T-cell Antigen Receptor Alpha Research Articles

Comparison of Genome-Wide and Gene-Specific DNA Methylation Profiling in First-Trimester Chorionic Villi From Pregnancies Conceived With Infertility Treatments.

Assisted reproductive technologies are associated with altered methylation in term placenta. However, it is unclear whether methylation patterns are the result of fertility treatments or intrauterine environment. Thus, we set out to determine whether there are differences in the first-trimester placenta that may be altered by the underlying fertility treatments. Genome-wide DNA methylation analyses from chorionic villus sampling (CVS) from matched singleton pregnancies conceived using in vitro fertilization (IVF), non-IVF fertility treatment (NIFT), or those conceived spontaneously were performed using Illumina Infinium HumanMethylation450 BeadChip from 15 matched CVS samples. Nanofluidic quantitative polymerase chain reaction (qPCR) of differently methylated genes was performed in a confirmatory cohort of 23 IVF conceptions and 24 NIFT conceptions. Global methylation was similar among the IVF, NIFT, and spontaneous conceptions. However, differential methylation from IVF and NIFT pregnancies was present at 34 CpG sites, which was significantly different. Of those, 14 corresponded to known genes, with methylation changes detected at multiple loci in 3 genes, anaphase-promoting complex subunit 2 ( ANAPC2), C-X-C motif chemokine ligand 14 ( CXCL14), and regulating synaptic membrane exocytosis 1 ( RIMS1). Nanofluidic qPCR of differentially methylated genes identified pre T-cell antigen receptor alpha ( PTCRA) to be significantly downregulated in IVF versus NIFT conceptions. Although global methylation patterns are similar, there are differences in methylation of specific genes in IVF compared to NIFT conceptions, leading to altered gene expression. PTCRA was differentially methylated and downregulated in IVF conceptions, warranting further investigation. It remains to be determined whether these changes affect placentation and whether it is due to the more profound underlying infertility requiring IVF, yet these data provide unique insight into the first-trimester placental epigenome.

Recognition of Microbial Glycolipids by Natural Killer T Cells

T cells can recognize microbial antigens when presented by dedicated antigen-presenting molecules. While peptides are presented by classical members of the major histocompatibility complex (MHC) family (MHC I and II), lipids, glycolipids, and lipopeptides can be presented by the non-classical MHC member, CD1. The best studied subset of lipid-reactive T cells are type I natural killer T (iNKT) cells that recognize a variety of different antigens when presented by the non-classical MHCI homolog CD1d. iNKT cells have been shown to be important for the protection against various microbial pathogens, including B. burgdorferi, the causative agents of Lyme disease, and S. pneumoniae, which causes pneumococcal meningitis and community-acquired pneumonia. Both pathogens carry microbial glycolipids that can trigger the T cell antigen receptor (TCR), leading to iNKT cell activation. iNKT cells have an evolutionary conserved TCR alpha chain, yet retain the ability to recognize structurally diverse glycolipids. They do so using a conserved recognition mode, in which the TCR enforces a conserved binding orientation on CD1d. TCR binding is accompanied by structural changes within the TCR binding site of CD1d, as well as the glycolipid antigen itself. In addition to direct recognition of microbial antigens, iNKT cells can also be activated by a combination of cytokines (IL-12/IL-18) and TCR stimulation. Many microbes carry TLR antigens, and microbial infections can lead to TLR activation. The subsequent cytokine response in turn lower the threshold of TCR-mediated iNKT cell activation, especially when weak microbial or even self-antigens are presented during the cause of the infection. In summary, iNKT cells can be directly activated through TCR triggering of strong antigens, while cytokines produced by the innate immune response may be necessary for TCR triggering and iNKT cell activation in the presence of weak antigens. Here, we will review the molecular basis of iNKT cell recognition of glycolipids, with an emphasis on microbial glycolipids.

Signal- and Development-Dependent Alternative Splicing of LEF1 in T Cells Is Controlled by CELF2

The HMG-box transcription factor LEF1 controls many developmentally regulated genes, including genes that activate expression of the T-cell antigen receptor alpha chain (TCR-alpha) in developing thymocytes. At least two distinct isoforms of LEF1 are expressed, resulting from variable inclusion of LEF1 exon 6; however, the expression pattern of these isoforms and mechanism of splicing regulation have not been explored. Here we demonstrate that inclusion of LEF1 exon 6 is increased during thymic development and in response to signaling in a cultured T-cell line in a manner which temporally correlates with increased expression of TCR-alpha. We further find that inclusion of exon 6 is dependent on the signal-induced increase in expression and binding of the splicing factor CELF2 to two intronic sequences flanking the regulated exon. Importantly, loss of exon 6 inclusion, through knockdown of CELF2 or direct block of the exon 6 splice site, results in reduced expression of TCR-alpha mRNA. Together, these data establish the mechanistic basis of LEF1 splicing regulation and demonstrate that LEF1 alternative splicing is a contributing determinant in the optimal expression of the TCR-alpha chain.

NMR study of the structure and self‐association of core peptide in aqueous solution and DPC micelles

Core peptide is a hydrophobic peptide derived from the T-cell antigen receptor-alpha chain (TCR-alpha) transmembrane region with therapeutic potential. The mechanism by which the peptide inserts into the membrane, including any requirements to change conformational or association states during the insertion, is unclear. Here, the self-association and secondary structure of Core peptide in aqueous solution and in dodecylphosphocholine (DPC) micelles were examined using various nuclear magnetic resonance (NMR) techniques. NMR diffusion measurements were performed on 0.05, 1, and 5 mM Core peptide in D2O. These samples had pH values varying from 3 to 4. A constant measured diffusion coefficient of 2 X 10(-10) m2 s(-1) was observed in these samples indicating that Core peptide was monomeric. Multidimensional NMR experiments (i.e., TOCSY and NOESY) revealed the formation of beta-strands in water at low pH and random coil in DPC micelles. The results of this study reveal that at relatively low pH, the insertion mechanism must involve Core peptide in the monomeric state but it undergoes a conformational transition during membrane insertion.

T-Cell Antigen Receptor Assembly and Cell Surface Expression Is Not Affected by Treatment with T-Cell Antigen Receptor-Alpha Chain Transmembrane Peptide

A synthetic peptide termed core peptide (CP), which corresponds to a specific sequence of the TCR-alpha chain transmembrane domain, is known to inhibit IL-2 production in antigen stimulated T-cells. The molecular mechanism of the TCR inhibition is not known. This study examined the effects of CP on TCR subunit assembly and TCR cell surface expression in vitro. Co-transfection experiments between TCR-alpha and CD3-delta using COS-7 cells, and the interaction between TCR-alpha and the CD3 proteins in a T-cell line (2B4) were analysed after incubation with CP or its conjugates. Results indicate that CP co-precipitates with CD3-delta and CD3-epsilon in vitro, without any effect on TCR-alpha/CD3-delta dimerisation or TCR multisubunit assembly and cell surface expression.

Immunoreceptor Transmembrane Peptides and Their Effect on Natural Killer (NK) Cell Cytotoxicity

Short peptides derived from the transmembrane sequence of NK activating receptors and associated molecules were tested in vitro for inhibition of NK cell cytotoxicity using a standard (51)Cr release assay in the absence or presence of peptides. NKL23 cell line was used as the NK effector and the target was the NKL23 sensitive 721.221 cell line. NKp46, NKp30, NKG2D and CD3-zeta peptides inhibited NK activity at higher concentration (100 microM) compared to controls by 6-13% (p<0.05). Modification of one non-effective peptide (NKP44) significantly enhanced inhibition by 30%, 17% and 11% at 100 microM, 50 microM and 10 microM respectively compared to controls. A T-cell antigen receptor-alpha chain transmembrane sequence derived peptide (CP) significantly inhibited NKL cell activation by 20-30% (p<0.05) at 50 microM and 100 microM concentrations compared to the control. The structural similarities between these immuno-receptors, and in particular the need for transmembrane electrostatic interactions for receptor function, provides the basis for current and future targeted therapeutic strategies.

T-Cell Antigen Receptor-alpha Chain Transmembrane Peptides: Correlation between Structure and Function

Core peptide (CP) is a unique peptide derived from the transmembrane sequence of T cell antigen receptor (TCR)-alpha chain that is capable of inhibiting the immune response both in vitro and in animal models of T cell mediated inflammation. CP contains two basic amino acids (lysine and arginine) in its sequence. The presence of these charged residues interspersed between hydrophobic amino acids is important for function. Here in an attempt to understand CP’s biophysical properties leading to activity we have synthesized a number of CP analogues and correlated their model structure with their biological activity. It became apparent that it is not only the charge of the amino acids but also the nature of the polar amino acids themselves and the topography and spacing between them by hydrophobic amino acids, creating a hydrophobic face, that are critical for CP function.

OLIG2 (BHLHB1), a bHLH transcription factor, contributes to leukemogenesis in concert with LMO1.

OLIG2 (originally designated BHLHB1) encodes a transcription factor that contains the basic helix-loop-helix motif. Although expression of OLIG2 is normally restricted to neural tissues, overexpression of OLIG2 has been shown in patients with precursor T-cell lymphoblastic lymphoma/leukemia (pre-T LBL). In the current study, we found that overexpression of OLIG2 was not only found in oligodendroglioma samples and normal neural tissue but also in a wide spectrum of malignant cell lines including leukemia, non-small cell lung carcinoma, melanoma, and breast cancer cell lines. To investigate whether enforced expression of OLIG2 is oncogenic, we generated transgenic mice that overexpressed OLIG2 in the thymus. Ectopic OLIG2 expression in the thymus was only weakly oncogenic as only 2 of 85 mice developed pre-T LBL. However, almost 60% of transgenic mice that overexpressed both OLIG2 and LMO1 developed pre-T LBL with large thymic tumor masses. Gene expression profiling of thymic tumors that developed in OLIG2/LMO1 mice revealed up-regulation of Notch1 as well as Deltex1 (Dtx1) and pre T-cell antigen receptor alpha (Ptcra), two genes that are considered to be downstream of Notch1. Of note, we found mutations in the Notch1 heterodimerization or proline-, glutamic acid-, serine-, and threonine-rich domain in three of six primary thymic tumors. In addition, growth of leukemic cell lines established from OLIG2/LMO1 transgenic mice was suppressed by a gamma-secretase inhibitor, suggesting that Notch1 up-regulation is important for the proliferation of OLIG2-LMO1 leukemic cells.

Morphologic transformation of the thymus in developing zebrafish

The morphologic transformation of the developing zebrafish thymus from 1 week postfertilization (wpf) to 15 wpf is described. The thymus overall morphology changed from a small pouch-like shape at 1 wpf to a conical shape between 2 and 3 wpf before acquiring a more complex shape from 4 wpf onward. Rapid growth rate along the lateral axis at the region near the pharyngeal epithelium occurred between 1 and 2 wpf, whereas rapid growth rate along the dorsal-ventral axis occurred between 3 and 6 wpf. Expansion of thymocyte population beginning from 1 wpf became more evident by 2 to 3 wpf, as indicated by the apparent increase of different sizes of lymphocytes, recombination activating gene-1 (rag-1), and T-cell antigen receptor alpha chain constant region (TCRAC) -positive cells. Tissue section in situ hybridization (ISH) analysis with rag-1 probe reveals that cortex-medullary regionalization has begun between 1 and 2 wpf as rag-1 expression clearly demarcated the cortex, whereas the medulla was rag-1 negative. The presence of TCRAC-positive cells in the medulla by 2-3 wpf, suggests that the thymic selection processes had begun. The zebrafish thymus is morphologically mature by 3 wpf. Early signs of thymic involution were observed in zebrafish aged 15 wpf.

Structure and diversity of T-lymphocyte antigen receptors alpha and gamma in Xenopus.

Short primer PCR directed at conserved regions of amino acid sequence within the T-cell antigen receptor (TCR) and immunoglobulin (Ig) light chain variable ( V) regions was used to amplify putative TCRgamma V region amplicons from stage 45 Xenopus laevis (African clawed frog) mRNA (cDNA). An adult Xenopus spleen cDNA library was screened using the Vgamma and a putative TCRValpha amplicon. Full copy length cDNAs containing the specific PCR-derived Valpha and Vgamma amplicons were recovered at relatively low frequency. Probes complementing the TCRalpha and TCRgamma constant ( C) regions were employed to isolate equivalent numbers of additional TCR alpha and TCR gamma cDNAs in an unbiased (non- V-based) manner. Few Vgamma genes appear to be expressed relative to the highly diverse expression of V alpha genes in equivalent numbers of cDNAs that were analyzed. Two TCRgamma C regions differ at only two positions; whereas two TCRalpha C regions differ at 33 coding positions as well as in their respective 3' untranslated regions, consistent with two independent loci. However, genomic Southern blots revealed considerably higher numbers of hybridizing bands when probed with C gamma than with C alpha. A potential novel mechanism of diversification is suggested by an unusual TCR alpha cDNA in which the V region can be translated in two frames through utilization of two closely linked V genes and an alternative splicing process. This process produces a translatable cDNA that is not readily predictable from the genomic locus utilizing normal recombination and splicing mechanisms.

The KDEL receptor mediates a retrieval mechanism that contributes to quality control at the endoplasmic reticulum.

Newly synthesized proteins in the endoplasmic reticulum (ER) must fold and assemble correctly before being transported to their final cellular destination. While some misfolded or partially assembled proteins have been shown to exit the ER, they fail to escape the early secretory system entirely, because they are retrieved from post-ER compartments to the ER. We elucidate a mechanistic basis for this retrieval and characterize its contribution to ER quality control by studying the fate of the unassembled T-cell antigen receptor (TCR) alpha chain. While the steady-state distribution of TCRalpha is in the ER, inhibition of retrograde transport by COPI induces the accumulation of TCRalpha in post-ER compartments, suggesting that TCRalpha is cycling between the ER and post-ER compartments. TCRalpha associates with BiP, a KDEL protein. Disruption of the ligand-binding function of the KDEL receptor releases TCRalpha from the early secretory system to the cell surface, so that TCRalpha is no longer subject to ER degradation. Thus, our findings suggest that retrieval by the KDEL receptor contributes to mechanisms by which the ER monitors newly synthesized proteins for their proper disposal.

Characterization of three isotypes of immunoglobulin light chains and T-cell antigen receptor alpha in zebrafish.

The zebrafish (Danio rerio) has become a significant model for understanding the developmental regulation of gene expression and holds considerable potential for characterizing the development of the immune system. Using a number of different approaches, including heterologous hybridization and short-primer PCR, cDNAs for three different classes of light-chain genes were identified and characterized. The zebrafish light chains are similar to trout type 1, trout type 2, and catfish type F, respectively. T-cell antigen receptor alpha (TCRalpha) was also identified and characterized. A high proportion of unusual transcripts including sterile transcripts, germline VJC transcripts, aberrant splice forms, and V-V transcripts were encountered in the immunoglobulin and TCR cDNAs examined. The light-chain and TCRalpha loci each consist of multiple families of V gene segments, apparent even from the small numbers of cDNAs of each isotype sequenced. The gene sequences reported provide an essential set of markers of both B- and T-cell lineages that will facilitate investigations of immune system development.

Structural requirements for antigen presentation by mouse CD1.

The structural basis for the T cell response to glycolipid antigens (Ags) remains poorly understood. T lymphocytes autoreactive for mouse CD1 (mCD1.1) or reactive for the glycosphingolipid alphagalactosylceramide (alpha-GalCer) presented by mCD1.1 have been described previously. In this paper it is shown that mutations at the top of the alpha helices and in the bottom of the Ag-binding groove can disrupt both mCD1.1 autoreactivity and alpha-GalCer recognition. The locations of the positions that affect T cell responses indicate that recognition of mCD1.1 is not likely to be unconventional or superantigen-like. Furthermore, the effects of the bottom of the pocket mutation suggest that the autoreactive response could require an autologous ligand, and they indicate that alpha-GalCer binds to the groove of mCD1.1, most likely with the shorter 18-carbon hydrophobic chain in the A' pocket. Natural killer T cell hybridomas with identical T cell antigen receptor (TCR) alpha chains and different beta chains respond differently to alpha-GalCer presented by mCD1.1 mutants. This finding indicates a role for TCR beta in defining natural killer T cell specificity, despite the more restricted diversity of the alpha chains in these cells. Overall, the data are consistent with a mode of lipoglycan recognition similar to that proposed for glycopeptides, in which the TCR alpha and beta chains survey a surface composed of both mCD1.1 and the carbohydrate portion of alpha-GalCer.

Accessibility control of variable region gene assembly during T-cell development.

T-cell development is a complex and ordered process that is regulated in part by the progressive assembly and expression of antigen receptor genes. T cells can be divided into two lineages based on expression of either an alpha beta or gamma delta T-cell antigen receptor (TCR). The genes that encode the TCR beta and gamma chains lie in distinct loci, whereas the genes that encode the TCR alpha and delta chains lie in a single locus (TCR alpha/delta locus). Assembly of TCR variable region genes is mediated by a site-specific recombination process that is common among all lymphocytes. Despite the common nature of this process, recombination of TCR genes is tightly regulated within the context of the developing T cell. TCR beta, gamma and delta variable region genes are assembled prior to TCR alpha variable region genes. Furthermore, assembly of TCR beta variable region genes is regulated within the context of allelic exclusion. The regulation of rearrangement and expression of genes within the TCR alpha/delta locus presents a complicated problem. TCR alpha and delta variable region genes are assembled at different stages of T-cell development, and fully assembled TCR alpha and delta variable region genes must be expressed in distinct lineages of T cells, alpha beta and gamma delta, respectively. We have developed several experimental approaches to assess the role of cis-acting elements in regulating recombination and expression of TCR genes. Here we describe these approaches and discuss our analyses of the regulation of accessibility of the TCR beta and TCR alpha/delta loci during T-cell development.

Rapid deletion of rearranged T cell antigen receptor (TCR) Valpha-Jalpha segment by secondary rearrangement in the thymus: role of continuous rearrangement of TCR alpha chain gene and positive selection in the T cell repertoire formation.

A rearranged T cell receptor (TCR) Valpha and Jalpha gene from a cytochrome c-specific T cell hybridoma was introduced into the genomic Jalpha region. The introduced TCR alpha chain gene is expressed in a majority of CD3 positive and CD4 CD8 double-negative immature thymocytes. However, only a few percent of the double-positive and single-positive thymocytes express this TCR alpha chain. This decrease is caused by a rearrangement of TCR alpha chain locus, which deletes the introduced TCR gene. Analysis of the mice carrying the introduced TCR alpha chain and the transgenic TCR beta chain from the original cytochrome c-specific T cell hybridoma revealed that positive selection efficiently rescues double-positive thymocytes from the loss of the introduced TCR alpha chain gene. In the mice with negatively selecting conditions, T cells expressing the introduced TCR alphabeta chains were deleted at the double-positive stage. However, a large number of thymocytes escape negative selection by using an endogenous TCR alpha chain created by secondary rearrangement maintaining normal thymocyte development. These results suggest that secondary rearrangements of the TCR alpha chain gene play an important role in the formation of the T cell repertoire.

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.

Quantitative analysis of T lymphocyte subsets in pregnant and nonpregnant human endometrium.

The purpose of this study was to characterize and quantify T lymphocyte populations and to investigate T cell antigen receptor (TcR) expression in human endometrium throughout the menstrual cycle and in early pregnancy. Frozen sections of endometrium were labeled using the highly sensitive avidinbiotin method and a panel of monoclonal antibodies. CD3-positive, CD8-positive, CD4-positive, TcR alpha beta-positive, and TcR gamma delta-positive cells were demonstrated in all phases of the menstrual cycle and in early pregnancy. There were no differences between pregnant and nonpregnant human endometrium in the proportion that each T cell subpopulation formed relative to the total CD3-positive T cell population. However, significantly fewer T cells were detected in early pregnant compared with nonpregnant endometrium. Our results clarify the TcR expression by T cells in pregnant and nonpregnant human endometrium and indicate that endometrial T cells are unlikely to play a significant role in implantation and the maintenance of human pregnancy since they decrease in number considerably in the first trimester of gestation.

Melanoma specific Th1 cytotoxic T lymphocyte lines in Vogt-Koyanagi-Harada disease.

To determine the functional properties and cytokine production profiles of melanoma specific cytotoxic T lymphocytes (CTLs) induced from peripheral blood leucocytes of two patients with Vogt-Koyanagi-Harada disease (VKH). Melanoma specific CTL lines were established by long term coculture with a human melanoma cell line (P-36). Cytotoxic activity against P-36 was measured by 51Cr release. The involvement of human leucocyte antigen (HLA) class I or class II molecules in the cytotoxicity of the CTL lines against P-36 was analysed using anti-HLA class I or anti-HLA class II monoclonal antibody (MAb). Surface molecules of CTL lines were analysed by flow cytometry using MAbs specific for CD4, CD8, CD16, CD25, CD56, HLA-DR, T cell antigen receptor (TCR) alpha beta and TCR gamma delta. Cytokine production and soluble interleukin 2 receptor (sIL-2R) secretion were determined by enzyme linked immunosorbent assays. mRNAs of cytokines were analysed using reverse transcription polymerase chain reaction (RT-PCR). CTLs showed strong cytotoxic activity against P-36. The CTL activity of the cell lines against P-36 was inhibited by the anti-HLA-DR MAb, whereas the MAb specific for monomorphic determinants of HLA-A, B, and C failed to block lytic activity. Flow cytometry identified the following surface molecules: CD4+, CD8-, CD16-, CD25+, CD56-, HLA-DR+, TCR alpha beta +, and TCR gamma delta-. CTLs constitutively produced a high level of IL-6. IL-6 production and sIL-2R secretion of CTLs were enhanced when CTLs were stimulated with P-36. CTLs also produced high levels of interferon gamma (IFN-gamma) and IL-2, but not IL-4. mRNAs of IL-2 and IFN-gamma were detected by RT-PCR in the CTLs. Melanoma specific HLADR restricted T helper 1 (Th1) CTLs may play a role in the immunopathogenesis of VKH.

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.