mutant mice or B and T cells in recombination-activating gene (RAG)-2(-/-) as well as SCID mutant mice was associated with a marked acceleration of epithelial cell turnover and an up-regulation of the expression of MHC class II molecules. No such effects were observed in T cell-deficient TCR-delta and -beta double-mutant mice. As far as the goblet cells of villous epithelium are concerned, absolute numbers of them remained the same among these mutant mice that have no B and/or T cells. Alymphoplasia (aly/aly) mutant mice that lacked Peyer's patches and Ig-producing cells in the lamina propria, but harbored a large number of intestinal mucosal T cells, also displayed a significant acceleration of epithelial cell turnover and, to some extent, up-regulated expression of MHC class II molecules. Notably, the accelerated epithelial cell turnover was not observed and returned to normalcy in the Ig mu-chain mutant mice that had been given antibiotic-containing water. These findings indicate that B cells down-regulate the generation and differentiation of intestinal epithelial cells in the normal wild-type condition and suggest that enteric microorganisms are implicated in the accelerated generation of epithelial cells in mice that have no B cells.

Multiple accumulation of Vdelta2+ gammadelta T-cell clonotypes in intestinal mucosa from patients with Crohn's disease.

The gammadeltaT cells have been known to play an important role in the regulation of the mucosal immune system, but the relationship between these cells and the pathogenesis of Crohn's disease (CD) has remained obscure. We now demonstrate the T-cell receptor (TCR) Vdelta2 gene transcripts characterize antigenic immune response in the intestinal mucosa from patients with CD. TCR Vdelta2 gene transcripts of six patients with CD and six controls were subcloned and 20 randomly selected clones from each sample were subjected to nucleotide sequencing. Sequence analysis demonstrated that the different clones in the intestinal mucosa of CD were significantly increased (11.833 +/- 0.946) compared to controls (7.167 +/- 1.470) (P = 0.0374). The presence of intraindividual dominance and oligoclonality of TCR Vdelta2 gene transcripts in normal controls appears reflect positive selection and expansion of specific gammadelta T cells in normal controls. By contrast TCR Vdelta2 gene transcripts in the intestinal mucosa of CD can express different clonotypes. We conclude that accumulation of multiple Vdelta2+ gammadelta T-cell clonotypes are involved in the pathogenesis of CD.

Both gamma delta T cells and NK cells inhibit the engraftment of xenogeneic rat bone marrow cells and the induction of xenograft tolerance in mice.

In murine allogeneic bone marrow transplantation recipients, treatment of the hosts with a nonmyeloablative regimen, including depleting anti-CD4 and anti-CD8 mAbs, allows establishment of long-term mixed chimerism and donor-specific tolerance. However, in the xenogeneic rat-to-mouse combination, additional anti-Thy1.2 and anti-NK1.1 mAbs are required. We have now attempted to identify the xenoresistant mouse cell populations that are targeted by anti-NK1.1 and anti-Thy1.2 mAbs. C57BL/6 (B6) wild-type, B6 TCRbeta(-/-), and B6 TCRdelta(-/-) mice received anti-CD4 and anti-CD8 mAbs, followed by 3 Gy of whole body irradiation, 7 Gy of thymic irradiation, and transplantation of T cell-depleted rat bone marrow cells. Anti-NK1.1 and anti-Thy1.2 mAbs were additionally administered to some groups. Increased rat chimerism was observed in TCRdelta(-/-) mice treated with anti-CD4, anti-CD8, and anti-NK1.1 mAbs compared with similarly treated TCRbeta(-/-) mice. In TCRbeta(-/-) mice, but not in TCR delta(-/-) mice, donor chimerism was increased by treatment with anti-Thy1.2 mAb, indicating that CD4(-)CD8(-)TCRgammadelta(+)Thy1. 2(+)NK1.1(-) cells (gammadelta T cells) are involved in the rejection of rat marrow. In addition, chimerism was enhanced in both TCRbeta(-/-) and TCRdelta(-/-) mice treated with anti-CD4, anti-CD8, and anti-Thy1.2 mAbs by the addition of anti-NK1.1 mAb to the conditioning regimen. Donor-specific skin graft prolongation was enhanced by anti-Thy1.2 and anti-NK1.1 mAbs in TCRdelta(-/-) mice. Therefore, in addition to CD4 and CD8 T cells, gammadelta T cells and NK cells play a role in resisting engraftment of rat marrow and the induction of xenograft tolerance in mice.

A developmental switch from TCR delta enhancer to TCR alpha enhancer function during thymocyte maturation.

V(D)J recombination and transcription within the TCR alpha/delta locus are regulated by three characterized cis-acting elements: the TCR delta enhancer (Edelta), TCR alpha enhancer (Ealpha), and T early alpha (TEA) promoter. Analysis of enhancer and promoter occupancy and function in developing thymocytes in vivo indicates Edelta and Ealpha to be developmental-stage-specific enhancers, with Edelta "on" and Ealpha "off" in double-negative III thymocytes and Edelta "off" and Ealpha "on" in double-positive thymocytes. Edelta downregulation reflects a loss of occupancy. Surprisingly, Ealpha and TEA are extensively occupied even prior to activation. TCR delta downregulation in double-positive thymocytes depends on two events, Edelta inactivation and removal of TCR delta from the influence of Ealpha by chromosomal excision.