RESUMO
Programming T cells to distinguish self from non-self is a vital, multi-step process that occurs in the thymus1-4. Signalling through the pre-T cell receptor (preTCR), a CD3-associated heterodimer comprising an invariant pTα chain and a clone-specific ß chain, is a critical early checkpoint in thymocyte development within the αß T cell lineage5,6. PreTCRs arrayed on CD4-CD8- double-negative thymocytes ligate peptides bound to major histocompatibility complex molecules (pMHC) on thymic stroma, similar to αß T cell receptors that appear on CD4+CD8+ double-positive thymocytes, but via a different molecular docking strategy7-10. Here we show the consequences of these distinct interactions for thymocyte progression using synchronized fetal thymic progenitor cultures that differ in the presence or absence of pMHC on support stroma, and single-cell transcriptomes at key thymocyte developmental transitions. Although major histocompatibility complex (MHC)-negative stroma fosters αß T cell differentiation, the absence of preTCR-pMHC interactions leads to deviant thymocyte transcriptional programming associated with dedifferentiation. Highly proliferative double-negative and double-positive thymocyte subsets emerge, with antecedent characteristics of T cell lymphoblastic and myeloid malignancies. Compensatory upregulation of diverse MHC class Ib proteins in B2m/H2-Ab1 MHC-knockout mice partially safeguards in vivo thymocyte progression, although disseminated double-positive thymic tumours may develop with ageing. Thus, as well as promoting ß chain repertoire broadening for subsequent αß T cell receptor utilization, preTCR-pMHC interactions limit cellular plasticity to facilitate normal thymocyte differentiation and proliferation that, if absent, introduce developmental vulnerabilities.
Assuntos
Desdiferenciação Celular , Antígenos de Histocompatibilidade Classe I , Receptores de Antígenos de Linfócitos T , Timócitos , Animais , Camundongos , Camundongos Knockout , Simulação de Acoplamento Molecular , Peptídeos/imunologia , Peptídeos/metabolismo , Timócitos/citologia , Timócitos/imunologia , Timo/citologia , Timo/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismoRESUMO
Initial molecular details of cellular activation following αßT cell antigen receptor (TCR) ligation by peptide-major histocompatibility complexes (pMHC) remain unexplored. We determined the nuclear magnetic resonance (NMR) structure of the TCRα subunit transmembrane (TM) domain revealing a bipartite helix whose segmentation fosters dynamic movement. Positively charged TM residues Arg251 and Lys256 project from opposite faces of the helix, with Lys256 controlling immersion depth. Their modification caused stepwise reduction in TCR associations with CD3ζζ homodimers and CD3εγ plus CD3εδ heterodimers, respectively, leading to an activated transcriptome. Optical tweezers revealed that Arg251 and Lys256 mutations altered αßTCR-pMHC bond lifetimes, while mutations within interacting TCRα connecting peptide and CD3δ CxxC motif juxtamembrane elements selectively attenuated signal transduction. Our findings suggest that mechanical forces applied during pMHC ligation initiate T cell activation via a dissociative mechanism, shifting disposition of those basic sidechains to rearrange TCR complex membrane topology and weaken TCRαß and CD3 associations.
Assuntos
Complexo CD3/metabolismo , Membrana Celular/metabolismo , Domínios Proteicos , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Sequência de Aminoácidos , Biomarcadores , Complexo CD3/química , Sequência Conservada , Perfilação da Expressão Gênica , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Receptores de Antígenos de Linfócitos T alfa-beta/química , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Transdução de Sinais , TranscriptomaRESUMO
High-acuity αßT cell receptor (TCR) recognition of peptides bound to major histocompatibility complex molecules (pMHCs) requires mechanosensing, a process whereby piconewton (pN) bioforces exert physical load on αßTCR-pMHC bonds to dynamically alter their lifetimes and foster digital sensitivity cellular signaling. While mechanotransduction is operative for both αßTCRs and pre-TCRs within the αßT lineage, its role in γδT cells is unknown. Here, we show that the human DP10.7 γδTCR specific for the sulfoglycolipid sulfatide bound to CD1d only sustains a significant load and undergoes force-induced structural transitions when the binding interface-distal γδ constant domain (C) module is replaced with that of αß. The chimeric γδ-αßTCR also signals more robustly than does the wild-type (WT) γδTCR, as revealed by RNA-sequencing (RNA-seq) analysis of TCR-transduced Rag2-/- thymocytes, consistent with structural, single-molecule, and molecular dynamics studies reflective of γδTCRs as mediating recognition via a more canonical immunoglobulin-like receptor interaction. Absence of robust, force-related catch bonds, as well as γδTCR structural transitions, implies that γδT cells do not use mechanosensing for ligand recognition. This distinction is consonant with the fact that their innate-type ligands, including markers of cellular stress, are expressed at a high copy number relative to the sparse pMHC ligands of αßT cells arrayed on activating target cells. We posit that mechanosensing emerged over â¼200 million years of vertebrate evolution to fulfill indispensable adaptive immune recognition requirements for pMHC in the αßT cell lineage that are unnecessary for the γδT cell lineage mechanism of non-pMHC ligand detection.
Assuntos
Mecanotransdução Celular , Receptores de Antígenos de Linfócitos T gama-delta/química , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Sequência de Aminoácidos , Animais , Perfilação da Expressão Gênica , Humanos , Ligantes , Camundongos , Domínios Proteicos , Estabilidade Proteica , Estrutura Secundária de Proteína , Receptores de Antígenos de Linfócitos T alfa-beta/química , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Transdução de Sinais , Imagem Individual de Molécula , Linfócitos T/metabolismo , Timócitos/metabolismo , Timo/metabolismo , Transcriptoma/genéticaRESUMO
Each [Formula: see text]T cell receptor (TCR) functions as a mechanosensor. The TCR is comprised of a clonotypic TCR[Formula: see text] ligand-binding heterodimer and the noncovalently associated CD3 signaling subunits. When bound by ligand, an antigenic peptide arrayed by a major histocompatibility complex molecule (pMHC), the TCR[Formula: see text] has a longer bond lifetime under piconewton-level loads. The atomistic mechanism of this "catch bond" behavior is unknown. Here, we perform molecular dynamics simulation of a TCR[Formula: see text]-pMHC complex and its variants under physiologic loads to identify this mechanism and any attendant TCR[Formula: see text] domain allostery. The TCR[Formula: see text]-pMHC interface is dynamically maintained by contacts with a spectrum of occupancies, introducing a level of control via relative motion between Vα and Vß variable domains containing the pMHC-binding complementarity-determining region (CDR) loops. Without adequate load, the interfacial contacts are unstable, whereas applying sufficient load suppresses Vα-Vß motion, stabilizing the interface. A second level of control is exerted by Cα and Cß constant domains, especially Cß and its protruding FG-loop, that create mismatching interfaces among the four TCR[Formula: see text] domains and with a pMHC ligand. Applied load enhances fit through deformation of the TCR[Formula: see text] molecule. Thus, the catch bond involves the entire TCR[Formula: see text] conformation, interdomain motion, and interfacial contact dynamics, collectively. This multilayered architecture of the machinery fosters fine-tuning of cellular response to load and pMHC recognition. Since the germline-derived TCR[Formula: see text] ectodomain is structurally conserved, the proposed mechanism can be universally adopted to operate under load during immune surveillance by diverse [Formula: see text]TCRs constituting the T cell repertoire.
Assuntos
Complexo Principal de Histocompatibilidade , Simulação de Dinâmica Molecular , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Humanos , Ligantes , Mecanotransdução Celular , Linfócitos T/metabolismoRESUMO
T lymphocytes discriminate between healthy and infected or cancerous cells via T-cell receptor-mediated recognition of peptides bound and presented by cell-surface-expressed major histocompatibility complex molecules (MHCs). Pre-T-cell receptors (preTCRs) on thymocytes foster development of αßT lymphocytes through their ß chain interaction with MHC displaying self-peptides on thymic epithelia. The specific binding of a preTCR with a peptide-MHC complex (pMHC) has been identified previously as forming a weak affinity complex with a distinct interface from that of mature αßTCR. However, a lack of appropriate tools has limited prior efforts to investigate this unique interface. Here we designed a small-scale linkage screening protocol using bismaleimide linkers for determining residue-specific distance constraints between transiently interacting protein pairs in solution. Employing linkage distance restraint-guided molecular modeling, we report the oriented solution docking geometry of a preTCRß-pMHC interaction. The linkage model of preTCRß-pMHC complex was independently verified with paramagnetic pseudocontact chemical shift (PCS) NMR of the unlinked protein mixtures. Using linkage screens, we show that the preTCR binds with differing affinities to peptides presented by MHC in solution. Moreover, the C-terminal peptide segment is a key determinant in preTCR-pMHC recognition. We also describe the process for future large-scale production and purification of the linked constructs for NMR, X-ray crystallography, and single-molecule electron microscopy studies.
Assuntos
Antígenos de Superfície/ultraestrutura , Ligação Proteica/genética , Receptores de Antígenos de Linfócitos T/ultraestrutura , Linfócitos T/ultraestrutura , Antígenos de Superfície/química , Antígenos de Superfície/genética , Humanos , Complexo Principal de Histocompatibilidade/genética , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/ultraestrutura , Ressonância Magnética Nuclear Biomolecular , Peptídeos/química , Peptídeos/genética , Domínios e Motivos de Interação entre Proteínas/genética , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T alfa-beta/química , Receptores de Antígenos de Linfócitos T alfa-beta/ultraestrutura , Linfócitos T/química , Linfócitos T/imunologia , Timócitos/química , Timócitos/ultraestruturaRESUMO
T lymphocytes use surface [Formula: see text] T-cell receptors (TCRs) to recognize peptides bound to MHC molecules (pMHCs) on antigen-presenting cells (APCs). How the exquisite specificity of high-avidity T cells is achieved is unknown but essential, given the paucity of foreign pMHC ligands relative to the ubiquitous self-pMHC array on an APC. Using optical traps, we determine physicochemical triggering thresholds based on load and force direction. Strikingly, chemical thresholds in the absence of external load require orders of magnitude higher pMHC numbers than observed physiologically. In contrast, force applied in the shear direction ([Formula: see text]10 pN per TCR molecule) triggers T-cell Ca2+ flux with as few as two pMHC molecules at the interacting surface interface with rapid positional relaxation associated with similarly directed motor-dependent transport via [Formula: see text]8-nm steps, behaviors inconsistent with serial engagement during initial TCR triggering. These synergistic directional forces generated during cell motility are essential for adaptive T-cell immunity against infectious pathogens and cancers.
Assuntos
Apresentação de Antígeno/imunologia , Ativação Linfocitária/imunologia , Mecanotransdução Celular/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Linfócitos T/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Antígenos/imunologia , Linhagem Celular , Camundongos , Camundongos Knockout , Pinças ÓpticasRESUMO
The pre-T cell receptor (pre-TCR) guides early thymocytes through maturation processes within the thymus via interaction with self-ligands displayed on thymic epithelial cells. The pre-TCR is a disulfide-linked heterodimer composed of an invariant pre-TCR α (pTα) subunit and a variable ß subunit, the latter of which is incorporated into the mature TCR in subsequent developmental progression. This interaction of pre-TCR with peptide-major histocompatibility complex (pMHC) molecules has recently been shown to drive robust pre-TCR signaling and thymocyte maturation. Although the native sequences of ß are properly folded and suitable for NMR studies in isolation, a tendency to self-associate rendered binding studies with physiological ligands difficult to interpret. Consequently, to structurally define this critical interaction, we have re-engineered the extracellular regions of ß, designated as ß-c1, for prokaryotic production to be used in NMR spectroscopy. Given the large size of the full extracellular domain of class I MHC molecules such as H-Kb, we produced a truncated form termed Kb-t harboring properties favorable for NMR measurements. This system has enabled robust measurement of a pre-TCR-pMHC interaction directly analogous to that of TCRαß-pMHC. Binding surface analysis identified a contact surface comparable in size to that of the TCRαß-pMHC but potentially with a rather distinct binding orientation. A tilting of the pre-TCRß when bound to the pMHC ligand recognition surface versus the upright orientation of TCRαß would alter the direction of force application between pre-TCR and TCR mechanosensors, impacting signal initiation.
Assuntos
Espectroscopia de Ressonância Magnética/métodos , Glicoproteínas de Membrana/química , Receptores de Antígenos de Linfócitos T alfa-beta/química , Complexo Principal de Histocompatibilidade/genética , Complexo Principal de Histocompatibilidade/fisiologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Mutagênese , Ligação Proteica , Dobramento de Proteína , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismoRESUMO
Early studies of T cell structural biology using X-ray crystallography, surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) focused on a picture of the αßT cell receptor (αßTCR) component domains and their cognate ligands (peptides bound to MHC molecules, i.e. pMHCs) as static interaction partners. Moving forward requires integrating this corpus of data with dynamic technologies such as NMR, molecular dynamics (MD) simulations and real-time single molecule (SM) studies exemplified by optical tweezers (OT). NMR bridges relevant timescales and provides the potential for an all-atom dynamic description of αßTCR components prior to and during interactions with binding partners. SM techniques have opened up vistas in understanding the non-equilibrium nature of T cell signaling through the introduction of force-mediated binding measurements into the paradigm for T cell function. In this regard, bioforces consequent to T-lineage cell motility are now perceived as placing piconewton (pN)-level loads on single receptor-pMHC bonds to impact structural change and αßT-lineage biology, including peptide discrimination, cellular activation, and developmental progression. We discuss herein essential NMR technologies in illuminating the role of ligand binding in the preT cell receptor (preTCR), the αßTCR developmental precursor, and convergence of NMR, SM and MD data in advancing our comprehension of T cell development. More broadly we review the central hypothesis that the αßTCR is a mechanosensor, fostered by breakthrough NMR-based structural insights. Collectively, elucidating dynamic aspects through the integrative use of NMR, SM, and MD shall advance fundamental appreciation of the mechanism of T cell signaling as well as inform translational efforts in αßTCR and chimeric T cell (CAR-T) immunotherapies and T cell vaccinology.
Assuntos
Antígenos de Histocompatibilidade/química , Ressonância Magnética Nuclear Biomolecular , Receptores de Antígenos de Linfócitos T/química , Antígenos de Histocompatibilidade/metabolismo , Humanos , Ligantes , Mecanotransdução Celular , Modelos Moleculares , Conformação Proteica , Receptores de Antígenos de Linfócitos T/metabolismo , Relação Estrutura-Atividade , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
The αß T-cell receptor (TCR) on each T lymphocyte mediates exquisite specificity for a particular foreign peptide bound to a major histocompatibility complex molecule (pMHC) displayed on the surface of altered cells. This recognition stimulates protection in the mammalian host against intracellular pathogens, including viruses, and involves piconewton forces that accompany pMHC ligation. Physical forces are generated by T-lymphocyte movement during immune surveillance as well as by cytoskeletal rearrangements at the immunological synapse following cessation of cell migration. The mechanistic explanation for how TCRs distinguish between foreign and self-peptides bound to a given MHC molecule is unclear: peptide residues themselves comprise few of the TCR contacts on the pMHC, and pathogen-derived peptides are scant among myriad self-peptides bound to the same MHC class arrayed on infected cells. Using optical tweezers and DNA tether spacer technology that permit piconewton force application and nanometer scale precision, we have determined how bioforces relate to self versus nonself discrimination. Single-molecule analyses involving isolated αß-heterodimers as well as complete TCR complexes on T lymphocytes reveal that the FG loop in the ß-subunit constant domain allosterically controls both the variable domain module's catch bond lifetime and peptide discrimination via force-driven conformational transition. In contrast to integrins, the TCR interrogates its ligand via a strong force-loaded state with release through a weakened, extended state. Our work defines a key element of TCR mechanotransduction, explaining why the FG loop structure evolved for adaptive immunity in αß but not γδTCRs or immunoglobulins.
Assuntos
Complexo Principal de Histocompatibilidade , Peptídeos/química , Receptores de Antígenos de Linfócitos T alfa-beta/química , Sequência de Aminoácidos , Modelos Moleculares , Pinças Ópticas , Homologia de Sequência de AminoácidosRESUMO
Adaptive cellular immunity requires accurate self- vs. nonself-discrimination to protect against infections and tumorous transformations while at the same time excluding autoimmunity. This vital capability is programmed in the thymus through selection of αßT-cell receptors (αßTCRs) recognizing peptides bound to MHC molecules (pMHC). Here, we show that the pre-TCR (preTCR), a pTα-ß heterodimer appearing before αßTCR expression, directs a previously unappreciated initial phase of repertoire selection. Contrasting with the ligand-independent model of preTCR function, we reveal through NMR and bioforce-probe analyses that the ß-subunit binds pMHC using Vß complementarity-determining regions as well as an exposed hydrophobic Vß patch characteristic of the preTCR. Force-regulated single bonds akin to those of αßTCRs but with more promiscuous ligand specificity trigger calcium flux. Thus, thymic development involves sequential ß- and then, αß-repertoire tuning, whereby preTCR interactions with self pMHC modulate early thymocyte expansion, with implications for ß-selection, immunodominant peptide recognition, and germ line-encoded MHC interaction.
Assuntos
Diferenciação Celular/imunologia , Regiões Determinantes de Complementaridade/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Timócitos/imunologia , Sequência de Aminoácidos , Animais , Cálcio/imunologia , Cálcio/metabolismo , Células Cultivadas , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/metabolismo , Citometria de Fluxo , Antígenos de Histocompatibilidade/química , Antígenos de Histocompatibilidade/imunologia , Antígenos de Histocompatibilidade/metabolismo , Ligantes , Ativação Linfocitária/imunologia , Espectroscopia de Ressonância Magnética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Imunológicos , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/imunologia , Peptídeos/metabolismo , Ligação Proteica/imunologia , Multimerização Proteica/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/química , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Homologia de Sequência de Aminoácidos , Timócitos/citologia , Timócitos/metabolismo , Timo/embriologia , Timo/imunologia , Timo/metabolismoRESUMO
The pre-T cell receptor (pre-TCR) is a pTα-ß heterodimer functioning in early αß T cell development. Although once thought to be ligand-autonomous, recent studies show that pre-TCRs participate in thymic repertoire formation through recognition of peptides bound to major histocompatibility molecules (pMHC). Using optical tweezers, we probe pre-TCR bonding with pMHC at the single molecule level. Like the αßTCR, the pre-TCR is a mechanosensor undergoing force-based structural transitions that dynamically enhance bond lifetimes and exploiting allosteric control regulated via the Cß FG loop region. The pre-TCR structural transitions exhibit greater reversibility than TCRαß and ordered force-bond lifetime curves. Higher piconewton force requires binding through both complementarity determining region loops and hydrophobic Vß patch apposition. This patch functions in the pre-TCR as a surrogate Vα domain, fostering ligand promiscuity to favor development of ß chains with self-reactivity but is occluded by α subunit replacement of pTα upon αßTCR formation. At the double negative 3 thymocyte stage where the pre-TCR is first expressed, pre-TCR interaction with self-pMHC ligands imparts growth and survival advantages as revealed in thymic stromal cultures, imprinting fundamental self-reactivity in the T cell repertoire. Collectively, our data imply the existence of sequential mechanosensor αßTCR repertoire tuning via the pre-TCR.
Assuntos
Regiões Determinantes de Complementaridade , Regulação da Expressão Gênica/fisiologia , Receptores de Antígenos de Linfócitos T alfa-beta , Timócitos , Animais , Regiões Determinantes de Complementaridade/biossíntese , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Camundongos , Camundongos Knockout , Estrutura Secundária de Proteína , Receptores de Antígenos de Linfócitos T alfa-beta/biossíntese , Receptores de Antígenos de Linfócitos T alfa-beta/química , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Timócitos/química , Timócitos/citologia , Timócitos/metabolismoRESUMO
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) αß 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.
Assuntos
Complexo CD3/química , Complexo CD3/metabolismo , Multimerização Proteica , Receptores de Antígenos de Linfócitos T/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Antígenos CD2/metabolismo , Membrana Celular/metabolismo , Citoplasma/metabolismo , Dissulfetos/química , Humanos , Células Jurkat , Ativação Linfocitária , Mecanotransdução Celular , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Oxirredução , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Mechanical force is critical for the interaction between an αß T cell receptor (TCR) and a peptide-bound major histocompatibility complex (pMHC) molecule to initiate productive T-cell activation. However, the underlying mechanism remains unclear. We use all-atom molecular dynamics simulations to examine the A6 TCR bound to HLA-A*02:01 presenting agonist or antagonist peptides under different extensions to simulate the effects of applied load on the complex, elucidating their divergent biological responses. We found that TCR α and ß chains move asymmetrically, which impacts the interface with pMHC, in particular the peptide-sensing CDR3 loops. For the wild-type agonist, the complex stabilizes in a load-dependent manner while antagonists destabilize it. Simulations of the Cß FG-loop deletion, which reduces the catch bond response, and simulations with in silico mutant peptides further support the observed behaviors. The present results highlight the combined role of interdomain motion, fluctuating forces, and interfacial contacts in determining the mechanical response and fine peptide discrimination by a TCR, thereby resolving the conundrum of nearly identical crystal structures of TCRαß-pMHC agonist and antagonist complexes.
Assuntos
Peptídeos , Receptores de Antígenos de Linfócitos T alfa-beta , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Ligação Proteica , Peptídeos/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Simulação de Dinâmica Molecular , Complexo Principal de Histocompatibilidade , Antígenos de Histocompatibilidade/metabolismoRESUMO
Protein nuclear magnetic resonance (NMR) spectroscopy relies on the ability to isotopically label polypeptides, which is achieved through heterologous expression in various host organisms. Most commonly, Escherichia coli is employed by leveraging isotopically substituted ammonium and glucose to uniformly label proteins with 15N and 13C, respectively. Moreover, E. coli can grow and express proteins in uniformly deuterium-substituted water (D2O), a strategy useful for experiments targeting high molecular weight proteins. Unfortunately, many proteins, particularly those requiring specific posttranslational modifications like disulfide bonding or glycosylation for proper folding and/or function, cannot be readily expressed in their functional forms using E. coli-based expression systems. One such class of proteins includes T-cell receptors and their related preT-cell receptors. In this study, we present an expression system for isotopic labeling of proteins using a nonadherent human embryonic kidney cell line, Expi293F, and a specially designed media. We demonstrate the application of this platform to the ß subunit common to both receptors. In addition, we show that this expression system and media can be used to specifically label amino acids Phe, Ile, Val, and Leu in this system, utilizing an amino acid-specific labeling protocol that allows targeted incorporation at high efficiency without significant isotopic scrambling. We demonstrate that this system can also be used to express proteins with fluorinated amino acids. We were routinely able to obtain an NMR sample with a concentration of 200 µM from 30 mL of culture media, utilizing less than 20 mg of the labeled amino acids.
Assuntos
Aminoácidos , Escherichia coli , Animais , Humanos , Escherichia coli/genética , Escherichia coli/metabolismo , Espectroscopia de Ressonância Magnética , Aminoácidos/química , Ressonância Magnética Nuclear Biomolecular/métodos , Receptores de Antígenos de Linfócitos T/metabolismo , MamíferosRESUMO
αß T cell receptors (TCRs) principally recognize aberrant peptides bound to major histocompatibility complex molecules (pMHCs) on unhealthy cells, amplifying specificity and sensitivity through physical load placed on the TCR-pMHC bond during immunosurveillance. To understand this mechanobiology, TCRs stimulated by abundantly and sparsely arrayed epitopes (NP366-374/Db and PA224-233/Db, respectively) following in vivo influenza A virus infection were studied with optical tweezers. While certain NP repertoire CD8 T lymphocytes require many ligands for activation, others are digital, needing just few. Conversely, all PA TCRs perform digitally, exhibiting pronounced bond lifetime increases through sustained, energizing volleys of structural transitioning. Optimal digital performance is superior in vivo, correlating with ERK phosphorylation, CD3 loss, and activation marker up-regulation in vitro. Given neoantigen array paucity, digital TCRs are likely critical for immunotherapies.
Assuntos
Linfócitos T CD8-Positivos , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Camundongos , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/química , Vírus da Influenza A/imunologia , Humanos , Ativação Linfocitária/imunologia , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Pinças ÓpticasRESUMO
αß T cells are mechanosensors that leverage bioforces during immune surveillance for highly sensitive and specific antigen discrimination. Single-molecule studies are used to profile the initial TCRαß-pMHC binding event, and various biophysical parameters can be identified. Isolating purified TCRαß and pMHC molecules on a coverslip allows for direct measurements of the kinetics and conformational changes in the system and removes cellular components along the load pathway that may interfere with or mask subtle changes. Optical tweezers provide high resolution position and force information that map the bonding profile, including catch bond, and the ability to measure distinct conformational changes driven by forces. The present method describes the single-molecule optical tweezers assay setup, considerations, and execution. This model can be used for various TCR-pMHC pairs or expanded to measure a wide variety of receptor-ligand interactions operative in multiple biological systems.
Assuntos
Pinças Ópticas , Linfócitos T , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Antígenos/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Ligação ProteicaRESUMO
Mechanical force is critical for the interaction between an αßT cell receptor (TCR) and a peptide-bound major histocompatibility complex (pMHC) molecule to initiate productive T-cell activation. However, the underlying mechanism remains unclear. We use all-atom molecular dynamics simulations to examine the A6 TCR bound to HLA-A*02:01 presenting agonist or antagonist peptides under different extensions to simulate the effects of applied load on the complex, elucidating their divergent biological responses. We found that TCR α and ß chains move asymmetrically, which impacts the interface with pMHC, in particular the peptide-sensing CDR3 loops. For the wild-type agonist, the complex stabilizes in a load-dependent manner while antagonists destabilize it. Simulations of the Cß FG-loop deletion, which reduces the catch bond response, and simulations with in silico mutant peptides further support the observed behaviors. The present results highlight the combined role of interdomain motion, fluctuating forces, and interfacial contacts in determining the mechanical response and fine peptide discrimination by a TCR, thereby resolving the conundrum of nearly identical crystal structures of TCRαß-pMHC agonist and antagonist complexes.
RESUMO
αß T-cell receptors (TCRs) recognize aberrant peptides bound to major histocompatibility complex molecules (pMHCs) on unhealthy cells, amplifying specificity and sensitivity through physical load placed on the TCR-pMHC bond during immunosurveillance. To understand this mechanobiology, TCRs stimulated by abundantly and sparsely arrayed epitopes (NP 366-374 /D b and PA 224-233 /D b , respectively) following in vivo influenza A virus infection were studied with optical tweezers. While certain NP repertoire CD8 T lymphocytes require many ligands for activation, others are digital, needing just few. Conversely, all PA TCRs perform digitally, exhibiting pronounced bond lifetime increases through sustained, energizing volleys of structural transitioning. Optimal digital performance is superior in vivo, correlating with ERK phosphorylation, CD3 loss, and activation marker upregulation in vitro . Given neoantigen array paucity, digital TCRs are likely critical for immunotherapies. One Sentence Summary: Quality of ligand recognition in a T-cell repertoire is revealed through application of physical load on clonal T-cell receptor (TCR)-pMHC bonds.
RESUMO
T-cell antigen receptors (TCRs) are mechanosensors, which initiate a signaling cascade upon ligand recognition resulting in T-cell differentiation, homeostasis, effector and regulatory functions. An optical trap combined with fluorescence permits direct monitoring of T-cell triggering in response to force application at various concentrations of peptide-bound major histocompatibility complex molecules (pMHC). The technique mimics physiological shear forces applied as cells crawl across antigen-presenting surfaces during immune surveillance. True single molecule studies performed on single cells profile force-bond lifetime, typically seen as a catch bond, and conformational change at the TCR-pMHC bond on the surface of the cell upon force loading. Together, activation and single molecule single cell studies provide chemical and physical triggering thresholds as well as insight into catch bond formation and quaternary structural changes of single TCRs. The present methods detail assay design, preparation, and execution, as well as data analysis. These methods may be applied to a wide range of pMHC-TCR interactions and have potential for adaptation to other receptor-ligand systems.
Assuntos
Pinças Ópticas , Receptores de Antígenos de Linfócitos T alfa-beta , Antígenos de Histocompatibilidade , Ligantes , Complexo Principal de Histocompatibilidade , Imagem Óptica , Peptídeos/química , Ligação Proteica , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/genéticaRESUMO
Self-discrimination, a critical but ill-defined molecular process programmed during thymocyte development, requires myriad pre-T cell receptors (preTCRs) and αßTCRs. Using x-ray crystallography, we show how a preTCR applies the concave ß-sheet surface of its single variable domain (Vß) to "horizontally" grab the protruding MHC α2-helix. By contrast, αßTCRs purpose all six complementarity-determining region (CDR) loops of their paired VαVß module to recognize peptides bound to major histocompatibility complex molecules (pMHCs) in "vertical" head-to-head binding. The preTCR topological fit ensures that CDR3ß reaches the peptide's featured C-terminal segment for pMHC sampling, establishing the subsequent αßTCR canonical docking mode. "Horizontal" docking precludes germline CDR1ß- and CDR2ß-MHC binding to broaden ß-chain repertoire diversification before αßTCR-mediated selection refinement. Thus, one subunit successively attunes the recognition logic of related multicomponent receptors.