Modification of the T cell antigen receptor (TCR) complex by UDP-glucose:glycoprotein glucosyltransferase. TCR folding is finalized convergent with formation of alpha beta delta epsilon gamma epsilon 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.     

Rescue of tumor-infiltrating lymphocytes from activation-induced cell death enhances the antitumor CTL response in CD5-deficient mice

The CD5 coreceptor is expressed on all T cells and on the B1a B cell subset. It is associated with TCR and BCR, and modulates intracellular signals initiated by both Ag receptor complexes. Human CD5 contributes to regulation of the antitumor immune response and susceptibility of specific CTL to activation-induced cell death (AICD) triggered by the tumor. In this study, we compared the T cell response to the B16F10 melanoma engrafted into CD5-deficient and wild-type C57BL/6 mice. Compared with wild-type mice, CD5 knockout animals displayed delayed tumor growth, associated with tumor infiltration by T cell populations exhibiting a more activated phenotype and enhanced antitumor effector functions. However, control of tumor progression in CD5(-/-) mice was transient due to increased AICD of CD8(+) tumor-infiltrating T lymphocytes. Remarkably, in vivo protection of T cells from TCR-mediated apoptosis by an adenovirus engineered to produce soluble Fas resulted in a dramatic reduction in tumor growth. Our data suggest that recruitment of tumor-specific T cells in the tumor microenvironment occurs at early stages of cancer development and that tumor-mediated AICD of tumor-infiltrating T lymphocytes is most likely involved in tumor escape from the immune system.     

The gamma and epsilon subunits of the CD3 complex inhibit pre-Golgi degradation of newly synthesized T cell antigen receptors

The T cell receptor for antigen (TCR) is composed of six different transmembrane proteins. T cells carefully control the intracellular transport of the receptor and allow only complete receptors to reach the plasma membrane. In an attempt to understand how T cells regulate this process, we used c-DNA transfection and subunit-specific antibodies to follow the intracellular transport of five subunits (alpha beta gamma delta epsilon) of the receptor. In particular, we assessed the intracellular stability of each chain. Our results showed that the chains were markedly different in their susceptibility to intracellular degradation. TCR alpha and beta and CD3 delta were degraded rapidly, whereas CD3 gamma and epsilon were stable. An analysis of the N-linked oligosaccharides of the glycoprotein subunits suggested that the chains were unable to reach the medial Golgi during the metabolic chase. This was supported by immunofluorescence micrographs that showed both the stable CD3 gamma and unstable CD3 delta chain localized in the endoplasmic reticulum. To study the effects of subunit associations on intracellular transport we used cotransfection to reconstitute precise combinations of subunits. Associations between stable and unstable subunits expressed in the same cell led to the formation of stable complexes. These complexes were retained in or close to the endoplasmic reticulum. The results suggested that the intracellular transport of the T cell receptor could be regulated by two mechanisms. The TCR alpha and beta and CD3 delta subunits were degraded rapidly and as a consequence failed to reach the plasma membrane. CD3 gamma or epsilon were stable but were retained inside the cell. The results also demonstrated that there was an interplay between the two pathways such that the CD3 gamma and epsilon subunits were able to protect labile chains from rapid intracellular degradation. In this way, they could seed subunit assembly in or close to the endoplasmic reticulum and allow a stable receptor to form before its transport to the plasma membrane.     

Self-tolerance checkpoints in CD4 T cells specific for a peptide derived from the B cell antigen receptor

Linked recognition of Ag by B and T lymphocytes is ensured in part by a state of tolerance acquired by CD4 T cells to germline-encoded sequences within the B cell Ag receptor (BCR). We sought to determine how such tolerance is attained when a peptide from the BCR variable (V) region is expressed by small numbers of B cells as it is in the physiological state. Mixed bone marrow (BM) chimeras were generated using donor BM from mice with B cells that expressed a transgene (Tg)-encoded κ L chain and BM from TCR Tg mice in which the CD4 T cells (CA30) were specific for a Vκ peptide encoded by the κTg. In chimeras where few B cells express the κTg, many CA30 cells were deleted in the thymus. However, a substantial fraction survived to the CD4 single-positive stage. Among single-positive CA30 thymocytes, few reached maturity and migrated to the periphery. Maturation was strongly associated with, and likely promoted by, expression of an endogenous TCR α-chain. CD4(+) CA30 cells that reached peripheral lymphoid tissues were Ag-experienced and anergic, and some developed into regulatory cells. These findings reveal several checkpoints and mechanisms that enforce a state of self-tolerance in developing T cells specific for BCR V region sequences, thus ensuring that T cell help to B cells occurs through linked recognition of foreign Ag.     

Analysis of T cell receptor transcripts using the polymerase chain reaction

The immune system is composed of two major types of lymphocytes, called B and T cells, that recognize foreign antigens. Recognition of antigens is accomplished through the generation of a large repertoire of different cell surface receptors, called immunoglobulins (Igs) on B cells and T cell receptors (TCRs) on T cells. The elucidation of Ig structure and molecular genetics preceded that of the TCR because of the greater abundance of Ig protein and mRNA. Although studies of TCRs have recently shed light on many of the issues of T cell recognition, the process of examining TCR gene structure has been tedious. Such analyses are also difficult because of the time required for the production, maintenance, and culturing of T cell clones. This report describes several strategies that use the polymerase chain reaction (PCR) to analyze very rapidly the structure of TCRs. Specific manipulations of the amplified material are discussed, as are the advantages of using the PCR to study TCR diversity.     

Assembly of the human T cell receptor-CD3 complex takes place in the endoplasmic reticulum and involves intermediary complexes between the CD3-gamma.delta.epsilon core and single T cell receptor alpha or beta chains

Functionally mature human T lymphocytes express a cell-surface receptor for antigen (T cell receptor (TCR)-CD3) composed of at least six polypeptides (TCR-alpha and -beta; T3-gamma, -delta, -epsilon, and -zeta). Immature thymocytes and variants of T cell lines lacking one of the TCR.CD3 polypeptide chains fail to express surface receptor and accumulate the other chains intracellularly. Here we show that the assembly of the TCR.CD3 complex within the endoplasmic reticulum (ER) began with a core of CD3-gamma, -delta, and -epsilon to which TCR-alpha and -beta bound. A recently described intracellular protein, CD3-omega, participated in the assembly since it was found to be associated with the free TCR-alpha or -beta chains or with the CD3 chains. CD3-omega dissociated as TCR.CD3 complexes were formed in the ER. Association of non-disulfide-linked TCR-alpha and -beta chains with CD3 was detected before that of disulfide-bridged TCR-alpha/beta heterodimers. These data suggest that during assembly, the association of TCR-alpha and -beta chains with the CD3 complex precedes the formation of a TCR-alpha/beta dimer. The existence of intermediates consisting of CD3-gamma, -delta, and -epsilon chains and a single TCR-alpha or -beta chain was also confirmed by using a series of variant T cell lines lacking the TCR-beta or -alpha chain, respectively. Once the single TCR-alpha and -beta chains were associated with CD3, disulfide linkages were formed, and a 70-kDa form of the TCR was detected within the ER. This intracellular precursor of the TCR.CD3 complex was subsequently processed into the mature 90-kDa TCR as the TCR.CD3 complex passed through the Golgi apparatus. Assembly of the TCR.CD3 complex is a rather rapid process, whereas export from the ER occurs at a slow rate. After 1 h, 75% of the receptor complex remained within the ER.     

Prolonged antigen persistence within nonterminal late endocytic compartments of antigen-specific B lymphocytes

Although Ag-specific B lymphocytes can process Ag and express peptide-class II complexes as little as 1 h after Ag exposure, it requires 3-5 days for the immune system to develop a population of Ag-specific effector CD4 T lymphocytes to interact with these complexes. Presently, it is unclear how B cells maintain the expression of cell surface antigenic peptide-class II complexes until effector CD4 T lymphocytes become available. Therefore, we investigated B cell receptor (BCR)-mediated Ag processing and presentation by normal B lymphocytes to determine whether these cells have a mechanism to prolong the cell surface expression of peptide-class II complexes derived from the processing of cognate AG: Interestingly, after transit of early endocytic compartments, internalized Ag-BCR complexes are delivered to nonterminal late endosomes where they persist for a prolonged period of time. In contrast, Ags internalized via fluid phase endocytosis are rapidly delivered to terminal lysosomes and degraded. Moreover, persisting Ag-BCR complexes within nonterminal late endosomes exhibit a higher degree of colocalization with the class II chaperone HLA-DM/H2-M than with the HLA-DM/H2-M regulator HLA-DO/H2-O. Finally, B cells harboring persistent Ag-BCR complexes exhibit prolonged cell surface expression of antigenic peptide-class II complexes. These results demonstrate that B lymphocytes possess a mechanism for prolonging the intracellular persistence of Ag-BCR complexes within nonterminal late endosomes and suggest that this intracellular Ag persistence allows for the prolonged cell surface expression of peptide-class II complexes derived from the processing of specific AG:     

The immunological synapse: the more you look the less you know..

Before T cells of the immune system can recognize pathogens, antigen presenting cells (APCs) must process pathogen-derived peptides and present them together with major histocompatibility complex molecules (MHC) to T lymphocytes. T lymphocytes then scan the surface of APCs and antigen-specific activation of the T cell will happen after interaction of T cell antigen receptor (TCR) with MHC-peptide complexes expressed at the membrane of APCs. This interaction takes place in a nanometer scale gap between the two cells, referred to as an immunological synapse. Recent three-dimensional fluorescence analysis of this synapse revealed a dynamic spatial organization of membrane receptors, cytoskeleton and intracellular signaling complexes on the T cell side showing specific patterns, which depend on the nature of the T cell:APC pair. Although it is obvious that establishment of an intimate contact between T cells and APCs will facilitate cell:cell communication it is not clear what is the role, if any, of this receptors patterning. This molecular reorganization has long been thought to enhance and/or sustain TCR signaling and thus T cell activation, but this is now a matter of controversy. Moreover, mechanisms controlling immunological synapse formation are still unraveled. Segregation of proteins may occur spontaneously as proposed by mathematical modeling taking into account membrane fluidity, protein size and receptor/ligand affinity. Alternatively patterning of the molecules at the cell:cell interface could be driven by active processes involving T cell signaling and/or specific features of the APC. These different questions will be discussed herein.     

Mammalian T-lymphocyte antigen receptor genes: genetic and nongenetic potential to generate variability

Summary T lymphocytes of higher vertebrates are able to specifically recognize a seemingly unlimited number of foreign antigens via their receptors, the T cell antigen receptors (TCRs). T lymphocytes mature by passing through the thymus and acquire antigen specificity by expressing the TCR molecules on their cell surface. Genetic and somatic diversification mechanisms give rise to the enormous degree of TCR variability observed in mature T cells: germline and combinatorial diversity as well as junctional and the so-called N-region diversity. In contrast to the situation in immunoglobulin genes somatic hypermutation does not seem to play a significant role in TCR diversification. It is argued here that the enzyme terminal nucleotidyl-transferase is potentially a major factor in generating the immense diversity. We propose furthermore that this enzyme ensures the flexibility of T cell responses to novel antigens by random insertion of so-called N-region nucleotides. Apart from the physiological functions of TCR genes any involvement in the etiology of T cell neoplasia remains to be proven.     

Antigen-B Cell Receptor Complexes Associate with Intracellular major histocompatibility complex (MHC) Class II Molecules

Antigen processing and MHC class II-restricted antigen presentation by antigen-presenting cells such as dendritic cells and B cells allows the activation of naïve CD4+ T cells and cognate interactions between B cells and effector CD4+ T cells, respectively. B cells are unique among class II-restricted antigen-presenting cells in that they have a clonally restricted antigen-specific receptor, the B cell receptor (BCR), which allows the cell to recognize and respond to trace amounts of foreign antigen present in a sea of self-antigens. Moreover, engagement of peptide-class II complexes formed via BCR-mediated processing of cognate antigen has been shown to result in a unique pattern of B cell activation. Using a combined biochemical and imaging/FRET approach, we establish that internalized antigen-BCR complexes associate with intracellular class II molecules. We demonstrate that the M1-paired MHC class II conformer, shown previously to be critical for CD4 T cell activation, is incorporated selectively into these complexes and loaded selectively with peptide derived from BCR-internalized cognate antigen. These results demonstrate that, in B cells, internalized antigen-BCR complexes associate with intracellular MHC class II molecules, potentially defining a site of class II peptide acquisition, and reveal a selective role for the M1-paired class II conformer in the presentation of cognate antigen. These findings provide key insights into the molecular mechanisms used by B cells to control the source of peptides charged onto class II molecules, allowing the immune system to mount an antibody response focused on BCR-reactive cognate antigen.     

An inositol 1,4,5-trisphosphate receptor-dependent cation entry pathway in DT40 B lymphocytes

We examined the roles of inositol 1,4,5-trisphosphate (IP3) receptors (IP3R) in calcium signaling using DT40 B lymphocytes, and a variant lacking the three IP3R isoforms (IP3R-KO). In wild-type cells, B cell receptor (BCR) stimulation activates a cation entry route that exhibits significantly greater permeability to Ba2+ than does capacitative calcium entry. This cation entry is absent in IP3R-KO cells. Expression of the type-3 IP3R (IP3R-3) in the IP3R-KO cells rescued not only agonist-dependent release of intracellular Ca2+, but also Ba2+ influx following receptor stimulation. Similar results were obtained with an IP3R-3 mutant carrying a conservative point mutation in the selectivity filter region of the channel (D2477E); however, an IP3R-3 mutant in which this same aspartate was replaced by alanine (D2477A) failed to restore either BCR-induced Ca2+ release or receptor-dependent Ba2+ entry. These results suggest that in DT40 B lymphocytes, BCR stimulation activates a novel cation entry across the plasma membrane that depends upon, or is mediated by, fully functional IP3R.     

The pathway of antigen uptake and processing dictates MHC class II-mediated B cell survival and activation

The influence of the pathway of Ag uptake and processing on MHC class II (CII)-mediated B cell function is unknown. In this study, we investigate in resting and activated (via the BCR or CD40) B cells the biological properties of CII-peptide complexes (CII-peptide) generated by either the BCR-mediated Ag processing (type I complex) or fluid phase Ag processing (type II complex). Compared with type I complex, ligation of type II complex by either specific Ab or the TCR in Ag-presenting assay results in significant decreases in B cell survival rate (50-100%) and expression levels of CII, CD86, and CD54. Loss of B cells following ligation of type II complex occurs in the presence of a comparatively good level of specific CD4(+) T cell division, indicating that B cell loss is a late event following T cell stimulation. Comparative analysis of T and B cell conjugates after Ab ligation of type I or II complex reveals decreased efficiency of the latter in forming conjugates. Neither initial differential levels of CII and other studied surface markers, B cell type inherent differences, BCR signaling, T cell proliferation, nor initial density of CII-peptide complexes could explain the T cell-induced B cell loss. We propose that the context in which CII-peptide complexes are present in the membrane following BCR uptake and processing leads to B cell survival. Thus, appropriate targeting of Ag ensures generation of relevant immune responses.     

T cell receptor assembly and expression in the absence of calnexin

Most subunits of the alphabeta deltaepsilon gammaepsilon zetazeta T cell antigen receptor (TCR) complex associate with the molecular chaperone calnexin shortly after their synthesis in the endoplasmic reticulum, including clonotypic TCRalpha,beta molecules and invariant CD3gamma,delta,epsilon chains. While calnexin interaction is suggested to be important for the stability of newly synthesized TCRalpha 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.NK(R). We found that CEM and CEM.NK(R) cells constitutively synthesized all TCR subunits except for TCRalpha and that CD3gamma,delta,epsilon components and CD3-beta complexes were effectively assembled together in both cell types. The stability and folding of core CD3epsilon chains were similar in CEM and CEM.NK(R) cells. Interestingly, TCRalpha synthesis was differentially induced by phorbol myristate acetate treatment in CEM and CEM.NK(R) cells and TCRalpha proteins synthesized in CEM.NK(R) cells showed reduced survival compared to those made in CEM cells. Importantly, these data show that TCR complexes were inducibly expressed on CEM.NK(R) 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 TCRalpha proteins.     

Non-genomic oestrogen receptor signal in B lymphocytes: An approach towards therapeutic interventions for infection,autoimmunity and cancer

The non-genomic membrane bound oestrogen receptor (mER) regulates intracellular signals through receptor-ligand interactions. The mER, along with G-protein coupled oestrogen receptor GPR 30 (GPER), induces diverse cell signalling pathways in murine lymphocytes. The mER isoform ER-alpha46 has recently been demonstrated in human B and T lymphocytes as an analogue receptor for chemokine CCL18, the signalling events of which are not clearly understood. Ligand-induced mER and GPER signalling events are shared with BCR, CD19 mediated intracellular signalling through phospholipase C, PIP2/IP3/PI3 mediated activation of Akt, MAP kinase, and mTOR. Oestrogen has the ability to induce CD40-mediated activation of B cells. The complete signalling pathways of mER, GPR30 and their interaction with other signals are targeted areas for novel drug development in B cells during infection, autoimmunity and cancer. Therefore, an in depth investigation is critical for determining shared signal outputs during B cell activation. Here, we focus on the mode of action of membrane bound ER in B cells as therapeutic checkpoints.     

T cells deficient in inositol 1,4,5-trisphosphate receptor are resistant to apoptosis.

The type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) calcium release channel is present on the endoplasmic reticulum of most cell types. T lymphocytes which have been made deficient in IP3R1 lack detectable IP3-induced intracellular calcium release and exhibit defective signaling via the T-cell receptor (TCR) (T. Jayaraman, E. Ondriasova, K. Ondrias, D. Harnick, and A. R. Marks, Proc. Natl. Acad. Sci. USA 92:6007-6011, 1995). We now show that IP3R1-deficient T cells are resistant to apoptosis induced by dexamethasone, TCR stimulation, ionizing radiation, and Fas. Resistance to TCR-mediated apoptosis in IP3R1-deficient cells is reversed by pharmacologically raising cytoplasmic calcium levels. TCR-mediated apoptosis can be induced in calcium-free media, indicating that extracellular calcium influx is not required. These findings suggest that intracellular calcium release via the IP3R1 is a critical mediator of apoptosis.     

Large‐scale proteomic analysis of tyrosine‐phosphorylation induced by T‐cell receptor or B‐cell receptor activation reveals new signaling pathways

Activation of the T‐cell receptor (TCR) and that of the B‐cell receptor (BCR) elicits tyrosine‐phosphorylation of proteins that belongs to similar functional categories, but result in distinct cellular responses. Large‐scale analyses providing an overview of the signaling pathways downstream of TCR or BCR have not been described, so it has been unclear what components of these pathways are shared and which are specific. We have now performed a systematic analysis and provide a comprehensive list of tyrosine‐phosphorylated proteins (PY proteome) with quantitative data on their abundance in T cell, B cell, and nonlymphoid cell lines. Our results led to the identification of novel tyrosine‐phosphorylated proteins and signaling pathways not previously implicated in immunoreceptor signal transduction, such as clathrin, zonula occludens 2, eukaryotic translation initiation factor 3, and RhoH, suggesting that TCR or BCR signaling may be linked to downstream processes such as endocytosis, cell adhesion, and translation. Thus comparative and quantitative studies of tyrosine‐phosphorylation have the potential to expand knowledge of signaling networks and to promote understanding of signal transduction at the system level.     

Developmental regulation of αβ T cell antigen receptor assembly in immature CD4+CD8+ thymocytes

Most lymphocytes of the T cell lineage develop along the CD4/CD8 pathway and express antigen receptors on their surfaces consisting of clonotypic αβ chains associated with invariant CD3-γδε components and ζ chains, collectively referred to as the T cell antigen receptor complex (TCR). Expression of the TCR complex is dynamically regulated during T cell development, with immature CD4⁺CD8⁺ thymocytes expressing only 10% of the number of αβ TCR complexes on their surfaces expressed by mature CD4⁺ and CD8⁺ T cells. Recent evidence demonstrates that low surface TCR density on CD4⁺CD8⁺ thymocytes results from the limited survival of a single TCR component within the ER, the TCRα chain, which has a half life of only 15 minutes in immature thymocytes, compared to >75 minutes in mature T cells. Instability of TCRα proteins in immature CD4⁺CD8⁺ thymocytes represents a novel mechanism by which expression of the multisubunit TCR complex is quantitatively regulated during T cell development. In the current review we discuss our recent findings concerning the assembly, intracellular transport, and expression of αβ TCR complexes in CD4⁺CD8⁺ thymocytes and comment on the functional significance of TCRα instability during T cell development.