TCRα Genes Research Articles

TCRα Genes Direct MHC Restriction in the Potent Human T Cell Response to a Class I-Bound Viral Epitope

The underlying generic properties of alphabeta TCRs that control MHC restriction remain largely unresolved. To investigate MHC restriction, we have examined the CTL response to a viral epitope that binds promiscuously to two human leukocyte Ags (HLAs) that differ by a single amino acid at position 156. Individuals expressing either HLA-B*3501 (156Leucine) or HLA-B*3508 (156Arginine) showed a potent CTL response to the 407HPVGEADYFEY417 epitope from EBV. Interestingly, the response was characterized by highly restricted TCR beta-chain usage in both HLA-B*3501+ and HLA-B*3508+ individuals; however, this conserved TRBV9+ beta-chain was associated with distinct TCR alpha-chains depending upon the HLA-B*35 allele expressed by the virus-exposed host. Functional assays confirmed that TCR alpha-chain usage determined the HLA restriction of the CTLs. Structural studies revealed significant differences in the mobility of the peptide when bound to HLA-B*3501 or HLA-B*3508. In HLA-B*3501, the bulged section of the peptide was disordered, whereas in HLA-B*3508 the bulged epitope adopted an ordered conformation. Collectively, these data demonstrate not only that mobile MHC-bound peptides can be highly immunogenic but can also stimulate an extremely biased TCR repertoire. In addition, TCR alpha-chain usage is shown to play a critical role in controlling MHC restriction between closely related allomorphs.

Development of CD4+T cells expressing a nominally MHC class I-restricted T cell receptor by two different mechanisms

Differences in T cell receptor (TCR) signaling initiated by interactions among TCRs, coreceptors, and self-peptide-MHC complexes determine the outcome of CD4 versus CD8 lineage of T cell differentiation. The H-2Ld and Kbm3 alloreactive 2C TCR is positively selected by MHC class I Kb and a yet-to-be identified nonclassical class I molecule to differentiate into CD8+ T cells. Here we describe two mechanisms by which CD4+ 2C T cells can be generated in 2C TCR-transgenic mice. In the RAG-/- background, development of CD4+ 2C T cells requires the expression of both I-Ab and the TAP genes, indicating that both MHC class I and II molecules are required for positive selection of these T cells. Notably, only some of the 2C+ RAG-/- mice (approximately 30%) develop CD4+ 2C T cells, with frequencies in individual mice varying from 0.5% to as high as approximately 50%. In the RAG+ background, where endogenous TCRalpha genes are rearranged and expressed, CD4+ 2C T cells are generated because these cells express the 2C TCR as well as additional TCRs, consisting of the 2C TCRbeta and endogenous TCRalpha chains. Similarly, T cells expressing the OT-1 TCR, which is nominally MHC class I-restricted, can also develop into CD4+ T cells through the same two mechanisms. Thus, expression of two TCRs by a single thymocyte, TCR recognition of multiple MHC molecules, and heterogeneity of TCR, coreceptors, and peptide-MHC interactions in the thymus all contribute to the outcome of CD4 versus CD8 lineage development.

Characterization and expression analysis of CD3? and CD3?/? in fugu, Takifugu rubripes

CD3 is an essential component of the CD3-TCR complex. In this report, we describe the cloning, characterization, and expression analysis of the CD3ɛ and CD3γ/δ chain genes from fugu, Takifugu rubripes. Two distinct CD3ɛ homologue cDNAs, designated as CD3ɛ-1 and CD3ɛ-2, and a CD3γ/δ homologue cDNA were isolated from the fugu thymus. The deduced amino acid sequences of these cDNAs exhibit conserved essential CD3 chain motifs and overall structures. RT-PCR analysis demonstrated that the CD3ɛ and CD3γ/δ genes were expressed in lymphoid organs (e.g. thymus, head kidney, trunk kidney and spleen), mucosal tissues (gill, skin, and intestine), and peripheral blood leucocytes (PBL). The CD3 and TCRα genes were expressed only in the surface IgM− population, which were separated from PBL using an anti-fugu IgM monoclonal antibody. In addition, in situ hybridization confirmed that CD3-expressing cells were distributed randomly in the head kidney, trunk kidney, and spleen, but in the thymus were restricted to the lymphoid outer zone and epithelioid inner zone only. Collectively, these results suggest that CD3 molecules are useful markers for the identification of T cells in teleost fish. The present study thus provides a critical step in identifying T cells in this model organism.

Analysis of recombination signal sequences in zebrafish

Recombination signal sequences (RSS) from immunoglobulin and TCRα genes of zebrafish were analyzed in comparison with RSS from human and species-specific features were revealed. In contrast to human RSS, in zebrafish RSS from both V H and TCRα genes the last nonamer position is not conserved. On the contrary, the fourth nonamer position, which is not conserved in human or mouse is conserved in zebrafish. The 12 bp spacers from human and zebrafish RSS contain 9 bp motif resembling nonamer sequence. Spacers in zebrafish 23 bp RSS from both immunoglobulins and TCRα contain 7 bp motif also resembling nonamer sequence while corresponding human sequences do not contain analogous motif. RSS are recognized by RAG1 protein, which also has specific features in teleost suggesting co-evolution of RAG1 with corresponding RSS.

Clonality Analysis as a Tool To Study the Immunologic Reconstitution with Leukemia Patients Following Allogeneic Hematopoietic Stem Cell Transplantation.

Introduction The extensive diversity of the mature T-cell receptor(TCR) is determined primarily by the complementarity-determining regions (CDR3) of the TCR. The CDR3 of both TCRα and TCRβ genes is generated by extensive rearrangement and fusion between the V,D,and J segments and by random insertion and deletion of junctional nucleotides, which yields final products that are quite heterogeneous in size. As a result of these gene rearrangements, each T cell has a unique TCR and the diversity of the T-cell repertoire at any specific time can be characterized by the examination of CDR3 within that population. Using CDR3 spectratying technique, normal individuals demonstrate a highly diverse and polyclonal The aim of our study was to evaluate to investigate restricted expansion of TCR Vβ gene repertoire and the reconstitution of T cell receptor repertoire following allogeneic hematopoietic stem cell transplantation.Methods Patients Ten patients(9 males, 1 females; median age 31 years,range18–45) with 6 chronic myeloid leukemia-chronic phase and 4 cases of acute myelogenous lenkemia(CR1) who underwent HLA-matching sibling or unrelated BMT and/or peripheral blood stem cell transplantation (PBSCT) at our department between July 1999 and May 2000 were considered evaluable restricted expansion of TCR Vβ gene repertoire, the reconstitution of T cell receptor repertoire and oligoclonal T Cell Expansion in Chronic Graft-Versus-Host Disease. RT-PCR and Genes scan analysis (CDR 3 length analysis).Results Only 2-18Vβ genes were found in samples from these ten patients within one year, and there are different distribution in different patients. TCR repertoire complexity was abnormal in all patients, parts of the genes were expansion and part of them were suppressed. Samples from 9 patients with GVHD show V β3 in 7 cases, V β 8 and V β 23 in 6 patients. The results of genescan show that the PCR production of peripheral blood samples from these patients disply oligoclonal. Only 5–22Vβ subfamily T cells were found in samples from these patients whose transplantation more than one year. TCR repertoire complexity was abnormal in all patients.Discussion Following allogeneic BMT, regeneration of T-cell populations with a diverse repertoire can occure by at least two mechanisms: One mechanism is a thymic-dependent pathway, which presumably involves both negative and positive selection and recapitulates fetal ontogeny. Alternatively, regeneration of peripheral T cells may occur through thymic-independent mechanisms. All patients had marked abnormalities in their spectratypes, only 5-22Vβ subfamily T cells were found in samples from these patients, most of it was influenced after transplant, although the number of circulating CD3+ T lymphocytes in these patients have restored at normal lever by flow cytometic analysis, but the CD4+ T cell subset returned slowly in these patients resulting in an inversion of the normal CD4/CD8 ratio for more than 1 year after tuansplantation. Therefore, the analysis of TCRVβ subfamily is a usuaful methods and techniques for monitoring immune reconstitution after transplant.

Recognition of the Peripheral Self by Naturally Arising CD25 + CD4 + T Cell Receptors

Naturally arising CD25 + CD4 + regulatory T cells (T R) play an important role in the prevention of autoimmunity. TCR specificity is thought to play a critical role in T R development and function, but the repertoire and specificity of T R TCRs remain largely unknown. We find by sequencing of TRAV14 (Vα2) TCRα chains associated with a transgenic TCRβ chain that the T R and CD25 − CD4 + TCR repertoires are similarly diverse, yet only partially overlapping. Retroviral expression of TCRα genes in TCR transgenic RAG-deficient T cells revealed that a high frequency of TCRs derived from CD25 + but not CD25 − CD4 + T cells confers the ability to rapidly expand upon transfer into a lymphopenic host. Thus, these data show that a large proportion of naturally arising T R have substantially more efficient interactions with MHC class II bound peptides from the peripheral self than CD25 − T cells.

Resistance to viral infection by intraepithelial lymphocytes in HIV-1 P18-I10-specific T-cell receptor transgenic mice

For the analysis of mucosal immunity to HIV-1, we have recently established a line of transgenic (Tg) mice expressing the TCRα and TCRβ genes of the murine CTL clone RT1 specific for P18-I10 (RGPGRAFVTI), an immunodominant gp160 envelope-derived epitope of IIIB isolate, restricted by the H-2D d MHC-I molecule. Here we examine those cells bearing specific TCR among the intraepithelial lymphocytes (IELs), with flow cytometric analysis using H-2D d/P18-I10 tetramers. We observed three distinct CD3 +, tetramer positive populations among the IELs: extra-thymic CD8αα +, αβTCR T-cells; CD8αα +, γδTCR T-cells; and thymus-derived CD8αβ +, αβTCR T-cells. Challenge of these Tg mice with P18-I10 encoded by a vaccinia virus vector, either intrarectally (i.r.) or intraperitoneally (i.p.), revealed that the intraepithelial compartment seems to be a major site for prevention of the spread of viral infection. Such immunity appears due to the thymus-derived, CD8αβ + antigen-specific CTLs together with CD8αα + γδ cells, which regulate virus spread. This model system for studying CTL based immunity at mucosal sites should prove helpful in developing rational approaches for HIV control.

Analysis of Secondary V(D)J Rearrangements in Mature, Peripheral T Cells of Ataxia-Telangiectasia Heterozygotes

Ataxia-telangiectasia (AT) is a rare recessive disease with pleiotropic involvement of the nervous and lymphoid systems. AT heterozygotes have a population frequency of about 1%, and although not manifesting any overt clinical symptoms, they have an increased mortality, mainly because of cancer and ischemic heart disease. We and others have described a mature T lymphocyte population with an altered T cell receptor surface expression (“TCR variant”) that reactivates the recombination activating genes (RAG) and is expanded in the blood of patients with AT. In view of the known role of V(D)J recombination in the onset of tumorigenic translocations, we proposed that the increased RAG activity was responsible for the predisposition of AT homozygotes to develop mature-type T leukemia/lymphoma. In the present report, we used cytofluorimetry to quantify the TCR variant population and the memory/naïve T-cell compartments in the blood of AT heterozygotes compared with AT patients and controls. We assessed the expression of different recombinase genes through RT-PCR/oligotyping and cytofluorometric analysis and searched for rearrangement intermediates by ligase-mediated PCR in T-cell lines from four heterozygous carriers. We found the TCR variant population was increased on average 2 × in AT heterozygotes (vs 10 × in homozygotes) compared with controls, and naïve CD4+ T lymphocytes were reduced on average 0.5 × (vs 0.1 × in homozygotes). We were able to demonstrate recombinase gene expression in all four heterozygous T-cell lines, and rearrangement intermediates, indicative of ongoing V(D)J recombination, in two. These rearrangements were compatible with V-gene replacement, a mechanism of receptor editing described for Ig and TCRα genes, to our knowledge not previously documented for TCRβ. In conclusion, we found that RAG reactivation and secondary V(D)J rearrangements, potential risk factors of mature-type leukemia in AT homozygotes, also take place in AT heterozygous carriers and might place this large population fraction at an increased risk of leukemia/lymphoma.

Analysis of T Cell Receptor Variable Regions and Complementarity Determining Region 3 of Infiltrating T Lymphocytes in the Liver of Patients with Chronic Type B Hepatitis

Objectives: T cell receptor (TCR) variable regions and complementarity determining regions 3 (CDR3) of infiltrating T cells in the livers of patients with chronic hepatitis B were examined in detail. Methods: TCR usage was examined by the inverse polymerase chain reaction in 2 hepatitis B e antigen-positive patients, one at an early stage and the other at an advanced stage. Results: In 61 productively rearranged clones bearing TCRα genes in the liver of the patient with early stage chronic hepatitis B, the Vα 7.2 gene segment was most frequently used (16.4%), while the other Vα gene segments were used less frequently. All but one of the clones with the Vα7.2 gene were joined with the Jα33 gene, and the CDR3 regions of these clones were the same length and similar in amino acid sequence. In contrast, in 68 productively rearranged clones from the liver of the patient with advanced stage disease, all Vα gene segments were used less frequently, and combinations of the Vα and Jα gene segments varied. Conclusions: Our data suggest a relatively restricted usage of TCR in the liver in early stage chronic hepatitis B.

Regulated T-cell development: a victim of multiple conspiracies.

Over about 400 million years there has been a conserved subdivision of T cells into those bearing a γδ T-cell receptor (TCR) and those bearing an αβ TCR. No one is sure why this is, but the two cell types have some interesting distinguishing characteristics, particularly their antigen specificities and their anatomical homing.1 In addition, the development of γδ cells seems always to precede that of αβ T cells. This is a good thing for γδ cells, because the TCRδ locus lies embedded within the TCRα locus, and when TCR Vα and Jα gene segments rearrange, they delete the TCRδ genes. Unchecked, this would substantially reduce the chances of making γδ cells.

Consequences of Intrathymic TCR Engagement by Partial Agonist on Selection Events and Peripheral T Cell Activation Program

Functions elicited from mature T cells depend on the nature of the Ag. Thus, an agonist induces a larger set of cytokine responses than a partial agonist. Additionally, Ags present in the thymus influence both the selection of TCRs generated by gene rearrangement and the potential functional program of developing thymocytes. This can be approached by analysing the development of T cells in mice expressing the same transgenic TCR (tgTCR) under different conditions of intrathymic selection. H-2Kbm8 was found to act as a partial agonist for CD8+ T cells expressing a tgTCR specific for the H-2Kb alloantigen. Intrathymic exposure to full or to partial agonist affected the development of thymocytes at different stages, consistent with the respective CD8-independent and -dependent characteristic of the tgTCR/Ag interaction. The presence of the partial agonist led to the accumulation of a major population of thymocytes (tgTCR(high) CD4- CD8(low)) originating from TCR engagement at the immature single-positive CD8(low) stage as evidenced by: 1) results from reaggregated thymic organ culture in the presence of H-2(k/bm8) thymic stromal cells; 2) the absence of CD4+ thymocytes, the development of which depends on rearrangements of endogenous TCR alpha genes; and 3) the identification of the CD8(low) thymocytes as cycling cells. Peripheral CD8(low) T cells selected in an H-2(k/bm8) thymus expressed a partial functional program in response to H-2Kb, akin to the response of CD8(high) T cells to a partial agonist. The analysis of the molecular bases for partial reactivity revealed a correlation with inefficient AP-1, but efficient NF-kappaB transactivation.

Homeostasis Limits the Development of Mature CD8+ But Not CD4+ Thymocytes

AbstractThe involvement of a variety of clonal selection processes during the development of T lymphocytes in the thymus has been well established. Less information, however, is available on how homeostatic mechanisms may regulate the generation and maturation of thymocytes. To investigate this question, mixed radiation bone marrow chimeras were established in which wild-type T cell precursors capable of full maturation were diluted with precursors deficient in maturation potential because of targeted mutations of the RAG1 or TCR-α genes. In chimeras in which the majority of thymocytes are blocked at the CD4−CD8−CD25+ stage (RAG1 deficient), and only a small proportion of T cell precursors are of wild-type origin, we observed no difference in the maturation of wild-type CD4−CD8−CD25+ cells to the CD4+CD8+ stage as compared with control chimeras. Therefore, the number of cell divisions occurring during this transition is fixed and not subject to homeostatic regulation. In contrast, in mixed chimeras in which the majority of thymocytes are blocked at the CD4+CD8+ stage (TCR-α deficient), an increased efficiency of development of wild-type mature CD8+ cells was observed. Surprisingly, the rate of generation of mature CD4+ thymocytes was not affected in these chimeras. Thus, the number of selectable CD8 lineage thymocytes apparently saturates the selection mechanism in normal mice while the development of CD4 lineage cells seems to be limited only by the expression of a suitable TCR. These data may open the way to the identification of homeostatic mechanisms regulating thymic output and CD4/CD8 lineage commitment, and the development of means to modulate it.

The αβ T Cell Receptor Can Replace the γδ Receptor in the Development of γδ Lineage Cells

In peripheral lymphoid tissues of TCR transgenic mice that express the nominal antigen (HY peptide plus H-2D b MHC) recognized by the transgenic TCR, there exist unusual CD4 −CD8 − and CD4 −CD8 low cells bearing the transgenic TCR. Here we show that, unlike TCRαβ T cells that are generated in the absence of nominal antigen, these unusual cells do not express endogenous TCRα genes, have maintained the TCRδ locus on both chromosomes, and can coexpress TCRαβ and TCRγδ chains on the cell surface. The latter is also true for CD4 −CD8 −, HSA + TCRαβ + thymocytes in male and female TCR transgenic mice. The number of TCRαβ and TCRγδ coexpressing cells is increased in pre-TCR-deficient mice. The data indicate that the TCRαβ can replace the TCRγδ in the development of γδ lineage cells and that the pre-TCR interferes with the generation of γδ-expressing cells.

T Cells with Two Tcrβ Chains and Reactivity to both MHC/Idiotypic Peptide and Superantigen

It is thought that Tcrβ genes are effectively allelically excluded, while Tcrα genes are not. We report here that endogenous Tcrβ genes are expressed on as much as 5–7% of CD4+T cells in 8-week-old Tcrαβ-transgenic mice. In this model, the transgenic Tcr recognizes residues 91–101 of a λ2315Ig light chain, presented on the I-Edclass II molecule. From such mice, a CD4+T cell clone was isolated which not only responded to λ2315, but also mobilized Ca2+and proliferated in response to Mls-1a. (Inadvertently we found that the C3H/Tif substrain, in contrast to C3H/HeJ, is Mls-1apositive.) The clone expressed the transgenic Tcr (Vα1 and Vβ8.2), and in addition an endogenous Vβ6 chain, conferring the Mls-1areactivity. On the population level, 1–2% of Tcr-transgenic lymph node cells displayed Vβ6, in addition to the transgenic β-chain. Such dual Tcrβ expressor cells could be preferentially expandedin vitroby first stimulating with DBA/2 spleen cells and then with λ2315-pulsed BALB/c antigen-presenting cells. In addition to demonstrating that allelic exclusion of Tcrβ-chain genes is substantial but not complete in this model, the data show that the double β-chain expressors can have two different specificities, and be signaled through both receptors, by physiological ligands. However, such dual-Tcr T cells appear to have reduced sensitivity to ligands, due to their decreased expression of each receptor. This holds true for both early (Ca2+mobilization) and late (proliferation) T cells activation parameters. Dual Tcr cells may have a role in the pathogenesis of autoimmune diseases: If naive T cells are first stimulated by (infectious) superantigen, they could later, as activated T cells, respond to self-peptide/MHC on costimulation-deficient cells and cause autoimmunity. As a corollary, dual Tcr Id-specific T cells could, once activated, directly regulate Id+B cells.

Rearrangement and Expression of T-Ceil Receptor Delta Genes in T-Cell Acute Lymphoblastic Leukemias

AbstractWe have analyzed T-cell receptor δ (TcR-δ) gene rearrangement and transcription in appropriately phenotyped mononuclear cells derived from 12 patients with T-cell acute lymphoblastic leukemia (T-ALL). The T-ALL cells were also analyzed for rearrangement and transcription of the T-cell receptor(TcR)-β and γ genes as well as for the presence of TcR-α gene transcripts. Four T-ALLs expressed TcR-γδ at the cell surface, while three expressed TcR-αβ. The other five T-ALLs did not express a TcR-CD3 complex on their cell membrane. The TcR-γδ+T-ALL had rearranged both TcR-δ gene alleles and contained mature 2.2 and 1.5 kb TcR-δ transcripts. In one case, immature 1.9 and 1.2 kb TcR-δ transcripts were also found. Furthermore they contained mature TcR-γ mRNA, mature or immature TcR-β mRNA, but no TcR-α mRNA. The three TcR-αβ+ T-ALLs contained mature α and β transcripts, but lacked TcR-δ transcripts as a result of deletion of both TcR-δ gene alleles. These data are in line with a mutually exclusive expression of TcR-α and δ genes, which may be important to ensure the presence of only one type of TcR per T cell. One of the five CD3− T-ALLs had germline TcR-β, γ, and δgenes. The other four CD3- T-ALLs had rearranged their TcR-β, γ, and δ genes and contained immature 1.9 and 1.2 kb TcR-δgene transcripts. Remarkably, one of these T-ALLs also contained TcR-α transcripts in addition to the immature TcR-δ transcripts, which was in line with the deletion of one TcR-δgene allele and rearrangement of the other allele. This suggests that prevention of dual receptor expression may not only be regulated by the presence of germline TcR-α genes in TcR-γδ+ cells or by deletion of both TcR-δgene alleles in TcR-αβ+ cells, but also via other regulation mechanisms. Finally, our data indicated that the combinatorial repertoire of the TcR-δ genes is limited, which has also been described for the TcR-γ genes.

The context of T cell receptor beta chain genes among wild and inbred mouse species.

The organization of the mouse T cell receptor (TCR) genes has been extensively reviewed (Hood 1985). The structure of the TCR alpha (α), beta (β) and gamma (γ) genes is analogous to the immunoglobulin (Ig) gene organization in the mouse. This homology probably reflects similar requirements for diversity in binding antigen. The major source of diversity for Ig genes comes from (1) multiple germline variable (VK, Vλ and VH) regions, diversity (DH) regions and joining (Jj(, JX and Jh) elements; (2) combinatorial joining of V, D and J; (3) somatic mutation and (4) multiple alleles present in inbred and wild mice (Tonegawa 1983). Preliminary findings among the TCRα and β chains of the mouse inbred strains indicate fewer germline variable (VTα, VTβ) genes are available (30–40 each) (Becker 1985; Barth 1985; Behlke 1985a) than VK and VH genes (100–300 each) (Valbuena 1978; Cory 1981; Brodeur and Riblet 1984). Furthermore, studies indicate that the frequency of somatic mutation in TCRα and TCRβ chain genes although present is as much as 10% or less than that of Ig genes (Augustin and Sim 1984; Ikuta 1985).