Analyse comparative des performances diagnostiques et pronostiques de sous-familles d'auto-anticorps associés à la polyarthrite rhumatoïde, dirigés contre divers épitopes immunodominants de la fibrine citrullinée et analyse de leurs réactivités croisées

La polyarthrite rhumatoïde (PR) est une maladie auto-immune caractérisée par une inflammation chronique des articulations synoviales qui peut aboutir à une érosion ostéo-cartilagineuse douloureuse et handicapante. Des facteurs génétiques et environnementaux et surtout des auto-anticorps, facteurs rhumatoïdes et auto-anticorps anti-protéines citrullinées (ACPA) sont associés à la PR. Les ACPA ciblent des antigènes porteurs de résidus citrullyl et en particulier, la fibrine citrullinée, auto-antigène majeur dans le tissu synovial. Ils sont très spécifiques de la maladie, apparaissent avant les signes cliniques et sont associés aux formes les plus sévères. Ils sont détectés dans le sérum des patients par des tests immuno-enzymatiques (ELISA) développés avec divers peptides/protéines citrullinés : avec le fibrinogène humain citrulliné sont détectés les AhFibA (anti-human citrullinated fibrinogen autoantibodies). Une cartographie épitopique de la fibrine citrullinée a permis d'identifier deux peptides, a36-50Cit38,42 et ß60-74Cit60,72,74, comme porteurs des épitopes immuno-dominants. L'objectif de ce travail était de caractériser ces épitopes, de développer des tests permettant de détecter spécifiquement les sous-familles d'AhFibA ciblant ces épitopes, d'évaluer la valeur diagnostique et pronostique de chaque sous-famille et d'analyser leur spécificité antigénique fine, comparativement à celle d'autres ACPA. Nous avons d'abord identifié les épitopes reconnus par les AhFibA sur a36-50Cit38,42 (a : VECit42HQ et a' : Cit38VVE) et ß60-74Cit60,72,74 (GYCit72ACit74) en utilisant de courts peptides synthétiques chevauchants (MultipinTM). Ensuite, à l'aide de constructions peptidiques originales, nous avons développé et optimisé diverses méthodes d'ELISA qui ont permis d'évaluer l'impact de la longueur et du mode de fixation des peptides (adsorption passive vs avidine-biotine), ainsi que celui de la distance entre épitope-cible et support, sur le titre et les index diagnostiques des auto-anticorps détectés. Le paramètre majeur dans la capacité d'un peptide à être reconnu par les sérums a été la distance entre épitope et biotine, indépendamment de la longueur du peptide et que celle-ci soit C- ou N-terminale. Par ailleurs a été confirmée la valeur diagnostique du peptide ß60-74Cit60,72,74 qui, sous forme biotinylée en N-terminal présente des performances égales à celles des AhFibA. A l'aide d'une une cohorte toulousaine de patients atteints de maladies rhumatologiques anciennes comportant 76% de PR AhFibA-positives (à la spécificité de 98,5%), nous avons montré que 41% des PR avaient des anti-a36-50Cit38,42 et 62 % des anti-ß60-74Cit60,72,74, plus de 90% des patients possédant l'un ou l'autre de ces anticorps. Des tests d'inhibition croisée ont permis de montrer qu'anti-a36-50Cit38,42 et anti-ß60-74Cit60,72,74 constituaient deux sous-familles d'auto-anticorps différentes. Nous avons également montré avec une cohorte camerounaise de PR anciennes, que la sensibilité diagnostique des AhFibA (73%), la proportion d'anti-a36-50Cit38,42 (45%) et celle d'anti-ß60-74Cit60,72,74 (73%) étaient similaires à celles de la cohorte toulousaine. Les caractéristiques des AhFibA dans la PR sont donc similaires chez les Noirs Africains et chez les Caucasiens montrant que cette réponse immune B liée à la maladie est largement indifférente aux facteurs génétiques et environnementaux. Parallèlement, à partir d'une cohorte française multicentrique de PR débutantes (ESPOIR), nous avons confirmé que les performances diagnostiques des AhFibA étaient similaires à celles des tests de références (anti-CCP2, anti-MCV) et nous avons montré que les proportions de sérums AhFibA-positifs contenant des anti-a36-50Cit38,42 et/ou anti-ß60-74Cit60,72,74 étaient similaires à celles des sérums de PR établies, révélant ainsi que la composition des AhFibA est homogène et indépendante du stade d'évolution de la maladie. A l'aide de la cohorte ESPOIR, nous avons aussi évalué les valeurs pronostiques des deux sous-familles d'anticorps, en analysant divers paramètres cliniques et radiologiques à trois ans d'évolution de la maladie. Les patients qui présentaient des auto-anticorps (AhFibA, anti-a36-50Cit38,42 ou anti-ß60-74Cit60,72,74) au moment du diagnostic, ont eu une érosion articulaire médiane deux fois plus rapide que les patients qui n'en avaient pas. Les AhFibA sont donc des marqueurs de mauvais pronostic et ce, quelle que soit leur spécificité fine. Sous-familles et AhFibA (présence/absence ou titre) sont associés de façon similaire avec les allèles HLA-DR de susceptibilité à la PR mais ne le sont pas avec le tabagisme. De nombreux autres peptides-cibles des ACPA ont été décrits mais leurs réactivités croisées avec les ACPA sont encore mal caractérisées. Nous avons développé un ELISA avec l'un de ces peptides citrullinés (EBNA35-58Cit) dérivé d'une protéine du virus Epstein-Barr (EBV). Nous avons montré que les patients atteints de PR qui sont EBNA35-58Cit-positifs (36% de 181 PR) constituent un sous-groupe des patients AhFibA-positifs et, par des expériences d'inhibition, que les anticorps anti-EBNA35-58Cit présentent une très forte réactivité croisée avec le peptide ß60-74Cit60,72,74. Ce résultat renforce l'hypothèse d'une implication du virus dans la physiopathologie de la PR. En conclusion, notre travail a contribué à la caractérisation des AhFibA en montrant qu'ils étaient principalement constitués de deux sous-familles d'auto-anticorps différentes dont l'une présente une forte réactivité croisée avec un peptide dérivé d'EBV. Les AhFibA et leurs sous-familles sont présentes dans les mêmes proportions chez les Caucasiens et chez les Noirs Africains, et que la PR soit établie ou débutante. L'hétérogénéité des résultats des multiples tests ELISA que nous avons développés à partir de divers peptides porteurs des mêmes épitopes immuno-dominants, montrent que toute comparaison de données publiées concernant la spécificité fine des ACPA doit être abordée avec la plus grande circonspection.Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of synovial joints often leading to painful and disabling join erosion. Genetic and environmental factors and, above all autoantibodies, rheumatoid factor and anti-citrullinated proteins autoantibodies (ACPA), are associated with RA. ACPA recognize antigens containing Citrullyl residues and particularly citrullinated fibrin, the major autoantigen in synovial tissues. They are highly specific of the disease, appear years before clinical onset and are associated with the more severe forms. ACPA can be assayed in sera by ELISAs developed with various citrullinated peptides/proteins, for example citrullinated human fibrinogen allowing the detection of anti-human citrullinated fibrinogen autoantibodies (AhFibA). An epitope mapping of citrullinated fibrin led to the identification of two peptides a36-50Cit38,42 and ß60-74Cit60,72,74 as bearing its immunodominant epitopes. The aim of the present work was to characterize those epitopes, to develop ELISAs that will specifically detect subfamilies of AhFibA directed to each epitope, to evaluate the diagnostic and prognostic value of each subfamily and to analyze their fine specificity comparatively to those of other ACPA. We first identified the epitopes recognized by AhFibA on a36-50Cit38,42 (a : VECit42HQ et a' : Cit38VVE) and ß60-74Cit60, 72,74 (GYCit72ACit74) using short overlapping synthetic peptides (MultipinTM). Then, with original peptide constructs we developed and optimized several ELISAs that allowed evaluating the impact of the length and the method of binding of peptides (passive adsorption vs avidin-biotin) as well as that of the distance between the targeted epitope and the solid phase, on the titres and the diagnostic indexes of the detected autoantibodies. The major parameter influencing the ability of a peptide to be recognized by sera was the distance between the epitope and the biotin, irrespective of the peptide length and the biotin position (C- or N-terminal). Besides, we confirmed the diagnostic value of the ß60-74Cit60,72,74 peptide that in a N-terminal biotinylated form presented diagnostic performances similar to those of AhFibA. With a cohort from Toulouse composed of patients with established rheumatic diseases, 76% of RA patients being AhFibA-positive (specificity 98.5%), we found that 41% of RA patients had anti-a36-50Cit38,42 and 62% anti-ß60-74Cit60,72,74 autoantibodies, 90% having one or both subfamilies of autoantibodies. Cross-inhibition experiments allowed demonstrating that anti-a36-50Cit38,42 and anti-ß60-74Cit60,72,74 constitute two different subfamilies of autoantibodies. We also showed, with a cohort of patients with established rheumatic diseases from Cameroon, that the diagnostic sensitivity of AhFibA (73%), the proportion of anti-a36-50Cit38,42 (45 %) and that of anti-ß60-74Cit60,72,74 (73%) were similar to those of the cohort from Toulouse. The AhFibA hallmarks in RA are thus identical in Black Africans and in Caucasians demonstrating that this RA-associated B-cell immune response is largely independent of environmental and genetic factors. In addition, using a French multicenter cohort of patients with early RA (ESPOIR) we confirmed that the diagnostic performance of AhFibA were similar to those of the reference tests (anti-CCP2, anti-MCV), and we showed that the proportions of AhFibA-positive sera containing anti-a36-50Cit38,42 and/or anti-ß60-74Cit60,72,74 were similar to those of established RA, revealing that AhFibA composition was homogeneous and independent of the disease stage. With the same cohort, we also assessed the prognostic value of the two autoantibody subfamilies, analyzing clinical and radiological parameters after three years of disease progression. Patients who had autoantibodies (AhFibA, anti-a36-50Cit38,42 or anti-ß60-74Cit60,72,74) at the time of the diagnosis, had a median variation of joint erosion, two times higher than those who did not. Thus, AhFibA are markers of poor prognosis regardless of their fine specificity. AhFibA and their subfamilies (presence/absence and titre) were also similarly associated with the HLA-DR susceptibility alleles but not with tobacco exposure. Other peptides have been described as ACPA targets, however their level of cross-reactivity with ACPA are not still well characterized. We developed an ELISA with one of those citrullinated peptide (EBNA35-58Cit) derived from a protein of the Epstein-Barr virus. We demonstrated that EBNA35-58Cit-positive RA patients (36% of 181 RA) constituted a subgroup of AhFibA-positive patients and, through cross-inhibition experiments, that anti-EBNA35-58Cit antibodies strongly cross-reacted with ß60-74Cit60,72,74. This result reinforces the hypothesis of a role for the virus in the pathophysiology of RA. In conclusion, our work contributed to characterize AhFibA demonstrating that they are mainly composed of two different subfamilies of autoantibodies, among which one strongly cross-react with a peptide derived from EBV. AhFibA and their subfamilies are present in the same proportions regardless of the disease stage (recent or established RA) and similarly in Caucasians and in Black Africans. Heterogeneity of the results obtained with ELISAs developed with various peptides bearing the same immunodominant epitopes show that the published data about ACPA fine specificity must be analyzed with extreme caution

Phenotypic diversity of human adipose tissue-resident NK cells in obesity

Natural killer (NK) cells have emerged as key mediators of obesity-related adipose tissue inflammation. However, the phenotype of NK cell subsets residing in human adipose tissue are poorly defined, preventing a detailed understanding of their role in metabolic disorders. In this study, we applied multicolor flow cytometry to characterize CD56bright and CD56dim NK cells in blood and adipose tissue depots in individuals with obesity and identified surface proteins enriched on adipose tissue-resident CD56bright NK cells. Particularly, we found that adipose tissue harbored clusters of tissue-resident CD56bright NK cells signatured by the expression of CD26, CCR5 and CD63, possibly reflecting an adaptation to the microenvironment. Together, our findings provide broad insights into the identity of NK cells in blood and adipose tissue in relation to obesity.publishedVersio

Retained NK cell phenotype and functionality in non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD), and the progressive stage non-alcoholic steatohepatitis (NASH), is the predominant cause of chronic liver disease globally. As part of the complex pathogenesis, natural killer (NK) cells have been implicated in the development of liver inflammation in experimental murine models of NASH. However, there is a lack of knowledge on how NK cells are affected in humans with this disease. Here, we explored the presence of disease-specific changes within circulating and tissue-resident NK cell populations, as well as within other major immune cell subsets, in patients with liver biopsy-confirmed NAFLD. Using 18-color-flow cytometry, substantial changes were observed in certain myeloid populations in patients as compared to controls. NK cell numbers, on the other hand, were not altered. Furthermore, only minor differences in expression of activating and inhibitory NK cell receptors were noted, with the exception of an increased expression of NKG2D on NK cells from patients with NASH. NK cell differentiation remained constant, and NK cells from these patients retain their ability to respond adequately upon stimulation. Instead, considerable alterations were observed between liver, adipose tissue, and peripheral blood NK cells, independently of disease status. Taken together, these results increase our understanding of the importance of the local microenvironment in shaping the NK cell compartment and stress the need for further studies exploring how NASH affects intrahepatic NK cells in humans.publishedVersio

Preoperative immunological plasma markers TRAIL, CSF1 and TIE2 predict survival after resection for biliary tract cancer

IntroductionSystemic inflammatory markers have been validated as prognostic factors for patients with biliary tract cancer (BTC). The aim of this study was to evaluate specific immunologic prognostic markers and immune responses by analyzing preoperative plasma samples from a large prospectively collected biobank.MethodsExpression of 92 proteins representing adaptive and innate immune responses was investigated in plasma from 102 patients undergoing resection for BTC 2009-2017 (perihilar cholangiocarcinoma n=46, intrahepatic cholangiocarcinoma n=27, gallbladder cancer n=29), by means of a high-throughput multiplexed immunoassay. Association with overall survival was analyzed by Cox regression, with internal validation and calibration. Tumor tissue bulk and single-cell gene expression of identified markers and receptors/ligands was analyzed in external cohorts.ResultsThree preoperative plasma markers were independently associated with survival: TRAIL, TIE2 and CSF1, with hazard ratios (95% confidence intervals) 0.30 (0.16-0.56), 2.78 (1.20-6.48) and 4.02 (1.40-11.59) respectively. The discrimination of a preoperative prognostic model with the three plasma markers was assessed with concordance-index 0.70, while the concordance-index of a postoperative model with histopathological staging was 0.66. Accounting for subgroup differences, prognostic factors were assessed for each type of BTC. TRAIL and CSF1 were prognostic factors in intrahepatic cholangiocarcinoma. In independent cohorts, TRAIL-receptor expression was higher in tumor tissue and seen in malignant cells, with TRAIL and CSF1 expressed by intra- and peritumoral immune cells. Intratumoral TRAIL-activity was decreased compared to peritumoral immune cells, while CSF1-activity was increased. The highest CSF1 activity was seen in intratumoral macrophages, while the highest TRAIL-activity was seen in peritumoral T-cells.DiscussionIn conclusion, three preoperative immunological plasma markers were prognostic for survival after surgery for BTC, providing good discrimination, even compared to postoperative pathology. TRAIL and CSF1, prognostic factors in intrahepatic cholangiocarcinoma, showed marked differences in expression and activity between intra- and peritumoral immune cells

Subtype-specific surface proteins on adipose tissue macrophages and their association to obesity-induced insulin resistance

A chronic low-grade inflammation, originating in the adipose tissue, is considered a driver of obesity-associated insulin resistance. Macrophage composition in white adipose tissue is believed to contribute to the pathogenesis of metabolic diseases, but a detailed characterization of pro- and anti-inflammatory adipose tissue macrophages (ATMs) in human obesity and how they are distributed in visceral- and subcutaneous adipose depots is lacking. In this study, we performed a surface proteome screening of pro- and anti-inflammatory ATMs in both subcutaneous- (SAT) and visceral adipose tissue (VAT) and evaluated their relationship with systemic insulin resistance. From the proteomics screen we found novel surface proteins specific to M1-like- and M2-like macrophages, and we identified depot-specific immunophenotypes in SAT and VAT. Furthermore, we found that insulin resistance, assessed by HOMA-IR, was positively associated with a relative increase in pro-inflammatory M1-like macrophages in both SAT and VAT.publishedVersio

NK cells are activated and primed for skin-homing during acute dengue virus infection in humans

10.1038/s41467-019-11878-3Nature Communications101389

Invasive fungal infection among hematopoietic stem cell transplantation patients with mechanical ventilation in the intensive care unit

<p>Abstract</p> <p>Background</p> <p>Invasive fungal infection (IFI) is associated with high morbidity and high mortality in hematopoietic stem cell transplantation (HSCT) patientsThe purpose of this study was to assess the characteristics and outcomes of HSCT patients with IFIs who are undergoing MV at a single institution in Taiwan.</p> <p>Methods</p> <p>We performed an observational retrospective analysis of IFIs in HSCT patients undergoing mechanical ventilation (MV) in an intensive care unit (ICU) from the year 2000 to 2009. The characteristics of these HSCT patients and risk factors related to IFIs were evaluated. The status of discharge, length of ICU stay, date of death and cause of death were also recorded.</p> <p>Results</p> <p>There were 326 HSCT patients at the Linkou Chang-Gung Memorial Hospital (Taipei, Taiwan) during the study period. Sixty of these patients (18%) were transferred to the ICU and placed on mechanical ventilators. A total of 20 of these 60 patients (33%) had IFIs. Multivariate analysis indicated that independent risk factors for IFI were admission to an ICU more than 40 days after HSCT, graft versus host disease (GVHD), and high dose corticosteroid (<it>p </it>< 0.01 for all). The overall ICU mortality rate was 88% (53 of 60 patients), and was not significantly different for patients with IFIs (85%) and those without IFIs (90%, <it>p </it>= 0.676).</p> <p>Conclusion</p> <p>There was a high incidence of IFIs in HSCT patients requiring MV in the ICU in our study cohort. The independent risk factors for IFI are ICU admission more than 40 days after HSCT, GVHD, and use of high-dose corticosteroid.</p

Robust T cell immunity in convalescent individuals with asymptomatic or mild COVID-19

SARS-CoV-2-specific memory T cells will likely prove critical for long-term immune protection against COVID-19. Here, we systematically mapped the functional and phenotypic landscape of SARS-CoV-2-specific T cell responses in unexposed individuals, exposed family members, and individuals with acute or convalescent COVID-19. Acute-phase SARS-CoV-2-specific T cells displayed a highly activated cytotoxic phenotype that correlated with various clinical markers of disease severity, whereas convalescent-phase SARS-CoV-2-specific T cells were polyfunctional and displayed a stem-like memory phenotype. Importantly, SARS-CoV-2-specific T cells were detectable in antibody-seronegative exposed family members and convalescent individuals with a history of asymptomatic and mild COVID-19. Our collective dataset shows that SARS-CoV-2 elicits broadly directed and functionally replete memory T cell responses, suggesting that natural exposure or infection may prevent recurrent episodes of severe COVID-19

In ACPA-positive RA patients, antibodies to EBNA35-58Cit, a citrullinated peptide from the Epstein–Barr nuclear antigen-1, strongly cross-react with the peptide β60-74Cit which bears the immunodominant epitope of citrullinated fibrin

International audienceAlthough several infectious agents and particularly Epstein-Barr virus (EBV) have been suspected to be involved in aetiology of rheumatoid arthritis (RA), their role still remains elusive. Almost 80% of RA sera contain antibodies to citrullinated proteins/peptides. Among them, the autoantibodies to citrullinated human fibrinogen (AhFibA) are composed of two non-cross-reactive subsets directed to immunodominant epitopes borne by the α36-50Cit and β60-74Cit fibrin peptides. RA sera also contain antibodies towards the citrullinated EBNA35-58Cit peptide derived from the EBNA-1 protein of EBV. Here, using a large cohort of RA patients and controls, we showed that for a diagnostic specificity of 98.5%, 47% of the AhFibA-positive patients were anti-EBNA35-58Cit-positive and that almost all (98.5%) the anti-EBNA35-58Cit-positive were AhFibA-positive, whereas 86% were anti-β60-74Cit-positive and only 43% anti-α36-50Cit-positive. AhFibA, anti-EBNA35-58Cit- and anti-β60-74Cit-antibody titres were significantly correlated. Competition assays showed that anti-EBNA35-58Cit antibodies are highly cross-reactive with the β60-74Cit peptide. The demonstration that a citrullinated peptide derived from the EBNA-1 protein of EBV presents a molecular mimicry with human citrullinated fibrin constitutes an additional argument for a possible role of EBV in RA aetiopathogeny

29-Color Flow Cytometry : Unraveling Human Liver NK Cell Repertoire Diversity

Recent studies have demonstrated extraordinary diversity in peripheral blood human natural killer (NK) cells and have suggested environmental control of receptor expression patterns on distinct subsets of NK cells. However, tissue localization may influence NK cell differentiation to an even higher extent and less is known about the receptor repertoire of human tissue-resident NK cells. Advances in single-cell technologies have allowed higher resolution studies of these cells. Here, the power of high-dimensional flow cytometry was harnessed to unravel the complexity of NK cell repertoire diversity in liver since recent studies had indicated high heterogeneity within liver NK cells. A 29-color flow cytometry panel allowing simultaneous measurement of surface tissue-residency markers, activating and inhibitory receptors, differentiation markers, chemokine receptors, and transcription factors was established. This panel was applied to lymphocytes across three tissues (liver, peripheral blood, and tonsil) with different distribution of distinct NK cell subsets. Dimensionality reduction of this data ordered events according to their lineage, rather than tissue of origin. Notably, narrowing the scope of the analysis to the NK cell lineage in liver and peripheral blood separated subsets according to tissue, enabling phenotypic characterization of NK cell subpopulations in individual tissues. Such dimensionality reduction, coupled with a clustering algorithm, identified CD49e as the preferred marker for future studies of liver-resident NK cell subsets. We present a robust approach for diversity profiling of tissue-resident NK cells that can be applied in various homeostatic and pathological conditions such as reproduction, infection, and cancer