Anti-CD45RC antibody immunotherapy prevents and treats experimental autoimmune polyendocrinopathy-candidiasis- ectodermal dystrophy syndrome

Targeted monoclonal antibody (mAb) therapies show great promise for the treatment of transplant rejection and autoimmune diseases by inducing more specific immunomodulatory effects than broadly immunosuppressive drugs routinely used. We recently described the therapeutic advantage of targeting CD45RC, expressed at high levels by conventional T (Tconv) cells (CD45RC(hi)), their precursors, and terminally differentiated T (TEMRA) cells, but not by regulatory T cells (Tregs; CD45RC(lo/-)). We demonstrated efficacy of anti-CD45RC mAb treatment in transplantation, but its potential has not been examined in autoimmune diseases. Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a rare genetic syndrome caused by loss-of-function mutations of autoimmune regulator (AIRE), a key central tolerance mediator, leading to abnormal autoreactive T cell responses and autoantibody production. Herein, we show that, in a rat model of APECED syndrome, anti-CD45RC mAb was effective for both prevention and treatment of autoimmune manifestations and inhibited autoantibody development. Anti-CD45RC mAb intervention depleted CD45RC(hi) T cells, inhibited CD45RC(hi) B cells, and restored the Treg/Tconv cell ratio and the altered Treg transcriptomic profile. In APECED patients, CD45RC was significantly increased in peripheral blood T cells, and lesioned organs from APECED patients were infiltrated by CD45RC(hi) cells. Our observations highlight the potential role for CD45RC(hi) cells in the pathogenesis of experimental and human APECED syndrome and the potential of anti-CD45RC antibody treatment.Peer reviewe

Portuguese Results of the ETICC Study: Impact of the Pandemic COVID-19 in the Diagnosis and Management of Colorectal Cancer in 2020 in Portuguese Hospitals

Introduction: The outbreak of coronavirus disease 2019 (COVID-19) had affected clinical practice in several ways, including the restriction of nonessential endoscopic procedures. Therefore, our aim was to evaluate how colorectal cancer (CRC) diagnosis and management was affected during the first year of pandemics in Portugal. Methods: This is a Portuguese substudy of the French retrospective multicentric study ETICC (Etude de l’Impact de la pandémie COVID-19 sur le diagnostic et la prise en charge du Cancer Colorectal). We compared patients’ characteristics, clinical manifestations, CRC staging at diagnosis, delay to first medical appointment, histological diagnosis, surgical and medical treatments between the year previous to the pandemics (control) and the first year of pandemics. Results: We included 766 patients: 496 in the control group and 270 in the COVID group. There was no significant difference in CRC staging at diagnosis between both groups, with 21% being diagnosed as metastatic in the control group and 22% in the first year of pandemics (p = 0.770). Contrary to what happened in France, there was a significant decrease in CRC diagnosis in asymptomatic patients (25–8.4%; p < 0.001) and after a positive fecal immunochemical test (20.8–11.3%; p = 0.002) during the pandemics. Although the increase in the overall complication rate at diagnosis was nonsignificant, in Portugal, there was a significant increase in diagnosis of abdominal occlusion (12.1–18.1%; p = 0.033). In Portugal, time between the beginning of symptoms and the first medical appointment significantly increased from a median of 50 days to 64 days during COVID (p < 0.001). On the contrary, time between histological diagnosis and tumor resection had significantly decreased from a median of 65 to 39 days (p < 0.001). Time between histological diagnosis and neoadjuvant treatment was not statistically different (median of 64–67 days; p = 0.590), as was time between histological diagnosis and palliative chemotherapy (median of 50–51 days; p = 1.000). Time from CRC resection and adjuvant treatment has significantly decreased from a median of 54 to 43 days (p = 0.001). Discussion: We found a significant impact in CRC diagnosis in the first year of pandemics, more pronounced than what was found in France. These are likely related not only with the closing of endoscopy units but also with the difficulties patients had in finding an appointment with their general practitioners. On the other hand, both in France and Portugal, the first year of pandemics did not worsen CRC staging at diagnosis and did not significantly affect medical and surgical treatments once the diagnosis was made

Investigation of the potential of anti-CD45RC antibody immunotherapy to treat the APECED autoimmune syndrome

AIRE (Autoimmune regulator) est un régulateur de la transcription essentiel qui permet la sélection négative des thymocytes en favorisant l’expression thymique d’antigènes spécifiques de tissus (TSAs). Chez l’Homme, la déficience pour AIRE induit le syndrome de l’Autoimmune PolyEndocrinopathy - Candidiasis - Ectodermal - Dystrophy (APECED), une maladie auto-immune létale caractérisée par la présence de lésions en périphérie, un défaut suppressif des lymphocytes T régulateurs (Tregs), et la production d’auto-anticorps. Notre équipe a généré le premier modèle de rat Aire-/- qui présente les caractéristiques principales de ce syndrome. Précédemment, nous avons démontré que l’injection d’anticorps anti-CD45RC permet d’induire la tolérance dans le cadre de la transplantation, la maladie du greffon contre l’hôte (GVHD) et la myopathie de Duchesne. Lors de cette thèse, nous avons évalué le potentiel de l’immunothérapie par injection d’anticorps anti-CD45RC pour traiter le syndrome de l’APECED. Nous avons démontré que ce traitement permet d’améliorer les symptômes auto-immuns des rats Aire-/-, en condition préventive et curative. Puis, que cet effet thérapeutique est médié par la déplétion spécifique des lymphocytes T effecteurs (Teffs), la diminution de la production d’autoanticorps et la modification transcriptionnelle des Tregs. Pour finir, nous avons démontré que les patients APECED possèdent une plus grande proportion de LTs CD45RChi et que ces cellules infiltrent les lésions auto-immunes. L’utilisation d’un anticorps humain anti- CD45RC permet de dépléter ces cellules, soulignant ainsi le potentiel thérapeutique de ce traitement.AIRE (Autoimmune regulator) is an essential transcriptional regulator that allows negative selection of thymocytes by promoting thymic expression of tissue-specific antigens (TSAs). In Human, AIRE deficiency induces the Autoimmune PolyEndocrinopathy - Candidiasis - Ectodermal - Dystrophy (APECED) syndrome, a lethal autoimmune disease characterized by the presence of peripheral lesions, a suppressive defect of regulatory T cells (Tregs), and the production of autoantibodies. Our team has generated the first Aire-/- rat model that harbors several key features of this syndrome. Previously, we have demonstrated that injection of anti-CD45RC antibodies can induce tolerance in transplantation, graft-versus-host disease (GVHD) and Duchesne's myopathy. In this thesis, we evaluated the potential of anti-CD45RC antibody immunotherapy to treat the APECED syndrome. We demonstrated that this treatment improves the autoimmune symptoms of Aire-/- rats, in both preventive and curative conditions. Then, that this therapeutic effect is mediated by the specific depletion of effector T cells (Teffs), the decrease of autoantibody production and the transcriptional modification of Tregs. Finally, we demonstrated that APECED patients have a higher proportion of CD45RChi T cells and that these cells infiltrate auto-immune lesions. The use of a human anti-CD45RC antibody depletes these cells, highlighting the therapeutic potential of this treatmen

AIRE deficiency, from preclinical models to human APECED disease

International audienceABSTRACT Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED) is a rare life-threatening autoimmune disease that attacks multiple organs and has its onset in childhood. It is an inherited condition caused by a variety of mutations in the autoimmune regulator (AIRE) gene that encodes a protein whose function has been uncovered by the generation and study of Aire-KO mice. These provided invaluable insights into the link between AIRE expression in medullary thymic epithelial cells (mTECs), and the broad spectrum of self-antigens that these cells express and present to the developing thymocytes. However, these murine models poorly recapitulate all phenotypic aspects of human APECED. Unlike Aire-KO mice, the recently generated Aire-KO rat model presents visual features, organ lymphocytic infiltrations and production of autoantibodies that resemble those observed in APECED patients, making the rat model a main research asset. In addition, ex vivo models of AIRE-dependent self-antigen expression in primary mTECs have been successfully set up. Thymus organoids based on pluripotent stem cell-derived TECs from APECED patients are also emerging, and constitute a promising tool to engineer AIRE-corrected mTECs and restore the generation of regulatory T cells. Eventually, these new models will undoubtedly lead to main advances in the identification and assessment of specific and efficient new therapeutic strategies aiming to restore immunological tolerance in APECED patients

A new automated 3D detection of synaptic contacts reveals the formation of cortico-striatal synapses upon cocaine treatment in vivo

International audienceAddiction can be considered as a form of neuronal adaptation within the reward circuitry. Upon psychostimulant administration, long-term behavioral adaptations are associated with synaptic plasticity and morphological changes of medium spiny neurons (MSN) from the striatum. Increased spine density onto MSN in response to chronic cocaine exposure in mice has been described for more than a decade, but no evidence indicates that these newly formed spines establish connections. We developed a method for labeling, automated detection and morphological analysis of synaptic contacts. Individual labeling of neurons in mice that express the Vesicular GLUtamate Transporter-1 fused to Venus allows visualization of both dendritic spines and axonal boutons. Automated three-dimensional segmentation and morphometric analysis retrieve information on thousands of synapses at high resolution. We used this method to demonstrate that new cortico-striatal connections are formed in the striatum upon chronic cocaine. We also show that the cortical input weight is preserved over other cerebral inputs and that the newly formed spines contact pre-existing axonal boutons. Our results pave the way for other studies, since our method can be applied to any other neuronal type as demonstrated herein for glutamatergic connections on pyramidal neurons and Purkinje cells

ANTI-CD45RC MABS IMMUNOTHERAPY CONTROLS THE DEVELOPPEMENT OF AUTO-IMMUNE SYMPTOMS IN AN APECED RAT MODEL OF AIRE DEFICIENCY

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Cross-Reactive Donor-Specific CD8+ Tregs Efficiently Prevent Transplant Rejection

International audienceTo reduce the use of non-specific immunosuppressive drugs detrimental to transplant patient health, therapies in development aim to achieve antigen-specific tolerance by promoting antigen-specific regulatory T cells (Tregs). However, identification of the natural antigens recognized by Tregs and the contribution of their dominance in transplantation has been challenging. We identify epitopes derived from distinct major histocompatibility complex (MHC) class II molecules, sharing a 7-amino acid consensus sequence positioned in a central mobile section in complex with MHC class I, recognized by cross-reactive CD8+ Tregs, enriched in the graft. Antigen-specific CD8+ Tregs can be induced in vivo with a 16-amino acid-long peptide to trigger transplant tolerance. Peptides derived from human HLA class II molecules, harboring the rat consensus sequence, also activate and expand human CD8+ Tregs, suggesting its potential in human transplantation. Altogether, this work should facilitate the development of therapies with peptide epitopes for transplantation and improve our understanding of CD8+ Treg recognition

Workflow for SNP genotyping using the Hi-Plex method v2

Many research questions in ecology and evolution require balancing sampling strategies between their spatial (how many populations? on which geographical, environmental gradients?), temporal (diachronic approaches), and genomic (how many and which loci?) dimensions. High-throughput molecular biology protocols often offer very good genomic coverage, but this is often only achievable at the expense of other sampling dimensions. This has led to the development of targeted genotyping strategies for SNP locus sets, in addition to whole or reduced genome sequencing strategies. We here present an adaptation of a protocol developed by the University of Melbourne for genotyping rare variants in human oncology to non model species for use in ecology and evolution. Hi-Plex is an amplicon sequencing technique (sensu Meek & Larson 2019) in which all loci are co-amplified in a multiplex reaction before Illumina or Ion Torrent sequencing (we used Illumina). Intermediate steps include dual indexing of individual samples used for demultiplexing

Breakdown of Immune Tolerance in AIRE-Deficient Rats Induces a Severe Autoimmune Polyendocrinopathy–Candidiasis–Ectodermal Dystrophy–like Autoimmune Disease

International audienceAutoimmune regulator (AIRE) deficiency in humans induces a life-threatening generalized autoimmune disease called autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), and no curative treatments are available. Several models of AIRE-deficient mice have been generated, and although they have been useful in understanding the role of AIRE in central tolerance, they do not reproduce accurately the APECED symptoms, and thus there is still a need for an animal model displaying APECED-like disease. We assessed, in this study, the potential of the rat as an accurate model for APECED. In this study, we demonstrate that in rat, AIRE is expressed by MHC class II (MCH-II)+ and MHC-II- medullary thymic epithelial cells in thymus and by CD4int conventional dendritic cells in periphery. To our knowledge, we generated the first AIRE-deficient rat model using zinc-finger nucleases and demonstrated that they display several of the key symptoms of APECED disease, including alopecia, skin depigmentation, and nail dystrophy, independently of the genetic background. We observed severe autoimmune lesions in a large spectrum of organs, in particular in the pancreas, and identified several autoantibodies in organs and cytokines such as type I IFNs and IL-17 at levels similar to APECED. Finally, we demonstrated a biased Ab response to IgG1, IgM, and IgA isotypes. Altogether, our data demonstrate that AIRE-deficient rat is a relevant APECED animal model, opening new opportunity to test curative therapeutic treatments

Synthesis of Mono- and Dihydroxylated Amino Acids with New α\alpha-Ketoglutarate-Dependent Dioxygenases: Biocatalytic Oxidation of C−-H Bonds

International audienceIron(II)/$\alpha$-ketoacid-dependent oxygenases ($\alpha$KAOs) are enzymesthat mainly catalyse hydroxylation reaction. By usinga genomic approach combining sequence comparison andprotein-domain sharing, a set of 131 $\alpha$KAO enzymes was prepared.The screening of various substrates revealed five new$\alpha$KAOs. Four $\alpha$KAOs were found to be active towards L-lysine,L-ornithine and L-arginine with total regio- and stereoselectivitiesand yielding the corresponding 3- or 4-hydroxyaminoacids. The enzymatic cascade reaction with two stereoselectiveregiodivergent $\alpha$ KAOs enabled the synthesis of 3,4-dihydroxy-L-lysine