T Cell Antigen Receptor Chains Research Articles

Phosphoantigen recognition by Vγ9Vδ2 T cells.

Vγ9Vδ2 T cells comprise 1-10% of human peripheral blood T cells. As multifunctional T cells with a strong antimicrobial and antitumor potential, they are of strong interest for immunotherapeutic development. Their hallmark is the eponymous Vγ9Vδ2 T-cell antigen receptor (TCR), which mediates activation by so-called "phosphoantigens" (PAg). PAg are small pyrophosphorylated intermediates of isoprenoid synthesis of microbial or host origin, with the latter elevated in some tumors and after administration of aminobisphosphonates. This review summarizes the progress in understanding PAg-recognition, with emphasis on the interaction between butyrophilins (BTN) and PAg and insights gained by phylogenetic studies on BTNs and Vγ9Vδ2 T cells, especially the comparison of human and alpaca. It proposes a composite ligand model in which BTN3A1-A2/A3-heteromers and BTN2A1 homodimers form a Vγ9Vδ2 TCR activating complex. An initiating step is the binding of PAg to the intracellular BTN3A1-B30.2 domain and formation of a complex with the B30.2 domains of BTN2A1. On the extracellular surface this results in BTN2A1-IgV binding to Vγ9-TCR framework determinants and BTN3A-IgV to additional complementarity determining regions of both TCR chains. Unresolved questions of this model are discussed, as well as questions on the structural basis and the physiological consequences of PAg-recognition.

T-Cell Engineering for Cancer Immunotherapy

The adoptive transfer of tumor-reactive cells is a promising approach for the treatment of melanoma and some other cancers. To remedy the difficulties associated with the isolation and expansion of tumor-reactive T cells in most cancer patients, peripheral blood T cells can be retargeted to any chosen tumor antigen by the genetic transfer of an antigen-specific receptor. The transduced receptors may be human leukocyte antigen-restricted, heterodimeric T-cell antigen receptor (TCRs), or chimeric antigen receptors (CARs), which typically recognize native cell-surface antigens. Considerable progress has been made in recent years to address the challenges posed by the transfer of either receptor type. Vector and protein modifications enable the expression of TCR chains in human T cells at functional levels and with a reduced risk of mis-pairing with endogenous TCR chains. The combinatorial inclusion of activating and costimulatory domains in CARs has dramatically enhanced the signaling properties of the chimeric receptors described over a decade ago. Based on the effective T-cell transduction and expansion procedures now available to support clinical investigation, improved designer TCRs and second generation CARs targeting an array of antigens are being evaluated in a range of hematological malignancies and solid tumors.

T-cell antigen receptors in Atlantic cod ( Gadus morhua L.): structure, organisation and expression of TCR α and β genes

By using short degenerate primers complementing conserved T-cell antigen receptor (TCR) variable and constant region segments for PCR, we were able to isolate putative TCRα and β chain full length cDNAs in Atlantic cod. The Vα and Vβ domains have the canonical features of known teleost and mammalian TCR V domains, including conserved residues in the beginning of FR2 and at the end of FR3. The Jα and Jβ region possess the conserved Phe-Gly-X-Gly motif found in nearly all TCR and immunoglobulin light chain J regions. Similar to other vertebrates, the Atlantic cod Cα and Cβ sequences exhibit distinct immunoglobulin, connecting peptide, transmembrane and cytoplasmic regions. The Atlantic cod Cβ sequence lacks a cysteine in its connecting peptide region, but other motifs proposed to be important for dimerisation and cell surface expression are observed. Four different cod Cβ sequences were identified, two of which share 3′ untranslated regions different from one of the other two sequences, suggesting the existence of isotypic gene variants of Cβ. Based on Southern blot analyses, the TCRα and β gene loci appear to be arranged in translocon organisation (as opposed to multicluster) with multiple V gene segments, some (D) and J gene segments and a single or few C gene segments. Northern blot analyses show expression of the TCRα and β chains in thymus, spleen and head kidney, expression of the TCRβ chain was also detected in the ovary. Interestingly, no expression was detected in intestine even though the existence of T-cells in intestine has been proposed in other teleost species.

Modification of the T Cell Antigen Receptor (TCR) Complex by UDP-glucose:Glycoprotein Glucosyltransferase: TCR FOLDING IS FINALIZED CONVERGENT WITH FORMATION OF αβδεγε COMPLEXES

Most T lymphocytes express on their surfaces a multisubunit receptor complex, the T cell antigen receptor (TCR) containing alpha, beta, gamma, delta, epsilon, and zeta molecules, that has been widely studied as a model system for protein quality control. Although the parameters of TCR assembly are relatively well established, little information exists regarding the stage(s) of TCR oligomerization where folding of TCR proteins is completed. Here we evaluated the modification of TCR glycoproteins by the endoplasmic reticulum folding sensor enzyme UDP-glucose:glycoprotein glucosyltransferase (GT) as a unique and sensitive indicator of how TCR subunits assembled into multisubunit complexes are perceived by the endoplasmic reticulum quality control system. These results demonstrate that all TCR subunits containing N-glycans were modified by GT and that TCR proteins were differentially reglucosylated during their assembly with partner TCR chains. Importantly, these data show that GT modification of most TCR subunits persisted until assembly of CD3alpha beta chains and formation of CD3-associated, disulfide-linked alpha beta heterodimers. These studies provide a novel evaluation of the folding status of TCR glycoproteins during their assembly into multisubunit complexes and are consistent with the concept that TCR folding is finalized convergent with formation of alpha beta delta epsilon gamma epsilon complexes.

Calnexin Associates with Monomeric and Oligomeric (Disulfide-linked) CD3δ Proteins in Murine T Lymphocytes

The antigen-binding receptor expressed on most T lymphocytes consists of disulfide-linked clonotypic alphabeta heterodimers noncovalently associated with monomeric CD3gamma,delta,epsilon proteins and disulfide-linked zeta zeta homodimers, collectively referred to as the T cell antigen receptor (TCR) complex. Here, we examined and compared the disulfide linkage status of newly synthesized TCR proteins in murine CD4(+)CD8(+) thymocytes and splenic T cells. These studies demonstrate that CD3delta proteins exist as both monomeric and oligomeric (disulfide-linked) species that differentially assemble with CD3epsilon subunits in CD4(+)CD8(+) thymocytes and splenic T cells. Interestingly, unlike previous results on glucose trimming and TCR assembly of CD3delta proteins in splenic T cells (Van Leeuwen, J. E. M., and K. P. Kearse (1996) J. Biol. Chem. 271, 9660-9665), we found that glucose residues were not invariably removed from CD3delta glycoproteins prior to their assembly with CD3epsilon subunits in CD4(+)CD8(+) thymocytes. Finally, these studies show that calnexin associates with both monomeric and disulfide-linked CD3delta proteins in murine T cells. The data in the current report demonstrate that CD3delta proteins exist as both monomeric and disulfide-linked molecules in murine T cells that differentially associate with partner TCR chains in CD4(+)CD8(+) thymocytes and splenic T cells. These results are consistent with the concept that folding and assembly of CD3delta proteins is a function of their oxidation state.

Mutation in splicing consensus sequences causes lack of TCR membrane expression due to exon excision.

T-cell antigen receptor (TCR) membrane-negative T-cell mutants can be divided into two groups: 1) those which lack one of the six TCR polypeptides and 2) those which contain a mutated TCR chain. The present experiments reveal a new mechanism for the development of TCR membrane-negative T-cell variants: mutations in splicing consensus motifs causing excision or misreading of an entire exon (exon 3 of the TCRAC or TCRBC genes). C27.15 cells transcribe a TCR alpha chain consisting of TCRAVJCexon1Cexon2-encoded amino acids plus six new amino acids. The assembly defect seems to be that the truncated alpha chain does not interact with CD3 delta molecules; consequently, no TCR alphabeta/CD3 deltaepsilongammaepsilon complexes are formed. E6.E12 cells transcribe a TCR beta chain composed of TCRBVDJCexon1Cexon2-encoded amino acids plus twenty-seven new amino acids, which seem not to form a transmembrane region. The truncated beta chain does associate with CD3 gammaepsilon heterodimers, yet no TCR alphabeta/CD3 deltaepsilongammaepsilon complexes are made. This may be due either to low assembly of TCR beta/CD3 gammaepsilon trimers or to lack of access of the mutated TCR beta/CD3 gammaepsilon trimers to the TCR alpha/CD3 deltaepsilon compartment in the endoplasmic reticulum.

CD3‐induced apoptosis of CD4+CD8+ thymocytes in the absence of clonotypic T cell antigen receptor

Clonal selection of T cells mediated through the T cell antigen receptor (TCR) mostly occurs at the CD4+CD8+ double positive thymocyte stage. Immature CD4+CD8+ thymocytes expressing self-reactive TCR are induced to die upon clonotypic engagement of TCR by self antigens. CD3 engagement by antibody of the surface TCR-CD3 complex is known to induce apoptosis of CD4+CD8+ thymocytes, a process that is generally thought to represent antigen-induced negative selection in the thymus. The present study shows that the CD3-induced apoptosis of CD4+CD8+ thymocytes can occur even in TCR alpha- mutant mice which do not express the TCR alpha beta/CD3 antigen receptor. Anti-CD3 antibody induces death of CD4+CD8+ thymocytes in TCR alpha- mice either in cell cultures or upon administration in vivo. Interestingly, most surface CD3 chains expressed on CD4+CD8+ thymocytes from TCR alpha- mice are not associated with clonotypic TCR chains, including TCR beta. Thus, apoptosis of CD4+CD8+ thymocytes appear to be induced through the CD3 complex even in the absence of clonotypic antigen receptor chains. These results shed light on previously unknown functions of the clonotype-independent CD3 complex expressed on CD4+CD8+ thymocytes, and suggest its function as an apoptotic receptor inducing elimination of developing thymocytes.

Calnexin Associates Exclusively with Individual CD3δ and T Cell Antigen Receptor (TCR) α Proteins Containing Incompletely Trimmed Glycans That Are Not Assembled into Multisubunit TCR Complexes

Most T lymphocytes express on their surfaces an oligomeric protein complex consisting of clonotypic alpha beta polypeptides associated with invariant CD3-gamma delta epsilon and zeta chains, designated the T cell antigen receptor (TCR) complex. Assembly and intracellular transport of nascent TCR proteins is believed to be assisted by their interaction with the molecular chaperone calnexin, which for certain molecules functions as a lectin for monoglucosylated glycans. However, as most of our knowledge about calnexin-TCR protein associations has been obtained under conditions of limited TCR assembly, the role of calnexin in the formation of nascent TCR complexes is unclear. Here, we studied the role of glucose (Glc) trimming and calnexin association in the oligomerization of TCR alpha and CD3 delta glycoproteins in murine splenic T lymphocytes, a model cell type for efficient assembly of complete TCR complexes. We show that removal of Glc residues from both CD3 delta proteins and TCR alpha proteins occurred prior to their association with any other TCR components and that calnexin specifically interacted with unassembled TCR alpha and CD3 delta proteins containing incompletely trimmed oligosaccharides. Interestingly, we found that removal of Glc residues from glycan chains was necessary for efficient association of calnexin with TCR alpha glycoproteins but not with CD3 delta glycoproteins. These studies define Glc trimming and calnexin association as initial molecular events in the translation of CD3 delta and TCR alpha proteins occurring coincident with or immediately after their translocation into the endoplasmic reticulum and preceding the ordered pairing of TCR chains. In addition, these data document that calnexin assembly with CD3 delta and TCR alpha glycoproteins involves both glycan-dependent and glycan-independent mechanisms.

The T cell antigen receptor α and β chains interact via distinct regions with CD3 chains

Selective pairwise interactions between CD3 chains and the clonotypic T cell antigen receptor (TCR)-alpha, -beta chains has recently been established. In this study, the region of interaction between clonotypic and CD3 chains involved with assembly was examined. To determine the site of protein interaction a variety of genetically altered TCR chains were constructed. These included: truncated proteins, lacking transmembrane and or cytosolic domains; chimeric proteins, in which extracellular, transmembrane or cytosolic domains were replaced with similar domains derived from either the Tac antigen or CD4; and point mutagenized TCR chains. COS-1 cells were transfected with cDNA, metabolically labeled, and immunoprecipitates analyzed using non-equilibrium pH gel electrophoresis (NEPHGE)-SDS/PAGE. The results demonstrated that assembly between TCR-alpha and TCR-beta chains occurred at the extracellular level. Assembly of the TCR-alpha chain with CD3-delta, and CD3-epsilon was localized to an eight-amino acid motif within the transmembrane domain of TCR-alpha. Site-specific mutations of the TCR-alpha charged residues within this motif (arginine, lysine) to leucine and similar point mutations of the transmembrane CD3-epsilon and CD3-delta charge groups resulted in the abrogation of assembly. In contrast, TCR-beta and CD3-epsilon binary complexes interacted via their extracellular domain. Analogous to TCR-alpha, the site of TCR-beta and CD3-delta assembly was at the transmembrane region. Despite multiple genetic manipulations on CD3-gamma and zeta these proteins failed to assemble with TCR-alpha. Similarly, there was no interaction between TCR-beta and zeta. These findings when coupled with the information on pairwise interactions and formation of higher order subcomplexes extend our model for the structure of the TCR complex.

T cell antigen receptor signal transduction: A tale of tails and cytoplasmic protein-tyrosine kinases

The mechanisms by which extracellular ligands induce receptor-regulated signal transduction events have been an area of intense investigation. Remarkable progress has been made in understanding howthe binding of extracellular ligands to protein-tyrosine kinase (PTK) growth factor receptors induces the activation of their kinase domains. In contrast, the mechanism whereby receptors without an intrinsic PTK domain activate cytoplasmic PTKs has been enigmatic. However, recent studies of receptors involved in antigen recognition by cells of the hematopoietic lineage provide insight into functionally important cytoplasmic sequence motifs of these receptors and the interaction of these motifs with cytoplasmic PTKs of the Src and non-Src types. This review focuses on mechanisms involved in PTK activation by the T cell antigen receptor (TCR), but similar mechanisms are likely to be relevant for the B cell antigen receptor, the mast cell and basophil high affinity Fc receptorfor immunoglobulin (lg) E (FcsR), and certain Fc receptors expressed on natural killer (NK) cells and myeloid cells. These receptors are involved in the initiation of cellular activation or differentiation but are not directly involved in cell proliferation. All of these receptors, which are responsible for antigen recognition, initiate cellular activation by regulating the function of cytoplasmic PTKs that control the activation of intracellular signaling molecules, such as phospholipase C (reviewed by Samelson and Klausner, 1992). Act/vat/on Motifs in the TCR CD3 and 4 Chains Regulate Cytoplasmic PTKs The TCR is a complex oligomer composed of the products of six genes (Figure l), all of which are required for efficient plasma membrane expression. Together, the a and 8 chains form the ligand-binding subunit responsible for recognition of an antigenic peptide bound to a major histocompatibility complex (MHC) molecule. The CD3 and 5 chains are responsible for signal transduction. Recent studies with chimeric molecules and reconstituted receptors have provided definitive evidence for a signal transduction function for the CD3 and c chains. The transmembrane and membrane-proximal segments of the TCR chains are responsible for the interchain associations and the assembly of the oligomeric receptor. This observation permitted the development of a strategy to study the functions of the individual cytoplasmic domains of CD3 or r;. Chimeric molecules containing the cytoplasmic domain of 6, its alternatively spliced product n, or the related FCER y chains fused to the extracellular and transmembrane domains of another receptor (including CD8, CD4, CD18, and the interleukin-2 [IL-21 receptor a chain) could be expressed independently of theTCR (Irving and Weiss, 1991; Letourneur and Klausner, 1991; Romeo and Seed, 1991). Stimulation of these chimeric receptors with natural ligands or monoclonal antibodies (MAbs) induce all the early and late events characteristically associated with the intact oligomeric TCR. These findings suggest that 1; or the related chains can function to couple the TCR to intracellular signal transduction machinery. Surprisingly, subsequent experiments suggested a similar function for the CD3 chains. Reconstitution of TCR expression on a T cell hybridoma with cDNAs encoding various TCR chains has revealed that a funcfionally active TCR can be restored with a r; chain that is essentially devoid of cytoplasmic sequences (Wegener et al., 1992). A signaling function for the CD3 chains was confirmed when a chimeric molecule incorporating the CD3 E chain cytoplasmic domain was shown to be capable of activating a T cell hybridoma (Letourneur and Klausner, 1992). Thus, the CD3 and r; chains, though structurally distinct, appear to have redundant functions in TCR signaling. Identification of the functional domains of the chimeric receptors (Irving et al., 1993; Letourneur and Klausner, 1992; Romeo et al., 1992) explained the redundancy of function of the CD3 and r; chains. A sequence motif, first noted by Reth (1989), which is triplicated in 5 and present as a single copy in each of the CD3 chains, is responsible for the signal transduction capability of these chains (Figures 1 and 2). This motif, referred to herein as the antigen recognition activation motif (ARAM), is based on tyrosine and leucine (or isoleucine) residues. ARAMs are also present in the cytoplasmic domains of the proteins (Iga and lgp) associated with immunoglobulin on B cells, the 8 and y chains of the FcsR on mast cells and basophils, and, interestingly, the envelope glycoprotein of the bovine leukemia virus that induces polyclonal B cell proliferation. Since the genomic structure of each ARAM within c, CD3, Iga, lgp, and the FcsR j3 and y chains is similar (each motif is

In vivo and in vitro clonal deletion of double-positive thymocytes.

To study the processes of thymic development, we have established transgenic mice expressing and alpha/beta T cell antigen receptor (TCR) specific for cytochrome c associated with class II major histocompatibility complex (MHC) molecules. The transgenic TCR chains are expressed by most of the thymocytes in these mice, and these cells have been shown to efficiently mature in association with Ek- and Ab-encoded class II MHC molecules. This report describes a characterization of the negative selection of these transgenic thymocytes in vivo that is associated with the expression of As molecules. Negative selection by As molecules appears to result in the deletion of a late stage of CD4/CD8 double-positive thymocytes in that there is a virtual absence of transgenic TCR bearing CD4 single-positive thymocytes. This phenotype is accompanied by the appearance of CD4/CD8 double-negative thymocytes and peripheral T cells that are functionally antigen reactive. The process of negative selection has also been investigated using an in vitro culture system. Upon presentation of cytochrome c by Eb-expressing nonthymic antigen-presenting cells, there occurs an antigen dose-dependent deletion of the majority of CD4/CD8 double-positive thymocytes. In contrast, presentation of Staphylococcal enterotoxin A by Eb in vitro results in minimal deletion of double-positive thymocytes. In addition, we use this in vitro model to examine the effects of cyclosporin A on negative selection. In contrast to its effects on mature T cells, and the findings of others in vivo, cyclosporin A does not inhibit antigen-induced deletion of double-positive thymocytes. Finally, a comparison of the antigen dose responses for thymocyte deletion and for peripheral T cell activation indicates that double-positive thymocyte recognition is more sensitive than mature T cells to antigen recognition.

Regulation of protein tyrosine kinase activation by the T-cell antigen receptor zeta chain.

Regulation of protein tyrosine kinase activation by the T-cell antigen receptor zeta chain.

Surface expression of alternative forms of the TCR/CD3 complex.

T-cell antigen receptor (TCR) heterodimers of both the alpha beta and gamma delta types are expressed at the surface of T cells only in association with a complex of invariant chains called CD3. The requirement for individual CD3 components to achieve TCR surface expression was examined by cotransfection of a non-T-cell line with TCR alpha and beta, as well as CD3 delta, epsilon, gamma, and zeta, cDNAs. Both transient and stable transfectants expressing TCR and CD3 epitopes at the cell surface were generated. By transfection of TCR and CD3 components in different combinations, the TCR chains, as well as the CD3 epsilon and zeta chains, were each shown to be essential for reconstituting surface expression. On the other hand, CD3 delta and gamma chains could be used alternatively, providing evidence for two different types of TCR/CD3 complexes.

The zeta chain is associated with a tyrosine kinase and upon T-cell antigen receptor stimulation associates with ZAP-70, a 70-kDa tyrosine phosphoprotein.

Stimulation of the T-cell antigen receptor (TCR) leads to tyrosine phosphorylation of a number of cellular proteins, including phospholipase C (PLC) gamma 1 and the TCR zeta chain. We describe here a 70-kDa tyrosine phosphoprotein (ZAP-70) that associates with zeta within 15 sec following TCR stimulation. The phosphorylation of ZAP-70 and its association with zeta is independent of the other TCR chains since stimulation of a functional CD8/zeta chimeric receptor in a TCR-negative T cell leads to coprecipitation of ZAP-70 with the chimeric protein. In a Jurkat cell expressing the TCR and the CD8/zeta chimeric protein, tyrosine phosphorylation and association of ZAP-70 occurs exclusively with the stimulated receptor complex. In addition, a tyrosine kinase that does not appear to be fyn associates with the cytoplasmic domain of zeta and phosphorylates zeta and ZAP-70 in vitro.

Pairwise, cooperative and inhibitory interactions describe the assembly and probable structure of the T-cell antigen receptor.

The T-cell antigen receptor (TCR) is a multi-subunit complex consisting of clonotypic heterodimers (TCR-alpha beta or TCR-gamma delta) that are non-covalently linked to at least four invariant chains (CD3-delta, -epsilon, -gamma; and zeta or eta). The ordered process of assembly and the final number of individual chains that comprise the TCR is unclear. In this study, we examined the molecular basis of subunit interactions and the component requirements leading to the formation of a complete TCR. Analysis of transient cotransfections in monkey kidney fibroblasts (COS cells) showed assembly between selective chain pairs. Multiple chain cotransfections demonstrated the formation of stable higher order partial complexes. Assembly of such subcomplexes was facilitated by cooperative interactions between clonotypic and invariant CD3 chains. When zeta was cotransfected with any TCR component, no pairwise interaction was detected. Only when there was coexpression of all of the other TCR chains (TCR-alpha, -beta, CD3- epsilon, -gamma, -delta) did zeta assemble with the TCR complex. Not all chain pairs formed stable heterodimers. For one such pair, lack of assembly is due to the inhibitory effects of negatively charged residues within their transmembrane domains. The combined effects of these interactions probably determine the assembly and the quaternary structure of the TCR complex.

Six chains of the human T cell antigen receptor.CD3 complex are necessary and sufficient for processing the receptor heterodimer to the cell surface.

The T cell antigen receptor (TCR) plays a key role in the process of antigen recognition. It is a complex of at least seven peptide chains (alpha beta gamma delta epsilon zeta-zeta). It is found on the surface of mature T cells and functions in antigen binding in the presence of the major histocompatibility complex. It has been known for some time that physical associations between the CD3 proteins and the TCR chains are essential for efficient transport of either component to the surface of T cells. For example, T cells that lack either the alpha, beta, or delta chains synthesize partial complexes that are eventually degraded. cDNAs encoding the six chains of receptor have become available recently. We have used transfection techniques to generate a panel of Chinese hamster ovary cells that contain partial receptor complexes of known composition and also cells that express all six subunits of the TCR.CD3 complex. Cells in this panel were analyzed for the ability to form alpha-beta heterodimers and also an ability to transport the synthesized chains to the plasma membrane. These studies have allowed us to define the minimum requirements for TCR.CD3 expression on the cell surface.

Requirements for cell surface expression of the human TCR/CD3 complex in non-T cells

The T-cell antigen receptor (TCR) consists of a glycoprotein heterodimer (alpha/beta or gamma/delta) which is non-covalently associated with at least four or five invariant polypeptides (CD3 gamma, delta, epsilon, zeta and eta). In T-cell variants lacking TCR alpha, beta or zeta, it has been shown that incomplete TCR/CD3 complexes are retained within the cell. To examine requirements for cell surface expression of TCR/CD3, we transfected COS monkey kidney cells with cDNAs encoding TCR alpha, beta and CD3 gamma, delta, epsilon and zeta. We report that cell surface appearance of TCR/CD3 on COS cells requires coordinate expression of all six proteins. In the absence of the zeta chain, subcomplexes comprising from two to five chains were readily demonstrable in COS cells, but they failed to reach the cell surface or to acquire N-linked oligosaccharide side chains indicating failure to reach the medial Golgi. Pulse-chase metabolic labelling of transfected COS cells showed that three chains (CD3 gamma, CD3 epsilon, and zeta) were stable while three (TCR alpha, TCR beta and CD3 delta) were rapidly degraded. In two- and three-chain co-transfections specific intracellular subcomplexes were formed between TCR alpha and CD3 gamma, TCR alpha and CD3 delta, or TCR beta and CD3 epsilon. Binary subcomplexes having at least one stable chain (CD3 epsilon - TCR beta) were stable while one formed by two unstable chains (TCR alpha - CD3 delta) was still degraded. Assembly of the TCR/CD3 complex in COS cells thus appears centered around the metabolically stable CD3 gamma and CD3 epsilon proteins. Site-specific mutations of the negatively-charged transmembrane amino acid of residues of the CD3 chains to alanines served to either abolish (for TCR alpha - CD3 delta and TCR beta - CD3 epsilon) or diminish (for TCR alpha -CD3 gamma) these TCR-CD3 interactions. These mutations had no effect, however, on CD3-CD3 interactions or upon synthesis, metabolism, or intracellular distributions of the CD3 proteins. The transmembrane domains of CD3 gamma, delta, and epsilon thus appear to play a major role in associations of CD3 with TCR chains.

Colocalized transmembrane determinants for ER degradation and subunit assembly explain the intracellular fate of TCR chains

The intracellular fate of T cell antigen receptor (TCR) subunits ( αβγδϵξ 2) is determined by their assembly in the endoplasmic reticulum (ER). To study the structural bases for this tight correlation between assembly and intracellular fate, we sought to define the nature of determinants for both ER degradation and subunit assembly within the TCR-α chain. We found that a 9 amino acid transmembrane sequence of the TCR-α chain, containing 2 critical charged residues, was sufficient to cause ER degradation when placed in the context of the Tac antigen, used here as a reporter protein. CD3-δ assembled with chimeric proteins containing this short transmembrane sequence, and this assembly resulted in abrogation of targeting for ER degradation. Thus, the colocalization of determinants for ER degradation and sites of subunit interactions explains how the fate of some newly synthesized TCR chains can be decided on the basis of their assembly status.

Novel post-translational regulation of TCR expression in CD4+CD8+ thymocytes influenced by CD4

Expression of the multicomponent T-cell antigen receptor (TCR) complex on the surface of thymocytes is developmentally controlled. Most immature CD4-CD8- 'double negative' and CD4+CD8+ 'double positive' thymocytes express either no or few TCR on their surface, and maturation to CD4+CD8- or CD4-CD8+ 'single positive' thymocytes is accompanied by a dramatic increase in the number of surface TCR complexes. Although the initial appearance of TCR during differentiation results from rearrangement and initiation of transcription of TCR genes in the thymus, the mechanisms regulating the quantitative changes in TCR expression during intrathymic differentiation are unknown. Surface TCR levels in T-hybridoma cells can be quantitatively regulated by a series of post-translational processes, including sorting to alternative intracellular compartments and degradation, which ensure that only fully and correctly assembled receptor complexes are efficiently transported to the cell surface. Quantitative increases in TCR expression on the surface of CD4+CD8+ thymocytes occur in vivo in response to anti-CD4 antibody treatment. Here we present evidence that immature CD4+CD8+ thymocytes normally retain and degrade in the endoplasmic reticulum greater than 90% of some endogenously synthesized TCR chains, and that the increased surface TCR expression on immature CD4+CD8+ thymocytes induced by anti-CD4 is due to an increase in the escape of newly synthesized receptor chains from the endoplasmic reticulum, and is not due to increases in RNA levels, translation, or assembly. Post-translational mechanisms therefore control the levels of TCR complexes on CD4+CD8+ thymocytes, and these mechanisms can be modulated by signalling through CD4 surface molecules.

Pre-Golgi degradation of newly synthesized T-cell antigen receptor chains: intrinsic sensitivity and the role of subunit assembly.

The T cell antigen receptor (TCR) is a multisubunit complex composed of at least seven transmembrane chains. The predominant species in most T cells has the composition alpha beta gamma delta epsilon zeta 2. The roles of subunit assembly in transport out of the ER and in the recently described process of pre-Golgi degradation of newly synthesized TCR chains were analyzed in a T-cell line deficient in the synthesis of delta chains (delta 2) and in COS-1 fibroblasts transfected with genes encoding individual TCR chains. Studies with the delta-deficient T-cell line showed that, in the absence of delta, the other TCR chains were synthesized at normal rates, but, instead of being transported to the cell surface, they were retained in the ER. Analysis of the fate of TCR chains retained in the ER showed that they were degraded at vastly different rates by a nonlysosomal pathway. Whereas the alpha chains were degraded rapidly, gamma, zeta, and epsilon were relatively long-lived. To analyze whether this selective degradation was because of different intrinsic susceptibility of the individual chains to degradation or to the formation of resistant oligomers, TCR chains were expressed alone or in combinations in COS-1 fibroblasts. These studies showed that (a) individual TCR chains were degraded at different rates when expressed alone in COS-1 cells, and (b) sensitive chains could be stabilized by coexpression with a resistant chain. Taken together, these observations indicate that both intrinsic sensitivity and subunit assembly play a role in determining the rates at which newly synthesized TCR chains are degraded in the ER.