T-cell Antigen Receptor Complex Research Articles

Enhanced cytotoxic CD8+ T cell (CTL) antigen recognition by LCK activation

Abstract The anti-tumor response of cytotoxic T cells requires detection of tumor-derived antigenic peptide-MHC complex (pMHC) by the T cell antigen receptor (TCR). Strategies that boost TCR signaling caused by weak pMHC could augment the recognition of poorly immunogenic tumors by T cells. Because LCK kinase phosphorylates the TCR complex and the recruited ZAP-70 kinase, increased LCK activity has been shown to increase TCR signaling in naïve T cells. Our previous study demonstrated that a small molecule inhibitor of CSK kinase, a negative regulator of LCK, increased LCK kinase activity and sensitized naïve T cells such that they became activated by weak antigens ex vivo. Our goal was to determine whether CD8+ cytotoxic T cells (CTL) could also be sensitized to detect weak antigens, and evaluate whether their capacity to recognize and kill cancer cells could be augmented. Because the Nur77-GFP reporter system responds to TCR-mediated signaling, we used it to determine that LCK activation sensitized OT-1 TCR transgenic CTLs to detect weak (low affinity) antigens displayed by splenocytes. We also observed increased phosphorylation of ZAP-70 and other signaling mediators upon TCR stimulation. We will next evaluate whether CTL-mediated effector functions can be enhanced by monitoring killing of MC38 cancer cells that display a weak antigen. Our findings will inform whether LCK kinase can be targeted to improve the recognition of cancer by T cells.

Mechanisms determining a differential threshold for sensing Src family kinase activity by B and T cell antigen receptors

Although signal transduction by immunoreceptors such as the T cell antigen receptor (TCR), B cell antigen receptor (BCR), and Fc receptors uses the same schematic and similar molecules, the threshold and the fine-tuning are set differently for each receptor. One manifestation of these differences is that inhibition of Src family kinases (SFK) blocks TCR but not BCR signaling. SFKs are key kinases phosphorylating immunoreceptor tyrosine-based activation motifs (ITAM) in both these receptors. However, it has been proposed that in B cells, downstream kinase SYK can phosphorylate ITAM sequences independently of SFK, allowing it to compensate for the loss of SFK activity, whereas its T cell paralog ZAP-70 is not capable of this compensation. To test this proposal, we examined signaling in SYK- and ZAP-70-deficient B and T cell lines expressing SYK or ZAP-70. We also analyzed signal transduction in T cells expressing BCR or B cells expressing part of the TCR complex. We show that when compared with ZAP-70, SYK lowered the threshold for SFK activity necessary to initiate antigen receptor signaling in both T and B cells. However, neither SYK nor ZAP-70 were able to initiate signaling independently of SFK. We further found that additional important factors are involved in setting this threshold. These include differences between the antigen receptor complexes themselves and the spatial separation of the key transmembrane adaptor protein LAT from the TCR. Thus, immunoreceptor sensing of SFK activity is a complex process regulated at multiple levels.

CD45 functions as a signaling gatekeeper in T cells.

T cells require the protein tyrosine phosphatase CD45 to detect and respond to antigen because it activates the Src family kinase Lck, which phosphorylates the T cell antigen receptor (TCR) complex. CD45 activates Lck by opposing the negative regulatory kinase Csk. Paradoxically, CD45 has also been implicated in suppressing TCR signaling by dephosphorylating the same signaling motifs within the TCR complex upon which Lck acts. We sought to reconcile these observations using chemical and genetic perturbations of the Csk/CD45 regulatory axis incorporated with computational analyses. Specifically, we titrated the activities of Csk and CD45 and assessed their influence on Lck activation, TCR-associated ζ-chain phosphorylation, and more downstream signaling events. Acute inhibition of Csk revealed that CD45 suppressed ζ-chain phosphorylation and was necessary for a regulatable pool of active Lck, thereby interconnecting the activating and suppressive roles of CD45 that tune antigen discrimination. CD45 suppressed signaling events that were antigen independent or induced by low-affinity antigen but not those initiated by high-affinity antigen. Together, our findings reveal that CD45 acts as a signaling "gatekeeper," enabling graded signaling outputs while filtering weak or spurious signaling events.

The TCR/CD3 complex in leukemogenesis and as a therapeutic target in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) arises from T cell precursors and is characterized by expression of many lineage-specific proteins. While T-cell antigen receptor (TCR) signaling and its strength are central for thymocyte development, mature T cell homeostasis and immune responses, their roles in T-ALL remain undetermined. Indeed, in contrast to mouse models, in which absence of TCR or major histocompatibility complex binding does not impact on leukemogenesis, other mouse models suggest that basal or weak signaling drives leukemia development. However, recent reports indicate that strong TCR signaling can be detrimental to leukemic cells. Indeed, sustained/high level TCR signaling, stimulated by antigen or CD3 antibody, is strongly anti-leukemic in both murine T-ALL expressing endogenous or transgenic TCR and diagnostic T-ALL cases. As discussed, further work should address the efficacy of T-ALL therapeutic targeting with either TCR/CD3 antibodies or TCR-directed chimeric antigen receptor T cells.

ZAP-70 in Signaling, Biology, and Disease.

T cells possess an array of functional capabilities important for host defense against pathogens and tumors. T cell effector functions require the T cell antigen receptor (TCR). The TCR has no intrinsic enzymatic activity, and thus signal transduction from the receptor relies on additional signaling molecules. One such molecule is the cytoplasmic tyrosine kinase ZAP-70, which associates with the TCR complex and is required for initiating the canonical biochemical signal pathways downstream of the TCR. In this article, we describe recent structure-based insights into the regulation and substrate specificity of ZAP-70, and then we review novel methods for determining the role of ZAP-70 catalytic activity-dependent and -independent signals in developing and mature T cells. Lastly, we discuss the disease states in mouse models and humans, which range from immunodeficiency to autoimmunity, that are caused by mutations in ZAP-70.

TCRα-TCRβ pairing controls recognition of CD1d and directs the development of adipose NKT cells.

The interaction between the T cell antigen receptor (TCR) expressed by natural killer T cells (NKT cells) and the antigen-presenting molecule CD1d is distinct from interactions between the TCR and major histocompatibility complex (MHC). Our molecular modeling suggested that a hydrophobic patch created after TCRα-TCRβ pairing has a role in maintaining the conformation of the NKT cell TCR. Disruption of this patch ablated recognition of CD1d by the NKT cell TCR but not interactions of the TCR with MHC. Partial disruption of the patch, while permissive to the recognition of CD1d, significantly altered NKT cell development, which resulted in the selective accumulation of adipose-tissue-resident NKT cells. These results indicate that a key component of the TCR is essential for the development of a distinct population of NKT cells.

Abstract B042: Regulation of Lck activity by a cryptic phosphosite

Abstract Src family kinases (SFKs) are initiators of receptor-mediated signaling events, enabling cells to perceive and interact with their environment. For immune cells SFKs are especially crucial because they couple antigen recognition to intracellular signaling pathways. In T cells, the SFK Lck phosphorylates the T cell antigen receptor (TCR) complex upon antigen encounter. Because Lck phosphorylates the TCR complex, its activity sets a critical threshold for T cell activation; yet how Lck activity is regulated in resting or stimulated cells is poorly understood. Through quantitative mass spectrometry (SILAC), we recently identified a cryptic Lck phosphosite (Y192) as sensitive to inhibition of the effector kinase Zap70. Because Zap70 is an Lck substrate, and dramatic reduction in phosphorylation of Y192 occurred upon Zap70 inhibition, we reasoned this site might function to regulate Lck in response to changes in Zap70 activity. To test this hypothesis we reconstituted Lck-deficient Jurkat T cells with Lck Y192 variants. Mutation of Y192 reveals that this site has a profound impact on Lck activity. Cells reconstituted with Y192E or Y192A show a marked reduction in Lck activity, characterized by hyperphosphorylation on Lck's inhibitory C-terminus (Y505) and a reduction in Lck autophosphorylation within the activation loop (Y394). Consistent with a defect in Lck activity, cells that express Lck Y192E/A variants, when assessed for hallmarks of T cell activation, reveal striking defects in TCR signaling. Interestingly, these defects are reminiscent of CD45-deficient T cells. Preliminary findings suggest that mutation of Y192 disrupts the ability of the phosphatase CD45 to dephosphorylate Lck's inhibitory C-terminus (Y505). Because of the profound effect that Y192 modification exerts on Lck activity, and therefore TCR signaling, we believe that phosphorylation of Y192 comprises a Zap70-dependent negative feedback mechanism that tunes Lck activity. Such a mechanism predicts that increases in Zap70 activity cause phosphorylation of Y192 within Lck's SH2 domain, favouring Lck inactivation by preventing CD45-mediated dephosphorylation of the inhibitory C-terminus. Citation Format: Adam H. Courtney, Hanna E. Sjolin-Goodfellow, Qinqin Ji, Theresa A. Kadlecek, Arthur R. Salomon, Arthur Weiss. Regulation of Lck activity by a cryptic phosphosite. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B042.

TCR ITAM multiplicity is required for the generation of follicular helper T-cells.

The T-cell antigen receptor (TCR) complex contains 10 copies of a di-tyrosine Immunoreceptor-Tyrosine-based-Activation-Motif (ITAM) that initiates TCR signalling by recruiting protein tyrosine kinases. ITAM multiplicity amplifies TCR signals, but the importance of this capability for T-cell responses remains undefined. Most TCR ITAMs (6 of 10) are contributed by the CD3ζ subunits. We generated ‘knock-in' mice that express non-signalling CD3ζ chains in lieu of wild-type CD3ζ. Here we demonstrate that ITAM multiplicity is important for the development of innate-like T-cells and follicular helper T-cells, events that are known to require strong/sustained TCR–ligand interactions, but is not essential for ‘general' T-cell responses including proliferation and cytokine production or for the generation of a diverse antigen-reactive TCR repertoire.

Reevaluating the biochemical valency of native TCR/CD3 complexes isolated from T cells (IRM7P.708)

Abstract The αβ T cell antigen Receptor (TCR) is a multi-protein complex expressed together with CD3 subunits (γδεζ). The conventional model has it as monovalent with a stochiometry of (αβγδε2ζ2); however, some previously published work reported observation of bivalent complexes with two αβ heterodimers. To reevaluate the valency of the TCR, we have subjected T cell digitonin-solubilized complexes to immunoprecipitation detected by flow cytometry (IP-FCM), size exclusion chromatography, and other methods for detecting complex size and subunit composition. Our results indicate that precisely bivalent TCRs are found among surface-expressed complexes independent of cross-linking by strong antigenic ligands. Their detection can be masked in the presence of Coomassie reagent from Blue-native PAGE conditions, or if antibodies used in detection assays are themselves bivalent. Therefore, bivalent TCR/CD3 complexes can be found under the biochemical conditions that have classically defined the minimally expressed TCR/CD3 unit (digitonin solubilization). Further investigation is required to determine the functional relevance of these bivalent TCR complexes, although greater antigenic binding and signaling is predicted over that of monovalent TCRs.

Detection of constant domain of human T cell antigen receptor alpha-chain via novel monoclonal antibody 7F18.

The αβ T cell antigen receptor (TCR) endows T lymphocytes with immune specificity and controls their effector functions. Each person possesses a vast repertoire of TCRs that is generated by the well-studied processes of somatic recombination and thymic selection. While many antibodies specific for TCRβ variable domains are available, antibodies specific for human TCRα are rare. We now report a novel monoclonal antibody, 7F18, which binds to human TCRα constant region, with specificity for a denatured epitope that can be visualized by SDS-PAGE followed by Western blot. Both immature and mature TCR α-chain products can be visualized, making 7F18 potentially applicable to various biochemical assays of multiprotein complex assembly and maturation. This new monoclonal antibody provides a tool that can potentially facilitate the biochemical analysis of comprehensive populations of human αβ TCR complexes that need not be limited to small subsets of the repertoire.

Toward a Molecular Understanding of Adaptive Immunity: A Chronology, Part III.

Early reports on T cell antigen receptor (TCR) signaling uncovered a rapid increase in intracellular calcium concentration and the activation of calcium-dependent protein kinase as necessary for T cell activation. Cytolytic T cell clones were instrumental in the discovery of intracellular cytolytic granules, and the isolation of the perforin and granzyme molecules as the molecular effectors of cell-mediated lysis of target cells via apoptosis. Cytolytic T cell clones and TCR cDNA clones were also instrumental for the generation of TCR transgenic animals, which provided definitive evidence for negative selection of self-reactive immature thymocytes. In addition, studies of TCR complex signaling of immature thymocytes compared with mature T cells were consistent with the interpretation that negative selection occurs as a consequence of the incapacity of immature cells to produce IL-2, resulting in cytokine deprivation apoptosis. By comparison, taking advantage of cloned TCRs derived from T cell clones reactive with male-specific molecules, using TCR transgenic mice it was possible to document positive selection of female thymocytes when the male-specific molecules were absent. Focusing on the molecular mechanisms of T cell “help” for the generation of antibody-forming cells following the path opened by the elucidation of the IL-2 molecule, several groups were successful in the identification, isolation, and characterization of three new interleukin molecules (IL-4, IL-5, and IL-6) that promote the proliferation and differentiation of B cells. In addition, the identification of a B cell surface molecule (CD40) that augmented B cell antigen receptor-stimulated proliferation and differentiation led to the discovery of a T cell activation surface molecule that proved to be the CD40-ligand, thus finally providing a molecular explanation for “linked or cognate” recognition when T cells and B cells interact physically. Accordingly, the decade after the generation of the first T cell clones saw the elucidation of the molecular mechanisms of T cell cytotoxicity and T cell help, thereby expanding the number of molecules responsible for adaptive T cell immunity.

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications. Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system (1,2). T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells (3,4). T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity (5). The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression (6,7). The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites (8). Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications. Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process (9,10). We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization (11). Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody (8,9), which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

Engagement of T-cell antigen receptor and CD4/CD8 co-receptors induces prolonged STAT activation through autocrine/paracrine stimulation in human primary T cells

Signal transducer and activator of transcription (STAT) proteins are key signaling molecules in response to cytokines and in regulating T cell biology. However, there are contradicting reports on whether STAT is involved in T-cell antigen receptor (TCR) signaling. To better define the role of STAT in TCR signaling, we activated the CD4/CD8-associated Lck kinase by co-crosslinking TCR and CD4/CD8 co-receptors in human peripheral blood T cells. Sequential STAT1, STAT3 and STAT5 activation was observed 1h after TCR stimulation suggesting that STAT proteins are not the immediate targets in the TCR complex. We further identified interferon-γ as the key cytokine in STAT1 activation upon TCR engagement. In contrast to transient STAT activation in cytokine response, this autocrine/paracrine-induced STAT activation was sustained. It correlated with the absence of two suppressors of cytokine signaling (SOCS) proteins, SOCS3 and cytokine-inducible SH2 containing protein that are negative feedback regulators of STAT signaling. Moreover, enforced expression of SOCS3 inhibited tyrosine phosphorylation of zeta-associated protein kinase of 70kD in TCR-stimulated human Jurkat T cells. This is the first report demonstrating delayed and prolonged STAT activation coordinated with the loss of SOCS expression in human primary T cells after co-crosslinking of TCR and CD4/CD8 co-receptors.

Protein kinase D2 has a restricted but critical role in T-cell antigen receptor signalling in mature T-cells

PKD (protein kinase D) 2 is a serine/threonine kinase activated by diacylglycerol in response to engagement of antigen receptors in lymphocytes. To explore PKD2 regulation and function in TCR (T-cell antigen receptor) signal transduction we expressed TCR complexes with fixed affinity for self antigens in the T-cells of PKD2-null mice or mice deficient in PKD2 catalytic activity. We also developed a single cell assay to quantify PKD2 activation as T-cells respond to developmental stimuli or engagement of α/β TCR complexes in vivo. Strikingly, PKD2 loss caused increases in thymic output, lymphadenopathy and splenomegaly in TCR transgenic mice. The precise magnitude and timing of PKD2 activation during T-cell development is thus critical to regulate thymic homoeostasis. PKD2-null T-cells that exit the thymus have a normal transcriptome, but show a limited and abnormal transcriptional response to antigen. Transcriptional profiling reveals the full consequences of PKD2 loss and maps in detail the selective, but critical, function for PKD2 in signalling by α/β mature TCR complexes in peripheral T-cells.

Toward a molecular understanding of adaptive immunity: a chronology – part II

By 1980 it was obvious that to more fully understand adaptive immunity, one needed to somehow reduce the tremendous complexity of antigen recognition by T cell populations. Thus, there were two developments that resulted in a paradigm shift in immunology, one being the generation of monoclonal antibodies (MoAbs), and the other the development of monoclonal functional antigen-specific T cell lines. For the first time, the cellular reagents became available to ask new questions as to how individual cells comprising the complex cell populations recognize and respond to changes in their molecular environments. The first successful generation of monoclonal T cells depended upon the understanding that antigen renders cells responsive to the antigen non-specific T cell growth factor that came to be termed interleukin-2 (IL-2), which could then be used in propagating large numbers of the progeny of single cells, which in turn could then be used for molecular analyses. Monoclonal functional human T cells were used to immunize mice to generate clone-specific (clonotypic) MoAbs, which then permitted the first biochemical characterizations of the antigen recognition elements of the T cell antigen receptor (TCR) complex. Moreover, the use of monoclonal cytolytic and helper/inducer human T cell clones essentially proved that the T cell-specific molecules T4 (CD4) and T8 (CD8) functioned as accessory molecules in antigen recognition by defining MHC class II or class I restriction respectively. As well, the expression of the T3 (CD3) molecules, found to be common to all T cells, were shown further to be obligatory for functional antigen-specific T cell signaling. The monoclonal IL-2-dependent T cells were also instrumental in the isolation and purification of the IL-2 molecule to homogeneity, the first interleukin molecule to be identified and characterized. These advances then led to the generation of pure radiolabeled IL-2 molecules that were used to identify the first interleukin cellular receptors, and as well the generation of the first MoAbs reactive with both IL-2 and IL-2 receptors. All of these advances led subsequently to the isolation of the first cDNA clones recognizing one of the two chains comprising the T cell antigen recognition elements (β-chain), as well cDNA clones encoding IL-2. Accordingly, armed with all of these unique cellular and molecular reagents, it was possible to determine that antigen triggering of the TCR complex initiates IL-2 production and IL-2 receptor expression, which in turn initiate the T cell clonal proliferative expansion, envisioned by Burnet in his formulation of the clonal selection theory. Thus, adaptive immunity receives antigen-specific activation signals from the environment and turns them into antigen non-specific endogenous action signals.

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.

Serine Residues in the Cytosolic Tail of the T-cell Antigen Receptor α-Chain Mediate Ubiquitination and Endoplasmic Reticulum-associated Degradation of the Unassembled Protein

The T-cell antigen receptor (TCR) alpha-chain (TCRalpha) is a type I integral membrane protein that becomes ubiquitinated and targeted to the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway when it fails to assemble into the heteromeric TCR complex. Remarkably, TCRalpha has a cytosolic tail of only five amino acid residues (i.e. RLWSS), none of which is the conventional ubiquitin acceptor, lysine. Herein we report that substitution of two conserved serine residues in the cytosolic tail of TCRalpha to alanine decreased ubiquitination, whereas placement of additional serine residues enhanced it. Moreover, replacement of the cytosolic serine residues by other ubiquitinatable residues (i.e. cysteine, threonine, or lysine) allowed ubiquitination to take place. Serine-dependent ubiquitination perfectly correlated with targeting of TCRalpha for ERAD. We also found that this ubiquitination was mediated by the ER-localized ubiquitin ligase, HRD1. These findings indicate that serine-dependent, HRD1-mediated ubiquitination targets TCRalpha to the ERAD pathway.

ITAM-mediated signaling by the T-cell antigen receptor.

Signal transduction by the T-cell antigen receptor (TCR) is initiated by phosphorylation of conserved motifs (ITAMs) contained within the cytoplasmic domains of the invariant subunits. TCR complexes contain a total of 10 ITAMs and this unusual configuration has prompted studies of the role of specific ITAMs, or of ITAM multiplicity, in regulating TCR-directed developmental and effector responses. Here, we summarize data generated during the past two decades and discuss how these findings have in some cases resolved, and in others complicated, outstanding questions relating to the function of TCR ITAMs.

Stoichiometry and intracellular fate of TRIM-containing TCR complexes.

BackgroundStudying the stoichiometry and intracellular trafficking of the T cell antigen receptor (TCR) is pivotal in understanding its mechanisms of activation. The αβTCR includes the antigen-binding TCRαβ heterodimer as well as the signal transducing CD3εγ, CD3εδ and ζ2 subunits. Although the TCR-interacting molecule (TRIM) is also part of the αβTCR complex, it has not been included in most reports so far.ResultsWe used the native antibody-based mobility shift (NAMOS) assay in a first dimension (1D) blue native (BN)-PAGE and a 2D BN-/BN-PAGE to demonstrate that the stoichiometry of the digitonin-solublized TRIM-containing αβTCR is TCRαβCD3ε2γδζ2TRIM2. Smaller αβTCR complexes possess a TCRαβ CD3ε2γδζ2 stoichiometry. Complexes of these sizes were detected in T cell lines as well as in primary human and mouse T cells. Stimulating the αβTCR with anti-CD3 antibodies, we demonstrate by confocal laser scanning microscopy that CD3ε colocalizes with ζ and both are degraded upon prolonged stimulation, possibly within the lysosomal compartment. In contrast, a substantial fraction of TRIM does not colocalize with ζ. Furthermore, TRIM neither moves to lysosomes nor is degraded. Immunoprecipitation studies and BN-PAGE indicate that TRIM also associates with the γδTCR.ConclusionsSmall αβTCR complexes have a TCRαβ CD3ε2γδζ2 stoichiometry; whereas those associated with one TRIM dimer are TCRαβ CD3ε2γδζ2TRIM2. TRIM is differentially processed compared to CD3 and ζ subunits after T cell activation and is not degraded. The γδTCR also associates with TRIM.