A Novel Flow Cytometric High Throughput Assay for a Systematic Study on Molecular Mechanisms Underlying T Cell Receptor-Mediated Integrin Activation

Lymphocyte function-associated antigen 1 (LFA-1), a member of β2-integrin family, exerts multiple roles in host T cell immunity and has been identified as a useful drug-development target for inflammatory and autoimmune diseases. Applying the findings that primary resting T cells absorb nanometric membrane vesicles derived from antigen presenting cells (APC) via dual receptor/ligand interactions of T cell receptor (TCR) with cognate peptide-major histocompatibility complex (MHC) complex (pMHC) and LFA-1 with its ligand, intercellular adhesion molecule-1 (ICAM-1), and that signaling cascades triggered by TCR/pMHC interaction take a part in the vesicle-absorption, we established a cell-based high throughput assay for systematic investigation, via isolation of small molecules modulating the level of vesicle-absorption, of molecular mechanisms underlying the T cell absorption of APC-derived vesicles, i.e., structural basis of TCR/pMHC and LFA-1/ICAM-1 interactions and TCR-mediated LFA-1 activation. As primary T cells along with physiological ligands expressed in biological membrane are used and also individual cells in assay samples are analyzed by flow cytometry, results obtained using the assay system hold superior physiological and therapeutic relevance as well as statistical precision.     

T Cell Receptor Engagement Triggers Its CD3ε and CD3ζ Subunits to Adopt a Compact,Locked Conformation

How the T cell antigen receptor (TCR) discriminates between molecularly related peptide/Major Histocompatibility Complex (pMHC) ligands and converts this information into different possible signaling outcomes is still not understood. One current model proposes that strong pMHC ligands, but not weak ones, induce a conformational change in the TCR. Evidence supporting this comes from a pull-down assay that detects ligand-induced binding of the TCR to the N-terminal SH3 domain of the adapter protein Nck, and also from studies with a neoepitope-specific antibody. Both methods rely on the exposure of a polyproline sequence in the CD3ε subunit of the TCR, and neither indicates whether the conformational change is transmitted to other CD3 subunits. Using a protease-sensitivity assay, we now show that the cytoplasmic tails of CD3ε and CD3ζ subunits become fully protected from degradation upon TCR triggering. These results suggest that the TCR conformational change is transmitted to the tails of CD3ε and CD3ζ, and perhaps all CD3 subunits. Furthermore, the resistance to protease digestion suggests that CD3 cytoplasmic tails adopt a compact structure in the triggered TCR. These results are consistent with a model in which transduction of the conformational change induced upon TCR triggering promotes condensation and shielding of the CD3 cytoplasmic tails.     

The ���� T Cell Receptor Is an Anisotropic Mechanosensor

Thymus-derived lymphocytes protect mammalian hosts against virus- or cancer-related cellular alterations through immune surveillance, eliminating diseased cells. In this process, T cell receptors (TCRs) mediate both recognition and T cell activation via their dimeric αβ, CD3ϵγ, CD3ϵδ, and CD3ζζ subunits using an unknown structural mechanism. Here, site-specific binding topology of anti-CD3 monoclonal antibodies (mAbs) and dynamic TCR quaternary change provide key clues. Agonist mAbs footprint to the membrane distal CD3ϵ lobe that they approach diagonally, adjacent to the lever-like Cβ FG loop that facilitates antigen (pMHC)-triggered activation. In contrast, a non-agonist mAb binds to the cleft between CD3ϵ and CD3γ in a perpendicular mode and is stimulatory only subsequent to an external tangential but not a normal force (∼50 piconewtons) applied via optical tweezers. Specific pMHC but not irrelevant pMHC activates a T cell upon application of a similar force. These findings suggest that the TCR is an anisotropic mechanosensor, converting mechanical energy into a biochemical signal upon specific pMHC ligation during immune surveillance. Activating anti-CD3 mAbs mimic this force via their intrinsic binding mode. A common TCR quaternary change rather than conformational alterations can better facilitate structural signal initiation, given the vast array of TCRs and their specific pMHC ligands.     

Regulation and Function of the CD3γ DxxxLL Motif: A Binding Site for Adaptor Protein-1 and Adaptor Protein-2 in Vitro

Several receptors are downregulated by internalization after ligand binding. Regulation of T cell receptor (TCR) expression is an important step in T cell activation, desensitization, and tolerance induction. One way T cells regulate TCR expression is by phosphorylation/dephosphorylation of the TCR subunit clusters of differentiation (CD)3γ. Thus, phosphorylation of CD3γ serine 126 (S126) causes a downregulation of the TCR. In this study, we have analyzed the CD3γ internalization motif in three different systems in parallel: in the context of the complete multimeric TCR; in monomeric CD4/CD3γ chimeras; and in vitro by binding CD3γ peptides to clathrin-coated vesicle adaptor proteins (APs). We find that the CD3γ D¹²⁷xxxLL^131/132 sequence represents one united motif for binding of both AP-1 and AP-2, and that this motif functions as an active sorting motif in monomeric CD4/ CD3γ molecules independently of S126. An acidic amino acid is required at position 127 and a leucine (L) is required at position 131, whereas the requirements for position 132 are more relaxed. The spacing between aspartic acid 127 (D127) and L131 is crucial for the function of the motif in vivo and for AP binding in vitro. Furthermore, we provide evidence indicating that phosphorylation of CD3γ S126 in the context of the complete TCR induces a conformational change that exposes the DxxxLL sequence for AP binding. Exposure of the DxxxLL motif causes an increase in the TCR internalization rate and we demonstrate that this leads to an impairment of TCR signaling. On the basis of the present results, we propose the existence of at least three different types of L-based receptor sorting motifs.     

Coronin-1A Links Cytoskeleton Dynamics to TCRαβ-Induced Cell Signaling

Actin polymerization plays a critical role in activated T lymphocytes both in regulating T cell receptor (TCR)-induced immunological synapse (IS) formation and signaling. Using gene targeting, we demonstrate that the hematopoietic specific, actin- and Arp2/3 complex-binding protein coronin-1A contributes to both processes. Coronin-1A-deficient mice specifically showed alterations in terminal development and the survival of αβT cells, together with defects in cell activation and cytokine production following TCR triggering. The mutant T cells further displayed excessive accumulation yet reduced dynamics of F-actin and the WASP-Arp2/3 machinery at the IS, correlating with extended cell-cell contact. Cell signaling was also affected with the basal activation of the stress kinases sAPK/JNK1/2; and deficits in TCR-induced Ca²⁺ influx and phosphorylation and degradation of the inhibitor of NF-κB (IκB). Coronin-1A therefore links cytoskeleton plasticity with the functioning of discrete TCR signaling components. This function may be required to adjust TCR responses to selecting ligands accounting in part for the homeostasis defect that impacts αβT cells in coronin-1A deficient mice, with the exclusion of other lympho/hematopoietic lineages.     

Broad T-Cell Receptor Repertoire in T-Lymphocytes Derived from Human Induced Pluripotent Stem Cells

Human induced pluripotent stem cells (hiPSCs) have enormous potential for the treatment of inherited and acquired disorders. Recently, antigen-specific T lymphocytes derived from hiPSCs have been reported. However, T lymphocyte populations with broad T cell receptor (TCR) diversity have not been generated. We report that hiPSCs derived from skin biopsy are capable of producing T lymphocyte populations with a broad TCR repertoire. In vitro T cell differentiation follows a similar developmental program as observed in vivo, indicated by sequential expression of CD7, intracellular CD3 and surface CD3. The γδ TCR locus is rearranged first and is followed by rearrangement of the αβ locus. Both γδ and αβ T cells display a diverse TCR repertoire. Upon activation, the cells express CD25, CD69, cytokines (TNF-α, IFN-γ, IL-2) and cytolytic proteins (Perforin and Granzyme-B). These results suggest that most, if not all, mechanisms required to generate functional T cells with a broad TCR repertoire are intact in our in vitro differentiation protocol. These data provide a foundation for production of patient-specific T cells for the treatment of acquired or inherited immune disorders and for cancer immunotherapy.     

CD8+ T cell activation is governed by TCR-peptide/MHC affinity, not dissociation rate

Binding of peptide/MHC (pMHC) complexes by TCR initiates T cell activation. Despite long interest, the exact relationship between the biochemistry of TCR/pMHC interaction (particularly TCR affinity or ligand off-rate) and T cell responses remains unresolved, because the number of complexes examined in each independent system has been too small to draw a definitive conclusion. To test the current models of T cell activation, we have analyzed the interactions between the mouse P14 TCR and a set of altered peptides based on the lymphocytic choriomeningitis virus epitope gp33-41 sequence bound to mouse class I MHC D(b). pMHC binding, TCR-binding characteristics, CD8+ T cell cytotoxicity, and IFN-gamma production were measured for the peptides. We found affinity correlated well with both cytotoxicity and IFN-gamma production. In contrast, no correlation was observed between any kinetic parameter of TCR-pMHC interaction and cytotoxicity or IFN-gamma production. This study strongly argues for an affinity threshold model of T cell activation.     

Viral Escape Mutant Epitope Maintains TCR Affinity for Antigen yet Curtails CD8 T Cell Responses

T cells have the remarkable ability to recognize antigen with great specificity and in turn mount an appropriate and robust immune response. Critical to this process is the initial T cell antigen recognition and subsequent signal transduction events. This antigen recognition can be modulated at the site of TCR interaction with peptide:major histocompatibility (pMHC) or peptide interaction with the MHC molecule. Both events could have a range of effects on T cell fate. Though responses to antigens that bind sub-optimally to TCR, known as altered peptide ligands (APL), have been studied extensively, the impact of disrupting antigen binding to MHC has been highlighted to a lesser extent and is usually considered to result in complete loss of epitope recognition. Here we present a model of viral evasion from CD8 T cell immuno-surveillance by a lymphocytic choriomeningitis virus (LCMV) escape mutant with an epitope for which TCR affinity for pMHC remains high but where the antigenic peptide binds sub optimally to MHC. Despite high TCR affinity for variant epitope, levels of interferon regulatory factor-4 (IRF4) are not sustained in response to the variant indicating differences in perceived TCR signal strength. The CD8+ T cell response to the variant epitope is characterized by early proliferation and up-regulation of activation markers. Interestingly, this response is not maintained and is characterized by a lack in IL-2 and IFNγ production, increased apoptosis and an abrogated glycolytic response. We show that disrupting the stability of peptide in MHC can effectively disrupt TCR signal strength despite unchanged affinity for TCR and can significantly impact the CD8+ T cell response to a viral escape mutant.     

Transfer of mRNA Encoding Invariant NKT Cell Receptors Imparts Glycolipid Specific Responses to T Cells and γδT Cells

Cell-based therapies using genetically engineered lymphocytes expressing antigen-specific T cell receptors (TCRs) hold promise for the treatment of several types of cancers. Almost all studies using this modality have focused on transfer of TCR from CD8 cytotoxic T lymphocytes (CTLs). The transfer of TCR from innate lymphocytes to other lymphocytes has not been studied. In the current study, innate and adaptive lymphocytes were transfected with the human NKT cell-derived TCRα and β chain mRNA (the Vα24 and Vβ11 TCR chains). When primary T cells transfected with NKT cell-derived TCR were subsequently stimulated with the NKT ligand, α-galactosylceramide (α-GalCer), they secreted IFN-γ in a ligand-specific manner. Furthermore when γδT cells were transfected with NKT cell-derived TCR mRNA, they demonstrated enhanced proliferation, IFN-γ production and antitumor effects after α-GalCer stimulation as compared to parental γδT cells. Importantly, NKT cell TCR-transfected γδT cells responded to both NKT cell and γδT cell ligands, rendering them bi-potential innate lymphocytes. Because NKT cell receptors are unique and universal invariant receptors in humans, the TCR chains do not yield mispaired receptors with endogenous TCR α and β chains after the transfection. The transfection of NKT cell TCR has the potential to be a new approach to tumor immunotherapy in patients with various types of cancer.     

Constitutively Oxidized CXXC Motifs within the CD3 Heterodimeric Ectodomains of the T Cell Receptor Complex Enforce the Conformation of Juxtaposed Segments

The CD3ϵγ and CD3ϵδ heterodimers along with the CD3ζζ homodimer are the signaling components of the T cell receptor (TCR). These invariant dimers are non-covalently associated on the T cell plasma membrane with a clone-specific (i.e. clonotypic) αβ heterodimer that binds its cognate ligand, a complex between a particular antigenic peptide, and an MHC molecule (pMHC). These four TCR dimers exist in a 1:1:1:1 stoichiometry. At the junction between the extracellular and transmembrane domains of each mammalian CD3ϵ, CD3γ, and CD3δ subunit is a highly conserved CXXC motif previously found to be important for thymocyte and T cell activation. The redox state of each CXXC motif is presently unknown. Here we show using LC-MS and a biotin switch assay that these CXXC segments are constitutively oxidized on resting and activated T cells, consistent with their measured reduction potential. NMR chemical shift perturbation experiments comparing a native oxidized CD3δ CXXC-containing segment with that of a mutant SXXS-containing CD3δ segment in LPPG (1-palmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt)) micelles show extensive chemical shift differences in residues within the membrane-proximal motif as well as throughout the transmembrane and cytoplasmic domains as a result of the elimination of the native disulfide. Likewise, direct comparison of the native CD3δ segment in oxidizing and reducing conditions reveals numerous spectral differences. The oxidized CXXC maintains the structure within the membrane-proximal stalk region as well as that of its contiguous transmembrane and cytoplasmic domain, inclusive of the ITAM (immunoreceptor tyrosine-based activation motif) involved in signaling. These results suggest that preservation of the CD3 CXXC oxidized state may be essential for TCR mechanotransduction.     

Interplay between TCR affinity and necessity of coreceptor ligation: high-affinity peptide-MHC/TCR interaction overcomes lack of CD8 engagement

CD8 engagement is believed to be a critical event in the activation of naive T cells. In this communication, we address the effects of peptide-MHC (pMHC)/TCR affinity on the necessity of CD8 engagement in T cell activation of primary naive cells. Using two peptides with different measured avidities for the same pMHC-TCR complex, we compared biochemical affinity of pMHC/TCR and the cell surface binding avidity of pMHC/TCR with and without CD8 engagement. We compared early signaling events and later functional activity of naive T cells in the same manner. Although early signaling events are altered, we find that high-affinity pMHC/TCR interactions can overcome the need for CD8 engagement for proliferation and CTL function. An integrated signal over time allows T cell activation with a high-affinity ligand in the absence of CD8 engagement.     

Bi-Allelic TCRα or β Recombination Enhances T Cell Development but Is Dispensable for Antigen Responses and Experimental Autoimmune Encephalomyelitis

Dual TCRα-expressing T cells outnumber dual TCRβ-expressing cells by ~10:1. As a result, efforts to understand how dual TCR T cells impact immunity have focused on dual TCRα expression; dual TCRβ expression remains understudied. We recently demonstrated, however, that dual TCRβ expression accelerated disease in a TCR transgenic model of autoimmune arthritis through enhanced positive selection efficiency, indicating that dual TCRβ expression, though rare, can impact thymic selection. Here we generated mice hemizygous for TCRα, TCRβ, or both on the C57BL/6 background to investigate the impact bi-allelic TCR chain recombination has on T cell development, repertoire diversity, and autoimmunity. Lack of bi-allelic TCRα or TCRβ recombination reduced αβ thymocyte development efficiency, and the absence of bi-allelic TCRβ recombination promoted γδ T cell development. However, we observed no differences in the numbers of naïve and expanded antigen-specific T cells between TCRα^+/-β^+/- and wildtype mice, and TCR repertoire analysis revealed only subtle differences in Vβ gene usage. Finally, the absence of dual TCR T cells did not impact induced experimental autoimmune encephalomyelitis pathogenesis. Thus, despite more stringent allelic exclusion of TCRβ relative to TCRα, bi-allelic TCRβ expression can measurably impact thymocyte development and is necessary for maintaining normal αβ/γδ T cell proportions.     

TCR Triggering by pMHC Ligands Tethered on Surfaces via Poly(Ethylene Glycol) Depends on Polymer Length

Antigen recognition by T cells relies on the interaction between T cell receptor (TCR) and peptide-major histocompatibility complex (pMHC) at the interface between the T cell and the antigen presenting cell (APC). The pMHC-TCR interaction is two-dimensional (2D), in that both the ligand and receptor are membrane-anchored and their movement is limited to 2D diffusion. The 2D nature of the interaction is critical for the ability of pMHC ligands to trigger TCR. The exact properties of the 2D pMHC-TCR interaction that enable TCR triggering, however, are not fully understood. Here, we altered the 2D pMHC-TCR interaction by tethering pMHC ligands to a rigid plastic surface with flexible poly(ethylene glycol) (PEG) polymers of different lengths, thereby gradually increasing the ligands’ range of motion in the third dimension. We found that pMHC ligands tethered by PEG linkers with long contour length were capable of activating T cells. Shorter PEG linkers, however, triggered TCR more efficiently. Molecular dynamics simulation suggested that shorter PEGs exhibit faster TCR binding on-rates and off-rates. Our findings indicate that TCR signaling can be triggered by surface-tethered pMHC ligands within a defined 3D range of motion, and that fast binding rates lead to higher TCR triggering efficiency. These observations are consistent with a model of TCR triggering that incorporates the dynamic interaction between T cell and antigen-presenting cell.     

Human and Mouse Type I Natural Killer T Cell Antigen Receptors Exhibit Different Fine Specificities for CD1d-Antigen Complex

Human and mouse type I natural killer T (NKT) cells respond to a variety of CD1d-restricted glycolipid antigens (Ags), with their NKT cell antigen receptors (NKT TCRs) exhibiting reciprocal cross-species reactivity that is underpinned by a conserved NKT TCR-CD1d-Ag docking mode. Within this common docking footprint, the NKT TCR recognizes, to varying degrees of affinity, a range of Ags. Presently, it is unclear whether the human NKT TCRs will mirror the generalities underpinning the fine specificity of the mouse NKT TCR-CD1d-Ag interaction. Here, we assessed human NKT TCR recognition against altered glycolipid ligands of α-galactosylceramide (α-GalCer) and have determined the structures of a human NKT TCR in complex with CD1d-4′,4″-deoxy-α-GalCer and CD1d-α-GalCer with a shorter, di-unsaturated acyl chain (C20:2). Altered glycolipid ligands with acyl chain modifications did not affect the affinity of the human NKT TCR-CD1d-Ag interaction. Surprisingly, human NKT TCR recognition is more tolerant to modifications at the 4′-OH position in comparison with the 3′-OH position of α-GalCer, which contrasts the fine specificity of the mouse NKT TCR-CD1d-Ag recognition (4′-OH > 3′-OH). The fine specificity differences between human and mouse NKT TCRs was attributable to differing interactions between the respective complementarity-determining region 1α loops and the Ag. Accordingly, germline encoded fine-specificity differences underpin human and mouse type I NKT TCR interactions, which is an important consideration for therapeutic development and NKT cell physiology.     

A Conserved CXXC Motif in CD3ε Is Critical for T Cell Development and TCR Signaling

Virtually all T cell development and functions depend on its antigen receptor. The T cell receptor (TCR) is a multi-protein complex, comprised of a ligand binding module and a signal transmission module. The signal transmission module includes proteins from CD3 family (CD3ε, CD3δ, CD3γ) as well as the ζ chain protein. The CD3 proteins have a short extracellular stalk connecting their Ig-like domains to their transmembrane regions. These stalks contain a highly evolutionarily conserved CXXC motif, whose function is unknown. To understand the function of these two conserved cysteines, we generated mice that lacked endogenous CD3ε but expressed a transgenic CD3ε molecule in which these cysteines were mutated to serines. Our results show that the mutated CD3ε could incorporate into the TCR complex and rescue surface TCR expression in CD3ε null mice. In the CD3ε mutant mice, all stages of T cell development and activation that are TCR-dependent were impaired, but not eliminated, including activation of mature naïve T cells with the MHCII presented superantigen, staphylococcal enterotoxin B, or with a strong TCR cross-linking antibody specific for either TCR-Cβ or CD3ε. These results argue against a simple aggregation model for TCR signaling and suggest that the stalks of the CD3 proteins may be critical in transmitting part of the activation signal directly through the membrane.     

T-cell Receptor (TCR)-Peptide Specificity Overrides Affinity-enhancing TCR-Major Histocompatibility Complex Interactions

αβ T-cell receptors (TCRs) engage antigens using complementarity-determining region (CDR) loops that are either germ line-encoded (CDR1 and CDR2) or somatically rearranged (CDR3). TCR ligands compose a presentation platform (major histocompatibility complex (MHC)) and a variable antigenic component consisting of a short “foreign” peptide. The sequence of events when the TCR engages its peptide-MHC (pMHC) ligand remains unclear. Some studies suggest that the germ line elements of the TCR engage the MHC prior to peptide scanning, but this order of binding is difficult to reconcile with some TCR-pMHC structures. Here, we used TCRs that exhibited enhanced pMHC binding as a result of mutations in either CDR2 and/or CDR3 loops, that bound to the MHC or peptide, respectively, to dissect the roles of these loops in stabilizing TCR-pMHC interactions. Our data show that TCR-peptide interactions play a strongly dominant energetic role providing a binding mode that is both temporally and energetically complementary with a system requiring positive selection by self-pMHC in the thymus and rapid recognition of non-self-pMHC in the periphery.