Functional role of T-cell receptor nanoclusters in signal initiation and antigen discrimination

Antigen recognition by the T-cell receptor (TCR) is a hallmark of the adaptive immune system. When the TCR engages a peptide bound to the restricting major histocompatibility complex molecule (pMHC), it transmits a signal via the associated CD3 complex. How the extracellular antigen recognition event leads to intracellular phosphorylation remains unclear. Here, we used single-molecule localization microscopy to quantify the organization of TCR–CD3 complexes into nanoscale clusters and to distinguish between triggered and nontriggered TCR–CD3 complexes. We found that only TCR–CD3 complexes in dense clusters were phosphorylated and associated with downstream signaling proteins, demonstrating that the molecular density within clusters dictates signal initiation. Moreover, both pMHC dose and TCR–pMHC affinity determined the density of TCR–CD3 clusters, which scaled with overall phosphorylation levels. Thus, TCR–CD3 clustering translates antigen recognition by the TCR into signal initiation by the CD3 complex, and the formation of dense signaling-competent clusters is a process of antigen discrimination.The activation of T cells orchestrates an adaptive immune response by translating antigen binding to the T-cell receptor (TCR) into appropriate cellular responses (1–4). The αβ TCR engages MHC molecules (or HLA) bound to antigenic peptides (pMHC) on the surface of antigen-presenting cells (5). The interaction of the TCR with pMHC is highly specific because T cells are able to distinguish rare foreign pMHC among abundant self pMHC molecules (6). TCR signaling is also extremely sensitive; even a single pMHC molecule is sufficient to trigger activation (7–9). TCRs are noncovalently coupled to the conserved multisubunit CD3 complex, comprising CD3εγ, CD3εδ, and CD3ζζ dimers (10), whose immunoreceptor tyrosine-based activation motifs (ITAMs) are phosphorylated upon pMHC engagement by the nonreceptor tyrosine kinase Lck (1, 2). ITAM phosphorylation is required for the recruitment and phosphorylation of the ζ-chain-associated protein kinase 70 kDa (Zap70) and the adaptor linker for activation of T cells (Lat) (11) to mediate downstream activation responses (12). Phosphorylation of the TCR–CD3 complex is one of the earliest detectable biochemical events in T-cell signaling and already at this level, important “activation decisions” are being made. For example, when the extent of ITAM phosphorylation was modulated through specific mutations, low levels of TCR–CD3 phosphorylation were sufficient for signaling through the Zap70–SLP-76–Lat pathway and cytokine production, whereas high levels of TCR–CD3 phosphorylation were required for Vav1-Numb-Notch signaling and T-cell proliferation (12–14). However, how the TCR–CD3 complex encodes both the quality and quantity of pMHC molecules and steers signaling activities toward appropriate cellular outcomes is not fully understood (1–4).Although many of the molecular players and TCR signaling pathways have been identified and characterized by biochemical and genetic approaches (12, 15), the precise mechanism by which the binding of the TCR to pMHC results in phosphorylation of the TCR–CD3 complex, referred to as TCR triggering, still remains contested (1, 16). There is increasing evidence that the spatial reorganization of the TCR into micrometer- and submicron-sized clusters is involved in regulating T-cell activation (2, 11, 17–19). With the advent of superresolution fluorescence microscopy, we have gained a much more nuanced picture of the spatial organization of TCR signaling proteins (3, 20). In particular, single-molecule localization microscopy [SMLM, including photoactivated localization microscopy (PALM) (21) and direct stochastic optical reconstruction microscopy (dSTORM) (22)] was used to report that at least a proportion of TCRs were organized into small clusters that were 30–300 nm in diameter, termed “nanoclusters” (23, 24). Similarly, Lat (23–25), Lck (26), and Zap70 (24, 27) were also found to reside in nanoclusters that are extensively remodeled during T-cell activation. The link between preexisting and pMHC-induced nanoclustering and signaling activities is not clear at present and is the focus of the present study.To identify the functional role of TCR nanoclusters, we used two-color SMLM data and integrated a cluster detection method, density-based spatial clustering of applications with noise (DBSCAN) (28) with a customized colocalization analysis (29). This process allowed us to distinguish phosphorylated from nonphosphorylated TCR–CD3 complex clusters in intact T cells and identify the spatial organization at which individual TCR–CD3 complexes had the highest signaling efficiency. We found that not all TCR–CD3 complexes had the same likelihood of being phosphorylated, even with excess doses of high-affinity pMHC molecules. The signaling efficiency of the TCR–CD3 complex was dependent upon the distance to neighboring complexes so that dense nanoclusters had the highest TCR triggering efficiency.     

Structural features underlying T-cell receptor sensitivity to concealed MHC class I micropolymorphisms

Polymorphic differences distinguishing MHC class I subtypes often permit the presentation of shared epitopes in conformationally identical formats but can affect T-cell repertoire selection, differentially impacting autoimmune susceptibilities and viral clearance in vivo. The molecular mechanisms underlying this effect are not well understood. We performed structural, thermodynamic, and functional analyses of a conserved T-cell receptor (TCR) which is frequently expanded in response to a HIV-1 epitope when presented by HLA-B*5701 but is not selected by HLA-B*5703, which differs from HLA-B*5701 by two concealed polymorphisms. Our findings illustrate that although both HLA-B*57 subtypes display the epitope in structurally conserved formats, the impact of their polymorphic differences occurs directly as a consequence of TCR ligation, primarily because of peptide adjustments required for TCR binding, which involves the interplay of polymorphic residues and water molecules. These minor differences culminate in subtype-specific differential TCR-binding kinetics and cellular function. Our data demonstrate a potential mechanism whereby the most subtle MHC class I micropolymorphisms can influence TCR use and highlight their implications for disease outcomes.The MHC class I (MHCI) locus is described consistently as a major host factor influencing disease outcome in the setting of HIV-1 infection (1). As MHCI molecules select the repertoires of viral epitopes presented to CD8⁺ T cells, they shape the immune response against the virus. A broad variety of distinct MHCI allotypes, which can demonstrate up to 10% amino acid diversity, allows extensive sampling of epitope repertoires, and these differences also influence the efficacy of viral control, as illustrated by the strong association of individual MHCI types with prolonged AIDS-free survival (1, 2). Less understood is the role of minor polymorphisms (termed “micropolymorphisms”) that distinguish closely related MHCI subtypes, often by as few as one amino acid change. Although such minimal changes frequently allow identical epitopes to be presented, they often influence the efficacy of viral clearance (3) and disease susceptibilities in vivo (4). These effects have important implications in the context of HIV-1 infection, where the delicate interplay between CD8⁺ T-cell selection, viral evolution, and fitness cost is assumed to shape the clinical course of disease (5).MHCI subtypes separated by micropolymorphisms frequently permit the presentation of shared epitopes, however, these differences can affect the conformation of peptides in their binding grooves (6–8) and/or the positioning of MHC α1/α2 helices (9, 10), with implications for T-cell receptor (TCR) usage in vivo. The protective HLA-B*5703 and B*5702 subtypes, for example, are distinguished by a single amino acid substitution that does not notably alter their peptide-binding motifs. However, the positioning of this polymorphism within the TCR footprint is likely to affect the use of the TCR repertoire and could explain the greater association of HLA-B*5703 with lower viral set point and immune control (11). Yet for other MHCI subtype–peptide combinations, minor polymorphic differences result in minimal, if any, conformational disparities (4) and the molecular processes underlying differential T-cell selection are not well understood. This is especially true for longer epitopes, which comprise an important group of ligands (12, 13) for which the diversity of their responding TCR repertoires may be further limited by their atypical structural conformations when presented by MHCI. Notable examples are the HLA-B*57 subtypes HLA-B*5701 and B*5703, which consistently are associated with prolonged AIDS-free survival (14–16). Despite two polymorphic amino acids distinguishing these subtypes at residues 114 (D-N) and 116 (S-Y), both bind an equivalent repertoire of peptides (17) and in HIV-1 infection share similar CD8⁺ T-cell immunodominance hierarchies from acute infection through to chronic disease (18–22). The contribution of these HLA-B*57 subtypes to successful viral control is thought to relate to the epitopes selected, most notably to three p24-derived capsid epitopes targeted. Two of these, the ISPRTLNAW (IW9) and TSTLQEQIGW (TW10) epitopes, are targeted in early infection, presumably contributing to rapid viral control (22, 23). The nature of T-cell–driven escape mutations that accrue for these epitopes are conserved in the presence of both B*57 subtypes, presumably reflecting shared modes of peptide presentation and shared T-cell recognition conformations in vivo. However, the subtype-specific differences appear to impact a third epitope, KAFSPEVIPMF (KF11), which dominates the B*57-restricted immune response in chronic disease (18, 21). Targeting of this epitope is important in patients in whom circulating IW9 and TW10 mutations lead to immune escape (20, 24, 25), and its recognition is associated with lower plasma viral load (26). In HLA-B*5703⁺ patients, diverse KF11 variants circulate, which frequently associate with elevated viral loads (20, 27). However, viruses harboring these mutations are rare in carriers of HLA-B*5701 (24, 28), a finding not readily explained by factors specific to the infecting viral clades (27). We and others have analyzed the KF11-specific TCR repertoire in HLA-B*57⁺ patients and have reported common use of a conserved and frequently immunodominant Vα5/Vβ19 TCR pair sharing highly conserved CDR3α and -β motifs in unrelated HLA-B*5701⁺ individuals (21, 27, 29). This “public” TCR displays cross-reactivity against broad KF11 variants (30). However, this receptor pair does not represent a KF11-specific clonotype in carriers of HLA-B*5703 and its absence might contribute to the higher incidence of circulating KF11 variants in HLA-B*5703⁺ individuals (21, 27, 31).Although the majority of MHCI restricted epitopes are 8–10 amino acids, peptides of noncanonical length up to 13 residues, and particularly viral peptides targeted in humans, represent an important category of epitopes, (32–34). Longer peptides bind MHCI molecules either by extending beyond the peptide-binding groove (PBG) (35) or, more frequently, by forming a central bulge that arches above the cleft while maintaining standard A, B, and F pocket binding (13, 33, 34, 36). Although many arched peptides remain mobile (33), others assume a rigid conformation due to stabilizing interactions that involve hydrophobic forces (36) and water-mediated and direct peptide–MHC hydrogen bonds (9). Having previously determined the structure of KF11 in complex with HLA-B*5703, we observed a central peptide bulge, with two P residues forming the basis of the stable peptide arch (36). Here we present a structural, thermodynamic, and functional study describing the molecular features underlying TCR-mediated recognition of the atypical KF11 epitope and highlight the vital role played by germline-encoded TCR α-chain residues. We also demonstrate how minimal differences between the HLA-B*57 subtypes involving subtle alterations in the interplay of polymorphic residues and specific networks of water molecules in the PBG are paramount in facilitating optimal TCR binding, influencing the kinetics of the TCR–pMHCI interactions and cellular function. Collectively, our findings illustrate how subtle subtype-specific polymorphic differences can have important implications for T-cell use, repertoire diversity, and presumably, disease outcomes.     

Signal strength regulates antigen-mediated T-cell deceleration by distinct mechanisms to promote local exploration or arrest

T lymphocytes are highly motile cells that decelerate upon antigen recognition. These cells can either completely stop or maintain a low level of motility, forming contacts referred to as synapses or kinapses, respectively. Whether similar or distinct molecular mechanisms regulate T-cell deceleration during synapses or kinapses is unclear. Here, we used microfabricated channels and intravital imaging to observe and manipulate T-cell kinapses and synapses. We report that high-affinity antigen induced a pronounced deceleration selectively dependent on Ca²⁺ signals and actin-related protein 2/3 complex (Arp2/3) activity. In contrast, low-affinity antigens induced a switch of migration mode that promotes T-cell exploratory behavior, characterized by partial deceleration and frequent direction changes. This switch depended on T-cell receptor binding but was largely independent of downstream signaling. We propose that distinct mechanisms of T-cell deceleration can be triggered during antigenic recognition to favor local exploration and signal integration upon suboptimal stimulus and complete arrest on the best antigen-presenting cells.Vigorous cellular motility is a critical property of T cells that constantly survey secondary lymphoid organs and peripheral tissues in search of cognate antigen. At steady state, T cells migrate in lymph nodes at 12–15 μm/min in a pattern best described as a “guided random walk,” moving among the fibroblastic reticular cell (FRC) network in an apparent stochastic manner (1, 2). Upon encounter with an antigen-presenting cell (APC) harboring cognate antigen, T cells can adopt two types of behavior (3, 4). Under certain conditions, T-cell receptor (TCR) stimulation can lead to the complete arrest of T-cell migration and subsequent stable T cell–APC conjugation. This contact, also referred to as synapse, may last several hours and is promoted by high intracellular calcium signals (5, 6), although Ca²⁺ elevation may not always be required for T-cell arrest (7). Under other circumstances, T cells decelerate upon antigen encounter but do not completely stop migrating, only maintaining brief contact with the APC for a few minutes. Such transient and dynamic interactions have been termed kinapses (3). Kinapses can predominate in the early phases of T-cell activation (8–10) and are favored by TCR ligands of low potency or low affinity (6, 11). Kinapses can also be observed in the late phase of activation when T cells have been visualized swarming antigen-bearing APCs (1, 9) as well as during interactions between follicular helper T cells and germinal center B cells (12). At least in some instances, kinapses can result in measurable TCR signaling, as visualized by TCR internalization, Ca²⁺ elevation, and shedding of CD62L (11–14). Therefore, T cells can effectively couple motility and integration of activation signals. Although the formation of stable T cell–APC immunological synapses has been studied in detail, the molecular mechanisms driving kinapse behavior remain to be fully understood. In particular, it is not known whether T-cell deceleration during kinapses and synapses relies on the same molecular mechanisms. It is also unclear whether kinapses simply reflect a slow version of T-cell steady-state migration or whether they are associated with a fundamentally different mode of motility.To address these issues, we visualized and manipulated T-cell kinapses and synapses using two complementary approaches: (i) fabricated microchannels (15) to provide a 3D confined environment favoring T-cell motility in vitro (16) and (ii) intravital two-photon imaging of lymph nodes to study T cells in their native environment.We found that high-affinity antigen triggered maximal T-cell deceleration that was selectively dependent on Ca²⁺ signals and actin-related protein 2/3 complex (Arp2/3) activity. By contrast, weak-affinity ligands promoted a switch of migration mode, characterized by partial deceleration and frequent direction changes that underlay the exploratory behavior of immunological kinapses. This switch required TCR binding to pMHC but was independent of intracellular TCR signals. Our results suggest that the action of distinct mechanisms tailors the level of T-cell deceleration to the antigenic stimulus to promote scanning of APCs with low stimulatory capacity and full arrest on highly stimulatory APCs.     

DNA-based nanoparticle tension sensors reveal that T-cell receptors transmit defined pN forces to their antigens for enhanced fidelity

T cells are triggered when the T-cell receptor (TCR) encounters its antigenic ligand, the peptide-major histocompatibility complex (pMHC), on the surface of antigen presenting cells (APCs). Because T cells are highly migratory and antigen recognition occurs at an intermembrane junction where the T cell physically contacts the APC, there are long-standing questions of whether T cells transmit defined forces to their TCR complex and whether chemomechanical coupling influences immune function. Here we develop DNA-based gold nanoparticle tension sensors to provide, to our knowledge, the first pN tension maps of individual TCR-pMHC complexes during T-cell activation. We show that naïve T cells harness cytoskeletal coupling to transmit 12–19 pN of force to their TCRs within seconds of ligand binding and preceding initial calcium signaling. CD8 coreceptor binding and lymphocyte-specific kinase signaling are required for antigen-mediated cell spreading and force generation. Lymphocyte function-associated antigen 1 (LFA-1) mediated adhesion modulates TCR-pMHC tension by intensifying its magnitude to values >19 pN and spatially reorganizes the location of TCR forces to the kinapse, the zone located at the trailing edge of migrating T cells, thus demonstrating chemomechanical crosstalk between TCR and LFA-1 receptor signaling. Finally, T cells display a dampened and poorly specific response to antigen agonists when TCR forces are chemically abolished or physically “filtered” to a level below ∼12 pN using mechanically labile DNA tethers. Therefore, we conclude that T cells tune TCR mechanics with pN resolution to create a checkpoint of agonist quality necessary for specific immune response.T-cell activation is a crucial step in adaptive immunity, offering defense against pathogens and cancer (1). During activation, the T-cell receptor (TCR) recognizes and binds to its ligand, the antigenic peptide-major histocompatibility complex (pMHC), which is expressed on the surface of antigen-presenting cells (APCs). Because T cells are continuously moving and scanning the surfaces of APCs for evidence of antigens, and TCR-ligand binding occurs at the junction between two cells, it is likely that the TCR experiences mechanical forces during normal T-cell function. Therefore, important questions in this area pertain to whether the TCR-pMHC complex experiences defined forces during T-cell activation, and whether these forces influence immune function (2).An elegant body of single-molecule experiments further underscores the connection between TCR signaling and mechanics. For example, Lang and Reinherz (3) used optical tweezers to demonstrate that the TCR responds to physical forces applied using an optically trapped bead. This team also showed that the TCR undergoes distinct structural transitions within its FG loop (a region formed by F and G strands) when the pMHC-TCR complex is strained at ∼15 pN (4). Complementary single-molecule force spectroscopy measurements using the biomembrane force probe by Zhu and Evavold (5) showed that the average TCR-pMHC bond lifetime (1/k_off rate) is enhanced when ∼10 pN of tension is applied through a specific antigen. Enhancement of bond lifetime under the influence of antigen mechanical strain (∼10 pN) was further demonstrated in CD4⁺ T cells (6) and for pre–TCR-pMHC interactions (7). These experiments specifically demonstrate an inherent TCR sensitivity to pN forces transmitted through its cognate pMHC ligand (3–8).The role of forces in modulating bond lifetimes is particularly striking given that the most widely accepted model of TCR activation invokes a kinetic proofreading mechanism, emphasizing the importance of the TCR-pMHC dissociation rate in boosting antigen specificity (2, 9). The implicit model is that a T cell actively regulates forces transmitted to its TCR-pMHC complex to fine-tune bond lifetimes, thereby enhancing selective and differential levels of TCR activation and regulating antigen discrimination and T-cell selection.The role of mechanics in T-cell activation remains controversial, however. For example, does the T cell itself transmit 10–15 pN of tension to its engaged TCR-pMHC complex during the early stages of antigen proofreading? Although traction force microscopy methods demonstrate that T cells generate contractile forces ∼5 min after activation (10, 11), there is no evidence showing that the TCR-pMHC complex experiences pN forces during initial antigen encounters. Such forces are beyond the spatial and temporal resolution of traction force microscopy. Therefore, new molecular approaches are needed to investigate intrinsic TCR mechanics and to determine the physical basis and physiological consequences of mechanics in immunology.     

The coreceptor CD4 is expressed in distinct nanoclusters and does not colocalize with T-cell receptor and active protein tyrosine kinase p56lck

CD4 molecules on the surface of T lymphocytes greatly augment the sensitivity and activation process of these cells, but how it functions is not fully understood. Here we studied the spatial organization of CD4, and its relationship to T-cell antigen receptor (TCR) and the active form of Src kinase p56lck (Lck) using single and dual-color photoactivated localization microscopy (PALM) and direct stochastic optical reconstruction microscopy (dSTORM). In nonactivated T cells, CD4 molecules are clustered in small protein islands, as are TCR and Lck. By dual-color imaging, we find that CD4, TCR, and Lck are localized in their separate clusters with limited interactions in the interfaces between them. Upon T-cell activation, the TCR and CD4 begin clustering together, developing into microclusters, and undergo a larger scale redistribution to form supramolecluar activation clusters (SMACs). CD4 and Lck localize in the inner TCR region of the SMAC, but this redistribution of disparate cluster structures results in enhanced segregation from each other. In nonactivated cells these preclustered structures and the limited interactions between them may serve to limit spontaneous and random activation events. However, the small sizes of these island structures also ensure large interfacial surfaces for potential interactions and signal amplification when activation is initiated. In the later activation stages, the increasingly larger clusters and their segregation from each other reduce the interfacial surfaces and could have a dampening effect. These highly differentiated spatial distributions of TCR, CD4, and Lck and their changes during activation suggest that there is a more complex hierarchy than previously thought.For helper T cells, CD4 has been termed a coreceptor based on its important role in antigen recognition class II major histocompatibility complex (MHC)–peptide complexes by the αβ T-cell receptor (TCR) as well as in signal transduction. Indeed, CD4 significantly increases T-cell sensitivity to antigen upon activation (1–4). This ability of CD4 to enhance antigen recognition has often been connected to the fact that the N-terminal Ig domain of CD4 has specific affinity to invariant sites on MHC class II molecules (5, 6). It has been suggested that CD4 stabilizes the molecular complex of TCR and peptide–MHC (pMHC) by binding to the same MHC either simultaneously with the TCR (7) or shortly after TCR–pMHC engagement (2, 3). However, from more recent 2D measurements, CD4 blockades showed no effect on the stability of TCR binding to agonist peptide–MHC complexes in a synapse (8). In terms of signal transduction, the role of CD4 has been studied based on the binding ability of a cysteine motif in the cytoplasmic tail of CD4 to Src kinase p56lck (Lck) (9), which is responsible for the phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) sequences in TCR–CD3 complex as the earliest observable biochemical event during T-cell activation (10). It has been proposed that CD4 mainly contributes to the sensitivity of T cells by facilitating the recruitment of Lck to TCR–CD3s that are actively engaged in ligand recognition (11, 12). Nevertheless, the absence of CD4 does not preclude T cells from being generated at the thymus or being activated by TCR–pMHC engagement (13, 14).It is now well appreciated that spatial reorganization and distribution of some of the membrane receptors and signaling molecules is one of the critical regulating mechanisms in T-cell activation. The molecular assembly and clusters such as supramolecular activation clusters (SMACs) (15) of immunological synapse (IS) (14), microclusters (16–20), and their roles in T-cell signaling have been widely studied. More recently, the presence and unique roles of smaller-sized protein clusters, termed “nanoclusters” or “protein islands,” of TCR–CD3 complex (21–24), linker for activation of T cell (LAT) (21, 22, 24, 25), Lck (26), and other signaling molecules (24) were revealed by electron microscopy and the newly available superresolution fluorescence microscopy.Considering that the TCR–CD3 complex, CD4, and Lck are constitutively expressed in nonactivated T cells, it is highly likely that the interaction dynamics between these components would also be controlled spatially during the T-cell activation process. Here, we studied the relative molecular distribution of these molecules using single- and dual-color photoactivated localization microscopy (PALM) (27) and direct stochastic optical reconstruction microscopy (dSTORM) (28, 29) in live and fixed T cells for both nonactivated and activating conditions. The corresponding spatial analyses were also used to quantitatively determine the sizes, degree of clustering, and degree of interactions of these clusters. We found that CD4 is also expressed in preclustered structures, separate from TCR–CD3 and LAT, and composed of three to six molecules per cluster. The interactions between these molecules occurred only in the interfaces between the clusters. Upon T-cell activation, the TCR–CD3 and CD4 molecules increased the size of their own clusters without appreciable mixing. Instead, their molecular segregation increased, whereas the T cell develops a synapse structure, often in the SMAC or “bull’s eye” pattern, with the TCR–CD3 in the central supramolecular activation cluster (cSMAC) with the CD4 and Lck clusters localizing around it. These observed clustering behaviors accompanying reorganization of spatial distributions of CD4, Lck, and TCR might be a general and effective mechanism to activate and regulate the T-cell signaling by controlling the magnitude of interfacial interactions between signaling components in each cluster.     

Neofunction of ACVR1 in fibrodysplasia ossificans progressiva

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease characterized by extraskeletal bone formation through endochondral ossification. FOP patients harbor point mutations in ACVR1 (also known as ALK2), a type I receptor for bone morphogenetic protein (BMP). Two mechanisms of mutated ACVR1 (FOP-ACVR1) have been proposed: ligand-independent constitutive activity and ligand-dependent hyperactivity in BMP signaling. Here, by using FOP patient-derived induced pluripotent stem cells (FOP-iPSCs), we report a third mechanism, where FOP-ACVR1 abnormally transduces BMP signaling in response to Activin-A, a molecule that normally transduces TGF-β signaling but not BMP signaling. Activin-A enhanced the chondrogenesis of induced mesenchymal stromal cells derived from FOP-iPSCs (FOP-iMSCs) via aberrant activation of BMP signaling in addition to the normal activation of TGF-β signaling in vitro, and induced endochondral ossification of FOP-iMSCs in vivo. These results uncover a novel mechanism of extraskeletal bone formation in FOP and provide a potential new therapeutic strategy for FOP.Heterotopic ossification (HO) is defined as bone formation in soft tissue where bone normally does not exist. It can be the result of surgical operations, trauma, or genetic conditions, one of which is fibrodysplasia ossificans progressiva (FOP). FOP is a rare genetic disease characterized by extraskeletal bone formation through endochondral ossification (1–6). The responsive mutation for classic FOP is 617G > A (R206H) in the intracellular glycine- and serine-rich (GS) domain (7) of ACVR1 (also known as ALK2), a type I receptor for bone morphogenetic protein (BMP) (8–10). ACVR1 mutations in atypical FOP patients have been found also in other amino acids of the GS domain or protein kinase domain (11, 12). Regardless of the mutation site, mutated ACVR1 (FOP-ACVR1) has been shown to activate BMP signaling without exogenous BMP ligands (constitutive activity) and transmit much stronger BMP signaling after ligand stimulation (hyperactivity) (12–25).To reveal the molecular nature of how FOP-ACVR1 activates BMP signaling, cells overexpressing FOP-ACVR1 (12–20), mouse embryonic fibroblasts derived from Alk2^R206H/+ mice (21, 22), and cells from FOP patients, such as stem cells from human exfoliated deciduous teeth (23), FOP patient-derived induced pluripotent stem cells (FOP-iPSCs) (24, 25) and induced mesenchymal stromal cells (iMSCs) from FOP-iPSCs (FOP-iMSCs) (26) have been used as models. Among these cells, Alk2^R206H/+ mouse embryonic fibroblasts and FOP-iMSCs are preferred because of their accessibility and expression level of FOP-ACVR1 using an endogenous promoter. In these cells, however, the constitutive activity and hyperactivity is not strong (within twofold normal levels) (22, 26). In addition, despite the essential role of BMP signaling in development (27–31), the pre- and postnatal development and growth of FOP patients are almost normal, and HO is induced in FOP patients after physical trauma and inflammatory response postnatally, not at birth (1–6). These observations led us to hypothesize that FOP-ACVR1 abnormally responds to noncanonical BMP ligands induced by trauma or inflammation.Here we show that FOP-ACVR1 transduced BMP signaling in response to Activin-A, a molecule that normally transduces TGF-β signaling (10, 32–34) and contributes to inflammatory responses (35, 36). Our in vitro and in vivo data indicate that activation of TGF-β and aberrant BMP signaling by Activin-A in FOP-cells is one cause of HO in FOP. These results suggest a possible application of anti–Activin-A reagents as a new therapeutic tool for FOP.     

Impaired NK-mediated regulation of T-cell activity in multiple sclerosis is reconstituted by IL-2 receptor modulation

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS) resulting from a breakdown in peripheral immune tolerance. Although a beneficial role of natural killer (NK)-cell immune-regulatory function has been proposed, it still needs to be elucidated whether NK cells are functionally impaired as part of the disease. We observed NK cells in active MS lesions in close proximity to T cells. In accordance with a higher migratory capacity across the blood–brain barrier, CD56^bright NK cells represent the major intrathecal NK-cell subset in both MS patients and healthy individuals. Investigating the peripheral blood and cerebrospinal fluid of MS patients treated with natalizumab revealed that transmigration of this subset depends on the α₄β₁ integrin very late antigen (VLA)-4. Although no MS-related changes in the migratory capacity of NK cells were observed, NK cells derived from patients with MS exhibit a reduced cytolytic activity in response to antigen-activated CD4⁺ T cells. Defective NK-mediated immune regulation in MS is mainly attributable to a CD4⁺ T-cell evasion caused by an impaired DNAX accessory molecule (DNAM)-1/CD155 interaction. Both the expression of the activating NK-cell receptor DNAM-1, a genetic alteration consistently found in MS-association studies, and up-regulation of the receptor’s ligand CD155 on CD4⁺ T cells are reduced in MS. Therapeutic immune modulation of IL-2 receptor restores impaired immune regulation in MS by increasing the proportion of CD155-expressing CD4⁺ T cells and the cytolytic activity of NK cells.Multiple sclerosis (MS) is a chronic inflammatory demyelinating autoimmune disease of the central nervous system (CNS) (1) and one of the major causes of neurological disability in young adults (2). MS is considered to be a primarily antigen-driven T cell-mediated disease with a complex genetic background influenced by environmental factors (1, 3) that is caused by an imbalanced immune-regulatory network (4). Among other well-known players of this network such as regulatory T cells and tolerogenic dendritic cells (DCs) (1), natural killer (NK) cells have been recently identified as additional factors in controlling homeostasis of antigen-activated T cells (5, 6).Originally discovered as antigen receptor-negative innate lymphocytes that play an important role in controlling virus-infected and tumor cells (7), NK cells have also been shown to suppress activated T cells through secretion of anti-inflammatory cytokines and/or cytolytic function (5, 6, 8–12). NK cells lyse target cells in a complex process depending on cell surface expression of certain inhibitory and activating receptors on NK cells and the corresponding ligands on target cells (13). Several activating NK-cell receptors–in particular, NKG2D (CD314) (5, 8, 9, 11, 14), the receptor for MIC-A/B and ULBP1-6, and DNAM-1 (DNAX accessory molecule, CD226) (6, 12, 15), the receptor for Nectin-2 (CD112) and poliovirus receptor (PVR/CD155)−have been proposed to be involved in NK cell-mediated lysis of activated T cells. Of note, polymorphisms in the gene encoding for DNAM-1 have been consistently found in MS-association studies (16–18). Both major NK-cell subsets, namely the CD56^brightCD16^dim/− and the CD56^dimCD16⁺ subsets (here referred to as CD56^bright and CD56^dim, respectively), seem to be capable of killing activated T cells (19). CD56^dim NK cells are the major NK-cell subset in the peripheral blood (PB) (90% of NK cells) and kill target cells without prior sensitization but only secrete low levels of cytokines (7, 20, 21), whereas CD56^bright NK cells are more abundant in secondary lymphoid tissues and inflammatory lesions (75–95% of NK cells), where they produce high amounts of immune-modulating cytokines but acquire cytolytic functions only after prolonged activation (7, 20, 21).Immune-modulating therapies targeting NK-cell frequencies and cytolytic functions among others such as IFN-β (22–24), glatiramer acetate (25), natalizumab (26, 27), fingolimod (28, 29), and daclizumab (10, 30, 31) point to an immune-protective role of both NK-cell subsets in MS. Daclizumab, a humanized antibody directed against the IL-2 receptor (IL-2R) α-chain (CD25) (reviewed in ref. 4) is a promising MS therapy, which recently showed superior efficacy compared with IFN-β in a phase III study (32). Expansion of peripheral (10, 33) as well as intrathecal (34) CD56^bright NK cells under daclizumab treatment correlated positively with therapeutic response (10, 30, 35). Nevertheless, it still remains to be elucidated whether NK-cell immune-regulatory functions are impaired as part of the disease process and whether modulation of the IL-2R with daclizumab restores these deficits or simply boosts NK-cell activity (4). Furthermore, the distribution and function of NK cells in active MS lesions is still poorly understood. Resolving the molecular basis of NK cell-mediated immune control and its potential impairment in MS is important for a better understanding of the role of NK cells in MS pathogenesis and the mechanism of action of NK cell-modulating therapies.The aim of the current study was to characterize the role of NK cells in the pathogenesis of MS by investigating the presence, distribution, and function of NK cells in three different compartments [CNS, cerebrospinal fluid (CSF), and PB]. Furthermore, a potential deficit in NK-cell immune-regulatory function, its underlying molecular mechanism, and the impact of IL-2R modulation by daclizumab high-yield process (DAC HYP) were explored by studying PB mononuclear cells (PBMCs) derived from clinically stable therapy-naïve MS patients and MS patients receiving daclizumab treatment in comparison with those derived from healthy individuals.     

From the Cover: Functional evidence for TCR-intrinsic specificity for MHCII

How T cells become restricted to binding antigenic peptides within class I or class II major histocompatibility complex molecules (pMHCI or pMHCII, respectively) via clonotypic T-cell receptors (TCRs) remains debated. During development, if TCR–pMHC interactions exceed an affinity threshold, a signal is generated that positively selects the thymocyte to become a mature CD4⁺ or CD8⁺ T cell that can recognize foreign peptides within MHCII or MHCI, respectively. But whether TCRs possess an intrinsic, subthreshold specificity for MHC that facilitates sampling of the peptides within MHC during positive selection or T-cell activation is undefined. Here we asked if increasing the frequency of lymphocyte-specific protein tyrosine kinase (Lck)-associated CD4 molecules in T-cell hybridomas would allow for the detection of subthreshold TCR–MHC interactions. The reactivity of 10 distinct TCRs was assessed in response to selecting and nonselecting MHCII bearing cognate, null, or “shaved” peptides with alanine substitutions at known TCR contact residues: Three of the TCRs were selected on MHCII and have defined peptide specificity, two were selected on MHCI and have a known pMHC specificity, and five were generated in vitro without defined selecting or cognate pMHC. Our central finding is that IL-2 was made when each TCR interacted with selecting or nonselecting MHCII presenting shaved peptides. These responses were abrogated by anti-CD4 antibodies and mutagenesis of CD4. They were also inhibited by anti-MHC antibodies that block TCR–MHCII interactions. We interpret these data as functional evidence for TCR-intrinsic specificity for MHCII.Positive and negative selection limit the αβT-cell repertoire to cells expressing clonotypic T-cell receptors (TCRs) that distinguish the antigenicity of peptides embedded within class I and class II major histocompatibility complex molecules (pMHCI or pMHCII, respectively) based on their source of origin (i.e., self or foreign) (1–4). Approximately 7.5% of CD4⁺CD8⁺ double-positive (DP) thymocytes express TCRs that interact with self-pMHC above an affinity threshold required for positive selection, whereas 7.5% cross a higher affinity threshold that mediates negative selection and the remaining TCRs fail to direct positive selection (5). The rules that restrict TCR recognition of antigenic peptides within MHCI or MHCII are unresolved.Two models have been proposed to explain MHC restriction. One posits that restriction is imposed by CD4 or CD8 during thymocyte development to eliminate TCRs that recognize non-MHC ligands (2, 6). Here, the CD4- and CD8-associated Src kinase, p56^Lck [lymphocyte-specific protein tyrosine kinase (Lck)], is sequestered away from the immunoreceptor tyrosine-based activation motifs (ITAMs) of the TCR-associated CD3δε, CD3γε, and CD3ζζ signaling modules. Positively selecting signals are then generated in thymocytes expressing TCRs that bind MHCII or MHCI together with CD4 or CD8, respectively, as this localizes Lck to the ITAMs. Those thymocytes expressing TCRs that do not bind MHCI or MHCII would fail to localize Lck to the ITAMs and die. In the second model, germ line-encoded complementary determining regions (CDR) 1 and 2 allow each clonotypic TCR to bind distinct classes and alleles of MHC molecules via unique yet specific recognition codons that impose a canonical docking polarity and MHC restriction (1, 3, 4, 7, 8). Although it is not obvious that these models are mutually exclusive, the key distinction is that in the first model the randomly generated preselection TCR repertoire would contain TCRs that do and do not bind pMHC, whereas in the second model most if not all TCRs would have a specificity for MHC that is germ line-encoded, regardless of the class or allele of MHC.The canonical docking polarity of TCRs on MHCI or MHCII observed in crystal structures, and the CDR1 and CDR2 contacts therein, provides evidence for germ line-encoded TCR–MHC interactions for positively selected TCRs (1, 3, 4, 7, 8). But this is taken as supporting either model, as germ line-encoded contacts are likely to be required to allow the formation of a TCR–CD3–pMHC–CD4/CD8 macrocomplex that situates the CD3 ITAMs and Lck in a functionally mandated orientation (1–4, 6, 9, 10). Structural insights from positively selected TCRs thus do not allow the basis of MHC restriction to be cleanly addressed, and functional data that support either model have been reported (11–15).An open question that can shed light on the similarities and differences between the two models is whether TCRs participate in subthreshold scanning of MHC (4, 16). Scanning would allow a TCR to dock on MHC and survey its contents for peptides that increase the duration of TCR–pMHC interactions, via contacts with clonotypic CDR3s, and allow the formation of a TCR–CD3–pMHC–CD4/CD8 macrocomplex that generates signals (4, 10). In the co-receptor imposed model, a diverse preselection repertoire would contain TCRs with no intrinsic capacity to bind MHC, TCRs that interact with pMHC by atypical modalities, and TCRs that interact with a composite pMHC surface in a canonical modality in a lock-and-key manner akin to antibody–antigen recognition (2, 6). Once selected, this last group of TCRs would be predicted to scan composite pMHC with shapes (i.e., topology and chemical characteristics) related to the selecting pMHC—presumably the same MHC, or similar allelic variant, presenting related peptides. In the germ line-encoded recognition model, TCR scanning of MHC via recognition codons would be intrinsic to most if not all TCRs, regardless of the class of MHC, allelic variants, or the peptide sequence therein (4). At present, functional evidence for TCR scanning of MHC is lacking, regardless of whether it is MHC class-, allele-, and peptide sequence-dependent.Recently, the frequency of Lck-associated CD4 molecules was proposed to influence if a TCR–pMHC interaction is of sufficient duration to direct a specific cell fate decision, such as negative selection (17). We thus hypothesized that genetically increasing the frequency of CD4–Lck association should allow for the detection of subthreshold TCR–pMHC interactions that are normally of insufficient duration to elicit a functional response. Here we show that T-cell hybridomas expressing 10 distinct TCRs along with a CD4–Lck fusion make IL-2 in response to APCs expressing selecting or nonselecting MHCII, regardless of the sequence of the presented peptide. These responses were independent of positive selection on MHCII, as TCRs that were positively selected on MHCI, or generated in vitro and thus not thymically selected, yielded similar responses. These data provide functional evidence for subthreshold TCR scanning of MHCII that is independent of the class of MHC, the allele, or the peptide sequence therein.     

Programmed cell death 1 inhibits inflammatory helper T-cell development through controlling the innate immune response

Programmed cell death 1 (PD-1) is an inhibitory coreceptor on immune cells and is essential for self-tolerance because mice genetically lacking PD-1 (PD-1^−/−) develop spontaneous autoimmune diseases. PD-1^−/− mice are also susceptible to severe experimental autoimmune encephalomyelitis (EAE), characterized by a massive production of effector/memory T cells against myelin autoantigen, the mechanism of which is not fully understood. We found that an increased primary response of PD-1^−/− mice to heat-killed mycobacteria (HKMTB), an adjuvant for EAE, contributed to the enhanced production of T-helper 17 (Th17) cells. Splenocytes from HKMTB-immunized, lymphocyte-deficient PD-1^−/− recombination activating gene (RAG)2^−/− mice were found to drive antigen-specific Th17 cell differentiation more efficiently than splenocytes from HKMTB-immunized PD-1^+/+ RAG2^−/− mice. This result suggested PD-1’s involvement in the regulation of innate immune responses. Mice reconstituted with PD-1^−/− RAG2^−/− bone marrow and PD-1^+/+ CD4⁺ T cells developed more severe EAE compared with the ones reconstituted with PD-1^+/+ RAG2^−/− bone marrow and PD-1^+/+ CD4⁺ T cells. We found that upon recognition of HKMTB, CD11b⁺ macrophages from PD-1^−/− mice produced very high levels of IL-6, which helped promote naive CD4⁺ T-cell differentiation into IL-17–producing cells. We propose a model in which PD-1 negatively regulates antimycobacterial responses by suppressing innate immune cells, which in turn prevents autoreactive T-cell priming and differentiation to inflammatory effector T cells.Autoimmune disease development is impacted by both genetic and environmental factors. Programmed cell death 1 (PD-1) is a type I membrane protein that delivers inhibitory signals to immune cells upon the binding of its ligand, PD-L1 or PD-L2 (1). PD-1 has been shown to be important for self-tolerance because spontaneous autoimmune diseases develop in PD-1^−/− mice (2–4). A single-nucleotide polymorphism that affects PD-1 expression is associated with autoimmune diseases in humans, such as systemic lupus erythematosus (5), type I diabetes (6), rheumatoid arthritis (7), and multiple sclerosis (MS) (8), suggesting that PD-1 deficiency may be a genetic factor involved in the development of autoimmunity.Experimental autoimmune encephalomyelitis (EAE) is a rodent model of T-cell–mediated inflammatory disease in the central nervous system (CNS), causing demyelination, axonal damage, and paralysis, and is a commonly used model for human MS. Previous reports suggested that PD-1 functions to attenuate EAE. PD-1 and its ligands were found to be strongly expressed on immune infiltrates in the CNS during the peak phase of EAE (9–11). In EAE studies, PD-1–deficient mice or the use of blocking antibodies that inhibit PD-1 engagement by ligands resulted in earlier disease onset, increased inflammatory infiltrates, and increased severity of clinical symptoms compared with normal disease progression (10–16). It has been demonstrated that ligand engagement of PD-1 inhibits T-cell activation, expansion, and cytokine production (17–19). Similarly, in EAE, PD-1 signaling in CNS-specific helper T cells may inhibit their expansion and secretion of inflammatory cytokines (10–12). Recently, T-helper 17 (Th17) cells were shown to be involved in EAE by producing IL-17 and GM-CSF (20, 21). Two reports showed that PD-1^−/− mice mount an augmented Th17 response to EAE induction (14, 16). However, the fundamental mechanisms by which PD-1 regulates antigen-specific Th17 cell differentiation, expansion, and effector function in EAE remain to be understood.To induce EAE, mice are immunized with myelin autoantigens in an emulsion of Mycobacterium tuberculosis (MTB)-derived adjuvants, causing a strong innate inflammatory response, leading to Th skewing (22). Curiously, recent studies showed that PD-1^−/− mice exhibited an altered response to infection with mycobacteria, characterized by uncontrolled bacterial burden; massive production of cytokines, termed “cytokine storm”; and early death (23–25). We wondered if this unique response of PD-1^−/− mice to mycobacteria contributed to their Th response in EAE.In this study, we took a combination of genetic and immunological approaches in which the innate response to MTB-derived adjuvant and antigen-specific T-cell polarization were separately analyzed. The present data suggest that an enhanced innate response of PD-1^−/− mice to MTB contributes to the susceptibility of these mice to severe EAE. We propose a previously undescribed function of PD-1 in controlling the basal state of the innate immune response, the failure of which can cause the activation of adaptive immune responses, provoking autoimmunity.     

From the Cover: Distinctive properties of identical twins' TCR repertoires revealed by high-throughput sequencing

Adaptive immunity in humans is provided by hypervariable Ig-like molecules on the surface of B and T cells. The final set of these molecules in each organism is formed under the influence of two forces: individual genetic traits and the environment, which includes the diverse spectra of alien and self-antigens. Here we assess the impact of individual genetic factors on the formation of the adaptive immunity by analyzing the T-cell receptor (TCR) repertoires of three pairs of monozygous twins by next-generation sequencing. Surprisingly, we found that an overlap between the TCR repertoires of monozygous twins is similar to an overlap between the TCR repertoires of nonrelated individuals. However, the number of identical complementary determining region 3 sequences in two individuals is significantly increased for twin pairs in the fraction of highly abundant TCR molecules, which is enriched by the antigen-experienced T cells. We found that the initial recruitment of particular TCR V genes for recombination and subsequent selection in the thymus is strictly determined by individual genetic factors. J genes of TCRs are selected randomly for recombination; however, the subsequent selection in the thymus gives preference to some α but not β J segments. These findings provide a deeper insight into the mechanism of TCR repertoire generation.Adaptive immunity is provided by B and T cells bearing B-cell receptors (BCRs) and Ig-like T-cell receptors (TCRs), respectively. These hypervariable molecules are the key part of the adaptive immune system as they can potentially recognize any alien agent and drive specific immune responses. The α/β TCRs recognize short peptides in the complex with major histocompatibility complex (MHC) molecules and play the key role in the targeted immune response. The total diversity of TCR molecules in an individual human organism is initially formed via genomic recombination with subsequent positive and negative selection at several stages of maturation and activation. The maximal theoretical diversity of TCRβ chain’s amino acid sequences in humans is estimated between 5 × 10¹¹ (1) and 10¹⁴ (2), whereas the maximal number of α/β pairs reaches 10¹⁸ (3). This huge number of variants is probably never achieved: the whole TCRβ chain repertoire size in a single human organism is estimated at 1–5 × 10⁶ (1, 4–6), although this is only a lower bound estimate. Two driving forces shape the final face of individual TCR repertoire: the individual genetics and the complexity of environmental factors. The genes coding for proteins involved in VDJ recombination, antigen processing and presentation, and products of genes participating in the immune response signaling belong to the first type of the repertoire-forming factors. The spectrum of the organism’s self-peptides presented in the thymus also depends on the individual’s set of the MHC molecules. Moreover, this spectrum of peptides is determined by the amino acid sequences of the organism’s proteins, which thus can also be considered a genetic factor. Furthermore, TCRs arising to the same alien antigenic peptides are known to be MHC restricted (7). The environmental factors include the whole range of pathogens met by the individual including disease-causing bacteria and viruses, as well as vaccines, symbionts, etc. The genetic component can potentially have a major impact on the initial recombination and selection in the thymus forming the naïve TCR repertoire, whereas the subsequent interference with antigens provides the selective expansion of some TCRs and forms the final repertoire structure. However, the particular impact of genetic factors on TCR repertoire structure and diversity is unknown.All genes of monozygous (MZ) twins are identical (including those responsible for the TCR repertoire formation), and therefore, MZ twins are widely used in the studies where the genetic impact is evaluated. Several studies of TCR repertoires were performed mainly focusing on diseases concordant and discordant MZ twins and using complementarity determining region 3 (CDR3) spectra-typing and/or low depth sequencing (8–11). Some of these studies reported the common use of particular V genes and common clonotypes. In recent years, the high-throughput sequencing technologies paved the way to whole-repertoire studies of individual TCRs that led to new findings in the field of adaptive immunity (1, 5, 6, 12–22). In this study, for the first time to the best of our knowledge, we obtain and compare the α and β chain TCR repertoires of three pairs of MZ twins using next-generation sequencing (NGS).     

Self-assembly of alkynylplatinum(II) terpyridine amphiphiles into nanostructures via steric control and metal–metal interactions

A series of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing the hydrophilic oligo(para-phenylene ethynylene) with two 3,6,9-trioxadec-1-yloxy chains was designed and synthesized. The mononuclear alkynylplatinum(II) terpyridine complex was found to display a very strong tendency toward the formation of supramolecular structures. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would lead to the formation of nanotubes or helical ribbons. These desirable nanostructures were found to be governed by the steric bulk on the platinum(II) terpyridine moieties, which modulates the directional metal−metal interactions and controls the formation of nanotubes or helical ribbons. Detailed analysis of temperature-dependent UV-visible absorption spectra of the nanostructured tubular aggregates also provided insights into the assembly mechanism and showed the role of metal−metal interactions in the cooperative supramolecular polymerization of the amphiphilic platinum(II) complexes.Square-planar d⁸ platinum(II) polypyridine complexes have long been known to exhibit intriguing spectroscopic and luminescence properties (1–54) as well as interesting solid-state polymorphism associated with metal−metal and π−π stacking interactions (1–14, 25). Earlier work by our group showed the first example, to our knowledge, of an alkynylplatinum(II) terpyridine system [Pt(tpy)(C ≡ CR)]⁺ that incorporates σ-donating and solubilizing alkynyl ligands together with the formation of Pt···Pt interactions to exhibit notable color changes and luminescence enhancements on solvent composition change (25) and polyelectrolyte addition (26). This approach has provided access to the alkynylplatinum(II) terpyridine and other related cyclometalated platinum(II) complexes, with functionalities that can self-assemble into metallogels (27–31), liquid crystals (32, 33), and other different molecular architectures, such as hairpin conformation (34), helices (35–38), nanostructures (39–45), and molecular tweezers (46, 47), as well as having a wide range of applications in molecular recognition (48–52), biomolecular labeling (48–52), and materials science (53, 54). Recently, metal-containing amphiphiles have also emerged as a building block for supramolecular architectures (42–44, 55–59). Their self-assembly has always been found to yield different molecular architectures with unprecedented complexity through the multiple noncovalent interactions on the introduction of external stimuli (42–44, 55–59).Helical architecture is one of the most exciting self-assembled morphologies because of the uniqueness for the functional and topological properties (60–69). Helical ribbons composed of amphiphiles, such as diacetylenic lipids, glutamates, and peptide-based amphiphiles, are often precursors for the growth of tubular structures on an increase in the width or the merging of the edges of ribbons (64, 65). Recently, the optimization of nanotube formation vs. helical nanostructures has aroused considerable interests and can be achieved through a fine interplay of the influence on the amphiphilic property of molecules (66), choice of counteranions (67, 68), or pH values of the media (69), which would govern the self-assembly of molecules into desirable aggregates of helical ribbons or nanotube scaffolds. However, a precise control of supramolecular morphology between helical ribbons and nanotubes remains challenging, particularly for the polycyclic aromatics in the field of molecular assembly (64–69). Oligo(para-phenylene ethynylene)s (OPEs) with solely π−π stacking interactions are well-recognized to self-assemble into supramolecular system of various nanostructures but rarely result in the formation of tubular scaffolds (70–73). In view of the rich photophysical properties of square-planar d⁸ platinum(II) systems and their propensity toward formation of directional Pt···Pt interactions in distinctive morphologies (27–31, 39–45), it is anticipated that such directional and noncovalent metal−metal interactions might be capable of directing or dictating molecular ordering and alignment to give desirable nanostructures of helical ribbons or nanotubes in a precise and controllable manner.Herein, we report the design and synthesis of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing hydrophilic OPEs with two 3,6,9-trioxadec-1-yloxy chains. The mononuclear alkynylplatinum(II) terpyridine complex with amphiphilic property is found to show a strong tendency toward the formation of supramolecular structures on diffusion of diethyl ether in dichloromethane or dimethyl sulfoxide (DMSO) solution. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would result in nanotubes or helical ribbons in the self-assembly process. To the best of our knowledge, this finding represents the first example of the utilization of the steric bulk of the moieties, which modulates the formation of directional metal−metal interactions to precisely control the formation of nanotubes or helical ribbons in the self-assembly process. Application of the nucleation–elongation model into this assembly process by UV-visible (UV-vis) absorption spectroscopic studies has elucidated the nature of the molecular self-assembly, and more importantly, it has revealed the role of metal−metal interactions in the formation of these two types of nanostructures.