ABSTRACT
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.
Subject(s)
Cell Dedifferentiation , Histocompatibility Antigens Class I , Receptors, Antigen, T-Cell , Thymocytes , Animals , Mice , Mice, Knockout , Molecular Docking Simulation , Peptides/immunology , Peptides/metabolism , Thymocytes/cytology , Thymocytes/immunology , Thymus Gland/cytology , Thymus Gland/immunology , Receptors, Antigen, T-Cell/immunology , Receptors, Antigen, T-Cell/metabolism , Histocompatibility Antigens Class I/immunology , Histocompatibility Antigens Class I/metabolismABSTRACT
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.
Subject(s)
CD3 Complex/metabolism , Cell Membrane/metabolism , Protein Domains , Receptors, Antigen, T-Cell, alpha-beta/metabolism , Amino Acid Sequence , Biomarkers , CD3 Complex/chemistry , Conserved Sequence , Gene Expression Profiling , Models, Molecular , Mutation , Protein Binding , Protein Conformation , Protein Multimerization , Receptors, Antigen, T-Cell, alpha-beta/chemistry , Receptors, Antigen, T-Cell, alpha-beta/genetics , Signal Transduction , TranscriptomeABSTRACT
T cell receptors (TCR) on cytolytic T lymphocytes (CTLs) recognize "foreign" antigens bound in the groove of major histocompatibility complex (MHC) molecules (H-2 in mouse and HLA in human) displayed on altered cells. These antigens are peptide fragments of proteins derived either from infectious pathogens or cellular transformations during cancer evolution. The conjoint ligand formed by the foreign peptide and MHC, termed pMHC, marks an aberrant cell as a target for CTL-mediated destruction. Recent data have provided compelling evidence that adaptive protection is achieved in a facile manner during immune surveillance when mechanical load consequent to cellular motion is applied to the bond formed between an αß TCR and its pMHC ligand arrayed on a disease-altered cell. Mechanobiology maximizes both TCR specificity and sensitivity in comparison to receptor ligation in the absence of force. While the field of immunotherapy has made advances to impact the survival of cancer patients, the latest information relevant to T cell targeting and mechanotransduction has yet to be applied for T cell monitoring and treatment of patients in the clinic. Here we review these data, and challenge scientists and physicians to apply critical biophysical parameters of TCR mechanobiology to the medical oncology field, broadening treatment success within and among various cancer types. We assert that TCRs with digital ligand-sensing performance capability directed at sparsely as well as luminously displayed tumor-specific neoantigens and certain tumor-associated antigens can improve effective cancer vaccine development and immunotherapy paradigms.
Subject(s)
Mechanotransduction, Cellular , Neoplasms , Humans , Mice , Animals , Ligands , Receptors, Antigen, T-Cell , Histocompatibility Antigens , Neoplasms/therapy , Antigens, Neoplasm , Medical Oncology , Receptors, Antigen, T-Cell, alpha-beta/metabolismABSTRACT
HIV-1 infection is incurable due to the persistence of the virus in a latent reservoir of resting memory CD4+ T cells. "Shock-and-kill" approaches that seek to induce HIV-1 gene expression, protein production, and subsequent targeting by the host immune system have been unsuccessful due to a lack of effective latency-reversing agents (LRAs) and kill strategies. In an effort to develop reagents that could be used to promote killing of infected cells, we constructed T cell receptor (TCR)-mimic antibodies to HIV-1 peptide-major histocompatibility complexes (pMHC). Using phage display, we panned for phages expressing antibody-like variable sequences that bound HIV-1 pMHC generated using the common HLA-A*02:01 allele. We targeted three epitopes in Gag and reverse transcriptase identified and quantified via Poisson detection mass spectrometry from cells infected in vitro with a pseudotyped HIV-1 reporter virus (NL4.3 dEnv). Sequences isolated from phages that bound these pMHC were cloned into a single-chain diabody backbone (scDb) sequence, such that one fragment is specific for an HIV-1 pMHC and the other fragment binds to CD3ε, an essential signal transduction subunit of the TCR. Thus, these antibodies utilize the sensitivity of T cell signaling as readouts for antigen processing and as agents to promote killing of infected cells. Notably, these scDbs are exquisitely sensitive and specific for the peptide portion of the pMHC. Most importantly, one scDb caused killing of infected cells presenting a naturally processed target pMHC. This work lays the foundation for a novel therapeutic killing strategy toward elimination of the HIV-1 reservoir.
Subject(s)
Antibodies, Bispecific , HIV Infections , HIV Seropositivity , HIV-1 , CD4-Positive T-Lymphocytes , Humans , Molecular Mimicry , Receptors, Antigen, T-Cell , Virus LatencyABSTRACT
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.
Subject(s)
Mechanotransduction, Cellular , Receptors, Antigen, T-Cell, gamma-delta/chemistry , Receptors, Antigen, T-Cell, gamma-delta/metabolism , Amino Acid Sequence , Animals , Gene Expression Profiling , Humans , Ligands , Mice , Protein Domains , Protein Stability , Protein Structure, Secondary , Receptors, Antigen, T-Cell, alpha-beta/chemistry , Receptors, Antigen, T-Cell, alpha-beta/metabolism , Signal Transduction , Single Molecule Imaging , T-Lymphocytes/metabolism , Thymocytes/metabolism , Thymus Gland/metabolism , Transcriptome/geneticsABSTRACT
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.
Subject(s)
Major Histocompatibility Complex , Molecular Dynamics Simulation , Receptors, Antigen, T-Cell, alpha-beta/metabolism , Humans , Ligands , Mechanotransduction, Cellular , T-Lymphocytes/metabolismABSTRACT
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.
Subject(s)
Antigens, Surface/ultrastructure , Protein Binding/genetics , Receptors, Antigen, T-Cell/ultrastructure , T-Lymphocytes/ultrastructure , Antigens, Surface/chemistry , Antigens, Surface/genetics , Humans , Major Histocompatibility Complex/genetics , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/ultrastructure , Nuclear Magnetic Resonance, Biomolecular , Peptides/chemistry , Peptides/genetics , Protein Interaction Domains and Motifs/genetics , Receptors, Antigen, T-Cell/chemistry , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell, alpha-beta/chemistry , Receptors, Antigen, T-Cell, alpha-beta/ultrastructure , T-Lymphocytes/chemistry , T-Lymphocytes/immunology , Thymocytes/chemistry , Thymocytes/ultrastructureABSTRACT
The membrane proximal external region (MPER) of HIV-1 envelope glycoprotein (gp) 41 is an attractive vaccine target for elicitation of broadly neutralizing antibodies (bNAbs) by vaccination. However, current details regarding the quaternary structural organization of the MPER within the native prefusion trimer [(gp120/41)3] are elusive and even contradictory, hindering rational MPER immunogen design. To better understand the structural topology of the MPER on the lipid bilayer, the adjacent transmembrane domain (TMD) was appended (MPER-TMD) and studied. Membrane insertion of the MPER-TMD was sensitive both to the TMD sequence and cytoplasmic residues. Antigen binding of MPER-specific bNAbs, in particular 10E8 and DH511.2_K3, was significantly impacted by the presence of the TMD. Furthermore, MPER-TMD assembly into 10-nm diameter nanodiscs revealed a heterogeneous membrane array comprised largely of monomers and dimers, as enumerated by bNAb Fab binding using single-particle electron microscopy analysis, arguing against preferential trimeric association of native MPER and TMD protein segments. Moreover, introduction of isoleucine mutations in the C-terminal heptad repeat to induce an extended MPER α-helical bundle structure yielded an antigenicity profile of cell surface-arrayed Env variants inconsistent with that found in the native prefusion state. In line with these observations, electron paramagnetic resonance analysis suggested that 10E8 inhibits viral membrane fusion by lifting the MPER N-terminal region out of the viral membrane, mandating the exposure of residues that would be occluded by MPER trimerization. Collectively, our data suggest that the MPER is not a stable trimer, but rather a dynamic segment adapted for structural changes accompanying fusion.
Subject(s)
Cell Membrane/virology , HIV Envelope Protein gp41/chemistry , HIV-1/immunology , Antibodies, Neutralizing/immunology , Cell Membrane/immunology , HIV Antibodies/immunology , HIV Envelope Protein gp41/genetics , HIV Envelope Protein gp41/immunology , HIV Infections/immunology , HIV Infections/virology , HIV-1/chemistry , HIV-1/genetics , Humans , Lipid Bilayers/chemistry , Lipid Bilayers/immunology , Protein DomainsABSTRACT
T lymphocytes use αß T cell receptors (TCRs) to recognize sparse antigenic peptides bound to MHC molecules (pMHCs) arrayed on antigen-presenting cells (APCs). Contrary to conventional receptor-ligand associations exemplified by antigen-antibody interactions, forces play a crucial role in nonequilibrium mechanosensor-based T cell activation. Both T cell motility and local cytoskeleton machinery exert forces (i.e., generate loads) on TCR-pMHC bonds. We review biological features of the load-dependent activation process as revealed by optical tweezers single molecule/single cell and other biophysical measurements. The findings link pMHC-triggered TCRs to single cytoskeletal motors; define the importance of energized anisotropic (i.e., force direction dependent) activation; and characterize immunological synapse formation as digital, revealing no serial requirement. The emerging picture suggests new approaches for the monitoring and design of cytotoxic T lymphocyte (CTL)-based immunotherapy.
Subject(s)
Cytoskeleton/metabolism , Immunotherapy, Adoptive/methods , Mechanotransduction, Cellular , Receptors, Antigen, T-Cell, alpha-beta/metabolism , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes/physiology , Animals , Anisotropy , Antigen Presentation , Antigens/metabolism , Histocompatibility Antigens/metabolism , Humans , Peptides/metabolism , Single-Cell AnalysisABSTRACT
Functional cognate T cell recognition is mediated via the interaction of a T cell receptor complex with its pMHC ligand. Adams et al. (2011) in this issue of Immunity provide evidence that docking geometry may impact 2D binding and T cell activation.
Subject(s)
Mechanotransduction, Cellular , Receptors, Antigen, T-Cell , CD3 Complex , Humans , T-LymphocytesABSTRACT
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.
Subject(s)
Antigen Presentation/immunology , Lymphocyte Activation/immunology , Mechanotransduction, Cellular/immunology , Receptors, Antigen, T-Cell, alpha-beta/immunology , T-Lymphocytes/immunology , Animals , Antigen-Presenting Cells/immunology , Antigens/immunology , Cell Line , Mice , Mice, Knockout , Optical TweezersABSTRACT
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.
Subject(s)
Magnetic Resonance Spectroscopy/methods , Membrane Glycoproteins/chemistry , Receptors, Antigen, T-Cell, alpha-beta/chemistry , Major Histocompatibility Complex/genetics , Major Histocompatibility Complex/physiology , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Mutagenesis , Protein Binding , Protein Folding , Receptors, Antigen, T-Cell, alpha-beta/genetics , Receptors, Antigen, T-Cell, alpha-beta/metabolismABSTRACT
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.
Subject(s)
Histocompatibility Antigens/chemistry , Nuclear Magnetic Resonance, Biomolecular , Receptors, Antigen, T-Cell/chemistry , Histocompatibility Antigens/metabolism , Humans , Ligands , Mechanotransduction, Cellular , Models, Molecular , Protein Conformation , Receptors, Antigen, T-Cell/metabolism , Structure-Activity Relationship , T-Lymphocytes/immunology , T-Lymphocytes/metabolismABSTRACT
To investigate the role of TCR-pMHC interaction in regulating lung CD8 tissue-resident T cell (TR ) differentiation, polyclonal responses were compared against NP366-374 /Db and PA224-233 /Db , two immunodominant epitopes that arise during influenza A infection in mice. Memory niches distinct from iBALTs develop within the lamina propria, supporting CD103+ and CD103- CD8 TR generation and intraepithelial translocation. Gene set enrichment analysis (GSEA) and weighted gene co-expression network analysis (WGCNA) identify dominant TCR, adherens junction, RIG-I-like and NOD-like pattern recognition receptor as well as TGF-ß signaling pathways and memory signatures among PA224-233 /Db T cells consistent with T resident memory (TRM ) status. In contrast, NP366-374 /Db T cells exhibit enrichment of effector signatures, upregulating pro-inflammatory mediators even among TRM . While NP366-374 /Db T cells manifest transcripts linked to canonical exhaustion pathways, PA224-233 /Db T cells exploit P2rx7 purinoreceptor attenuation. The NP366-374 /Db CD103+ subset expresses the antimicrobial lactotransferrin whereas PA224-233 /Db CD103+ utilizes pore-forming mpeg-1, with <22% of genes correspondingly upregulated in CD103+ (or CD103- ) subsets of both specificities. Thus, TCR-pMHC interactions among TR and antigen presenting cells in a tissue milieu strongly impact CD8 T cell biology.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Epitopes, T-Lymphocyte/immunology , Influenza A Virus, H1N1 Subtype/immunology , Receptors, Antigen, T-Cell, alpha-beta/immunology , T-Lymphocyte Subsets/immunology , Animals , Antigens, CD/biosynthesis , Cell Differentiation/immunology , DEAD Box Protein 58/metabolism , Female , Immunologic Memory/immunology , Integrin alpha Chains/biosynthesis , Lung/cytology , Lung/immunology , Mice , Mice, Inbred C57BL , NLR Proteins/metabolism , Orthomyxoviridae Infections/immunology , Receptors, Antigen, T-Cell, alpha-beta/genetics , Transforming Growth Factor beta/metabolismABSTRACT
A 1983 Immunology Today rostrum hypothesized that each T cell has two recognition units: a T cell receptor (TCR) complex, which binds antigen associated with a polymorphic region of a MHC molecule (pMHC), and a CD4 or CD8 molecule that binds to a conserved region of that same MHC gene product (class II or I, respectively). Structural biology has since precisely revealed those bidentate pMHC interactions. TCRαß ligates the membrane-distal antigen-binding MHC platform, whereas CD8 clamps a membrane-proximal MHCI α3 domain loop and CD4 docks to a hydrophobic crevice between MHCII α2 and ß2 domains. Here, we review how MHC class-restricted binding impacts signaling and lineage commitment, discussing TCR force-driven conformational transitions that may optimally expose the co-receptor docking site on MHC.
Subject(s)
Histocompatibility Antigens/immunology , Histocompatibility Antigens/metabolism , Major Histocompatibility Complex/physiology , Receptors, Antigen, T-Cell/metabolism , Animals , Histocompatibility Antigens/chemistry , Humans , Models, Biological , Models, Molecular , Protein Binding , Protein Conformation , Protein Interaction Domains and Motifs , Protein Multimerization , Receptors, Antigen, T-Cell/chemistry , T-Lymphocytes/immunology , T-Lymphocytes/metabolismABSTRACT
Germinal centers (GCs) are anatomic sites where B cells undergo secondary diversification to produce high-affinity, class-switched Abs. We hypothesized that proliferating B cells in GCs create a hypoxic microenvironment that governs their further differentiation. Using molecular markers, we found GCs to be predominantly hypoxic. Compared to normoxia (21% O2), hypoxic culture conditions (1% O2) in vitro accelerated class switching and plasma cell formation and enhanced expression of GL-7 on B and CD4+ T cells. Reversal of GC hypoxia in vivo by breathing 60% O2 during immunization resulted in reduced frequencies of GC B cells, T follicular helper cells, and plasmacytes, as well as lower expression of ICOS on T follicular helper cells. Importantly, this reversal of GC hypoxia decreased Ag-specific serum IgG1 and reduced the frequency of IgG1+ B cells within the Ag-specific GC. Taken together, these observations reveal a critical role for hypoxia in GC B cell differentiation.
Subject(s)
Cell Hypoxia , Germinal Center/immunology , Germinal Center/metabolism , Immunoglobulin Class Switching , Recombination, Genetic , Animals , B-Lymphocytes/immunology , B-Lymphocytes/physiology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/physiology , Cell Differentiation , Germinal Center/cytology , Immunoglobulin G/blood , Immunoglobulin G/immunology , Immunoglobulins/metabolism , Lymphocyte Activation , Mice , Mice, Inbred C57BL , Oxygen/metabolism , Plasma Cells/immunology , Plasma Cells/physiologyABSTRACT
Vaccines eliciting immunity against influenza A viruses (IAVs) are currently antibody-based with hemagglutinin-directed antibody titer the only universally accepted immune correlate of protection. To investigate the disconnection between observed CD8 T-cell responses and immunity to IAV, we used a Poisson liquid chromatography data-independent acquisition MS method to physically detect PR8/34 (H1N1), X31 (H3N2), and Victoria/75 (H3N2) epitopes bound to HLA-A*02:01 on human epithelial cells following in vitro infection. Among 32 PR8 peptides (8-10mers) with predicted IC50 < 60 nM, 9 were present, whereas 23 were absent. At 18 h postinfection, epitope copies per cell varied from a low of 0.5 for M13-11 to a high of >500 for M1(58-66) with PA, HA, PB1, PB2, and NA epitopes also detected. However, aside from M1(58-66), natural CD8 memory responses against conserved presented epitopes were either absent or only weakly observed by blood Elispot. Moreover, the functional avidities of the immunodominant M1(58-66)/HLA-A*02:01-specific T cells were so poor as to be unable to effectively recognize infected human epithelium. Analysis of T-cell responses to primary PR8 infection in HLA-A*02:01 transgenic B6 mice underscores the poor avidity of T cells recognizing M1(58-66). By maintaining high levels of surface expression of this epitope on epithelial and dendritic cells, the virus exploits the combination of immunodominance and functional inadequacy to evade HLA-A*02:01-restricted T-cell immunity. A rational approach to CD8 vaccines must characterize processing and presentation of pathogen-derived epitopes as well as resultant immune responses. Correspondingly, vaccines may be directed against "stealth" epitopes, overriding viral chicanery.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Epitopes/analysis , Influenza A virus/immunology , Lung/virology , Chromatography, Liquid , Epithelial Cells/immunology , Epithelial Cells/virology , Epitopes/immunology , Humans , Lung/immunologyABSTRACT
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.
Subject(s)
Cell Differentiation/immunology , Complementarity Determining Regions/immunology , Receptors, Antigen, T-Cell, alpha-beta/immunology , Thymocytes/immunology , Amino Acid Sequence , Animals , Calcium/immunology , Calcium/metabolism , Cells, Cultured , Complementarity Determining Regions/chemistry , Complementarity Determining Regions/metabolism , Flow Cytometry , Histocompatibility Antigens/chemistry , Histocompatibility Antigens/immunology , Histocompatibility Antigens/metabolism , Ligands , Lymphocyte Activation/immunology , Magnetic Resonance Spectroscopy , Mice, Inbred C57BL , Mice, Knockout , Models, Immunological , Models, Molecular , Molecular Sequence Data , Peptides/immunology , Peptides/metabolism , Protein Binding/immunology , Protein Multimerization/immunology , Receptors, Antigen, T-Cell, alpha-beta/chemistry , Receptors, Antigen, T-Cell, alpha-beta/metabolism , Sequence Homology, Amino Acid , Thymocytes/cytology , Thymocytes/metabolism , Thymus Gland/embryology , Thymus Gland/immunology , Thymus Gland/metabolismABSTRACT
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.