Molecular architecture of the αβ T cell receptor–CD3 complex

αβ T-cell receptor (TCR) activation plays a crucial role for T-cell function. However, the TCR itself does not possess signaling domains. Instead, the TCR is noncovalently coupled to a conserved multisubunit signaling apparatus, the CD3 complex, that comprises the CD3εγ, CD3εδ, and CD3ζζ dimers. How antigen ligation by the TCR triggers CD3 activation and what structural role the CD3 extracellular domains (ECDs) play in the assembled TCR–CD3 complex remain unclear. Here, we use two complementary structural approaches to gain insight into the overall organization of the TCR–CD3 complex. Small-angle X-ray scattering of the soluble TCR–CD3εδ complex reveals the CD3εδ ECDs to sit underneath the TCR α-chain. The observed arrangement is consistent with EM images of the entire TCR–CD3 integral membrane complex, in which the CD3εδ and CD3εγ subunits were situated underneath the TCR α-chain and TCR β-chain, respectively. Interestingly, the TCR–CD3 transmembrane complex bound to peptide–MHC is a dimer in which two TCRs project outward from a central core composed of the CD3 ECDs and the TCR and CD3 transmembrane domains. This arrangement suggests a potential ligand-dependent dimerization mechanism for TCR signaling. Collectively, our data advance our understanding of the molecular organization of the TCR–CD3 complex, and provides a conceptual framework for the TCR activation mechanism.T cells are key mediators of the adaptive immune response. Each αβ T cell contains a unique αβ T-cell receptor (TCR), which binds antigens (Ags) displayed by major histocompatibility complexes (MHCs) and MHC-like molecules (1). The TCR serves as a remarkably sensitive driver of cellular function: although TCR ligands typically bind quite weakly (1–200 μM), even a handful of TCR ligands are sufficient to fully activate a T cell (2, 3). The TCR does not possess intracellular signaling domains, uncoupling Ag recognition from T-cell signaling. The TCR is instead noncovalently associated with a multisubunit signaling apparatus, consisting of the CD3εγ and CD3εδ heterodimers and the CD3ζζ homodimer, which collectively form the TCR–CD3 complex (4, 5). The CD3γ/δ/ε subunits each consist of a single extracellular Ig domain and a single immunoreceptor tyrosine-based activation motif (ITAM), whereas CD3ζ has a short extracellular domain (ECD) and three ITAMs (6–11). The TCR–CD3 complex exists in 1:1:1:1 stoichiometry for the αβTCR:CD3εγ:CD3εδ:CD3ζζ dimers (12). Phosphorylation of the intracellular CD3 ITAMs and recruitment of the adaptor Nck lead to T-cell activation, proliferation, and survival (13, 14). Understanding the underlying principles of TCR–CD3 architecture and T-cell signaling is of therapeutic interest. For example, TCR–CD3 is the target of therapeutic antibodies such as the immunosuppressant OKT3 (15), and there is increasing interest in manipulating T cells in an Ag-dependent manner by using naturally occurring and engineered TCRs (16).Assembly of the TCR–CD3 complex is primarily driven by each protein’s transmembrane (TM) region, enforced through the interaction of evolutionarily conserved, charged, residues in each TM region (4, 5, 12). What, if any, role interactions between TCR and CD3 ECDs play in the assembly and function of the complex remains controversial (5): there are several plausible proposed models of activation, which are not necessarily mutually exclusive (5, 17–19). Although structures of TCR–peptide–MHC (pMHC) complexes (2), TCR–MHC-I–like complexes (1), and the CD3 dimers (6–10) have been separately determined, how the αβ TCR associates with the CD3 complex is largely unknown. Here, we use two independent structural approaches to gain an understanding of the TCR–CD3 complex organization and structure.     

Molecular mechanism underlying β1 regulation in voltage- and calcium-activated potassium (BK) channels

Being activated by depolarizing voltages and increases in cytoplasmic Ca²⁺, voltage- and calcium-activated potassium (BK) channels and their modulatory β-subunits are able to dampen or stop excitatory stimuli in a wide range of cellular types, including both neuronal and nonneuronal tissues. Minimal alterations in BK channel function may contribute to the pathophysiology of several diseases, including hypertension, asthma, cancer, epilepsy, and diabetes. Several gating processes, allosterically coupled to each other, control BK channel activity and are potential targets for regulation by auxiliary β-subunits that are expressed together with the α (BK)-subunit in almost every tissue type where they are found. By measuring gating currents in BK channels coexpressed with chimeras between β1 and β3 or β2 auxiliary subunits, we were able to identify that the cytoplasmic regions of β1 are responsible for the modulation of the voltage sensors. In addition, we narrowed down the structural determinants to the N terminus of β1, which contains two lysine residues (i.e., K3 and K4), which upon substitution virtually abolished the effects of β1 on charge movement. The mechanism by which K3 and K4 stabilize the voltage sensor is not electrostatic but specific, and the α (BK)-residues involved remain to be identified. This is the first report, to our knowledge, where the regulatory effects of the β1-subunit have been clearly assigned to a particular segment, with two pivotal amino acids being responsible for this modulation.High-conductance voltage- and calcium-activated potassium (BK) channels are homotetrameric proteins of α-subunits encoded by the slo1 gene (1). These channels are expressed in virtually all mammalian tissues, where they detect and integrate membrane voltage and calcium concentration changes dampening the responsiveness of cells when confronted with excitatory stimuli. They are abundant in the CNS and nonneuronal tissues, such as smooth muscle or hair cells. This wide distribution is associated with an outstandingly large functional diversity, in which BK channel activity appears optimally adapted to the particular physiological demands of each cell type (2). On the other hand, small alterations in BK channel function may contribute to the pathophysiology of hypertension, asthma, cancer, epilepsy, diabetes, and other conditions in humans (3–8). Alternative splicing, posttranslational modifications, and regulation by auxiliary proteins have been proposed to contribute to this functional diversity (1, 2, 9–16).The BK channel α-subunit is formed by a single polypeptide of about 1,200 amino acids that contains all of the key structural elements for ion permeation, gating, and modulation by ions and other proteins. Tetramers of α-subunits form functional BK channels. Each subunit has seven hydrophobic transmembrane segments (S0–S6), where the voltage-sensor domain (VSD) and pore domain (PD) reside (2). The N terminus faces the extracellular side of the membrane, whereas the C terminus is intracellular. The latter contains four hydrophobic α-helices (S7–S10) and the main Ca²⁺ binding sites (2). VSDs formed by segments S1–S4 harbor a series of charged residues across the membrane that contributes to voltage sensing (2). Upon membrane depolarization, each VSD undergoes a rearrangement (17) that prompts the opening of a highly K⁺-selective pore formed by the four PDs that come together at the symmetry center of the tetramer.Although BK channel expression is ubiquitous, in most physiological scenarios their functioning is provided by their coassembly with auxiliary proteins, such as β-subunits. This coassembly brings channel activity into the proper cell/tissue context (11, 13). Four different β-subunits have been cloned (β1–β4) (18–24), all of which have been observed to modify BK channel function. Albeit to a different extent, all β-subunits modify the Ca²⁺ sensitivity, voltage dependence, and gating properties of BK channels, hence modifying plasma membrane excitability balance. Regarding auxiliary β-subunits, β1- and β2-subunits increase apparent Ca²⁺ sensitivity and decelerate macroscopic current kinetics (14, 20, 21, 25–30); β2 and β3 induce fast inactivation as well as an instantaneous outward rectification (20, 21, 24, 31, 32); and β4 slows down activation and deactivation kinetics (12, 23) and modifies Ca²⁺ sensitivity (12, 33, 34).It should be kept in mind that β-subunits are potential targets for different molecules that modulate channel function, such as alcohol (35), estrogens (15), hormones (36), and fatty acids (37, 38). Additionally, scorpion toxin affinity in BK channels would tend to increase when β1 is coexpressed with the α-subunit (22).To identify the molecular elements that give β1 the ability to modulate the voltage sensor of BK channels, we constructed chimeric proteins of β1/β2- and β1/β3-subunits by swapping their N and C termini, the transmembrane (TM) segments, and the extracellular loops and recorded their gating currents. Two lysine residues that are unique to the N terminus of β1 were identified to be sufficient for BK voltage-sensor modulation.     

α-Synuclein assembles into higher-order multimers upon membrane binding to promote SNARE complex formation

Physiologically, α-synuclein chaperones soluble NSF attachment protein receptor (SNARE) complex assembly and may also perform other functions; pathologically, in contrast, α-synuclein misfolds into neurotoxic aggregates that mediate neurodegeneration and propagate between neurons. In neurons, α-synuclein exists in an equilibrium between cytosolic and membrane-bound states. Cytosolic α-synuclein appears to be natively unfolded, whereas membrane-bound α-synuclein adopts an α-helical conformation. Although the majority of studies showed that cytosolic α-synuclein is monomeric, it is unknown whether membrane-bound α-synuclein is also monomeric, and whether chaperoning of SNARE complex assembly by α-synuclein involves its cytosolic or membrane-bound state. Here, we show using chemical cross-linking and fluorescence resonance energy transfer (FRET) that α-synuclein multimerizes into large homomeric complexes upon membrane binding. The FRET experiments indicated that the multimers of membrane-bound α-synuclein exhibit defined intermolecular contacts, suggesting an ordered array. Moreover, we demonstrate that α-synuclein promotes SNARE complex assembly at the presynaptic plasma membrane in its multimeric membrane-bound state, but not in its monomeric cytosolic state. Our data delineate a folding pathway for α-synuclein that ranges from a monomeric, natively unfolded form in cytosol to a physiologically functional, multimeric form upon membrane binding, and show that only the latter but not the former acts as a SNARE complex chaperone at the presynaptic terminal, and may protect against neurodegeneration.α-Synuclein is an abundant presynaptic protein that physiologically acts to promote soluble NSF attachment protein receptor (SNARE) complex assembly in vitro and in vivo (1–3). Point mutations in α-synuclein (A30P, E46K, H50Q, G51D, and A53T) as well as α-synuclein gene duplications and triplications produce early-onset Parkinson''s disease (PD) (4–10). Moreover, α-synuclein is a major component of intracellular protein aggregates called Lewy bodies, which are pathological hallmarks of neurodegenerative disorders such as PD, Lewy body dementia, and multiple system atrophy (11–14). Strikingly, neurotoxic α-synuclein aggregates propagate between neurons during neurodegeneration, suggesting that such α-synuclein aggregates are not only intrinsically neurotoxic but also nucleate additional fibrillization (15–18).α-Synuclein is highly concentrated in presynaptic terminals where α-synuclein exists in an equilibrium between a soluble and a membrane-bound state, and is associated with synaptic vesicles (19–22). The labile association of α-synuclein with membranes (23, 24) suggests that binding of α-synuclein to synaptic vesicles, and its dissociation from these vesicles, may regulate its physiological function. Membrane-bound α-synuclein assumes an α-helical conformation (25–32), whereas cytosolic α-synuclein is natively unfolded and monomeric (refs. 25, 26, 31, and 32; however, see refs. 33 and 34 and Discussion for a divergent view). Membrane binding by α-synuclein is likely physiologically important because in in vitro experiments, α-synuclein remodels membranes (35, 36), influences lipid packing (37, 38), and induces vesicle clustering (39). Moreover, membranes were found to be important for the neuropathological effects of α-synuclein (40–44).However, the relation of membrane binding to the in vivo function of α-synuclein remains unexplored, and it is unknown whether α-synuclein binds to membranes as a monomer or oligomer. Thus, in the present study we have investigated the nature of the membrane-bound state of α-synuclein and its relation to its physiological function in SNARE complex assembly. We found that soluble monomeric α-synuclein assembles into higher-order multimers upon membrane binding and that membrane binding of α-synuclein is required for its physiological activity in promoting SNARE complex assembly at the synapse.     

Rab3-interacting molecules 2α and 2β promote the abundance of voltage-gated CaV1.3 Ca2+ channels at hair cell active zones

Ca²⁺ influx triggers the fusion of synaptic vesicles at the presynaptic active zone (AZ). Here we demonstrate a role of Ras-related in brain 3 (Rab3)–interacting molecules 2α and β (RIM2α and RIM2β) in clustering voltage-gated Ca_V1.3 Ca²⁺ channels at the AZs of sensory inner hair cells (IHCs). We show that IHCs of hearing mice express mainly RIM2α, but also RIM2β and RIM3γ, which all localize to the AZs, as shown by immunofluorescence microscopy. Immunohistochemistry, patch-clamp, fluctuation analysis, and confocal Ca²⁺ imaging demonstrate that AZs of RIM2α-deficient IHCs cluster fewer synaptic Ca_V1.3 Ca²⁺ channels, resulting in reduced synaptic Ca²⁺ influx. Using superresolution microscopy, we found that Ca²⁺ channels remained clustered in stripes underneath anchored ribbons. Electron tomography of high-pressure frozen synapses revealed a reduced fraction of membrane-tethered vesicles, whereas the total number of membrane-proximal vesicles was unaltered. Membrane capacitance measurements revealed a reduction of exocytosis largely in proportion with the Ca²⁺ current, whereas the apparent Ca²⁺ dependence of exocytosis was unchanged. Hair cell-specific deletion of all RIM2 isoforms caused a stronger reduction of Ca²⁺ influx and exocytosis and significantly impaired the encoding of sound onset in the postsynaptic spiral ganglion neurons. Auditory brainstem responses indicated a mild hearing impairment on hair cell-specific deletion of all RIM2 isoforms or global inactivation of RIM2α. We conclude that RIM2α and RIM2β promote a large complement of synaptic Ca²⁺ channels at IHC AZs and are required for normal hearing.Tens of Ca_V1.3 Ca²⁺ channels are thought to cluster within the active zone (AZ) membrane underneath the presynaptic density of inner hair cells (IHCs) (1–4). They make up the key signaling element, coupling the sound-driven receptor potential to vesicular glutamate release (5–7). The mechanisms governing the number of Ca²⁺ channels at the AZ as well as their spatial organization relative to membrane-tethered vesicles are not well understood. Disrupting the presynaptic scaffold protein Bassoon diminishes the numbers of Ca²⁺ channels and membrane-tethered vesicles at the AZ (2, 8). However, the loss of Bassoon is accompanied by the loss of the entire synaptic ribbon, which makes it challenging to distinguish the direct effects of gene disruption from secondary effects (9).Among the constituents of the cytomatrix of the AZ, RIM1 and RIM2 proteins are prime candidates for the regulation of Ca²⁺ channel clustering and function (10, 11). The family of RIM proteins has seven identified members (RIM1α, RIM1β, RIM2α, RIM2β, RIM2γ, RIM3γ, and RIM4γ) encoded by four genes (RIM1–RIM4). All isoforms contain a C-terminal C₂ domain but differ in the presence of additional domains. RIM1 and RIM2 interact with Ca²⁺ channels, most other proteins of the cytomatrix of the AZ, and synaptic vesicle proteins. They interact directly with the auxiliary β (Ca_Vβ) subunits (12, 13) and pore-forming Ca_Vα subunits (14, 15). In addition, RIMs are indirectly linked to Ca²⁺ channels via RIM-binding protein (14, 16, 17). A regulation of biophysical channel properties has been demonstrated in heterologous expression systems for RIM1 (12) and RIM2 (13).A role of RIM1 and RIM2 in clustering Ca²⁺ channels at the AZ was demonstrated by analysis of RIM1/2-deficient presynaptic terminals of cultured hippocampal neurons (14), auditory neurons in slices (18), and Drosophila neuromuscular junction (19). Because α-RIMs also bind the vesicle-associated protein Ras-related in brain 3 (Rab3) via the N-terminal zinc finger domain (20), they are also good candidates for molecular coupling of Ca²⁺ channels and vesicles (18, 21, 22). Finally, a role of RIMs in priming of vesicles for fusion is the subject of intense research (18, 21–27). RIMs likely contribute to priming via disinhibiting Munc13 (26) and regulating vesicle tethering (27). Here, we studied the expression and function of RIM in IHCs. We combined molecular, morphologic, and physiologic approaches for the analysis of RIM2α knockout mice [RIM2α SKO (28); see Methods] and of hair cell-specific RIM1/2 knockout mice (RIM1/2 cDKO). We demonstrate that RIM2α and RIM2β are present at IHC AZs of hearing mice, positively regulate the number of synaptic Ca_V1.3 Ca²⁺ channels, and are required for normal hearing.     

Transforming growth factor-β signaling is constantly shaping memory T-cell population

The long-term maintenance of memory T cells is essential for successful vaccines. Both the quantity and the quality of the memory T-cell population must be maintained. The signals that control the maintenance of memory T cells remain incompletely identified. Here we used two genetic models to show that continuous transforming growth factor-β signaling to antigen-specific T cells is required for the differentiation and maintenance of memory CD8⁺ T cells. In addition, both infection-induced and microbiota-induced inflammation impact the phenotypic and functional identity of memory CD8⁺ T cells.Infectious diseases pose a significant public health burden, accounting for nearly one-fifth of annual deaths worldwide. Vaccines remain the most effective way to prevent infectious diseases. Functionally sustained memory T cells are the ideal cell population to be generated by T-cell–based vaccines. Considerable efforts have been made to elucidate the mechanisms that mediate the establishment of long-lived immunologic memory (1–6); however, the signals that control the differentiation and maintenance of memory T cells remain incompletely identified.During the early stages of an immune response, proinflammatory cytokines IL-12 and type I IFN promote the expansion of effector CD8⁺ T cells by sustaining the expression of the high-affinity IL-2 receptor CD25 (7, 8). In addition to its role in T-cell proliferation, IL-2 also functions as a differentiation factor for effector CD8⁺ T cells by promoting the differentiation of short-lived effector cells [SLECs; IL-7Rα⁻KLRG1⁺ (killer cell lectin-like receptor subfamily G, member 1)] and inhibiting the differentiation of memory precursor effector cells (MPECs; IL-7Rα⁺KLRG1⁻) (9–12). Furthermore, IL-10 and IL-21 signals promote MPEC differentiation through a STAT3-dependent mechanism (13, 14). During the late stages of an immune response, IL-15 and IL-7 are required to maintain the population of memory CD8⁺ T cells (15, 16); however, after the clearance of an infection, whether memory CD8⁺ T cells require any additional signals to maintain their phenotypic and functional identity remains unknown.Recent findings have revealed that effector and memory CD8⁺ T cells display nearly endless diversity based on the expression of surface and intracellular molecules that serve as the markers of antigen-experienced T cells (17). Thus, it is conceivable that memory CD8⁺ T cells might not be a fixed cell lineage, but instead represent an active differentiation state. Even in the absence of cognate antigens, memory CD8⁺ T cells may constantly receive diverse environmental signals; however, how memory T cells maintain their relatively stable characters under such circumstances remains unexplored.Here we show that TGF-β signaling to CD8⁺ T cells controls the differentiation of memory T cells at both early and late stages. By deleting TGF-β receptor in antigen-specific T cells at different time points following an acute infection, we demonstrate that during the effector phase of an immune response, TGF-β restrains the inflammatory signals associated with the infection. At the memory phase, both the TGF-β signal and the basal inflammation induced by microbiota cooperate to shape the memory T-cell population. Taken together, our findings show that continuous TGF-β signaling is required to maintain the identity of memory CD8⁺ T cells following acute infections.     

Osteopontin expression by CD103? dendritic cells drives intestinal inflammation

Intestinal CD103⁻ dendritic cells (DCs) are pathogenic for colitis. Unveiling molecular mechanisms that render these cells proinflammatory is important for the design of specific immunotherapies. In this report, we demonstrated that mesenteric lymph node CD103⁻ DCs express, among other proinflammatory cytokines, high levels of osteopontin (Opn) during experimental colitis. Opn expression by CD103⁻ DCs was crucial for their immune profile and pathogenicity, including induction of T helper (Th) 1 and Th17 cell responses. Adoptive transfer of Opn-deficient CD103⁻ DCs resulted in attenuated colitis in comparison to transfer of WT CD103⁻ DCs, whereas transgenic CD103⁻ DCs that overexpress Opn were highly pathogenic in vivo. Neutralization of secreted Opn expressed exclusively by CD103⁻ DCs restrained disease severity. Also, Opn deficiency resulted in milder disease, whereas systemic neutralization of secreted Opn was therapeutic. We determined a specific domain of the Opn protein responsible for its CD103⁻ DC-mediated proinflammatory effect. We demonstrated that disrupting the interaction of this Opn domain with integrin α9, overexpressed on colitic CD103⁻ DCs, suppressed the inflammatory potential of these cells in vitro and in vivo. These results add unique insight into the biology of CD103⁻ DCs and their function during inflammatory bowel disease.Inflammatory bowel diseases (IBDs), including Crohn disease (CD) and ulcerative colitis (UC), are caused by excessive inflammatory responses to commensal microflora and other antigens present in the intestinal lumen (1). Intestinal dendritic cells (DCs) contribute to these inflammatory responses during human IBD, as well as in murine colitis models (2). DCs that reside in draining mesenteric lymph nodes (MLNs) are also crucial mediators of colitis induction (3) and may be grouped based on their surface CD103 (integrin αE) expression as CD11c^highCD103⁺ (CD103⁺ DCs) and CD11c^highCD103⁻ (CD103⁻ DCs) (4–6). CD103⁺ DCs are considered important mediators of gut homeostasis in steady state (4, 5, 7–9), and their tolerogenic properties are conserved between mice and humans (5). However, their role during intestinal inflammation is not well defined. Instead, CD103⁻ DC function has been described mostly during chronic experimental colitis (10–12). These cells secrete IL-23, IL-6, and IL-12 (10–12), contributing to the development of T helper (Th) 17 and Th1 cells, and are highly inflammatory during CD4⁺ T-cell transfer colitis (12) and during 2,4,6 trinitrobenzene sulfonic acid (TNBS)-induced chronic colitis (11). MLN CD103⁻ DCs cultured in the presence of LPS, a Toll-like receptor (TLR) 4 agonist, or R848, a TLR7 agonist, express higher levels of TNF-α and IL-6 (7, 12). In fact, these cells secrete IL-23 and IL-12 even in the absence of TLR stimulation (10). Both MLN CD103⁻ and CD103⁺ DC subsets are present in acute colitis (11, 13); however, their function, as well as their cytokine profile, during this phase of disease, reflecting colitis initiation, remains unknown.Recent studies suggest a proinflammatory role for the cytokine osteopontin (Opn) in TNBS- and dextran sulfate sodium (DSS)-induced colitis (14, 15), which are the models for CD and UC, respectively. Opn is expressed by DCs and other immune cell types, such as lymphocytes, during autoimmune responses (16–22), and its expression by DCs during autoimmunity contributes to disease severity (17–19, 21, 23). In addition, Opn expression is highly up-regulated in intestinal immune and nonimmune cells and in the plasma of patients with CD and UC (24–29), as well as in the colon and plasma of mice with experimental colitis (14, 15, 27, 30). Increased plasma Opn levels are related to the severity of CD inflammation (29), and certain Opn gene (Spp1) haplotypes are modifiers of CD susceptibility (31), indicating that Opn could be used as an IBD biomarker (27). In general, Opn affects DC biology during several inflammatory conditions (17–21, 32–37) and could be a potential therapeutic target in IBD.In this study, we initially asked whether Opn was expressed by MLN CD103⁻ and CD103⁺ DCs during colitis. We found that CD103⁻ DCs express excessive levels of Opn in addition to other proinflammatory cytokines. Conversely, CD103⁺ DCs express profoundly lower levels of Opn and are noninflammatory. Using adoptive transfer of purified specific DC subsets, we determined that MLN CD103⁻ DCs are critical mediators of acute intestinal inflammation and that their Opn expression is essential for their proinflammatory properties in both acute and chronic colitis. Furthermore, Opn-deficient and Opn-neutralized mice developed significantly milder disease. In addition, we constructed transgenic (Tg) mice overexpressing Opn only in DCs. These mice developed exaggerated colitis, and adoptive transfer of their CD103⁻ DCs into recipient mice dramatically exacerbated disease. Because Opn protein contains several domains interacting with various receptors, we defined a specific Opn domain significant for inducing proinflammatory properties in CD103⁻ DCs. Blockade of the interaction of this Opn domain [containing functional Ser-Leu-Ala-Tyr-Gly-Leu-Arg (SLAYGLR) sequence] with integrin α9 expressed on CD103⁻ DCs abrogated their proinflammatory profile and colitogenic effects in vivo.     

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.     

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.