A chimeric TCR-beta chain confers increased susceptibility to EAE

Autoreactive myelin-specific CD4(+) T cells play an important role in CNS demyelination observed in MS and EAE. Consequently, it is important to understand the mechanisms of T cell receptor signalling leading to the activation of autoreactive T cells. We have previously generated a chimeric T cell receptor beta-chain (betaIII) displaying increased antigen sensitivity by exchanging most of the transmembrane and the intracellular domain of the TCR-beta chain with the corresponding TCR-gamma sequence. To investigate the effect of this "super-signalling" TCR in an autoimmune setting, we generated MOG(35-55) specific TCR transgenic mice expressing either the wild-type or the chimeric betaIII TCR-beta chain. We found that na?ve transgenic T cells expressing the chimeric betaIII chain proliferated more extensively than wild-type cells in response to MOG(35-55)in vitro. Likewise, betaIII T cells skewed into a TH1 phenotype maintained the proliferative advantage over wild-type TH1 T cells at low antigen concentration. However, when skewed into a TH2 phenotype, there was no difference in proliferation between wild-type and betaIII T cells. Blocking of Fas-mediated cell death evenly affected wild-type and betaIII TH1 T cells and resulted in increased proliferation of both subsets, suggesting that betaIII T cells did not show defective Fas-FasL signalling. Finally, we found that betaIII TCR transgenic mice are more susceptible to EAE than wild-type TCR transgenic mice. We conclude that the change in the transmembrane domain of the TCR-beta chain affects TH1 T cells and the susceptibility to EAE, but does not affect TH2 cells. Investigating the molecular interaction within the TCR complex will help us to identify signalling pathways that can be manipulated to stop the progression of MS.     

The connecting peptide domain of pT alpha dictates weak association of the pre-T cell receptor with the TCR zeta subunit.

Signals delivered through the pre-TCR, a heterodimer of pT alpha and TCR beta chains, are crucial for the maturation and proliferation of immature alphabeta lineage thymocytes from the CD4- CD8- to the CD4+ CD8+ stage. To gain insight into the structural and functional properties of the pre-TCR, chimeric TCR alpha chains were generated by replacing domains of the alpha chain cytoplasmic, transmembrane and constant regions with homologous domains from the pT alpha chain. All chimeric TCR could be expressed stably at the cell surface and induce Ca2+ mobilization as well as phosphorylation of several protein substrates on tyrosine residues. However, chimeras wherein the connecting peptide of TCR alpha chain was substituted by the one from pT alpha, were weakly associated with the TCR zeta chain, showing that functional but not physical interactions were preserved in such chimeras. In contrast, introduction of the connecting peptide of TCR alpha in the pT alpha chain was insufficient to confer stable association with the TCR zeta chain. These results demonstrate that the inability of the pre-TCR to interact strongly with TCR zeta is attributable to amino acid residues present throughout the region comprised between the intrachain Cys and the transmembrane domain. It remains to be determined whether the weak physical interaction between the pre-TCR alphand the zeta2 homodimer prevents the activation of specific TCR zeta-dependent signaling pathways, and thus confers unique signaling properties upon the pre-TCR. In addition, this structural difference between the pT alpha/beta and alphabeta TCR might constitute a means to regulate the expression of these receptors at the surface of thymocytes, at different stages of their maturation.     

The polymerase chain reaction in the demonstration of monoclonality in T cell lymphomas.

AIMS--To evaluate polymerase chain reaction (PCR) amplification of T cell receptor (TCR) beta and gamma chain genes as a means of demonstrating monoclonality in T cell lymphomas using histological samples; to compare the performance of PCR with Southern blot analysis. METHODS--TCR-beta, TCR-gamma and immunoglobulin heavy chain (IGH) genes were analysed using PCR in 55 cases of T cell lymphoma (28 frozen tissue and 27 paraffin wax embedded samples), diagnosed using morphological and immunohistochemical criteria. The 28 frozen samples were subjected to Southern blot analysis using TCR-beta, TCR-gamma and IGH gene probes. Twenty five B cell lymphomas and 21 non-neoplastic lymphoid tissue samples were used as controls. RESULTS--Using TCR-beta PCR, monoclonality was detected in 24 (44%) of 55 T cell lymphomas compared with 43 (78%) of 55 using TCR-gamma PCR and in 82% with both techniques. Five (9%) of 55 T cell lymphomas were IGH PCR positive. None of the non-neoplastic lymphoid control samples were PCR positive. All B cell lymphomas showed a polyclonal pattern with TCR-beta PCR while a single B cell lymphoma was positive using TCR-gamma primers. With TCR-beta PCR, a monoclonal result was seen in 12 (43%) of 28 frozen samples of T cell lymphoma, compared with 23 (82%) of 28 using Southern blot analysis. With TCR-gamma PCR, 19 (68%) of 28 frozen tissue samples were positive, compared with 26 (93%) of 28 using Southern blot analysis. A single case showed IGH rearrangement by Southern blot analysis. CONCLUSION--TCR-gamma PCR should be the method of choice for analysis of clonality in paraffin wax embedded sections of lymphoproliferative lesions, as TCR-beta PCR has a high false negative rate. Southern blot analysis remains the most successful technique when sufficient fresh tissue samples and resources are available.     

Chimeric immune receptors (CIRs) specific to JC virus for immunotherapy in progressive multifocal leukoencephalopathy (PML)

Progressive multifocal leukoencephalopathy (PML) is a deadly brain disease caused by the polyomavirus JC (JCV). The aim of this study is to develop 'designer T cells' armed with anti-JCV TCR-based chimeric immune receptors (CIRs) by gene modification for PML immunotherapy. Two T cell lines specific to two dominant CTL epitopes derived from JCV VP1 protein (termed p36 and p100) from an HLA-A0201+ PML survivor were generated for TCR cloning. Two distinct dominant TCR alpha chains (Valpha6 and Valpha12) and a unique TCR beta chain (Vbeta5.1) were cloned from the p36-specific cell line, while only one alpha (Valpha8.6) and one beta (Vbeta2) chains were dominant in the p100-specific line. Retroviral constructs encoding CIRs were created with the extracellular domains of TCR alpha and beta chains fused to the transmembrane and cytoplasmic portions of CD3zeta (ValphaCalphaCD3zeta or VbetaCbetaCD3zeta). Cellular expression and screening for binding specific peptide-HLA-A0201 tetramer confirmed the reactivity of the p100 TCRalphabeta and of one of the two pairs of p36 TCRalphabeta (Valpha12 and Vbeta5.1). Functional tests confirmed CIR-expressing T cells secreted cytokines and expressed potent cytotoxicity on contact with A0201+ B-lymphoblastoid line loaded with peptides and/or with HLA-A0201+ cells expressing native JCV VP1 protein. In conclusion, anti-JCV designer T cells were generated.     

Transmembrane polar residues of TCR beta chain are required for signal transduction

Mutagenic analyses have identified structural motifs important for TCR- mediated signaling in the antigen-binding chains, CD3 and zeta subunits of the TCR complex. In this study, we altered selected residues in the transmembrane and extracellular constant regions of the TCR beta chain and expressed the mutants in a T hybridoma line bearing endogenous receptor. We measured cytokine production and apoptosis in response to antigen or antibody. We found that mutation of one or both of the transmembrane tyrosine residues in the TCR beta chain caused a marked reduction in responsiveness. Mutation of the transmembrane serine to alanine also reduced responses, although less markedly. Immunoprecipitation analyses showed that the TCR beta mutations did not alter association with zeta. These experiments identify a signaling role for the transmembrane domain of the TCR beta chain.      

Chimeric beta2 microglobulin/CD3zeta polypeptides expressed in T cells convert MHC class I peptide ligands into T cell activation receptors: a potential tool for specific targeting of pathogenic CD8(+) T cells

CD8(+) T cells are key mediators of transplant rejection and graft-versus-host disease and contribute to the pathogenesis of autoimmune diseases. We tested whether TCR ligands can be converted into T cell activation receptors, redirecting genetically modified T cells at pathogenic CD8(+) T cells. For this purpose we exploited the ability of the non-polymorphic beta(2) microglobulin light chain to pair with all MHC class I heavy chains. In this report we describe the design and expression in a T cell hybridoma of two modalities of beta(2) microglobulin polypeptides, fused with the transmembrane and intracellular portion of CD3zeta chain. In the absence of a particular antigenic peptide, the chimeric product associates with different endogenous MHC class I heavy chains and triggers T cell activation upon heavy chain cross-linking. When an antigenic peptide is covalently attached to the N-terminus of the chimeric polypeptide, transfectants express high level of surface peptide-class I complexes and respond to antibodies and target T cells in a peptide-specific manner. Our results provide the basis for a universal genetic approach aimed at antigen-specific immunotargeting of pathogenic CD8(+) T cells.     

Crippling of CD3-zeta ITAMs does not impair T cell receptor signaling

We evaluated the importance of CD3-zeta ITAMs in T cell responses by breeding the P14 transgenic TCR into mice in which CD3-zeta chains lacking all or part of their ITAMs were genetically substituted for wild-type CD3-zeta chains. In contrast to the H-Y TCR, the P14 TCR permitted the development of peripheral CD8+ T cells harboring signaling-defective CD3-zeta subunits. The absence of functional CD3-zeta ITAMs did not reduce the spectrum of activation events and effector functions that constitute the normal attributes of mature CD8+ T cells. The only detectable differences were quantitative and noted only when T cells were challenged with suboptimal peptide concentrations. Therefore, the ITAMs present in the CD3-gammadeltaepsilon module are sufficient for qualitatively normal TCR signaling and those present in CD3-zeta have no exclusive role during T cell activation.     

Hypothesis: TCR signal transduction--A novel tri-modular signaling system

Antigenic peptides initiate an immune response in T cells via the T cell receptor (TCR). The TCR itself is widely regarded as one of the most complex receptors in nature, as it is comprised of at least six different subunits, the antigen recognizing TCRalpha and beta chains, and the signal transmitting CD3deltavarepsilon, gammaepsilon, and zeta2 dimers. In order for a signal to be transmitted from the TCR to the cytoplasm, the CD3 chains must "sense" that an antigenic peptide has been presented to the TCRalpha and beta subunits. After accomplishing this, there are a total of 10 different immunoreceptor tyrosine activation motifs (ITAMs) present within the CD3 chains which effectively activate the T cell and hence the immune response. The importance of each CD3 chain and subsequently each ITAM has been the focus of intense research. However, the precise role(s) played by each CD3 chain has remained elusive. Using the immunomodulatory peptide termed core peptide (CP), which is proposed to inhibit TCR activation by disrupting TCR-CD3 interactions, a tri-modular signaling system for T cell activation is proposed. By contrast to the existing two distinct signaling model (zeta2, CD3epsilongamma/epsilondelta), in this model each of the three dimers, CD3gammaepsilon, deltaepsilon, and zeta2, are proposed to act as three separate and distinct signaling modules, performing both specific and redundant functions.     

Reactivity of mouse T-cell hybridomas expressing human Vbeta gene segments with staphylococcal and streptococcal superantigens.

A panel of 15 mouse T-cell hybridomas, each expressing a different human Vbeta gene segment (hVbeta) in an otherwise mouse T-cell receptor (i.e., mouse alpha chain and CD3 complex), was constructed by transfection of hVbeta/mouse Cbeta chimeric T-cell receptor (TCR)-beta genes into a mouse T-cell hybridoma recipient lacking the endogenous TCR-beta chain. Several qualities that are conferred by the hVbeta chain of the TCR are retained in the chimeric human-mouse TCR complex: a large panel of hVbeta-specific antibodies specifically stained the hVbeta expressed by the mouse T-cell hybridomas. Moreover, hVbeta-transfected mouse cells could readily produce interleukin 2 when stimulated by superantigens presented by antigen-presenting cells. These characteristics made it possible to refine the reactivity of 17 superantigen preparations with the available transfected Vbetas. Each superantigen gave a characteristic pattern of reactivity on the transfectants. Positive reactivities with some of these transfectants, which differ only by the expressed hVbeta, demonstrate unambiguously the superantigenic character of a protein or fraction and its potential to react with the corresponding Vbetas. Therefore, these hVbeta-transfected cells constituted a valuable tool for determining "specificity fingerprints" of known or putative superantigens. First, commonly used, commercially available superantigens such as staphylococcal enterotoxin B and toxic shock syndrome toxin-1 (TSST-1) showed additional Vbeta reactivities, compared with those of their recombinant counterparts. This stresses the importance of using defined preparations of superantigens for the definition of Vbeta specificities. Second, the stimulatory pattern of a strain of Streptococcus pyogenes demonstrated that this strain, unlike others, produces a potent Vbeta 8-specific superantigen that is an yet undefined at the molecular level.     

TCR-beta chains derived from peripheral gammadelta T cells can take part in alphabeta T-cell development

Between 10 and 20% of the peripheral gammadelta T cells express cytoplasmic TCR-beta proteins, but whether such TCR-beta chains can partake in alphabeta T-cell development has never been systematically investigated. Therefore, we reconstituted the T-cell compartment of CD3epsilon-deficient mice with Pax5-TCR-beta deficient proB cells expressing, via a retroviral vector, TCR-beta chains from either peripheral gammadelta or alphabeta T cells. Recipient thymi reconstituted with proB cells containing empty vector were small (<15x10(6) cells), contained few gammadelta T but no alphabeta T cells. In contrast, thymi from mice receiving proB cells containing gammadelta or alphabeta T-cell-derived TCR-beta chains contained 80-130x10(6) cells, and showed a normal CD4, CD8 and alphabeta TCR expression pattern. However, regardless of the source of TCR-beta chain, reconstituted mice rapidly showed signs of autoimmunity dying 5-15 wk following reconstitution. Autoimmune disease induction could be prevented by co-transfer of Treg cells thereby allowing the functionality of the generated T cells to be assessed. Results obtained show that TCR-beta chains from gammadelta T cells can efficiently take part in alphabeta T-cell development. The implications of these findings for gammadelta T-cell development will be discussed.     

Structure/function analysis of the invariant subunits of the T cell antigen receptor.

The T cell antigen receptor (TCR) is a multimeric surface receptor on T cells responsible for recognizing MHC-restricted antigens and initiating the cellular immune response. The clonotypic nature of the TCR resides in the antigen binding TCR-alpha beta or TCR-gamma delta heterodimer. The CD3 complex of gamma, delta and epsilon and the zeta-family disulfide dimer comprise the invariant TCR chains. Assembly of the mature TCR complex requires specific subunit interactions, the detailed nature of which is becoming more evident. Surface targetting of the TCR appears dependent on a region of the zeta chain near its transmembrane domain. A functional role attributable to zeta residing in the cytoplasmic tail is the capacity to couple antigen stimulation to IL-2 secretion, likely through activation of a tyrosine kinase. Assignment of functional roles for the remaining CD3 chains is not as clear; the removal of the cytoplasmic tail of CD3-delta does not affect TCR-mediated IL-2 signalling. Mounting evidence indicates that the structural complexity of the TCR is further enhanced by the various zeta family disulfide dimer pairs that can associate with the other TCR chains. This subunit diversification may offer the possibility of multiple coupling pathways available to the TCR as it responds to foreign antigens in various contexts.     

Conserved transmembrane tyrosine residues of the TCR beta chain are required for TCR expression and function in primary T cells and hybridomas

The T cell receptor (TCR) beta chain transmembrane domain contains two evolutionarily conserved tyrosines (Y). In this study, the functional basis for the evolutionary conservation is addressed by mutation of the residues, expression of the mutants in hybridoma and primary T cells, and examination of TCR signaling function. We find that the phenotype of the mutants, both surface expression and ability to signal for IL-2 production, is highly variable in different mouse T hybridoma lines. Although we have not been able to determine the basis for these differences in the hybridomas, expression of the mutants in primary T cells provides a definitive assessment of mutant phenotype. We show that mutation of the N-terminal Y to either leucine (L) or alanine (A) results in low surface expression in primary T cells, while mutation of both N- and C-terminal Y to A or L abrogates surface expression. However, the more conservative mutation of both transmembrane Y to phenylalanine maintained receptor surface expression and assembly while severely disrupting signaling in primary T cells. Our data demonstrate that TCR beta chain transmembrane Y are essential for TCR signal transduction as well as complex assembly. These findings suggest that protein-protein interactions involving membrane-spanning domains are likely relevant for TCR signal transduction mechanisms.     

Self-recognition and the biased mature repertoire in TCR beta transgenic mice: the exception that supports the rule

Detailed sequence analysis of the companion alpha chains in several T-cell receptor (TCR) beta chain transgenic mice have shown that the mature alpha chain repertoires of CD4+ T cells are biased toward the parental TCR alpha chain sequences. These studies further indicate that it is self-peptide-self-MHC recognition during positive intrathymic selection that biases the structure of the mature TCR alpha chain repertoire. To further establish the causative relationship, it is important to examine whether such a bias can be abolished when positive selection is absent. The human collagen IV-I-A(s) complex-specific KB TCR cannot be positively selected on the self-peptide-self-MHC complexes present in the I-A(s) strain. Therefore, the KB TCR beta chain transgenic mice offer a unique opportunity for addressing this issue.     

Identification of αβ and γδ T Cell Receptor-Positive Cells

Two lineages of T lymphocytes bearing the CD3 antigen can be defined on the basis of the nature of the heterodimeric receptor chain (alpha beta or gamma delta T cell receptor (TCR) expressed. Precise identification of alpha beta and gamma delta TCR+ cells is essential when studying the tissue distribution and function of these different T cells. In immunofluorescence studies gamma delta TCR+ cells have been identified as CD3+WT-31- or CD3+CD4-CD8- cells. However, this may not be the optimal procedure because gamma delta TCR+ cells are weakly WT-31+, and some are CD8+. The aim of this study was to evaluate a panel of monoclonal antibodies (MoAb) directed against different chains of the TCR-T3 complex for a more precise identification of alpha beta+ and gamma delta TCR+ cells in flow cytometric studies. We found that the MoAb anti-Ti-gamma A and delta-TCS-1, recognizing the TCR-gamma and the TCR-delta chain respectively, only reacted with a subpopulation of gamma delta TCR+ cells, whereas another TCR-delta chain recognizing MoAb anti-TCR-delta 1 reacted with all gamma delta TCR+ cells. All MoAb reported to belong to the CD3 group reacted with both alpha beta TCR+ and gamma delta TCR+ cells as expected. Our results indicate that all gamma delta TCR+ cells can be identified with the MoAb anti-TCR-delta 1. Because no MoAb recognizing the TCR-alpha or TCR-beta chains at the cell surface of intact cells are yet available, we suggest that alpha beta TCR+ cells could be identified as CD3+ anti-TCR-delta 1-cells.     

Signaling function of reconstituted CD16: zeta: gamma receptor complex isoforms.

Natural killer cells express an Fc receptor for IgG (CD16) in association with disulfide-linked dimers composed of two homologous subunits: the zeta chain of the T cell antigen receptor complex and the gamma chain of the mast cell/basophil Fc receptor for IgE. The ability of zeta and gamma to transduce CD16-mediated activation signals was compared by reconstituting distinct CD16 receptor isoforms composed of various combinations of zeta- and gamma-containing dimers. Stably transformed non-hematopoietic and hematopoietic cell lines were established that expressed chimeric molecules comprising the extracellular domain of CD16 joined to the transmembrane and intracellular domains of zeta or gamma. Reconstituted CD16 receptor complexes triggered Ca2+ influx, tyrosine phosphorylation, and IL-2 production in stable transformants of the Jurkat T cell line. However, cross-linking of the CD16/gamma chimera induced a specific pattern of tyrosine phosphorylation and was more efficient at signal transduction than a CD16, zeta-zeta complex, suggesting that zeta and gamma cytoplasmic domains may be coupled to distinct tyrosine kinase pathways that differentially regulate CD16-mediated activation signals. By contrast, both CD16/zeta and CD16/gamma chimeric molecules were not functional in stable transformants of the fibroblast Chinese Hamster Ovary cell line, indicating a requirement for downstream signaling components present in hematopoietic cells. Finally, the zeta transmembrane domain appears to preferentially associate with CD16 rather than the CD3:TCR complex, suggesting that a hierarchy of molecular interactions governs NK and T cell differentiation.     

Structural mutations in the constant region of the T-cell antigen receptor (TCR)beta chain and their effect on TCR alpha and beta chain interaction.

The region responsible for T-cell receptor (TCR)alpha and beta chain assembly has previously been shown to reside in their extracellular domains. In an attempt to delineate further the structural requirements for TCR alpha and beta chain assembly, chimeric TCR beta chains with increasing length of constant (C) region and mutant TCR beta chains with C-domain point mutations were constructed. Their ability to assemble with wild-type TCR alpha chain was evaluated in non-T (COS cells) or T cells. The results reveal that the C beta domain is the binding region to TCR alpha chain, whereas the intact variable (V), diversity (D) and joining (J) regions with a short C-domain of beta chain are not sufficient for the TCR alpha and beta chain assembly. The unique interchain disulphide bond between TCR alpha and beta chains is not required for the TCR alpha beta heterodimer formation.     

Specific elimination of IgE production using T cell lines expressing chimeric T cell receptor genes

B cells that are destined to secrete IgE express a membrane-bound form of IgE (mIgE) on their cell surface. Thus, elimination of such mIgE-positive cells should result in the suppression of IgE production, thereby alleviating the symptoms of IgE-mediated allergy. In this study, we examined, in a model system, whether IgE-specific effector T cells can be used specifically to eradicate IgE-producing B cells. To this end, we endowed T cells with anti-IgE specificity using chimeric T cell receptors (cTCR) containing the variable region domain (Fv) of the 84.1c non-anaphylactic anti-mouse IgE monoclonal antibody (mAb). Two configurations of chimeric receptor were used: in the first, we combined the heavy and light variable region chains of 84.1c with the constant (C) regions of the TCR α and β chains. The second construct consisted of a chimeric single-chain receptor (scFvR), composed of a single-chain Fv region of the 84.1c antibody and the Cβ domain of the TCR. Following transfection of the cTCR or the scFvR genes into the murine MD.45 cytotoxic T cell hybridoma or the Jurkat human T cell line, functional expression of IgE-specific chimeric receptors was detected on the cell surface. The transfected cells secreted interleukin-2 upon stimulation with immobilized IgE or fixed IgE-producing hybridoma cells. Moreover, cytotoxic T cell hybridomas expressing the chimeric receptor genes specifically eliminated IgE-secreting B cells in vitro, resulting in isotype-specific suppression of IgE production.     

Development of T cells expressing an altered TCR complex

Normal mouse T cells may express alternative TCR complexes containing the FcepsilonR gamma chain (FcRgamma) rather than the zeta homodimer that is present in conventional TCR complexes. While these T cells could play critical roles in regulating immunity, the role of alternative TCR complexes and their requirement for signaling molecules in T cell development remains unknown. We show thatexpression of an FcRgamma transgene in zeta chain-deficient mice (FcRgammaTG, zetaKO mice) reduced the percentage and number of CD4(+) T cells present in these animals, when compared to C57BL/6 mice. Further studies of FcRgammaTG, zetaKO mice expressing the DO11.10 TCR (DOTCR) transgene showed that, when compared to mice expressing conventional TCR complexes, the development of CD4(+), DOTCR(+) thymocytes was altered in mice of different MHC backgrounds and required the presence of zeta-associated protein (ZAP)-70 and lck kinases. The CD4(+), DOTCR(+) T cells bearing alternative TCR complexes have impaired Ca(2+) flux and proliferative response to stimulation. Altogether, these results suggest that the altered development of CD4(+) T cells is not due to qualitative differences in TCR-mediated signals, but more consistent with the hypothesis that it is due to reduced signaling strength mediated through the FcRgamma chain containing only one immunoreceptor tyrosine-based activation motif.     

Redirecting T lymphocyte specificity using T cell receptor genes

Redirecting T cells by transferring T cell receptor (TCR) genes from tumor-associated antigen (TAA)-reactive T cell clones into human peripheral blood lymphocytes (PBL) has therapeutic potential for the treatment of diseases, including cancer. T cell specificity can be altered using retroviruses encoding TCRalpha and TCRbeta chain genes, or chimeric immunoglobulin (cIg) genes containing signaling domains of CD3 zeta or Fc epsilon RI-gamma. This review evaluates recent studies using TCRs and cIgs to redirect T cell specificity and discusses some of the technical and biological hurdles that need to be addressed before these approaches can be successfully used to treat patients.