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.

Unconventional human CD61 pairing with CD103 promotes TCR signaling and antigen-specific T cell cytotoxicity.

Cancer remains one of the leading causes of mortality worldwide, leading to increased interest in utilizing immunotherapy strategies for better cancer treatments. In the past decade, CD103+ T cells have been associated with better clinical prognosis in patients with cancer. However, the specific immune mechanisms contributing toward CD103-mediated protective immunity remain unclear. Here, we show an unexpected and transient CD61 expression, which is paired with CD103 at the synaptic microclusters of T cells. CD61 colocalization with the T cell antigen receptor further modulates downstream T cell antigen receptor signaling, improving antitumor cytotoxicity and promoting physiological control of tumor growth. Clinically, the presence of CD61+ tumor-infiltrating T lymphocytes is associated with improved clinical outcomes, mediated through enhanced effector functions and phenotype with limited evidence of cellular exhaustion. In conclusion, this study identified an unconventional and transient CD61 expression and pairing with CD103 on human immune cells, which potentiates a new target for immune-based cellular therapies.

RTN4/NoGo-receptor binding to BAI adhesion-GPCRs regulates neuronal development.

RTN4-binding proteins were widely studied as "NoGo" receptors, but their physiological interactors and roles remain elusive. Similarly, BAI adhesion-GPCRs were associated with numerous activities, but their ligands and functions remain unclear. Using unbiased approaches, we observed an unexpected convergence: RTN4 receptors are high-affinity ligands for BAI adhesion-GPCRs. A single thrombospondin type 1-repeat (TSR) domain of BAIs binds to the leucine-rich repeat domain of all three RTN4-receptor isoforms with nanomolar affinity. In the 1.65 Å crystal structure of the BAI1/RTN4-receptor complex, C-mannosylation of tryptophan and O-fucosylation of threonine in the BAI TSR-domains creates a RTN4-receptor/BAI interface shaped by unusual glycoconjugates that enables high-affinity interactions. In human neurons, RTN4 receptors regulate dendritic arborization, axonal elongation, and synapse formation by differential binding to glial versus neuronal BAIs, thereby controlling neural network activity. Thus, BAI binding to RTN4/NoGo receptors represents a receptor-ligand axis that, enabled by rare post-translational modifications, controls development of synaptic circuits.

A Human IgSF Cell-Surface Interactome Reveals a Complex Network of Protein-Protein Interactions.

Cell-surface protein-protein interactions (PPIs) mediate cell-cell communication, recognition, and responses. We executed an interactome screen of 564 human cell-surface and secreted proteins, most of which are immunoglobulin superfamily (IgSF) proteins, using a high-throughput, automated ELISA-based screening platform employing a pooled-protein strategy to test all 318,096 PPI combinations. Screen results, augmented by phylogenetic homology analysis, revealed ∼380 previously unreported PPIs. We validated a subset using surface plasmon resonance and cell binding assays. Observed PPIs reveal a large and complex network of interactions both within and across biological systems. We identified new PPIs for receptors with well-characterized ligands and binding partners for "orphan" receptors. New PPIs include proteins expressed on multiple cell types and involved in diverse processes including immune and nervous system development and function, differentiation/proliferation, metabolism, vascularization, and reproduction. These PPIs provide a resource for further biological investigation into their functional relevance and may offer new therapeutic drug targets.

An immunodominant NP105-113-B*07:02 cytotoxic T cell response controls viral replication and is associated with less severe COVID-19 disease.

NP105-113-B*07:02-specific CD8+ T cell responses are considered among the most dominant in SARS-CoV-2-infected individuals. We found strong association of this response with mild disease. Analysis of NP105-113-B*07:02-specific T cell clones and single-cell sequencing were performed concurrently, with functional avidity and antiviral efficacy assessed using an in vitro SARS-CoV-2 infection system, and were correlated with T cell receptor usage, transcriptome signature and disease severity (acute n = 77, convalescent n = 52). We demonstrated a beneficial association of NP105-113-B*07:02-specific T cells in COVID-19 disease progression, linked with expansion of T cell precursors, high functional avidity and antiviral effector function. Broad immune memory pools were narrowed postinfection but NP105-113-B*07:02-specific T cells were maintained 6 months after infection with preserved antiviral efficacy to the SARS-CoV-2 Victoria strain, as well as Alpha, Beta, Gamma and Delta variants. Our data show that NP105-113-B*07:02-specific T cell responses associate with mild disease and high antiviral efficacy, pointing to inclusion for future vaccine design.

Isolation of a Structural Mechanism for Uncoupling T Cell Receptor Signaling from Peptide-MHC Binding.

TCR-signaling strength generally correlates with peptide-MHC binding affinity; however, exceptions exist. We find high-affinity, yet non-stimulatory, interactions occur with high frequency in the human T cell repertoire. Here, we studied human TCRs that are refractory to activation by pMHC ligands despite robust binding. Analysis of 3D affinity, 2D dwell time, and crystal structures of stimulatory versus non-stimulatory TCR-pMHC interactions failed to account for their different signaling outcomes. Using yeast pMHC display, we identified peptide agonists of a formerly non-responsive TCR. Single-molecule force measurements demonstrated the emergence of catch bonds in the activating TCR-pMHC interactions, correlating with exclusion of CD45 from the TCR-APC contact site. Molecular dynamics simulations of TCR-pMHC disengagement distinguished agonist from non-agonist ligands based on the acquisition of catch bonds within the TCR-pMHC interface. The isolation of catch bonds as a parameter mediating the coupling of TCR binding and signaling has important implications for TCR and antigen engineering for immunotherapy.

Antigen Identification for Orphan T Cell Receptors Expressed on Tumor-Infiltrating Lymphocytes.

The immune system can mount T cell responses against tumors; however, the antigen specificities of tumor-infiltrating lymphocytes (TILs) are not well understood. We used yeast-display libraries of peptide-human leukocyte antigen (pHLA) to screen for antigens of "orphan" T cell receptors (TCRs) expressed on TILs from human colorectal adenocarcinoma. Four TIL-derived TCRs exhibited strong selection for peptides presented in a highly diverse pHLA-A∗02:01 library. Three of the TIL TCRs were specific for non-mutated self-antigens, two of which were present in separate patient tumors, and shared specificity for a non-mutated self-antigen derived from U2AF2. These results show that the exposed recognition surface of MHC-bound peptides accessible to the TCR contains sufficient structural information to enable the reconstruction of sequences of peptide targets for pathogenic TCRs of unknown specificity. This finding underscores the surprising specificity of TCRs for their cognate antigens and enables the facile indentification of tumor antigens through unbiased screening.

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.

Autoimmunity-associated T cell receptors recognize HLA-B*27-bound peptides.

Human leucocyte antigen B*27 (HLA-B*27) is strongly associated with inflammatory diseases of the spine and pelvis (for example, ankylosing spondylitis (AS)) and the eye (that is, acute anterior uveitis (AAU))1. How HLA-B*27 facilitates disease remains unknown, but one possible mechanism could involve presentation of pathogenic peptides to CD8+ T cells. Here we isolated orphan T cell receptors (TCRs) expressing a disease-associated public ß-chain variable region-complementary-determining region 3ß (BV9-CDR3ß) motif2-4 from blood and synovial fluid T cells from individuals with AS and from the eye in individuals with AAU. These TCRs showed consistent α-chain variable region (AV21) chain pairing and were clonally expanded in the joint and eye. We used HLA-B*27:05 yeast display peptide libraries to identify shared self-peptides and microbial peptides that activated the AS- and AAU-derived TCRs. Structural analysis revealed that TCR cross-reactivity for peptide-MHC was rooted in a shared binding motif present in both self-antigens and microbial antigens that engages the BV9-CDR3ß TCRs. These findings support the hypothesis that microbial antigens and self-antigens could play a pathogenic role in HLA-B*27-associated disease.

Clonally expanded B cells in multiple sclerosis bind EBV EBNA1 and GlialCAM.

Multiple sclerosis (MS) is a heterogenous autoimmune disease in which autoreactive lymphocytes attack the myelin sheath of the central nervous system. B lymphocytes in the cerebrospinal fluid (CSF) of patients with MS contribute to inflammation and secrete oligoclonal immunoglobulins1,2. Epstein-Barr virus (EBV) infection has been epidemiologically linked to MS, but its pathological role remains unclear3. Here we demonstrate high-affinity molecular mimicry between the EBV transcription factor EBV nuclear antigen 1 (EBNA1) and the central nervous system protein glial cell adhesion molecule (GlialCAM) and provide structural and in vivo functional evidence for its relevance. A cross-reactive CSF-derived antibody was initially identified by single-cell sequencing of the paired-chain B cell repertoire of MS blood and CSF, followed by protein microarray-based testing of recombinantly expressed CSF-derived antibodies against MS-associated viruses. Sequence analysis, affinity measurements and the crystal structure of the EBNA1-peptide epitope in complex with the autoreactive Fab fragment enabled tracking of the development of the naive EBNA1-restricted antibody to a mature EBNA1-GlialCAM cross-reactive antibody. Molecular mimicry is facilitated by a post-translational modification of GlialCAM. EBNA1 immunization exacerbates disease in a mouse model of MS, and anti-EBNA1 and anti-GlialCAM antibodies are prevalent in patients with MS. Our results provide a mechanistic link for the association between MS and EBV and could guide the development of new MS therapies.

Dairying enabled Early Bronze Age Yamnaya steppe expansions.

During the Early Bronze Age, populations of the western Eurasian steppe expanded across an immense area of northern Eurasia. Combined archaeological and genetic evidence supports widespread Early Bronze Age population movements out of the Pontic-Caspian steppe that resulted in gene flow across vast distances, linking populations of Yamnaya pastoralists in Scandinavia with pastoral populations (known as the Afanasievo) far to the east in the Altai Mountains1,2 and Mongolia3. Although some models hold that this expansion was the outcome of a newly mobile pastoral economy characterized by horse traction, bulk wagon transport4-6 and regular dietary dependence on meat and milk5, hard evidence for these economic features has not been found. Here we draw on proteomic analysis of dental calculus from individuals from the western Eurasian steppe to demonstrate a major transition in dairying at the start of the Bronze Age. The rapid onset of ubiquitous dairying at a point in time when steppe populations are known to have begun dispersing offers critical insight into a key catalyst of steppe mobility. The identification of horse milk proteins also indicates horse domestication by the Early Bronze Age, which provides support for its role in steppe dispersals. Our results point to a potential epicentre for horse domestication in the Pontic-Caspian steppe by the third millennium BC, and offer strong support for the notion that the novel exploitation of secondary animal products was a key driver of the expansions of Eurasian steppe pastoralists by the Early Bronze Age.

Triangulation supports agricultural spread of the Transeurasian languages.

The origin and early dispersal of speakers of Transeurasian languages-that is, Japanese, Korean, Tungusic, Mongolic and Turkic-is among the most disputed issues of Eurasian population history1-3. A key problem is the relationship between linguistic dispersals, agricultural expansions and population movements4,5. Here we address this question by 'triangulating' genetics, archaeology and linguistics in a unified perspective. We report wide-ranging datasets from these disciplines, including a comprehensive Transeurasian agropastoral and basic vocabulary; an archaeological database of 255 Neolithic-Bronze Age sites from Northeast Asia; and a collection of ancient genomes from Korea, the Ryukyu islands and early cereal farmers in Japan, complementing previously published genomes from East Asia. Challenging the traditional 'pastoralist hypothesis'6-8, we show that the common ancestry and primary dispersals of Transeurasian languages can be traced back to the first farmers moving across Northeast Asia from the Early Neolithic onwards, but that this shared heritage has been masked by extensive cultural interaction since the Bronze Age. As well as marking considerable progress in the three individual disciplines, by combining their converging evidence we show that the early spread of Transeurasian speakers was driven by agriculture.

Immune receptor inhibition through enforced phosphatase recruitment.

Antibodies that antagonize extracellular receptor-ligand interactions are used as therapeutic agents for many diseases to inhibit signalling by cell-surface receptors1. However, this approach does not directly prevent intracellular signalling, such as through tonic or sustained signalling after ligand engagement. Here we present an alternative approach for attenuating cell-surface receptor signalling, termed receptor inhibition by phosphatase recruitment (RIPR). This approach compels cis-ligation of cell-surface receptors containing ITAM, ITIM or ITSM tyrosine phosphorylation motifs to the promiscuous cell-surface phosphatase CD452,3, which results in the direct intracellular dephosphorylation of tyrosine residues on the receptor target. As an example, we found that tonic signalling by the programmed cell death-1 receptor (PD-1) results in residual suppression of T cell activation, but is not inhibited by ligand-antagonist antibodies. We engineered a PD-1 molecule, which we denote RIPR-PD1, that induces cross-linking of PD-1 to CD45 and inhibits both tonic and ligand-activated signalling. RIPR-PD1 demonstrated enhanced inhibition of checkpoint blockade compared with ligand blocking by anti-PD1 antibodies, and increased therapeutic efficacy over anti-PD1 in mouse tumour models. We also show that the RIPR strategy extends to other immune-receptor targets that contain activating or inhibitory ITIM, ITSM or ITAM motifs; for example, inhibition of the macrophage SIRPα 'don't eat me' signal with a SIRPα-CD45 RIPR molecule potentiates antibody-dependent cellular phagocytosis beyond that of SIRPα blockade alone. RIPR represents a general strategy for direct attenuation of signalling by kinase-activated cell-surface receptors.

Opposing T cell responses in experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis is a model for multiple sclerosis. Here we show that induction generates successive waves of clonally expanded CD4+, CD8+ and γδ+ T cells in the blood and central nervous system, similar to gluten-challenge studies of patients with coeliac disease. We also find major expansions of CD8+ T cells in patients with multiple sclerosis. In autoimmune encephalomyelitis, we find that most expanded CD4+ T cells are specific for the inducing myelin peptide MOG35-55. By contrast, surrogate peptides derived from a yeast peptide major histocompatibility complex library of some of the clonally expanded CD8+ T cells inhibit disease by suppressing the proliferation of MOG-specific CD4+ T cells. These results suggest that the induction of autoreactive CD4+ T cells triggers an opposing mobilization of regulatory CD8+ T cells.

Economic evaluations of medical devices in paediatrics: a systematic review and a quality appraisal of the literature.

BACKGROUND: Although economic evaluations (EEs) have been increasingly applied to medical devices, little discussion has been conducted on how the different health realities of specific populations may impact the application of methods and the ensuing results. This is particularly relevant for pediatric populations, as most EEs on devices are conducted in adults, with specific aspects related to the uniqueness of child health often being overlooked. This study provides a review of the published EEs on devices used in paediatrics, assessing the quality of reporting, and summarising methodological challenges. METHODS: A systematic literature search was performed to identify peer-reviewed publications on the economic value of devices used in paediatrics in the form of full EEs (comparing both costs and consequences of two or more devices). After the removal of duplicates, article titles and abstracts were screened. The remaining full-text articles were retrieved and assessed for inclusion. In-vitro diagnostic devices were not considered in this review. Study descriptive and methodological characteristics were extracted using a structured template. The Consolidated Health Economic Evaluation Reporting Standards (CHEERS) 2022 checklist was used to assess the quality of reporting. A narrative synthesis of the results was conducted followed by a critical discussion on the main challenges found in the literature. RESULTS: 39 full EEs were eligible for review. Most studies were conducted in high-income countries (67%) and focused on high-risk therapeutic devices (72%). Studies comprised 25 cost-utility analyses, 13 cost-effectiveness analyses and 1 cost-benefit analysis. Most of the studies considered a lifetime horizon (41%) and a health system perspective (36%). Compliance with the CHEERS 2022 items varied among the studies. CONCLUSIONS: Despite the scant body of evidence on EEs focusing on devices in paediatrics results highlight the need to improve the quality of reporting and advance methods that can explicitly incorporate the multiple impacts related to the use of devices with distinct characteristics, as well as consider specific child health realities. The design of innovative participatory approaches and instruments for measuring outcomes meaningful to children and their families should be sought in future research.

Assessment of Cardiorespiratory and Metabolic Contributions in an Extreme Intensity CrossFit® Benchmark Workout.

Our purpose was to characterize the oxygen uptake kinetics (VO2), energy systems contributions and total energy expenditure during a CrossFit® benchmark workout performed in the extreme intensity domain. Fourteen highly trained male CrossFitters, aged 28.3 ± 5.4 years, with height 177.8 ± 9.4 cm, body mass 87.9 ± 10.5 kg and 5.6 ± 1.8 years of training experience, performed the Isabel workout at maximal exertion. Cardiorespiratory variables were measured at baseline, during exercise and the recovery period, with blood lactate and glucose concentrations, including the ratings of perceived exertion, measured pre- and post-workout. The Isabel workout was 117 ± 10 s in duration and the VO2 peak was 47.2 ± 4.7 mL·kg-1·min-1, the primary component amplitude was 42.0 ± 6.0 mL·kg-1·min-1, the time delay was 4.3 ± 2.2 s and the time constant was 14.2 ± 6.0 s. The accumulated VO2 (0.6 ± 0.1 vs. 4.8 ± 1.0 L·min-1) value post-workout increased substantially when compared to baseline. Oxidative phosphorylation (40%), glycolytic (45%) and phosphagen (15%) pathways contributed to the 245 ± 25 kJ total energy expenditure. Despite the short ~2 min duration of the Isabel workout, the oxygen-dependent and oxygen-independent metabolism energy contributions to the total metabolic energy release were similar. The CrossFit® Isabel requires maximal effort and the pattern of physiological demands identifies this as a highly intensive and effective workout for developing fitness and conditioning for sports.

Improved MAIT cell functions following fecal microbiota transplantation for metastatic renal cell carcinoma.

Strategies to modify the gut microbiome in cancer patients using fecal microbiota transplantation (FMT) have gained momentum as a therapeutic intervention. However, how FMT impacts innate-like, antimicrobial T lymphocytes is unclear. In this study, we assessed peripheral blood (PB) mucosa-associated invariant T (MAIT) cell frequencies and functions in patients with metastatic renal cell carcinoma (mRCC) before and seven days after they received FMT as part of a clinical trial. We found comparable MAIT cell frequencies in healthy controls and mRCC patients. In contrast, γδ T cells exhibited a numerical decline in mRCC, which was partially reversed by FMT. We also found a significant increase in the PB CD4+ MAIT cell compartment of mRCC patients with or without FMT. Paired sample analyses revealed CD69 upregulation on MAIT cells accompanied by decreased PD-1 levels post-FMT. These changes were unique to MAIT cells as non-MAIT T lymphocytes showed either no trend or a trend in the opposite direction. Importantly, FMT did not render MAIT cells exhausted as also judged by their stable expression of TIM-3, LAG-3, BTLA, CTLA-4, TIGIT and VISTA. These findings were corroborated in functional assays in which MAIT cells were stimulated with MR1 ligands or with a combination of IL-12 and IL-18 to produce inflammatory cytokines and granzyme B. Indeed, when stimulated ex vivo with IL-12 and IL-18, MAIT cells mounted a more rigorous TNF-α response post-FMT. In conclusion, FMT improves MAIT cell functions, which should serve patients well in subsequent microbial challenges in the face of cancer-elicited immunosuppression. Trial Registration: https://clinicaltrials.gov/ Identifier: NCT04163289 (registration date: November 14, 2019).

Implementation of a centralized pharmacovigilance system in academic pan-European clinical trials: Experience from EU-Response and conect4children consortia.

Setting-up a high quality, compliant and efficient pharmacovigilance (PV) system in multi-country clinical trials can be more challenging for academic sponsors than for companies. To ensure the safety of all participants in academic studies and that the PV system fulfils all regulations, we set up a centralized PV system that allows sponsors to delegate work on PV. This initiative was put in practice by our Inserm-ANRS MIE PV department in two distinct multinational European consortia with 19 participating countries: conect4children (c4c) for paediatrics research and EU-Response for Covid-19 platform trials. The centralized PV system consists of some key procedures to harmonize the complex safety processes, creation of a local safety officer (LSO) network and centralization of all safety activities. The key procedures described the safety management plan for each trial and how tasks were shared and delegated between all stakeholders. Processing of serious adverse events (SAEs) in a unique database guaranteed the full control of the safety data and continuous evaluation of the risk-benefit ratio. The LSO network participated in efficient regulatory compliance across multiple countries. In total, there were 1312 SAEs in EU-Response and 83 SAEs in c4c in the four trials. We present here the lessons learnt from our experience in four clinical trials. We managed heterogeneous European local requirements and implemented efficient communication with all trial teams. Our approach builds capacity for PV that can be used by multiple academic sponsors.