T Cell Antigen Receptor Proteins Research Articles

The T Cell Receptor Triggering Apparatus Is Composed of Monovalent or Monomeric Proteins

Understanding the component stoichiometry of the T cell antigen receptor (TCR) triggering apparatus is essential for building realistic models of signal initiation. Recent studies suggesting that the TCR and other signaling-associated proteins are preclustered on resting T cells relied on measurements of the behavior of membrane proteins at interfaces with functionalized glass surfaces. Using fluorescence recovery after photobleaching, we show that, compared with the apical surface, the mobility of TCRs is significantly reduced at Jurkat T cell/glass interfaces, in a signaling-sensitive manner. Using two biophysical approaches that mitigate these effects, bioluminescence resonance energy transfer and two-color coincidence detection microscopy, we show that, within the uncertainty of the methods, the membrane components of the TCR triggering apparatus, i.e. the TCR complex, MHC molecules, CD4/Lck and CD45, are exclusively monovalent or monomeric in human T cell lines, implying that TCR triggering depends only on the kinetics of TCR/pMHC interactions. These analyses also showed that constraining proteins to two dimensions at the cell surface greatly enhances random interactions versus those between the membrane and the cytoplasm. Simulations of TCR-pMHC complex formation based on these findings suggest how unclustered TCR triggering-associated proteins might nevertheless be capable of generating complex signaling outputs via the differential recruitment of cytosolic effectors to the cell membrane.

T Cell Receptor Assembly and Expression in the Absence of Calnexin

Most subunits of the αβδϵγϵζζ T cell antigen receptor (TCR) complex associate with the molecular chaperone calnexin shortly after their synthesis in the endoplasmic reticulum, including clonotypic TCRα,β molecules and invariant CD3γ,δ,ϵ chains. While calnexin interaction is suggested to be important for the stability of newly synthesized TCRα subunits, the role of calnexin in the survival and assembly of remaining TCR components is unknown. Here we evaluated the expression of TCR proteins in CEM T cells and the calnexin-deficient CEM variant CEM.NKR. We found that CEM and CEM.NKR cells constitutively synthesized all TCR subunits except for TCRα and that CD3-γ,δ,ϵ components and CD3-β complexes were effectively assembled together in both cell types. The stability and folding of core CD3ϵ chains were similar in CEM and CEM.NKR cells. Interestingly, TCRα synthesis was differentially induced by phorbol myristate acetate treatment in CEM and CEM.NKR cells and TCRα proteins synthesized in CEM.NKR cells showed reduced survival compared to those made in CEM cells. Importantly, these data show that TCR complexes were inducibly expressed on CEM.NKR cells in the absence of calnexin synthesis. These results demonstrate that TCR complexes can be expressed in the absence of calnexin and suggest that the role of calnexin in the quality control of TCR assembly is primarily restricted to the stabilization of newly synthesized TCRα proteins.

A comprehensive characterization of the T‐cell antigen receptor complex composition by microcapillary liquid chromatography‐tandem mass spectrometry

It has become apparent that many intracellular signaling processes involve the dynamic reorganization of cellular proteins into complex signaling assemblies that have a specific subunit composition, function, and subcellular location. Since the elements of such assemblies interact physically, multiprotein signaling complexes can be isolated and analyzed. Recent technical advances in highly sensitive protein identification by electrospray-tandem mass spectrometry have dramatically increased the sensitivity with which such analyses can be performed. The T-cell antigen receptor (TCR) is an oligomeric transmembrane protein complex that is essential to T-cell recognition and function. The extracellular protein domains are responsible for ligand binding while intracellular domains generate and transduce signals in response to specific receptor-ligand interactions. We used microbore capillary chromatography-tandem mass spectrometry to investigate the composition of the TCR protein complex isolated from resting and activated cells of the murine T-cell line CD11.3. We identified all the previously known subunits of the TCR/CD3 complex as well as proteins previously not known to associate with the TCR. The catalytic activities of some of these proteins could potentially be used to interfere pharmacologically with TCR signaling.

A comprehensive characterization of the T-cell antigen receptor complex composition by microcapillary liquid chromatography-tandem mass spectrometry

It has become apparent that many intracellular signaling processes involve the dynamic reorganization of cellular proteins into complex signaling assemblies that have a specific subunit composition, function, and subcellular location. Since the elements of such assemblies interact physically, multiprotein signaling complexes can be isolated and analyzed. Recent technical advances in highly sensitive protein identification by electrospray-tandem mass spectrometry have dramatically increased the sensitivity with which such analyses can be performed. The T-cell antigen receptor (TCR) is an oligomeric transmembrane protein complex that is essential to T-cell recognition and function. The extracellular protein domains are responsible for ligand binding while intracellular domains generate and transduce signals in response to specific receptor-ligand interactions. We used microbore capillary chromatography-tandem mass spectrometry to investigate the composition of the TCR protein complex isolated from resting and activated cells of the murine T-cell line CD11.3. We identified all the previously known subunits of the TCR/CD3 complex as well as proteins previously not known to associate with the TCR. The catalytic activities of some of these proteins could potentially be used to interfere pharmacologically with TCR signaling.

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.

The Related Molecular Chaperones Calnexin and Calreticulin Differentially Associate with Nascent T Cell Antigen Receptor Proteins within the Endoplasmic Reticulum

Assembly of the multisubunit T cell antigen receptor (TCR) complex is an intricate process requiring coordinated regulation of at least six different gene products (alpha, beta, gamma, delta, epsilon, and zeta) and the ordered pairing of partner chains within the endoplasmic reticulum (ER). To date, two proteins have been implicated as functioning as molecular chaperones in the assembly of nascent TCR proteins: calnexin, a resident ER transmembrane protein, which associates with all TCR components except zeta, and T cell receptor-associated protein, which selectively associates with CD3gammaepsilon pairs. In this study, we examined the association of calreticulin, a soluble protein with significant sequence homology to calnexin, with newly synthesized TCR proteins. Analogous to calnexin, processing of glycan chains by glucosidase enzymes was required for initial association of TCRalpha and -beta proteins with calreticulin; however, several major differences were noted regarding interaction of calnexin and calreticulin chaperones with TCR proteins. First, TCRalpha and -beta proteins showed prolonged association with calnexin molecules compared with calreticulin; interaction of TCRalpha proteins with calreticulin was particularly transient, with most calreticulin-TCRalpha protein complexes dissociating within 15 min of their initial assembly. Second, we found that, unlike calnexin, which associated with clonotypic TCRalpha and -beta proteins and invariant CD3delta and -epsilon polypeptides, calreticulin associated specifically with clonotypic TCRalpha and -beta proteins. These studies identify calreticulin as a molecular chaperone for nascent clonotypic TCRalpha and -beta proteins and demonstrate that calreticulin and calnexin differentially associate with newly synthesized TCR proteins within the ER.

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.

Hierarchy of T cell antigen receptor assembly

The formation of partial complexes in an African green monkey kidney cell line (COS cells) has been used to examine assembly of newly synthesized T cell antigen receptor (TCR) chains. The identification of assembly interactions between multiple subunits and the formation of higher order subcomplexes has led to the development of a model for the hierarchy of subunit assembly leading to a complete TCR. These assembly interactions suggest likely nearest neighbour relationships in the assembling structure and probably reflect the quarternary structure of the TCR complex. This may be important when examining the association of other molecules with TCR proteins or when trying to discern structural components involved with signal transduction.

Stoichiometry of the T cell antigen receptor (TCR) complex: each TCR/CD3 complex contains one TCR alpha, one TCR beta, and two CD3 epsilon chains.

The stoichiometry of the subunits that comprise the T cell antigen receptor (TCR) complex is not completely known. In particular, it is uncertain whether TCR alpha and TCR beta proteins are present in the TCR complex as one or multiple heterodimeric pairs. In this study we have used mice transgenic for two different TCR alpha and two different TCR beta proteins to determine the number of TCR alpha and TCR beta chains in a single TCR complex. Individual thymocytes and splenic T cells from double TCR transgenic mice simultaneously expressed all four transgenic TCR proteins on their surfaces. Because the individual TCR alpha and individual TCR beta proteins were biochemically distinguishable, we were able to examine association among the transgenic TCR products. We found that each TCR alpha chain paired with each TCR beta chain, but that each TCR complex contained only one TCR alpha and one TCR beta protein. Furthermore, quantitative immunofluorescence revealed that T cells expressed twice as many CD3 epsilon as TCR beta proteins. These findings demonstrate that there are precisely one TCR alpha, one TCR beta, and two CD3 epsilon chains in each TCR/CD3 complex expressed on the surfaces of both thymocytes and mature T cells.

Persistence of glucose residues on core oligosaccharides prevents association of TCR alpha and TCR beta proteins with calnexin and results specifically in accelerated degradation of nascent TCR alpha proteins within the endoplasmic reticulum.

The alpha beta T-cell antigen receptor (TCR) is a multisubunit transmembrane complex composed of at least six different proteins (alpha, beta, gamma, delta, epsilon and zeta) that are assembled in the endoplasmic reticulum (ER). In this report we have examined the role of oligosaccharide processing on survival and assembly of nascent TCR proteins within the ER and their associations with molecular chaperone proteins important in TCR assembly. We found that treatment of BW5147 T cells with the glucosidase inhibitor castanospermine resulted in markedly accelerated degradation of nascent TCR alpha proteins with a half-life of approximately 20 min. Accelerated degradation was unique to TCR alpha proteins, as the stability of nascent TCR beta and CD3 gamma,epsilon chains was unaltered. Consistent with a requirement for glucose (Glc) trimming for survival of nascent TCR alpha proteins within the ER, we found that newly synthesized TCR alpha chains were innately unstable in the glucosidase II-deficient BW5147 mutant cell line PHAR2.7. In addition to destabilizing nascent TCR alpha proteins we found that persistence of Glc residues on core oligosaccharides markedly interfered with association of both TCR alpha and TCR beta glycoproteins with the molecular chaperone calnexin. Finally, using 2B4 T hybridoma cells in which TCR complexes are efficiently assembled, we found that rapid degradation of nascent TCR alpha proteins induced by impaired Glc trimming severely limits assembly of TCR alpha proteins with TCR beta proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

The detection of a breast cancer antigen on MCF-7 cells reactive with the TCR (alpha) of a specific killer T cell line.

We established several immortalized human T cell lines by cotransfecting primary lymphocytes derived from breast cancer patients with human c-myc and human c-Ha-ras oncogenes. A CD8 positive (CD8+) killer T cell line, FT-8, exhibited in vitro specific cytotoxicity to a human breast cancer cell line, MCF-7. The FT-8 cells suppressed the growth of MCF-7 cells transplanted to athymic mice. The cytotoxic reaction was caused via T cell antigen receptor (TCR) on MCF-7 cells, because monoclonal antibodies against the TCR inhibited the cytotoxicity of FT-8 cells. The TCR (alpha) cDNA of FT-8 was cloned by using a PCR amplification technique and expressed by a cell-free in vitro translation system. The TCR (alpha) protein recognized a target antigen of 32 KDa on MCF-7 cells.

Immunocytometry and Gene Rearrangement Analysis in the Diagnosis of Lymphoma in an Idiopathic Pleural Effusion

We report a patient with an idiopathic pleural effusion in whom the diagnosis of non-Hodgkin's lymphoma was established by immunocytometry of pleural fluid and confirmed by the detection of B-cell immunoglobulin gene rearrangement. Immunocytometry is a rapid, semi-automated laboratory method for phenotyping lymphoid cells by determining immunoglobulin and other cell surface antigen expression. This method defines the cell lineage (T or B cells) and the clonality (monoclonal or polyclonal) of a population of lymphocytes. The presence of a monoclonal population of lymphocytes can also be confirmed by recently developed molecular biologic techniques (e.g., Southern blotting) that provide the ability to detect rearrangements of the genes that encode either B-cell immunoglobulin proteins or T-cell antigen receptor proteins. To our knowledge, this case represents the first reported application of immunophenotypic and gene rearrangement analysis in a previously undiagnosed pleural effusion to establish the diagnosis of lymphoma. These relatively new laboratory methods may have a role in the evaluation of idiopathic lymphocytic pleural effusions.

In Vivo modulation of the distribution of thymocyte subsets: Effects of estrogen on the expression of different T cell receptor Vβ gene families in CD4 −, CD8 − thymocytes

Estrogen treatment of mice has been shown to deplete CD4 +, CD8 + double-positive (DP) thymocytes and to alter the relative proportion of CD4 + and CD8 + single-positive (SP) thymocytes. In this work, we have studied the effect of the steroid hormone 17 β-estradiol (E2) on the different subsets of CD4 −/CD8 − double-negative (DN) thymocytes by analyzing the expression of CD5, CD3-ϵ and of several Vβ gene family products of the T cell antigen receptor (TCR). After in vivo administration of E2 a significant decrease in the number and proportion of dull CD5 +, CD3 −, β-TCR − DN thymocytes was observed. In contrast E2 treatment significantly increased the proportion of bright CD5 +, CD3 +, β-TCR + DN cells. The E2-induced increase in DN/TCR + cells was observed for subsets expressing Vβ6, Vβ8, and Vβ11, but not Vβ3 gene products of the TCR. Thus, estrogen administration results in a selective inbalance of the DN thymocyte subsets by depleting an immature, dull CD5 +, CD3 −, TCRβ − DN subset, while enriching a mature, bright CD5 +, CD3 +, TCRβ + DN subset of cells. In addition to TCRβ + DN thymocytes, an increased proportion of CD4 + and CD8 + SP thymocytes expressing Vν8, Vβ6, and Vβ11, but not Vβ3, TCR proteins was also observed after E2 administration. An involvement of intrathymic cytokine production in mediating the hormone action is suggested by the ability of estrogen to increase the levels of IL-la mRNA of intact thymus. Our data suggest that estrogen exerts its effects on a broad range of immature cells, including dull CD5 +, CD3 −, β-TCR − DN and DP thymocytes.

Confirmation of Lymphomatous Pulmonary Involvement by Immunophenotypic and Gene Rearrangement Analysis of Bronchoalveolar Lavage Fluid

Pathologic diagnosis of pulmonary involvement with lymphoma has traditionally necessitated open-lung biopsy in most cases. Specimens large enough to allow recognition of characteristic cytologic and architectural changes are usually not obtained bronchoscopically. Even when adequate biopsy specimens are available, however, unequivocal differentiation of lymphoma from benign inflammatory proliferative lesions (for example, pseudolymphoma or lymphocytic interstitial pneumonitis) is not possible on the basis of light microscopic findings alone. Pathologists have relied on immunohistologic examinations in which antibodies directed against B-cell and T-cell surface antigens are used to help distinguish benign from malignant lymphoid proliferations. Unfortunately, even immunohistologic findings may be nondiagnostic, particularly in T-cell lymphomas and in cases in which lymphoma is surrounded by a benign reactive lymphocytosis. Recent development of molecular biologic techniques (for example, Southern blotting) has provided the ability to detect a monoclonal population of cells based on detection of rearrangements of the genes that encode either B-cell immunoglobulin proteins or T-cell antigen receptor proteins. This technique is capable of detecting a clone of cells even when they constitute as little as 5% of a lymphoid infiltrate. Bronchoalveolar lavage can provide samples of sufficient size to facilitate Southern blotting. We present the first case wherein pulmonary involvement with a B-cell lymphoma was confirmed by both immunohistologic and molecular biologic studies of bronchoalveolar lavage fluid.

Molecular characterization of T-cell antigen receptor expression by subsets of CD4- CD8- murine thymocytes.

Precursors of all T-lineage cells are found in a population of thymocytes that lack the CD4 and CD8 surface glycoproteins. These "double-negative" thymocytes are markedly heterogeneous in their expression of other surface markers and include cells at various stages of development. In this study, CD4- CD8- adult murine thymocytes were separated into subsets based on the expression of the "heat stable antigen" (HSA) and of Ly 1 (CD5). The sorted subsets were analyzed directly (without prior expansion in culture) for T-cell antigen receptor (TcR) gene rearrangement and mRNA expression and for TcR and CD3 cell-surface protein expression. Very little surface CD3 or TcR expression was detected on the major HSA+ Ly 1low subset. However, the HSA+ Ly 1high, HSA- Ly 1high, and HSA- Ly 1low subsets all contained cells with surface expression of CD3 and TcR. In contrast to previous studies, we found no subset that exclusively expressed either the alpha beta or gamma delta heterodimer, although the ratio of alpha beta+ to gamma delta+ varied widely. Two of these three subsets (HSA- Ly 1low and HSA- Ly 1high) showed very high usage of V beta 8 gene products in the alpha beta heterodimer, but nevertheless included approximately equal to 15% non-V beta 8 alpha beta forms. All CD4- CD8- subsets were found to have extensively rearranged their TcR gamma genes and to express gamma mRNA. Expression of a high ratio of mature [1.3 kilobases (kb)] to truncated (1.0 kb) beta message and presence of alpha message was largely restricted to subsets with TcR alpha beta surface expression.