The αβ 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 αβ-heterodimers as well as complete TCR complexes on T lymphocytes reveal that the FG loop in the β-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 αβ but not γδTCRs or immunoglobulins.mechanosensor | T-cell receptor | peptide discrimination | optical tweezers | catch bond A ntigen recognition by T lymphocytes is a crucial feature of adaptive immunity. This process requires the interaction of a clone-specific T-cell receptor (TCR) via its membrane distal variable module with a cognate peptide bound to a major histocompatibility complex molecule (pMHC) (refs. 1 and 2 and references therein). "Foreign" peptide antigens derived from infectious or other cell-altering processes including oncogenic transformation are presented either on a surface of the perturbed cell directly or indirectly via cross-presentation on antigen-presenting cells (APC). In either case, ligation of the relevant TCRαβ heterodimer initiates a cascade of T-cell signaling events following exposure of the immunoreceptor tyrosine-based activation motif (ITAM) elements in the cytoplasmic tail of the noncovalently associated subunits (CD3eγ, CD3eδ, and CD3ζζ) composing the TCR complex in 1:1:1:1 dimer stoichiometry. This accessibility allows the active kinase, Lck, to bind and phosphorylate ITAMs followed by recruitment and activation of a second tyrosine kinase, ZAP-70 (3-6). In turn, multiple downstream pathways are engaged, including transcriptional regulators controlling activation and differentiation of T cells (7,8). Thym...

Interleukin-2 tyrosine kinase (Itk) is a nonreceptor protein tyrosine kinase of the Tec family that participates in the intracellular signaling events leading to T cell activation. Tec family members contain the conserved SH3, SH2, and catalytic domains common to many kinase families, but they are distinguished by unique sequences outside of this region. The mechanism by which Itk and related Tec kinases are regulated is not well understood. Our studies indicate that Itk catalytic activity is inhibited by the peptidyl prolyl isomerase activity of cyclophilin A (CypA). NMR structural studies combined with mutational analysis show that a prolinedependent conformational switch within the Itk SH2 domain regulates substrate recognition and mediates regulatory interactions with the active site of CypA. CypA and Itk form a stable complex in Jurkat T cells that is disrupted by treatment with cyclosporin A. Moreover, the phosphorylation levels of Itk and a downstream substrate of Itk, PLC␥1, are increased in Jurkat T cells that have been treated with cyclosporin A. These findings support a novel mode of tyrosine kinase regulation for a Tec family member and provide a molecular basis for understanding a cellular function of the ubiquitous peptidyl prolyl isomerase, CypA.N ormal cell growth depends on the precise control of protein tyrosine kinase activity (1). For certain families of kinases, the mechanism of catalytic regulation is well understood. Structures of Src tyrosine kinases (2, 3) reveal intramolecular interactions mediated by the Src homology 2 (SH2) and Src homology 3 (SH3) domains that control catalytic activity of the neighboring kinase domain. Specifically, distortion of the Src kinase active site is achieved in part by SH2 binding to a phosphorylated tyrosine residue in the C-terminal tail of Src (4). For other families of kinases, the mechanistic details of catalytic regulation remain elusive. In particular, the Tec family of nonreceptor tyrosine kinases (5) displays distinguishing characteristics that point to an alternative mode of regulation. The Tec family kinases modulate hematopoietic cellular responses to external stimuli (6). The T cell-specific Tec family member, interleukin-2 tyrosine kinase (Itk) (7,8), plays a role in the maturation of thymocytes, is required for intracellular signaling following T cell receptor (TCR) crosslinking, and is involved in generation of second messengers that mediate cytoskeletal reorganization (9). Itk is homologous to Src in the region spanning the SH3, SH2, and kinase domain but lacks the Src C-terminal tail that contains the regulatory tyrosine. However, activation of Itk depends on SH2-mediated interactions with phosphorylated signaling partners (9) such as Slp-76 (10) and LAT (11), suggesting a regulatory role for the Itk SH2 domain despite the absence of a Src-like C-terminal regulatory tyrosine residue.The Src regulatory mechanism highlights the role of molecular switches in controlling cellular signaling pathways. Well studied covalent modifications such as...

T lymphocytes use surface αβ 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 (∼10 pN per TCR molecule) triggers T-cell Ca 2+ 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 ∼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.mechanosensor | T-cell receptor | T-cell activation | optical tweezers | cellular force relaxation T he T-cell receptor (TCR) expressed on T lymphocytes of the adaptive immune system is a stout and squat (12-nm wide × 8-nm tall) multisubunit surface complex with a ligand binding moiety that is an αβ disulfide-linked heterodimer buttressed by the associated invariant CD3 subunits (1-3). The αβ chains are each encoded by V and J gene segments and in the case of the β, a D segment as well (4). The clone-specific TCR unique to each T lymphocyte endows mammals with the capacity to detect perturbations in host cellular function resulting from myriad infectious pathogens, physical damage (thermal, irradiation, etc.), or premalignant or malignant cellular transformations while averting strong self-reactivities that could induce autoimmunity (5).Signaling is initiated through ligation of the clonotype by its cognate antigenic peptide bound to an MHC molecule (pMHC) and displayed on the surface of antigen-presenting cells (APCs) (6, 7). Ligation impacts disposition and function of the associated CD3 dimers (CD3 γ, CD3 δ, and CD3ζζ) as well as the transmembrane domains of the TCR heterodimer and CD3 subunits that interdigitate in the membrane to signal into the cytoplasm (8, 9). A cascade of intracellular events involving phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) on the CD3 cytoplasmic domains with subsequent ZAP70 activation and downstream signaling follows (10, 11). Membrane-associated CD8 and CD4 coreceptors, marking cytotoxic T lymphocytes (CTLs) and helper T cells, respectively, function to bring the membrane-anchored Src family tyrosine kinase Lck to the TCR-pMHC complex for the initiation of ITAM phosphorylation. Signaling, in turn, leads to a transient rise of cytosolic Ca 2+ and other biochemical events resulting in transcriptional activation, ultimately resulting in developmental decisions or effector functi...

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 αβT-cell receptors (αβTCRs) recognizing peptides bound to MHC molecules (pMHC). Here, we show that the pre-TCR (preTCR), a pTα-β heterodimer appearing before αβTCR expression, directs a previously unappreciated initial phase of repertoire selection. Contrasting with the ligandindependent model of preTCR function, we reveal through NMR and bioforce-probe analyses that the β-subunit binds pMHC using Vβ complementarity-determining regions as well as an exposed hydrophobic Vβ patch characteristic of the preTCR. Force-regulated single bonds akin to those of αβTCRs but with more promiscuous ligand specificity trigger calcium flux. Thus, thymic development involves sequential β-and then, αβ-repertoire tuning, whereby preTCR interactions with self pMHC modulate early thymocyte expansion, with implications for β-selection, immunodominant peptide recognition, and germ line-encoded MHC interaction.pre-T-cell receptor | NMR spectroscopy | biomembrane force probe | thymic development | repertoire selection

Interleukin-2 tyrosine kinase (Itk) is a T cell-specific kinase required for a proper immune response following T cell receptor engagement. In addition to the kinase domain, Itk is composed of several noncatalytic regulatory domains, including a Src homology 2 (SH2) domain that contains a conformationally heterogeneous Pro residue. Cis-trans isomerization of a single prolyl imide bond within the SH2 domain mediates conformer-specific ligand recognition that may have functional implications in T cell signaling. To better understand the mechanism by which a proline switch regulates ligand binding, we have used NMR spectroscopy to determine two structures of Itk SH2 corresponding to the cis and trans imide bond-containing conformers. The structures indicate that the heterogeneous Pro residue acts as a hinge that modulates ligand recognition by controlling the relative orientation of protein-binding surfaces.

Edited by Peter CresswellThe pre-T cell receptor (pre-TCR) is a pT␣-␤ heterodimer functioning in early ␣␤ T cell development. Although once thought to be ligand-autonomous, recent studies show that preTCRs 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 ␣␤TCR, the pre-TCR is a mechanosensor undergoing force-based structural transitions that dynamically enhance bond lifetimes and exploiting allosteric control regulated via the C␤ FG loop region. The pre-TCR structural transitions exhibit greater reversibility than TCR␣␤ and ordered force-bond lifetime curves. Higher piconewton force requires binding through both complementarity determining region loops and hydrophobic V␤ patch apposition. This patch functions in the pre-TCR as a surrogate V␣ domain, fostering ligand promiscuity to favor development of ␤ chains with self-reactivity but is occluded by ␣ subunit replacement of pT␣ upon ␣␤TCR 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 ␣␤TCR repertoire tuning via the pre-TCR.The mammalian adaptive immune system protects its host against infectious diseases as well as tumors in a highly specific manner. At the core of ␣␤ T lymphocyte recognition is self-versus non-self-discrimination, a functionality endowed by clonal cell-surface T cell receptors (TCRs) 4 (1-3). In the mammalian thymus, the millions of distinct TCRs expressed create a repertoire that is refined to eliminate unwanted autoreactive specificities prior to export into the peripheral lymphoid compartment (Ref. 4 and references therein).The earliest thymocytes, termed double negative (DN1-4), lack both CD4 and CD8 and expression of ␣␤TCR complexes (hereafter termed ␣␤TCRs) (5). Within the DN3 stage, a pre-TCR complex is generated comprised of a variable TCR␤ chain disulfide-linked to the invariant pT␣ subunit. In turn, the pT␣-␤ heterodimer is noncovalently complexed with the same CD3 dimers as found in the ␣␤TCR, namely CD3⑀␥, CD3⑀␦, and CD3 (1, 2). This pre-TCR complex triggers cellular survival and expansion and, importantly, induces expression of CD4 and CD8 co-receptors so that the thymocytes transit to the DP (CD4 ϩ CD8 ϩ ) thymocyte stage where rearrangement of the TCR␣ gene occurs. Only at the DP stage is the ␣␤TCR expressed. The pre-TCR signaling process, termed ␤ selection, also controls allelic exclusion of the TCR␤ locus in a given cell (6). Pre-TCR signaling components include tyrosine kinases Lck, Fyn, with Notch-1, Notch-1 ligand DL4, interleukin 7, and CXCR4 supporting pre-TCR function (5, 10). Although ␣␤TCR DP thymocyte selection processes involve pMHC-dependent posit...

Each αβT cell receptor (TCR) functions as a mechanosensor. The TCR is comprised of a clonotypic TCRαβ 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 TCRαβ 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 TCRαβ-pMHC complex and its variants under physiologic loads to identify this mechanism and any attendant TCRαβ domain allostery. The TCRαβ-pMHC interface is dynamically maintained by contacts with a spectrum of occupancies, introducing a level of control via relative motion between Vα and Vβ 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-Vβ motion, stabilizing the interface. A second level of control is exerted by Cα and Cβ constant domains, especially Cβ and its protruding FG-loop, that create mismatching interfaces among the four TCRαβ domains and with a pMHC ligand. Applied load enhances fit through deformation of the TCRαβ molecule. Thus, the catch bond involves the entire TCRαβ 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 TCRαβ ectodomain is structurally conserved, the proposed mechanism can be universally adopted to operate under load during immune surveillance by diverse αβTCRs constituting the T cell repertoire.

The αβTCR was recently revealed to function as a mechanoreceptor. That is, it leverages mechanical energy generated during immune surveillance and at the immunological synapse to drive biochemical signaling following ligation by a specific foreign peptide–MHC complex (pMHC). Here, we review the structural features that optimize this transmembrane (TM) receptor for mechanotransduction. Specialized adaptations include (1) the CβFG loop region positioned between Vβ and Cβ domains that allosterically gates both dynamic T cell receptor (TCR)–pMHC bond formation and lifetime; (2) the rigid super β-sheet amalgams of heterodimeric CD3εγ and CD3εδ ectodomain components of the αβTCR complex; (3) the αβTCR subunit connecting peptides linking the extracellular and TM segments, particularly the oxidized CxxC motif in each CD3 heterodimeric subunit that facilitates force transfer through the TM segments and surrounding lipid, impacting cytoplasmic tail conformation; and (4) quaternary changes in the αβTCR complex that accompany pMHC ligation under load. How bioforces foster specific αβTCR-based pMHC discrimination and why dynamic bond formation is a primary basis for kinetic proofreading are discussed. We suggest that the details of the molecular rearrangements of individual αβTCR subunit components can be analyzed utilizing a combination of structural biology, single-molecule FRET, optical tweezers, and nanobiology, guided by insightful atomistic molecular dynamic studies. Finally, we review very recent data showing that the pre-TCR complex employs a similar mechanobiology to that of the αβTCR to interact with self-pMHC ligands, impacting early thymic repertoire selection prior to the CD4+CD8+ double positive thymocyte stage of development.