Structure of a fully assembled tumor-specific T cell receptor ligated by pMHC.

The T cell receptor (TCR) expressed by T lymphocytes initiates protective immune responses to pathogens and tumors. To explore the structural basis of how TCR signaling is initiated when the receptor binds to peptide-loaded major histocompatibility complex (pMHC) molecules, we used cryogenic electron microscopy to determine the structure of a tumor-reactive TCRαß/CD3δγε2ζ2 complex bound to a melanoma-specific human class I pMHC at 3.08 Å resolution. The antigen-bound complex comprises 11 subunits stabilized by multivalent interactions across three structural layers, with clustered membrane-proximal cystines stabilizing the CD3-εδ and CD3-εγ heterodimers. Extra density sandwiched between transmembrane helices reveals the involvement of sterol lipids in TCR assembly. The geometry of the pMHC/TCR complex suggests that efficient TCR scanning of pMHC requires accurate pre-positioning of T cell and antigen-presenting cell membranes. Comparisons of the ligand-bound and unliganded receptors, along with molecular dynamics simulations, indicate that TCRs can be triggered in the absence of spontaneous structural rearrangements.

Differences in CD80 and CD86 transendocytosis reveal CD86 as a key target for CTLA-4 immune regulation.

CD28 and CTLA-4 (CD152) play essential roles in regulating T cell immunity, balancing the activation and inhibition of T cell responses, respectively. Although both receptors share the same ligands, CD80 and CD86, the specific requirement for two distinct ligands remains obscure. In the present study, we demonstrate that, although CTLA-4 targets both CD80 and CD86 for destruction via transendocytosis, this process results in separate fates for CTLA-4 itself. In the presence of CD80, CTLA-4 remained ligand bound, and was ubiquitylated and trafficked via late endosomes and lysosomes. In contrast, in the presence of CD86, CTLA-4 detached in a pH-dependent manner and recycled back to the cell surface to permit further transendocytosis. Furthermore, we identified clinically relevant mutations that cause autoimmune disease, which selectively disrupted CD86 transendocytosis, by affecting either CTLA-4 recycling or CD86 binding. These observations provide a rationale for two distinct ligands and show that defects in CTLA-4-mediated transendocytosis of CD86 are associated with autoimmunity.

Antibody agonists trigger immune receptor signaling through local exclusion of receptor-type protein tyrosine phosphatases.

Antibodies can block immune receptor engagement or trigger the receptor machinery to initiate signaling. We hypothesized that antibody agonists trigger signaling by sterically excluding large receptor-type protein tyrosine phosphatases (RPTPs) such as CD45 from sites of receptor engagement. An agonist targeting the costimulatory receptor CD28 produced signals that depended on antibody immobilization and were sensitive to the sizes of the receptor, the RPTPs, and the antibody itself. Although both the agonist and a non-agonistic anti-CD28 antibody locally excluded CD45, the agonistic antibody was more effective. An anti-PD-1 antibody that bound membrane proximally excluded CD45, triggered Src homology 2 domain-containing phosphatase 2 recruitment, and suppressed systemic lupus erythematosus and delayed-type hypersensitivity in experimental models. Paradoxically, nivolumab and pembrolizumab, anti-PD-1-blocking antibodies used clinically, also excluded CD45 and were agonistic in certain settings. Reducing these agonistic effects using antibody engineering improved PD-1 blockade. These findings establish a framework for developing new and improved therapies for autoimmunity and cancer.

Monomeric TCRs drive T cell antigen recognition.

T cell antigen recognition requires T cell antigen receptors (TCRs) engaging MHC-embedded antigenic peptides (pMHCs) within the contact region of a T cell with its conjugated antigen-presenting cell. Despite micromolar TCR:pMHC affinities, T cells respond to even a single antigenic pMHC, and higher-order TCRs have been postulated to maintain high antigen sensitivity and trigger signaling. We interrogated the stoichiometry of TCRs and their associated CD3 subunits on the surface of living T cells through single-molecule brightness and single-molecule coincidence analysis, photon-antibunching-based fluorescence correlation spectroscopy and Förster resonance energy transfer measurements. We found exclusively monomeric TCR-CD3 complexes driving the recognition of antigenic pMHCs, which underscores the exceptional capacity of single TCR-CD3 complexes to elicit robust intracellular signaling.

Membrane nanoclusters-tails of the unexpected.

The existence, nature, and role of highly ordered membrane domains, often referred to as lipid rafts, have been highly debated by cell biologists for many years. In this issue, Raghupathy et al. describe molecular mechanisms leading to the formation of ordered lipid-protein clusters.

Initiation of T cell signaling by CD45 segregation at 'close contacts'.

It has been proposed that the local segregation of kinases and the tyrosine phosphatase CD45 underpins T cell antigen receptor (TCR) triggering, but how such segregation occurs and whether it can initiate signaling is unclear. Using structural and biophysical analysis, we show that the extracellular region of CD45 is rigid and extends beyond the distance spanned by TCR-ligand complexes, implying that sites of TCR-ligand engagement would sterically exclude CD45. We also show that the formation of 'close contacts', new structures characterized by spontaneous CD45 and kinase segregation at the submicron-scale, initiates signaling even when TCR ligands are absent. Our work reveals the structural basis for, and the potent signaling effects of, local CD45 and kinase segregation. TCR ligands have the potential to heighten signaling simply by holding receptors in close contacts.

Mineral carbonation of peridotite fueled by magmatic degassing and melt impregnation in an oceanic transform fault.

Most of the geologic CO2 entering Earth's atmosphere and oceans is emitted along plate margins. While C-cycling at mid-ocean ridges and subduction zones has been studied for decades, little attention has been paid to degassing of magmatic CO2 and mineral carbonation of mantle rocks in oceanic transform faults. We studied the formation of soapstone (magnesite-talc rock) and other magnesite-bearing assemblages during mineral carbonation of mantle peridotite in the St. Paul's transform fault, equatorial Atlantic. Clumped carbonate thermometry of soapstone yields a formation (or equilibration) temperature of 147 ± 13 °C which, based on thermodynamic constraints, suggests that CO2(aq) concentrations of the hydrothermal fluid were at least an order of magnitude higher than in seawater. The association of magnesite with apatite in veins, magnesite with a δ13C of -3.40 ± 0.04‰, and the enrichment of CO2 in hydrothermal fluids point to magmatic degassing and melt-impregnation as the main source of CO2. Melt-rock interaction related to gas-rich alkali olivine basalt volcanism near the St. Paul's Rocks archipelago is manifested in systematic changes in peridotite compositions, notably a strong enrichment in incompatible elements with decreasing MgO/SiO2. These findings reveal a previously undocumented aspect of the geologic carbon cycle in one of the largest oceanic transform faults: Fueled by magmatism in or below the root zone of the transform fault and subsequent degassing, the fault constitutes a conduit for CO2-rich hydrothermal fluids, while carbonation of peridotite represents a vast sink for the emitted CO2.

Hippocampal Functions Modulate Transfer-Appropriate Cortical Representations Supporting Subsequent Memory.

The hippocampus plays a central role as a coordinate system or index of information stored in neocortical loci. Nonetheless, it remains unclear how hippocampal processes integrate with cortical information to facilitate successful memory encoding. Thus, the goal of the current study was to identify specific hippocampal-cortical interactions that support object encoding. We collected fMRI data while 19 human participants (7 female and 12 male) encoded images of real-world objects and tested their memory for object concepts and image exemplars (i.e., conceptual and perceptual memory). Representational similarity analysis revealed robust representations of visual and semantic information in canonical visual (e.g., occipital cortex) and semantic (e.g., angular gyrus) regions in the cortex, but not in the hippocampus. Critically, hippocampal functions modulated the mnemonic impact of cortical representations that are most pertinent to future memory demands, or transfer-appropriate representations Subsequent perceptual memory was best predicted by the strength of visual representations in ventromedial occipital cortex in coordination with hippocampal activity and pattern information during encoding. In parallel, subsequent conceptual memory was best predicted by the strength of semantic representations in left inferior frontal gyrus and angular gyrus in coordination with either hippocampal activity or semantic representational strength during encoding. We found no evidence for transfer-incongruent hippocampal-cortical interactions supporting subsequent memory (i.e., no hippocampal interactions with cortical visual/semantic representations supported conceptual/perceptual memory). Collectively, these results suggest that diverse hippocampal functions flexibly modulate cortical representations of object properties to satisfy distinct future memory demands.Significance Statement The hippocampus is theorized to index pieces of information stored throughout the cortex to support episodic memory. Yet how hippocampal processes integrate with cortical representation of stimulus information remains unclear. Using fMRI, we examined various forms of hippocampal-cortical interactions during object encoding in relation to subsequent performance on conceptual and perceptual memory tests. Our results revealed novel hippocampal-cortical interactions that utilize semantic and visual representations in transfer-appropriate manners: conceptual memory supported by hippocampal modulation of frontoparietal semantic representations, and perceptual memory supported by hippocampal modulation of occipital visual representations. These findings provide important insights into the neural mechanisms underlying the formation of information-rich episodic memory and underscore the value of studying the flexible interplay between brain regions for complex cognition.

Visual Recognition Memory of Scenes Is Driven by Categorical, Not Sensory, Visual Representations.

When we perceive a scene, our brain processes various types of visual information simultaneously, ranging from sensory features, such as line orientations and colors, to categorical features, such as objects and their arrangements. Whereas the role of sensory and categorical visual representations in predicting subsequent memory has been studied using isolated objects, their impact on memory for complex scenes remains largely unknown. To address this gap, we conducted an fMRI study in which female and male participants encoded pictures of familiar scenes (e.g., an airport picture) and later recalled them, while rating the vividness of their visual recall. Outside the scanner, participants had to distinguish each seen scene from three similar lures (e.g., three airport pictures). We modeled the sensory and categorical visual features of multiple scenes using both early and late layers of a deep convolutional neural network. Then, we applied representational similarity analysis to determine which brain regions represented stimuli in accordance with the sensory and categorical models. We found that categorical, but not sensory, representations predicted subsequent memory. In line with the previous result, only for the categorical model, the average recognition performance of each scene exhibited a positive correlation with the average visual dissimilarity between the item in question and its respective lures. These results strongly suggest that even in memory tests that ostensibly rely solely on visual cues (such as forced-choice visual recognition with similar distractors), memory decisions for scenes may be primarily influenced by categorical rather than sensory representations.

Colonic epithelial cell diversity in health and inflammatory bowel disease.

The colonic epithelium facilitates host-microorganism interactions to control mucosal immunity, coordinate nutrient recycling and form a mucus barrier. Breakdown of the epithelial barrier underpins inflammatory bowel disease (IBD). However, the specific contributions of each epithelial-cell subtype to this process are unknown. Here we profile single colonic epithelial cells from patients with IBD and unaffected controls. We identify previously unknown cellular subtypes, including gradients of progenitor cells, colonocytes and goblet cells within intestinal crypts. At the top of the crypts, we find a previously unknown absorptive cell, expressing the proton channel OTOP2 and the satiety peptide uroguanylin, that senses pH and is dysregulated in inflammation and cancer. In IBD, we observe a positional remodelling of goblet cells that coincides with downregulation of WFDC2-an antiprotease molecule that we find to be expressed by goblet cells and that inhibits bacterial growth. In vivo, WFDC2 preserves the integrity of tight junctions between epithelial cells and prevents invasion by commensal bacteria and mucosal inflammation. We delineate markers and transcriptional states, identify a colonic epithelial cell and uncover fundamental determinants of barrier breakdown in IBD.

A late Middle Pleistocene Denisovan mandible from the Tibetan Plateau.

Denisovans are members of a hominin group who are currently only known directly from fragmentary fossils, the genomes of which have been studied from a single site, Denisova Cave1-3 in Siberia. They are also known indirectly from their genetic legacy through gene flow into several low-altitude East Asian populations4,5 and high-altitude modern Tibetans6. The lack of morphologically informative Denisovan fossils hinders our ability to connect geographically and temporally dispersed fossil hominins from Asia and to understand in a coherent manner their relation to recent Asian populations. This includes understanding the genetic adaptation of humans to the high-altitude Tibetan Plateau7,8, which was inherited from the Denisovans. Here we report a Denisovan mandible, identified by ancient protein analysis9,10, found on the Tibetan Plateau in Baishiya Karst Cave, Xiahe, Gansu, China. We determine the mandible to be at least 160 thousand years old through U-series dating of an adhering carbonate matrix. The Xiahe specimen provides direct evidence of the Denisovans outside the Altai Mountains and its analysis unique insights into Denisovan mandibular and dental morphology. Our results indicate that archaic hominins occupied the Tibetan Plateau in the Middle Pleistocene epoch and successfully adapted to high-altitude hypoxic environments long before the regional arrival of modern Homo sapiens.

The kinase occupancy of T cell coreceptors reconsidered.

The sensitivity of the αß T cell receptor (TCR) is enhanced by the coreceptors CD4 and CD8αß, which are expressed primarily by cells of the helper and cytotoxic T cell lineages, respectively. The coreceptors bind to major histocompatibility complex (MHC) molecules and associate intracellularly with the Src-family kinase Lck, which catalyzes TCR phosphorylation during receptor triggering. Although coreceptor/kinase occupancy was initially believed to be high, a recent study suggested that most coreceptors exist in an Lck-free state, and that this low occupancy helps to effect TCR antigen discrimination. Here, using the same method, we found instead that the CD4/Lck interaction was stoichiometric (~100%) and that the CD8αß/Lck interaction was substantial (~60%). We confirmed our findings in live cells using fluorescence cross-correlation spectroscopy (FCCS) to measure coreceptor/Lck codiffusion in situ. After introducing structurally guided mutations into the intracellular domain of CD4, we used FCCS to also show that stoichiometric coupling to Lck required an amphipathic α-helix present in CD4 but not CD8α. In double-positive cells expressing equal numbers of both coreceptors, but limiting amounts of kinase, CD4 outcompeted CD8αß for Lck. In T cells, TCR signaling induced CD4/Lck oligomerization but did not affect the high levels of CD4/Lck occupancy. These findings help settle the question of kinase occupancy and suggest that the binding advantages that CD4 has over CD8 could be important when Lck levels are limiting.

Differential Mnemonic Contributions of Cortical Representations during Encoding and Retrieval.

Several recent fMRI studies of episodic and working memory representations converge on the finding that visual information is most strongly represented in occipito-temporal cortex during the encoding phase but in parietal regions during the retrieval phase. It has been suggested that this location shift reflects a change in the content of representations, from predominantly visual during encoding to primarily semantic during retrieval. Yet, direct evidence on the nature of encoding and retrieval representations is lacking. It is also unclear how the representations mediating the encoding-retrieval shift contribute to memory performance. To investigate these two issues, in the current fMRI study, participants encoded pictures (e.g., picture of a cardinal) and later performed a word recognition test (e.g., word "cardinal"). Representational similarity analyses examined how visual (e.g., red color) and semantic representations (e.g., what cardinals eat) support successful encoding and retrieval. These analyses revealed two novel findings. First, successful memory was associated with representational changes in cortical location (from occipito-temporal at encoding to parietal at retrieval) but not with changes in representational content (visual vs. semantic). Thus, the representational encoding-retrieval shift cannot be easily attributed to a change in the nature of representations. Second, in parietal regions, stronger representations predicted encoding failure but retrieval success. This encoding-retrieval "flip" in representations mimics the one previously reported in univariate activation studies. In summary, by answering important questions regarding the content and contributions to the performance of the representations mediating the encoding-retrieval shift, our findings clarify the neural mechanisms of this intriguing phenomenon.

The CD6 interactome orchestrates ligand-independent T cell inhibitory signaling.

BACKGROUND: T-cell membrane scaffold proteins are pivotal in T cell function, acting as versatile signaling hubs. While CD6 forms a large intracellular signalosome, it is distinguished from typical scaffolds like LAT or PAG by possessing a substantial ectodomain that binds CD166, a well-characterized ligand expressed on most antigen-presenting cells (APC), through the third domain (d3) of the extracellular region. Although the intact form of CD6 is the most abundant in T cells, an isoform lacking d3 (CD6∆d3) is transiently expressed on activated T cells. Still, the precise character of the signaling transduced by CD6, whether costimulatory or inhibitory, and the influence of its ectodomain on these activities are unclear. METHODS: We expressed CD6 variants with extracellular deletions or cytosolic mutations in Jurkat cells containing eGFP reporters for NF-κB and NF-AT transcription factor activation. Cell activation was assessed by eGFP flow cytometry following Jurkat cell engagement with superantigen-presenting Raji cells. Using imaging flow cytometry, we evaluated the impact of the CD6-CD166 pair on cell adhesiveness during the antigen-dependent and -independent priming of T cells. We also examined the role of extracellular or cytosolic sequences on CD6 translocation to the immunological synapse, using immunofluorescence-based imaging. RESULTS: Our investigation dissecting the functions of the extracellular and cytosolic regions of CD6 revealed that CD6 was trafficked to the immunological synapse and exerted tonic inhibition wholly dependent on its cytosolic tail. Surprisingly, however, translocation to the synapse occurred independently of the extracellular d3 and of engagement to CD166. On the other hand, CD6 binding to CD166 significantly increased T cell:APC adhesion. However, this activity was most evident in the absence of APC priming with superantigen, and thus, in the absence of TCR engagement. CONCLUSIONS: Our study identifies CD6 as a novel 'on/off' scaffold-receptor capable of modulating responsiveness in two ways. Firstly, and independently of ligand binding, it establishes signaling thresholds through tonic inhibition, functioning as a membrane-bound scaffold. Secondly, CD6 has the capacity for alternative splicing-dependent variable ligand engagement, modulating its checkpoint-like activity.

Structure-based design and optimization of a new class of small molecule inhibitors targeting the P-stalk binding pocket of ricin.

Ricin, a category-B agent for bioterrorism, and Shiga toxins (Stxs), which cause food poisoning bind to the ribosomal P-stalk to depurinate the sarcin/ricin loop. No effective therapy exists for ricin or Stx intoxication. Ribosome binding sites of the toxins have not been targeted by small molecules. We previously identified CC10501, which inhibits toxin activity by binding the P-stalk pocket of ricin toxin A subunit (RTA) remote from the catalytic site. Here, we developed a fluorescence polarization assay and identified a new class of compounds, which bind P-stalk pocket of RTA with higher affinity and inhibit catalytic activity with submicromolar potency. A lead compound, RU-NT-206, bound P-stalk pocket of RTA with similar affinity as a five-fold larger P-stalk peptide and protected cells against ricin and Stx2 holotoxins for the first time. These results validate the P-stalk binding site of RTA as a critical target for allosteric inhibition of the active site.

Trapping or slowing the diffusion of T cell receptors at close contacts initiates T cell signaling.

T cell activation is initiated by T cell receptor (TCR) phosphorylation. This requires the local depletion of large receptor-type phosphatases from "close contacts" formed when T cells interact with surfaces presenting agonistic TCR ligands, but exactly how the ligands potentiate signaling is unclear. It has been proposed that TCR ligands could enhance receptor phosphorylation and signaling just by holding TCRs in phosphatase-depleted close contacts, but this has not been directly tested. We devised simple methods to move the TCR in and out of close contacts formed by T cells interacting with supported lipid bilayers (SLBs) and to slow the receptor's diffusion in the contacts, using a series of anti-CD3ε Fab- and ligand-based adducts of the receptor. TCRs engaging a Fab extended with the large extracellular region of CD45 were excluded from contacts and produced no signaling. Conversely, allowing the extended Fab to become tethered to the SLB trapped the TCR in the close contacts, leading to very strong signaling. Importantly, attaching untethered anti-CD3ε Fab or peptide/MHC ligands, each of which were largely inactive in solution but both of which reduced TCR diffusion in close contacts approximately fivefold, also initiated signaling during cell/SLB contact. Our findings indicate that holding TCRs in close contacts or simply slowing their diffusion in phosphatase-depleted regions of the cell surface suffices to initiate signaling, effects we could reproduce in single-particle stochastic simulations. Our study shows that the TCR is preconfigured for signaling in a way that allows it to be triggered by ligands acting simply as receptor "traps."

Structural Basis of Antibody-Mediated Inhibition of Ricin Toxin Attachment to Host Cells.

Monoclonal antibodies, JB4 and SylH3, neutralize ricin toxin (RT) by inhibiting the galactose-specific lectin activity of the B subunit of the toxin (RTB), which is required for cell attachment and entry. It is not immediately apparent how the antibodies accomplish this feat, considering that RTB consists of two globular domains (D1, D2) each divided into three homologous subdomains (α, ß, γ) with the two functional galactosyl-specific carbohydrate recognition domains (CRDs) situated on opposite poles (subdomains 1α and 2γ). Here, we report the X-ray crystal structures of JB4 and SylH3 Fab fragments bound to RTB in the context of RT. The structures revealed that neither Fab obstructed nor induced detectable conformational alterations in subdomains 1α or 2γ. Rather, JB4 and SylH3 Fabs recognize nearly identical epitopes within an ancillary carbohydrate recognition pocket located in subdomain 1ß. Despite limited amino acid sequence similarity between SylH3 and JB4 Fabs, each paratope inserts a Phe side chain from the heavy (H) chain complementarity determining region (CDR3) into the 1ß CRD pocket, resulting in local aromatic stacking interactions that potentially mimic a ligand interaction. Reconciling the fact that stoichiometric amounts of SylH3 and JB4 are sufficient to disarm RTB's lectin activity without evidence of allostery, we propose that subdomain 1ß functions as a "coreceptor" required to stabilize glycan interactions principally mediated by subdomains 1α and 2γ. Further investigation into subdomain 1ß will yield fundamental insights into the large family of R-type lectins and open novel avenues for countermeasures aimed at preventing toxin uptake into vulnerable tissues and cells.

Single-domain antibodies neutralize ricin toxin intracellularly by blocking access to ribosomal P-stalk proteins.

During ricin intoxication in mammalian cells, ricin's enzymatic (RTA) and binding (RTB) subunits disassociate in the endoplasmic reticulum. RTA is then translocated into the cytoplasm where, by virtue of its ability to depurinate a conserved residue within the sarcin-ricin loop (SRL) of 28S rRNA, it functions as a ribosome-inactivating protein. It has been proposed that recruitment of RTA to the SRL is facilitated by ribosomal P-stalk proteins, whose C-terminal domains interact with a cavity on RTA normally masked by RTB; however, evidence that this interaction is critical for RTA activity within cells is lacking. Here, we characterized a collection of single-domain antibodies (VHHs) whose epitopes overlap with the P-stalk binding pocket on RTA. The crystal structures of three such VHHs (V9E1, V9F9, and V9B2) in complex with RTA revealed not only occlusion of the ribosomal P-stalk binding pocket but also structural mimicry of C-terminal domain peptides by complementarity-determining region 3. In vitro assays confirmed that these VHHs block RTA-P-stalk peptide interactions and protect ribosomes from depurination. Moreover, when expressed as "intrabodies," these VHHs rendered cells resistant to ricin intoxication. One VHH (V9F6), whose epitope was structurally determined to be immediately adjacent to the P-stalk binding pocket, was unable to neutralize ricin within cells or protect ribosomes from RTA in vitro. These findings are consistent with the recruitment of RTA to the SRL by ribosomal P-stalk proteins as a requisite event in ricin-induced ribosome inactivation.