The expansion and selection of T cell receptor αβ intestinal intraepithelial T cell clones

In conventional mice, the T cell receptor (TCR)αβ⁺ CD8αα⁺ and CD8αβ⁺ subsets of the intestinal intraepithelial lymphocytes (IEL) constitute two subpopulations. Each comprise a few hundred clones expressing apparently random receptor repertoires which are different in individual genetically identical mice (Regnault, A., Cumano, A., Vassalli, P., Guy-Grand, D. and Kourilsky, P., J. Exp. Med. 1994. 180: 1345). We analyzed the repertoire diversity of sorted CD8αα and CD8αβ⁺ IEL populations from the small intestine of individual germ-free mice that contain ten times less TCRαβ⁺ T cells than conventional mice. The TCRβ repertoire of the CD8αα and the CD8αβ IEL populations of germ-free adult mice shows the same degree of oligoclonality as that of conventional mice. These results show that the intestinal microflora is not responsible for the repertoire oligoclonality of TCRαβ⁺ IEL. The presence of the microflora leads to an expansion of clones which arise independently of bacteria. To evaluate the degree of expansion of IEL clones in conventional mice, we went on to measure their clone sizes in vivo by quantitative PCR in the total and in adjacent sections of the small intestine of adult animals. We found that both the CD8αα and the CD8αβ TCRαβ IEL clones have a heterogeneous size pattern, with clones containing from 3 × 10³ cells up to 1.2 × 10⁶ cells, the clones being qualitatively and quantitatively different in individual mice. Cells from a given IEL clone are not evenly distributed throughout the length of the small intestine. The observation that the TCRαβ IEL populations comprise a few hundred clones of very heterogeneous size and distribution suggests that they arise from a limited number of precursors, which may be slowly but continuously renewed, and undergo extensive clonal expansion in the epithelium.     

αβ Lineage-committed thymocytes can be rescued by the γδ T cell receptor (TCR) in the absence of TCR β chain

Commitment of the αβ and γδ T cell lineages within the thymus has been studied in T cell receptor (TCR)-transgenic and TCR mutant murine strains. TCRγδ-transgenic or TCRβ knockout mice, both of which are unable to generate TCRαβ-positive T cells, develop phenotypically αβ-like thymocytes in significant proportions. We provide evidence that in the absence of functional TCRβ protein, the γδTCR can promote the development of αβ-like thymocytes, which, however, do not expand significantly and do not mature into γδ T cells. These results show that commitment to the αβ lineage can be determined independently of the isotype of the TCR, and suggest that αβ versus γδ T cell lineage commitment is principally regulated by mechanisms distinct from TCR-mediated selection. To accommodate our data and those reported previously on the effect of TCRγ and δ gene rearrangements on αβ T cell development, we propose a model in which lineage commitment occurs independently of TCR gene rearrangement.     

A transgenic T cell receptor restores thymocyte differentiation in interleukin-7 receptor α chain-deficient mice

Interleukin-7 (IL-7) receptor α chain-deficient (IL-7Rα^-/-) mice have severely depleted lymphocyte populations and thymocyte development is arrested at the double-negative (DN) stage. We show that thymocyte development in these mice can be reconstituted by the introduction of a transgenic T cell receptor (TCR), implying that one function of the IL-7Rα chain is to initiate TCR gene rearrangement. Expression of the recombinase-activating genes RAG1 and RAG2 was greatly reduced in the IL-7Rα^-/- thymuses, and in DN thymocytes from the TCR transgenic IL-7Rα^-/- mice, but was restored in double-positive thymocytes from the TCR transgenic IL-7Rα^-/- mice. These data suggest that the IL-7Rα chain controls RAG expression and initiation of TCRβ chain VDJ rearrangement in DN cells. In contrast, once cells have progressed beyond the DN stage of development the IL-7Rα chain becomes no longer essential for RAG expression.     

T cell receptor β chain dimers on immature thymocytes from normal mice

During T cell development the T cell receptor (TCR) β chain is expressed before the TCRα chain. Experiments in TCRβ transgenic severe combined immune deficiency (SCID) mice have shown that the TCRβ protein can be expressed on the cell surface of immature thymocytes in the absence of the TCRα chain and that the TCRβ protein controls T cell development with regard to cell number, CD4/CD8 expression and allelic exclusion of the TCRβ chain. Subsequent experiments have shown that on the surface of thymocytes fromTCRβ transgenic SCID mice the TCRβ protein can be expressed in a monomelic and dimeric form whereas only the dimeric form was found on the surface of a TCRβ-transfected, immature T cell line. The results presented here show that normal thymocytes from 16-day-old fetuses likewise express only the dimeric form and that the monomelic form on the surface of thymocytes from transgenic mice results from glycosyl phosphatidylinositol linkage. Our results show for the first time that under physiological conditions a TCRβ dimer can be expressed on the cell surface without the TCRα chain.     

A novel subset of adult γδ thymocytes that secretes a distinct pattern of cytokines and expresses a very restricted T cell receptor repertoire

We have characterized the function, phenotype, ontogenic development, and T cell receptor (TCR) repertoire of a subpopulation of γδ thymocytes, initially defined by expressing low levels of Thy-1, that represents around 5 % and 30 % of total γδ thymocytes in adult C57BL/6 and DBA/2 mice, respectively. Activation of FACS-sorted Thy-1^dull γδ thymocytes from DBA/2 mice with anti-γδ monoclonal antibodies in the presence of interleukin-2 (IL-2) results in the secretion of high levels of several cytokines, including interferon-γ (IFN-γ), IL-4, IL-10, and IL-3. In contrast, only IFN-γ was detected in parallel cultures of Thy-1^bright γδ thymocytes. Virtually all Thy-1^dull γδ thymocytes express high levels of CD44 and low levels of the heat-stable antigen and CD62 ligand, while around half of them express the NK1.1 marker. Thy-1^dull γδ thymocytes are barely detectable in newborn animals, and their representation increases considerably during the first 2 weeks of postnatal life. The majority of Thy-1^dull γδ thymocytes from DBA/2 mice express TCR encoded by the Vγ1 gene and a novel Vδ6 gene named Vδ6.4. Sequence analysis of these functionally rearranged γ and δ genes revealed highly restricted Vδ-Dδ-Jδ junctions, and somewhat more diverse Vγ-Jγ junctions. We conclude that Thy-1^dull γδ thymocytes exhibit properties that are equivalent to those of natural killer TCRαβ T cells. Both cell populations produce the same distinct pattern of cytokines upon activation, share a number of phenotypic markers originally defined for activated or memory T cells, display similar postnatal kinetics of appearance in the thymus and express a very restricted TCR repertoire.     

T cell receptor α gene rearrangement and transcription in adult thymic γδ cells

T cells belong to two separate lineages based on surface expression of αβ or γδ T cell receptors (TCR). Since during thymus development TCR β, γ, and δ genes rearrange before α genes, and γδ cells appear earlier than αβ cells, it has been assumed that αδ cells are devoid of TCR α rearrangements. We show here that this is not the case, since mature adult, but not fetal, thymic γδ cells undergo VJα rearrangements more frequently than immature αβ lineage thymic precursors. Sequence analysis shows VJα rearrangements in γδ cells to be mostly (70 %) nonproductive. Furthermore, VJα rearrangements in γδ cells are transcribed normally and, as shown by analysis of TCR β-/- mice, occur independently of productive VDJβ rearrangements. These data are interpreted in the context of a model in which precursors of αβ and γδ cells differ in their ability to express a functional pre-TCR complex.     

T cell receptor-γδ-expressing fetal mouse thymocytes are generated without T cell receptor Vβ selection

We investigated whether fetal mouse T cell receptor (TCR) γδ cells have been subjected to so-called TCRβ selection at the CD25 stage of thymus development. To this end, we carried out a comparative three-color flow microfluorimetric analysis of TCRβδ cells developing in the fetal, neonatal and adult thymus using monoclonal antibodies to CD2, CD8, CD24, CD25 and CD44. Day-15 fetal TCRγδ cells were CD2⁺, suggesting an origin at a post-CD25 stage. Molecular analysis of TCRβ rearrangements were also carried out. Thus, by semi-quantitative polymerase chain reaction (PCR) amplification of Vβ6 and Vβ8 to Jβ2 rearrangements day-15 fetal TCRγδ showed extensive TCRβ rearrangements, a finding confirmed by PCR amplification from single micromanipulated cells. Finally, sequencing analysis of 104 PCR-amplified TCR VDJβ2 fragments showed that the majority (58%) were rearranged out of frame. Taken together, these phenotypic and molecular analyses suggest that fetal TCRγδ cells have not been subject to TCRβ selection.     

Characteristics of fetal thymus-derived T cell receptor γδ intestinal intraepithelial lymphocytes

We have previously demonstrated that grafting of CBF1(H-2^b/d) fetal thymus (FTG) under the kidney capsule of congenitally athymic nude mice of BALB/c background (H-2^d) generates a substantial number of T cell receptor (TCR) γδ intestinal intraepithelial lymphocytes (IEL) that were of FTG origin (H-2^b+) (see accompanying report). Here we investigated the characteristics of these FTG-derived TCR γδ IEL and compared them to the extrathymically derived TCR γδ IEL found in nude mice. Phenotypically, FTG-derived TCR γδ IEL were similar to their extrathymically derived counterparts in that most were Thy-1 ^?, CD5^? and CD8αα (homodimer). Vγ and Vδ gene usage in thymus-derived and extrathymically derived TCR γδ IEL were found to be virtually the same. Functionally, FTG-derived TCR γδ IEL were similar to the TCR γδ IEL found in euthymic mice as both were relatively anergic to TCR cross-linking in vitro. However, FTG-derived TCR γδ IEL differed slightly from extrathymically derived TCR γδ IEL, which were completely nonresponsive to the same in vitro stimulation. Overall, these findings support the view that FTG-derived and extrathymically derived TCR γδ IEL are almost indistinguishable. Lastly, we demonstrate that despite their thymic origin, development of FTG-derived TCR γδ IEL partially takes place extrathymically; that is positive selection of FTG-derived Vδ4 IEL occurs extrathymically. In addition, we demonstrate that the CD8 molecule is not necessary for development and homing of FTG-derived TCR γδ IEL. This later finding suggests that the CD8αα molecule develops extrathymically for FTG-derived CD8αα TCR γδ IEL.     

Systematic study of human αβ T cell receptor V segments shows allelic variations resulting in a large number of distinct T cell receptor haplotypes

The variation of the αβ T cell receptor (TCR) results mainly from rearrangements of germ-line V, D and J elements combined with the processes of N- and P-region addition. In addition to this extensive diversity, diallelic polymorphism is also recognized in V regions of β loci. Four such polymorphisms have previously been defined, but the full extent of such variation has not yet been established. To investigate allelic polymorphism, we used a strategy based on V locus-specific polymerase chain reaction and single-strand conformation polymorphisms. Studying the two Vβ 2 loci and the Vα8.1 locus, we found that all exhibited a coding polymorphism. One of the Vβ 2 loci proved to be the first multiallele segment to be recognized, with three common variants. The second Vβ 2 locus, for which none of the two alleles has been identified in cDNA, appeared in fact to be a Vβ orphon, in abnormal location on the chromosome 9. A yeast artificial chromosome containing part of the TCRB locus allowed us to place the first Vβ 2 segment on the known map to define haplotypes with two other polymorphic segments: Vβ 1 and Vβ 6.7. Multiple distinct haplotypes result from combinations between these polymorphic loci, showing that Vβ regions are highly variable between individuals. Two alleles exist at the Vα8.1 segment and both are expressed. This represents the first example of a frequent coding polymorphism for TCRA gene. The distribution of allele frequencies for these segments suggest the action of balancing selection. These data add a further dimension to TCR polymorphism and suggest new candidates to explore TCR-encoded susceptibility to autoimmune diseases.     

Evidence that productive rearrangements of TCR γ genes influence the commitment of progenitor cells to differentiate into αβ or γδ T cells

Two models have been considered to account for the differentiation of γδ and αβ T cells from a common hematopoietic progenitor cell. In one model, progenitor cells commit to a lineage before T cell receptor (TCR) rearrangement occurs. In the other model, progenitor cells first undergo rearrangement of TCRγ, δ, or both genes, and cells that succeed in generating a functional receptor commit to the γδ lineage, while those that do not proceed to attempt complete β and subsequently α gene rearrangements. A prediction of the latter model is that TCRγ rearrangements present in αβ T cells will be nonproductive. We tested this hypothesis by examining Vγ2-Jγ1Cγ1 rearrangements, which are commonly found in αβ T cells. The results indicate that Vγ2-Jγ1Cγ1 rearrangements in purified αβ T cell populations are almost all nonproductive. The low frequency of productive rearrangements of Vγ2 in αβ T cells is apparently not due to a property of the rearrangement machinery, because a transgenic rearrangement substrate, in which the Vγ2 gene harbored a frame-shift mutation that prevents expression at the protein level, was often rearranged in a productive configuration in αβ T cells. The results suggest that progenitor cells which undergo productive rearrangement of their endogenous Vγ2 gene are selectively excluded from the αβ T cell lineage.     

Restricted onset of T cell receptor α gene rearrangement in fetal and neonatal thymocytes

The initial T cell receptor (TCR) α gene rearrangements were analyzed in fetal and neonatal thymocyte hybridomas by Southern blotting. Interestingly, in 30% of all thymocyte hybridomas and in all fetal day 16 thymocyte hybridomas the most proximal Jα50 (ΨJα) gene was rearranged. This rearrangement was found on one chromosome only and mostly in association with a δ rearrangement on the homologous chromosome. Jα50 was rearranged to multiple target genes based on the variable size of the restriction fragments. In addition, δ rearrangement was found with a concomitant α rearrangement in the majority of hybridomas and it was not only associated with Jα50 but with several other rearranged Jα genes as well. Our results clearly demonstrate that T cell precursors are not pre-committed to either δ or α rearrangement but a flexible progenitor responds to multiple regulatory signals during T cell differentiation and they do not support the notion that δrec-ΨJα rearrangement is required for cell commitment to TCR α gene rearrangement.     

T cells expressing the γδ T cell receptor are not required for egg granuloma formation in schistosomiasis

Immunopathology in schistosomiasis consists of a granulomatous response around parasite eggs. It has been established that granuloma formation is mediated by CD4⁺ T helper cells. However, the role of T cells bearing the γδ T cell receptor (TCR) has not been determined. In this study we utilized mutant mice that lack either αβ or γδ T cells as a result of gene targeting to investigate the relative roles of αβ and γδ T cells in the induction of immunopathology related to schistosomiasis. Mutant and control mice were infected with Schistosoma mansoni and granuloma formation as well as lymph node cell proliferative responses to egg antigens were analyzed after 8 weeks. TCR δ mutant mice (lacking γδ T cells) displayed vigorous formation of egg granulomas that were not significantly different from those observed in normal controls, both in terms of granuloma size and cellular composition. In contrast, TCR α and TCR β mutant mice (lacking αβ T cells) were unable to form granulomas. Moreover, mesenteric lymph node cells from TCR δ mutant and control mice responded strongly to egg antigens in vitro, while TCR α and β mutant mice did not. Our studies show that in schistosomiasis granuloma formation and proliferative responses to egg antigens are strictly dependent on αβ T cells. They also suggest that γδ T cells by themselves can neither mediate a granulomatous inflammation, nor significantly modify one mediated by αβ T cells.     

Peripheral blood T lymphocytes in systemic vasculitis: increased T cell receptor Vβ2 gene usage in microscopic polyarteritis

Antigen recognition by T lymphocytes is mediated by cell surface receptors. T cell specificity depends on the variable, diversity and junctional (VDJ) regions of the α and β polypeptide chains of the T cell receptor (TCR). The expression of the variable region genes of the β chain (Vβ) has been analysed to study the involvement of peripheral blood T cells in systemic vasculitis. RNA was extracted from peripheral blood lymphocytes of 12 patients with microscopic polyarteritis, 10 with Wegener's granulomatosis, six with unclassified vasculitis, and 28 healthy age- and sex-matched individuals. Complementary DNA was made from RNA and amplified by the anchored polymerase chain reaction (PCR) using redundant oligonucleotide primers for the TCR Vβ genes. To determine if the dominant usage of a Vβ gene family reflected the presence of particular T cell clones, cDNA was amplified with primers for the specific Vβ gene family. The product was screened for sequence homogeneity by single-stranded conformational polymorphism (SSCP) and cloned to sequence the adjoining TCR (Dβ)Jβ region. A significant increase in the mean percentage expression of the Vβ 2.1 gene was seen in vasculitis patients (11·4+1·0% (mean + s.e.m.)) compared with controls (6·6 + 0·6%; P < 0·003). The most marked increase was seen in microscopic polyarteritis (13·9 + 1·7%; P < 0·0001). There were also increases in the expression of Vβ3, 13 and 14 in peripheral blood of vasculitis patients compared with controls. SSCP analysis of Vβ 2.1 amplified products indicated the presence of oligoclonal bands in a smaller proportion of patients (8/27) than controls (12/28). There was no strong evidence for the conservation of the TCR Vβ 2.1 junctional region sequence data from a sample group of three patients with oligoclonal bands. Thus, a subset of patients with systemic vasculitis, particularly those with microscopic polyarteritis, have increased TCR Vβ 2.1 gene expression in their peripheral blood T cell repertoire. As superantigens binding Vβ 2.1 are postulated to activate T cells with diverse CDR3 sequences, it is proposed that a superantigen is involved in the immunopathogenesis of vasculitis.     

A TCR α chain transgene induces maturation of CD4− CD8− α β+ T cells from γ δ T cell precursors

The proportion of CD4⁻ CD8⁻ double-negative (DN) α β T cells is increased both in the thymus and in peripheral lymphoid organs of TCR α chain-transgenic mice. In this report we have characterized this T cell population to elucidate its relationship to α β and γ δ T cells. We show that the transgenic DN cells are phenotypically similar to γ δ T cells but distinct from DN NK T cells. The precursors of DN cells have neither rearranged endogenous TCRα genes nor been negatively selected by the Mls^a antigen, suggesting that they originate from a differentiation stage before the onset of TCR α chain rearrangements and CD4/CD8 gene expression. Neither in-frame VδDδJδ nor VγJγ rearrangements are over-represented in this population. However, since peripheral γ δ T cells with functional TCRβ gene rearrangements have been depleted in the transgenics, we propose that the transgenic DN population, at least partially, originates from the precursors of those cells. The present data lend support to the view that maturation signals to γ δ lineage-committed precursors can be delivered via TCR α β heterodimers.