RESUMEN
The engagement of the T-cell receptor (TCR) by a specific peptide-MHC ligand initiates transmembrane signaling to induce T-cell activation, a key step in most adaptive immune responses. Previous studies have indicated that TCR signaling is tightly regulated by cholesterol and its sulfate metabolite, cholesterol sulfate (CS), on the membrane. Here, we report a novel mechanism by which CS modulates TCR signaling through a conformational change of CD3 subunits. We found that the negatively charged CS interacted with the positively charged cytoplasmic domain of CD3ε (CD3εCD) to enhance its binding to the cell membrane and induce a stable secondary structure. This secondary structure suppressed the release of CD3εCD from the membrane in the presence of Ca2+, which in turn inhibited TCR phosphorylation and signaling. When a point mutation (I/A) was introduced to the intracellular immunoreceptor tyrosine-based activation motifs (YxxI-x6-8-YxxL) of CD3ε subunit, it reduced the stability of the secondary structure and regained sensitivity to Ca2+, which abolished CS-mediated inhibition and enhanced the signaling of the TCR complex. Notably, the I/A mutation could be applied to both murine and human TCR-T cell therapy to improve the antitumor efficacy. Our study reveals insights into the regulatory mechanism of TCR signaling and provides a strategy to functionally engineer the TCR/CD3 complex for T cell-based cancer immunotherapy.
Asunto(s)
Calcio , Linfocitos T , Animales , Humanos , Ratones , Calcio/metabolismo , Complejo Receptor-CD3 del Antígeno de Linfocito T/genética , Complejo Receptor-CD3 del Antígeno de Linfocito T/química , Complejo CD3/genética , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Fosforilación , Lípidos/análisisRESUMEN
The T cell receptor (TCR-CD3) initiates T cell activation by binding to peptides of Major Histocompatibility Complexes (pMHC). The TCR-CD3 topology is well understood but the arrangement and dynamics of its cytoplasmic tails remains unknown, limiting our grasp of the signalling mechanism. Here, we use molecular dynamics simulations and modelling to investigate the entire TCR-CD3 embedded in a model membrane. Our study demonstrates conformational changes in the extracellular and transmembrane domains, and the arrangement of the TCR-CD3 cytoplasmic tails. The cytoplasmic tails formed highly interlaced structures while some tyrosines within the immunoreceptor tyrosine-based activation motifs (ITAMs) penetrated the hydrophobic core of the membrane. Interactions between the cytoplasmic tails and phosphatidylinositol phosphate lipids in the inner membrane leaflet led to the formation of a distinct anionic lipid fingerprint around the TCR-CD3. These results increase our understanding of the TCR-CD3 dynamics and the importance of membrane lipids in regulating T cell activation.
Asunto(s)
Modelos Moleculares , Complejo Receptor-CD3 del Antígeno de Linfocito T/química , Complejo Receptor-CD3 del Antígeno de Linfocito T/metabolismo , Biología Computacional , Simulación por Computador , Microscopía por Crioelectrón , Citoplasma/química , Citoplasma/metabolismo , Humanos , Activación de Linfocitos , Lípidos de la Membrana/química , Lípidos de la Membrana/metabolismo , Simulación de Dinámica Molecular , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Complejo Receptor-CD3 del Antígeno de Linfocito T/ultraestructura , Electricidad Estática , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
The impact of ultrasmall nanoparticles (<10-nm diameter) on the immune system is poorly understood. Recently, ultrasmall silica nanoparticles (USSN), which have gained increasing attention for therapeutic applications, were shown to stimulate T lymphocytes directly and at relatively low-exposure doses. Delineating underlying mechanisms and associated cell signaling will hasten therapeutic translation and is reported herein. Using competitive binding assays and molecular modeling, we established that the T cell receptor (TCR):CD3 complex is required for USSN-induced T cell activation, and that direct receptor complex-particle interactions are permitted both sterically and electrostatically. Activation is not limited to αß TCR-bearing T cells since those with γδ TCR showed similar responses, implying that USSN mediate their effect by binding to extracellular domains of the flanking CD3 regions of the TCR complex. We confirmed that USSN initiated the signaling pathway immediately downstream of the TCR with rapid phosphorylation of both ζ-chain-associated protein 70 and linker for activation of T cells protein. However, T cell proliferation or IL-2 secretion were only triggered by USSN when costimulatory anti-CD28 or phorbate esters were present, demonstrating that the specific impact of USSN is in initiation of the primary, nuclear factor of activated T cells-pathway signaling from the TCR complex. Hence, we have established that USSN are partial agonists for the TCR complex because of induction of the primary T cell activation signal. Their ability to bind the TCR complex rapidly, and then to dissolve into benign orthosilicic acid, makes them an appealing option for therapies targeted at transient TCR:CD3 receptor binding.
Asunto(s)
Activación de Linfocitos/efectos de los fármacos , Nanopartículas/química , Complejo Receptor-CD3 del Antígeno de Linfocito T/efectos de los fármacos , Complejo Receptor-CD3 del Antígeno de Linfocito T/metabolismo , Dióxido de Silicio/química , Dióxido de Silicio/farmacología , Antígenos CD28/metabolismo , Complejo CD3/química , Complejo CD3/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Humanos , Interleucina-2/metabolismo , Modelos Moleculares , Fosforilación , Complejo Receptor-CD3 del Antígeno de Linfocito T/química , Complejo Receptor-CD3 del Antígeno de Linfocito T/genética , Transducción de Señal/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
The anti-tumor immune response is considered to be due to the T-cell receptor (TCR) binding to tumor antigens, which can be either wild-type, early stem cell proteins, presumably foreign to a developed immune system; or mutant peptides, foreign to the immune system because of a mutant amino acid (aa) or otherwise somatically altered aa sequence. Recently, very large numbers of TCR complementarity-determining region-3 (CDR3) aa sequences obtained from tumor specimens have become available. We developed a novel algorithm for assessing the complementarity of tumor mutant peptides and TCR CDR3s, based on the retrieval of TCR CDR3 aa sequences from both tumor specimen and patient blood exomes and by using an automated process of assessing CDR3 and mutant aa electrical charges. Results indicated many instances where high electrostatic complementarity was associated with a higher survival rate. In particular, our approach led to the identification of specific genes contributing significantly to the complementary, TCR CDR3-mutant aa. These results suggest a novel approach to tumor immunoscoring and may lead to the identification of high-priority neo-antigen, peptide vaccines; or to the identification of ex vivo stimulants of tumor-infiltrating lymphocytes.
Asunto(s)
Algoritmos , Antígenos de Neoplasias/química , Neoplasias de la Mama/genética , Regiones Determinantes de Complementariedad/química , Neoplasias Pulmonares/genética , Complejo Receptor-CD3 del Antígeno de Linfocito T/química , Neoplasias Cutáneas/genética , Secuencia de Aminoácidos , Aminoácidos , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Sitios de Unión , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/mortalidad , Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/inmunología , Exoma , Femenino , Expresión Génica , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/mortalidad , Masculino , Mutación , Pronóstico , Unión Proteica , Complejo Receptor-CD3 del Antígeno de Linfocito T/genética , Complejo Receptor-CD3 del Antígeno de Linfocito T/inmunología , Proyectos de Investigación , Neoplasias Cutáneas/diagnóstico , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/mortalidad , Electricidad Estática , Tasa de Supervivencia , Linfocitos T/inmunología , Linfocitos T/patologíaRESUMEN
The αß T cell receptor (TCR), in association with the CD3γε-CD3δε-CD3ζζ signalling hexamer, is the primary determinant of T cell development and activation, and of immune responses to foreign antigens. The mechanism of assembly of the TCR-CD3 complex remains unknown. Here we report a cryo-electron microscopy structure of human TCRαß in complex with the CD3 hexamer at 3.7 Å resolution. The structure contains the complete extracellular domains and all the transmembrane helices of TCR-CD3. The octameric TCR-CD3 complex is assembled with 1:1:1:1 stoichiometry of TCRαß:CD3γε:CD3δε:CD3ζζ. Assembly of the extracellular domains of TCR-CD3 is mediated by the constant domains and connecting peptides of TCRαß that pack against CD3γε-CD3δε, forming a trimer-like structure proximal to the plasma membrane. The transmembrane segment of the CD3 complex adopts a barrel-like structure formed by interaction of the two transmembrane helices of CD3ζζ with those of CD3γε and CD3δε. Insertion of the transmembrane helices of TCRαß into the barrel-like structure via both hydrophobic and ionic interactions results in transmembrane assembly of the TCR-CD3 complex. Together, our data reveal the structural basis for TCR-CD3 complex assembly, providing clues to TCR triggering and a foundation for rational design of immunotherapies that target the complex.
Asunto(s)
Modelos Moleculares , Complejo Receptor-CD3 del Antígeno de Linfocito T/química , Microscopía por Crioelectrón , Humanos , Dominios Proteicos , Estructura Cuaternaria de Proteína , Complejo Receptor-CD3 del Antígeno de Linfocito T/metabolismoRESUMEN
Understanding the binding of nanoparticles to receptors on biomembranes is critical to the development and screening of therapeutic materials. A prevailing understanding is that multivalent ligand-receptor binding leads to slower and confined translational motion of nanoparticles. In contrast, we report in this study distinct types of rotational dynamics of nanoparticles during their seemingly similar translational confinements in ligand-receptor binding. Our nanoparticles are fluorescently anisotropic and camouflaged with T cell membranes. As they bind to ligands on planar lipid bilayers, the particles transition from back-and-forth rocking motion to circling and eventually confined circling motion, while "hopping" between translational confinements. Both rotational and translational motions of the nanoparticles become more confined at higher ligand density. The time-dependent changes in particle rotation reveal different stages in the progression of multivalent binding between the cell-membrane coated nanoparticles and their ligands. Our work also demonstrates the promise of using combined rotational and translational single particle tracking to resolve biological interactions that could be "hidden" in translational measurements alone.
Asunto(s)
Membrana Celular/química , Membrana Dobles de Lípidos/química , Nanopartículas/química , Complejo Receptor-CD3 del Antígeno de Linfocito T/química , Membrana Celular/metabolismo , Difusión , Polarización de Fluorescencia , Colorantes Fluorescentes/química , Humanos , Células Jurkat , Cinética , Ligandos , Imagen Óptica/métodos , Poliestirenos/química , Complejo Receptor-CD3 del Antígeno de Linfocito T/metabolismo , Propiedades de Superficie , Linfocitos T/química , Linfocitos T/metabolismoRESUMEN
Activation of the T cell receptor (TCR) on the T cell through ligation with antigen-MHC complex of an antigen-presenting cell (APC) is an essential process in the activation of T cells and induction of the subsequent adaptive immune response. Upon activation, the TCR, together with its associated co-receptor CD3 complex, assembles in signaling microclusters that are transported to the center of the organizational structure at the T cell-APC interface termed the immunological synapse (IS). During IS formation, local cell surface receptors and associated intracellular molecules are reorganized, ultimately creating the typical bull's eye-shaped pattern of the IS. CD6 is a surface glycoprotein receptor, which has been previously shown to associate with CD3 and co-localize to the center of the IS in static conditions or stable T cell-APC contacts. In this study, we report the use of different experimental set-ups analyzed with microscopy techniques to study the dynamics and stability of CD6-TCR/CD3 interaction dynamics and stability during IS formation in more detail. We exploited antibody spots, created with microcontact printing, and antibody-coated beads, and could demonstrate that CD6 and the TCR/CD3 complex co-localize and are recruited into a stimulatory cluster on the cell surface of T cells. Furthermore, we demonstrate, for the first time, that CD6 forms microclusters co-localizing with TCR/CD3 microclusters during IS formation on supported lipid bilayers. These co-localizing CD6 and TCR/CD3 microclusters are both radially transported toward the center of the IS formed in T cells, in an actin polymerization-dependent manner. Overall, our findings further substantiate the role of CD6 during IS formation and provide novel insight into the dynamic properties of this CD6-TCR/CD3 complex interplay. From a methodological point of view, the biophysical approaches used to characterize these receptors are complementary and amenable for investigation of the dynamic interactions of other membrane receptors.
Asunto(s)
Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/metabolismo , Fenómenos Biofísicos , Complejo CD3/metabolismo , Linfocitos T/fisiología , Actinas/química , Actinas/metabolismo , Antígenos CD/química , Antígenos de Diferenciación de Linfocitos T/química , Línea Celular Tumoral , Técnica del Anticuerpo Fluorescente , Humanos , Sinapsis Inmunológicas/fisiología , Unión Proteica , Multimerización de Proteína , Transporte de Proteínas , Complejo Receptor-CD3 del Antígeno de Linfocito T/química , Complejo Receptor-CD3 del Antígeno de Linfocito T/metabolismoRESUMEN
T cells generate adaptive immune responses mediated by the T cell receptor (TCR)-CD3 complex comprising an αß TCR heterodimer noncovalently associated with three CD3 dimers. In early T cell activation, αß TCR engagement by peptide-major histocompatibility complex (pMHC) is first communicated to the CD3 signaling apparatus of the TCR-CD3 complex, but the underlying mechanism is incompletely understood. It is possible that pMHC binding induces allosteric changes in TCR conformation or dynamics that are then relayed to CD3. Here, we carried out NMR analysis and molecular dynamics (MD) simulations of both the α and ß chains of a human antiviral TCR (A6) that recognizes the Tax antigen from human T cell lymphotropic virus-1 bound to the MHC class I molecule HLA-A2. We observed pMHC-induced NMR signal perturbations in the TCR variable (V) domains that propagated to three distinct sites in the constant (C) domains: 1) the Cß FG loop projecting from the Vß/Cß interface; 2) a cluster of Cß residues near the Cß αA helix, a region involved in interactions with CD3; and 3) the Cα AB loop at the membrane-proximal base of the TCR. A biological role for each of these allosteric sites is supported by previous mutational and functional studies of TCR signaling. Moreover, the pattern of long-range, ligand-induced changes in TCR A6 revealed by NMR was broadly similar to that predicted by the MD simulations. We propose that the unique structure of the TCR ß chain enables allosteric communication between the TCR-binding sites for pMHC and CD3.
Asunto(s)
Productos del Gen tax/metabolismo , Antígeno HLA-A2/metabolismo , Complejo Receptor-CD3 del Antígeno de Linfocito T/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Regulación Alostérica , Animales , Sitios de Unión , Productos del Gen tax/química , Antígeno HLA-A2/química , Virus Linfotrópico T Tipo 1 Humano/química , Humanos , Ratones , Simulación de Dinámica Molecular , Unión Proteica , Conformación Proteica , Complejo Receptor-CD3 del Antígeno de Linfocito T/química , Receptores de Antígenos de Linfocitos T alfa-beta/químicaRESUMEN
The diversity of the T cell receptor (TCR) complementarity determining region 3 (CDR3) repertoire is the result of random combinations, insertions and deletions during recombination of the germline V, D and J gene fragments. During evolution, some human TCR beta chain variable (TRBV) pseudogenes have been retained. Many previous studies have focused on functional TRBV genes, while little attention has been given to TRBV pseudogenes. To describe the compositional characteristics of TRBV pseudogene rearrangements, we compared and analysed TRBV pseudogenes, TRBV open reading frames (ORFs) and functional TRBV genes via high-throughput sequencing of DNA obtained from the peripheral blood of 4 healthy volunteers and 4 patients. Our results revealed several differences in J and D gene usage. The V deletion distribution profile of the pseudogenes was significantly different from that of the ORFs and functional genes. In addition, arginine, lysine and cysteine were more frequently used in putative CDR3 pseudogene rearrangements, while functional rearrangements used more leucine. This study presents a comprehensive description of the compositional characteristics of peripheral TRBV pseudogene rearrangements, which will provide a reference for further research on TRBV pseudogenes.
Asunto(s)
Regiones Determinantes de Complementariedad/genética , Seudogenes/genética , Complejo Receptor-CD3 del Antígeno de Linfocito T/genética , Receptores de Antígenos de Linfocitos T/genética , Regiones Determinantes de Complementariedad/sangre , Genes Codificadores de la Cadena beta de los Receptores de Linfocito T/genética , Voluntarios Sanos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Complejo Receptor-CD3 del Antígeno de Linfocito T/sangre , Complejo Receptor-CD3 del Antígeno de Linfocito T/químicaRESUMEN
CD4+ T cells convert the time that T cell receptors (TCRs) interact with peptides embedded within class II major histocompatibility complex molecules (pMHCII) into signals that direct cell-fate decisions. In principle, TCRs relay information to intracellular signaling motifs of the associated CD3 subunits, while CD4 recruits the kinase Lck to those motifs upon coincident detection of pMHCII. But the mechanics by which this occurs remain enigmatic. In one model, the TCR and CD4 bind pMHCII independently, while in another, CD4 interacts with a composite surface formed by the TCR-CD3 complex bound to pMHCII. Here, we report that the duration of TCR-pMHCII interactions impact CD4 binding to MHCII. In turn, CD4 increases TCR confinement to pMHCII via reciprocal interactions involving membrane distal and proximal CD4 ectodomains. The data suggest that a precisely assembled macrocomplex functions to reliably convert TCR-pMHCII confinement into reproducible signals that orchestrate adaptive immunity.
Asunto(s)
Antígenos CD4/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Activación de Linfocitos/inmunología , Complejo Receptor-CD3 del Antígeno de Linfocito T/metabolismo , Inmunidad Adaptativa/inmunología , Animales , Antígenos CD4/química , Antígenos CD4/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Femenino , Antígenos de Histocompatibilidad Clase II/inmunología , Masculino , Ratones , Ratones Transgénicos , Dominios Proteicos , Complejo Receptor-CD3 del Antígeno de Linfocito T/química , Complejo Receptor-CD3 del Antígeno de Linfocito T/inmunología , Transducción de Señal/inmunologíaRESUMEN
The T-cell receptor (TCR)-CD3 complex, expressed on T cells, determines the outcome of a T-cell response. It consists of the TCR-αß heterodimer and the non-covalently associated signalling dimers of CD3εγ, CD3εδ and CD3ζζ. TCR-αß binds specifically to a cognate peptide antigen bound to an MHC molecule, whereas the CD3 subunits transmit the signal into the cytosol to activate signalling events. Recruitment of proteins to specialized localizations is one mechanism to regulate activation and termination of signalling. In the last 25 years a large number of signalling molecules recruited to the TCR-CD3 complex upon antigen binding to TCR-αß have been described. Here, we review knowledge about five of those interaction partners: Lck, ZAP-70, Nck, WASP and Numb. Some of these proteins have been targeted in the development of immunomodulatory drugs aiming to treat patients with autoimmune diseases and organ transplants.
Asunto(s)
Proteínas Portadoras/metabolismo , Complejo Receptor-CD3 del Antígeno de Linfocito T/metabolismo , Transducción de Señal , Subgrupos de Linfocitos T/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Complejo CD3/química , Complejo CD3/genética , Complejo CD3/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/genética , Humanos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Proteínas de la Membrana/metabolismo , Mutación , Proteínas del Tejido Nervioso/metabolismo , Proteínas Oncogénicas/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Complejo Receptor-CD3 del Antígeno de Linfocito T/química , Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/inmunología , Proteína del Síndrome de Wiskott-Aldrich/metabolismo , Proteína Tirosina Quinasa ZAP-70/metabolismoRESUMEN
The T-cell antigen receptor (TCR) is an assembly of eight type I single-pass membrane proteins that occupies a central position in adaptive immunity. Many TCR-triggering models invoke an alteration in receptor complex structure as the initiating event, but both the precise subunit organization and the pathway by which ligand-induced alterations are transferred to the cytoplasmic signaling domains are unknown. Here, we show that the receptor complex transmembrane (TM) domains form an intimately associated eight-helix bundle organized by a specific interhelical TCR TM interface. The salient features of this core structure are absolutely conserved between αß and γδ TCR sequences and throughout vertebrate evolution, and mutations at key interface residues caused defects in the formation of stable TCRαß:CD3δε:CD3γε:ζζ complexes. These findings demonstrate that the eight TCR-CD3 subunits form a compact and precisely organized structure within the membrane and provide a structural basis for further investigation of conformationally regulated models of transbilayer TCR signaling.
Asunto(s)
Complejo CD3/química , Membrana Celular/ultraestructura , Subunidades de Proteína/química , Complejo Receptor-CD3 del Antígeno de Linfocito T/química , Receptores de Antígenos de Linfocitos T alfa-beta/química , Receptores de Antígenos de Linfocitos T gamma-delta/química , Secuencia de Aminoácidos , Sitios de Unión , Complejo CD3/genética , Complejo CD3/inmunología , Membrana Celular/química , Membrana Celular/inmunología , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Humanos , Simulación de Dinámica Molecular , Unión Proteica , Conformación Proteica en Hélice alfa , Dominios y Motivos de Interacción de Proteínas , Subunidades de Proteína/genética , Subunidades de Proteína/inmunología , Complejo Receptor-CD3 del Antígeno de Linfocito T/genética , Complejo Receptor-CD3 del Antígeno de Linfocito T/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de Señal , Linfocitos T/química , Linfocitos T/inmunologíaRESUMEN
Antigen recognition of peptide-major histocompatibility complexes (pMHCs) by T cells, a key step in initiating adaptive immune responses, is performed by the T cell receptor (TCR) bound to CD3 heterodimers. However, the biophysical basis of the transmission of TCR-CD3 extracellular interaction into a productive intracellular signaling sequence remains incomplete. Here we used nuclear magnetic resonance (NMR) spectroscopy combined with mutational analysis and computational docking to derive a structural model of the extracellular TCR-CD3 assembly. In the inactivated state, CD3γε interacts with the helix 3 and helix 4-F strand regions of the TCR Cß subunit, whereas CD3δε interacts with the F and C strand regions of the TCR Cα subunit in this model, placing the CD3 subunits on opposing sides of the TCR. This work identifies the molecular contacts between the TCR and CD3 subunits, identifying a physical basis for transmitting an activating signal through the complex.
Asunto(s)
Complejo CD3/química , Modelos Moleculares , Complejo Receptor-CD3 del Antígeno de Linfocito T/química , Receptores de Antígenos de Linfocitos T/química , Complejo CD3/genética , Complejo CD3/metabolismo , Humanos , Mutagénesis Sitio-Dirigida , Resonancia Magnética Nuclear Biomolecular , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Complejo Receptor-CD3 del Antígeno de Linfocito T/metabolismo , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismoRESUMEN
The T cell receptor (TCR)-CD3 complex is composed of a genetically diverse αß TCR heterodimer associated noncovalently with the invariant CD3 dimers CD3ϵγ, CD3ϵδ, and CD3ζζ. The TCR mediates peptide-MHC recognition, whereas the CD3 molecules transduce activation signals to the T cell. Although much is known about downstream T cell signaling pathways, the mechanism whereby TCR engagement by peptide-MHC initiates signaling is poorly understood. A key to solving this problem is defining the spatial organization of the TCR-CD3 complex and the interactions between its subunits. We have applied solution NMR methods to identify the docking site for CD3 on the ß chain of a human autoimmune TCR. We demonstrate a low affinity but highly specific interaction between the extracellular domains of CD3 and the TCR constant ß (Cß) domain that requires both CD3ϵγ and CD3ϵδ subunits. The mainly hydrophilic docking site, comprising 9-11 solvent-accessible Cß residues, is relatively small (â¼400 Å(2)), consistent with the weak interaction between TCR and CD3 extracellular domains, and devoid of glycosylation sites. The docking site is centered on the αA and αB helices of Cß, which are located at the base of the TCR. This positions CD3ϵγ and CD3ϵδ between the TCR and the T cell membrane, permitting us to distinguish among several possible models of TCR-CD3 association. We further correlate structural results from NMR with mutational data on TCR-CD3 interactions from cell-based assays.
Asunto(s)
Complejo CD3/química , Subunidades de Proteína/química , Complejo Receptor-CD3 del Antígeno de Linfocito T/química , Receptores de Antígenos de Linfocitos T alfa-beta/química , Secuencia de Aminoácidos , Complejo CD3/genética , Complejo CD3/inmunología , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Humanos , Espectroscopía de Resonancia Magnética , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Mutación , Pliegue de Proteína , Multimerización de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína/genética , Subunidades de Proteína/inmunología , Complejo Receptor-CD3 del Antígeno de Linfocito T/genética , Complejo Receptor-CD3 del Antígeno de Linfocito T/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Linfocitos T/química , Linfocitos T/inmunologíaRESUMEN
αß T-cell receptor (TCR) activation plays a crucial role for T-cell function. However, the TCR itself does not possess signaling domains. Instead, the TCR is noncovalently coupled to a conserved multisubunit signaling apparatus, the CD3 complex, that comprises the CD3εγ, CD3εδ, and CD3ζζ dimers. How antigen ligation by the TCR triggers CD3 activation and what structural role the CD3 extracellular domains (ECDs) play in the assembled TCR-CD3 complex remain unclear. Here, we use two complementary structural approaches to gain insight into the overall organization of the TCR-CD3 complex. Small-angle X-ray scattering of the soluble TCR-CD3εδ complex reveals the CD3εδ ECDs to sit underneath the TCR α-chain. The observed arrangement is consistent with EM images of the entire TCR-CD3 integral membrane complex, in which the CD3εδ and CD3εγ subunits were situated underneath the TCR α-chain and TCR ß-chain, respectively. Interestingly, the TCR-CD3 transmembrane complex bound to peptide-MHC is a dimer in which two TCRs project outward from a central core composed of the CD3 ECDs and the TCR and CD3 transmembrane domains. This arrangement suggests a potential ligand-dependent dimerization mechanism for TCR signaling. Collectively, our data advance our understanding of the molecular organization of the TCR-CD3 complex, and provides a conceptual framework for the TCR activation mechanism.
Asunto(s)
Complejo Receptor-CD3 del Antígeno de Linfocito T/química , Secuencias de Aminoácidos , Antígenos/química , Membrana Celular/metabolismo , Células HEK293 , Humanos , Ligandos , Microscopía Electrónica , Modelos Moleculares , Péptidos/química , Multimerización de Proteína , Estructura Terciaria de Proteína , Receptores de Antígenos de Linfocitos T alfa-beta/química , Dispersión de Radiación , Transducción de Señal , Linfocitos T/química , Rayos XRESUMEN
Activation of the T cell receptor (TCR) by antigen is the key step in adaptive immunity. In the αßTCR, antigen induces a conformational change at the CD3 subunits (CD3 CC) that is absolutely required for αßTCR activation. Here, we demonstrate that the CD3 CC is not induced by antigen stimulation of the mouse G8 or the human Vγ9Vδ2 γδTCR. We find that there is a fundamental difference between the activation mechanisms of the αßTCR and γδTCR that map to the constant regions of the TCRαß/γδ heterodimers. Enforced induction of CD3 CC with a less commonly used monoclonal anti-CD3 promoted proximal γδTCR signaling but inhibited cytokine secretion. Utilizing this knowledge, we could dramatically improve in vitro tumor cell lysis by activated human γδ T cells. Thus, manipulation of the CD3 CC might be exploited to improve clinical γδ T cell-based immunotherapies.
Asunto(s)
Complejo CD3/química , Citotoxicidad Inmunológica , Receptores de Antígenos de Linfocitos T gamma-delta/química , Animales , Complejo CD3/inmunología , Línea Celular , Humanos , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Unión Proteica , Conformación Proteica , Complejo Receptor-CD3 del Antígeno de Linfocito T/química , Complejo Receptor-CD3 del Antígeno de Linfocito T/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Linfocitos T/inmunologíaRESUMEN
In mammals, the αßT cell receptor (TCR) signaling complex is composed of a TCRαß heterodimer that is noncovalently coupled to three dimeric signaling molecules, CD3εδ, CD3εγ, and CD3ζζ. The nature of the TCR signaling complex and subunit arrangement in different species remains unclear however. Here we present a structural and biochemical analysis of the more primitive ancestral form of the TCR signaling complex found in chickens. In contrast to mammals, chickens do not express separate CD3δ and CD3γ chains but instead encode a single hybrid chain, termed CD3δ/γ, that is capable of pairing with CD3ε. The NMR structure of the chicken CD3εδ/γ heterodimer revealed a unique dimer interface that results in a heterodimer with considerable deviation from the distinct side-by-side architecture found in human and murine CD3εδ and CD3εγ. The chicken CD3εδ/γ heterodimer also contains a unique molecular surface, with the vast majority of surface-exposed, nonconserved residues being clustered to a single face of the heterodimer. Using an in vitro biochemical assay, we demonstrate that CD3εδ/γ can assemble with both chicken TCRα and TCRß via conserved polar transmembrane sites. Moreover, analogous to the human TCR signaling complex, the presence of two copies of CD3εδ/γ is required for ζζ assembly. These data provide insight into the evolution of this critical receptor signaling apparatus.
Asunto(s)
Proteínas Aviares/química , Complejo CD3/química , Pollos/metabolismo , Complejo Receptor-CD3 del Antígeno de Linfocito T/química , Secuencia de Aminoácidos , Animales , Complejo CD3/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Multimerización de Proteína , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Alineación de SecuenciaRESUMEN
Bioactive nanoscale arrays were constructed to ligate activating cell surface receptors on T cells (the CD3 component of the TCR complex) and natural killer (NK) cells (CD16). These arrays are formed from biofunctionalized gold nanospheres with controlled interparticle spacing in the range 25-104 nm. Responses to these nanoarrays were assessed using the extent of membrane-localized phosphotyrosine in T cells stimulated with CD3-binding nanoarrays and the size of cell contact area for NK cells stimulated with CD16-binding nanoarrays. In both cases, the strength of response decreased with increasing spacing, falling to background levels by 69 nm in the T cell/anti-CD3 system and 104 nm for the NK cell/anti-CD16 system. These results demonstrate that immune receptor triggering can be influenced by the nanoscale spatial organization of receptor/ligand interactions.
Asunto(s)
Nanopartículas/química , Nanotecnología , Complejo Receptor-CD3 del Antígeno de Linfocito T/química , Receptores de Células Asesinas Naturales/química , Complejo CD3/química , Complejo CD3/inmunología , Humanos , Células Asesinas Naturales/química , Células Asesinas Naturales/inmunología , Complejo Receptor-CD3 del Antígeno de Linfocito T/inmunología , Receptores de Superficie Celular/química , Receptores de Superficie Celular/inmunología , Receptores de IgG/química , Receptores de IgG/inmunología , Receptores de Células Asesinas Naturales/inmunología , Linfocitos T/química , Linfocitos T/inmunologíaRESUMEN
The role of the multifunctional accessory Nef protein in the immunopathogenesis of HIV-2 infection is currently poorly understood. Here, we performed comprehensive functional analyses of 50 nef genes from 21 viremic (plasma viral load, >500 copies/ml) and 16 nonviremic (<500) HIV-2-infected individuals. On average, nef alleles from both groups were equally active in modulating CD4, TCR-CD3, CD28, MHC-I, and Ii cell surface expression and in enhancing virion infectivity. Thus, many HIV-2-infected individuals efficiently control the virus in spite of efficient Nef function. However, the potency of nef alleles in downmodulating TCR-CD3 and CD28 to suppress the activation and apoptosis of T cells correlated with high numbers of CD4(+) T cells in viremic patients. No such correlations were observed in HIV-2-infected individuals with undetectable viral load. Further functional analyses showed that the Nef-mediated downmodulation of TCR-CD3 suppressed the induction of Fas, Fas-L, PD-1, and CTLA-4 cell surface expression as well as the secretion of gamma interferon (IFN-γ) by primary CD4(+) T cells. Moreover, we identified a single naturally occurring amino acid variation (I132T) in the core domain of HIV-2 Nef that selectively disrupts its ability to downmodulate TCR-CD3 and results in functional properties highly reminiscent of HIV-1 Nef proteins. Taken together, our data suggest that the efficient Nef-mediated downmodulation of TCR-CD3 and CD28 help viremic HIV-2-infected individuals to maintain normal CD4(+) T cell homeostasis by preventing T cell activation and by suppressing the induction of death receptors that may affect the functionality and survival of both virally infected and uninfected bystander cells.
Asunto(s)
Antígenos CD28/metabolismo , Recuento de Linfocito CD4 , Infecciones por VIH/inmunología , VIH-2/metabolismo , Complejo Receptor-CD3 del Antígeno de Linfocito T/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Sustitución de Aminoácidos , Apoptosis/genética , Línea Celular , Regulación hacia Abajo/inmunología , Expresión Génica , Orden Génico , Infecciones por VIH/metabolismo , VIH-2/genética , VIH-2/inmunología , Humanos , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/virología , Activación de Linfocitos/genética , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Unión Proteica/inmunología , Conformación Proteica , Provirus/genética , Complejo Receptor-CD3 del Antígeno de Linfocito T/química , Receptores de Superficie Celular/metabolismo , Receptores de Muerte Celular/metabolismo , Linfocitos T/inmunología , Viremia/inmunología , Viremia/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/química , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
The last few years have seen the transfer of two decades of research into Chimeric Antigen Receptors (CARs) into clinical trials. Despite this extensive research, there is still a great deal of debate into the optimal design strategy for these, primarily, anti-cancer entities. The archetypal CAR consists of a single-chain antibody fragment, specific to a tumour-associated antigen, fused to a component of the T-cell receptor complex (typically CD3zeta) which on antigen binding primes the engrafted T-cell for anti-tumour activity. The modular nature of these artificial receptors has enabled researchers to modify aspects of their structure, including the extracellular spacer, transmembrane and cytoplasmic domain, to achieve laboratory defined optimal activity. Despite this there is no consensus on the optimal structure, a problem exacerbated by conflicting results using identical receptors. In this review, we provide a structural overview of CAR development and highlight areas that require further refinement. We also attempt to identify possible reasons for conflicting results in the hope that this information will inspire future rational design strategies for optimal tumour targeting using CARs.