Methods for high-dimensonal analysis of cells dissociated from cyropreserved synovial tissue

Background: Detailed molecular analyses of cells from rheumatoid arthritis (RA) synovium hold promise in identifying cellular phenotypes that drive tissue pathology and joint damage. The Accelerating Medicines Partnership RA/SLE Network aims to deconstruct autoimmune pathology by examining cells within target tissues through multiple high-dimensional assays. Robust standardized protocols need to be developed before cellular phenotypes at a single cell level can be effectively compared across patient samples. Methods: Multiple clinical sites collected cryopreserved synovial tissue fragments from arthroplasty and synovial biopsy in a 10% DMSO solution. Mechanical and enzymatic dissociation parameters were optimized for viable cell extraction and surface protein preservation for cell sorting and mass cytometry, as well as for reproducibility in RNA sequencing (RNA-seq). Cryopreserved synovial samples were collectively analyzed at a central processing site by a custom-designed and validated 35-marker mass cytometry panel. In parallel, each sample was flow sorted into fibroblast, T-cell, B-cell, and macrophage suspensions for bulk population RNA-seq and plate-based single-cell CEL-Seq2 RNA-seq. Results: Upon dissociation, cryopreserved synovial tissue fragments yielded a high frequency of viable cells, comparable to samples undergoing immediate processing. Optimization of synovial tissue dissociation across six clinical collection sites with ~ 30 arthroplasty and ~ 20 biopsy samples yielded a consensus digestion protocol using 100 μg/ml of Liberase™ TL enzyme preparation. This protocol yielded immune and stromal cell lineages with preserved surface markers and minimized variability across replicate RNA-seq transcriptomes. Mass cytometry analysis of cells from cryopreserved synovium distinguished diverse fibroblast phenotypes, distinct populations of memory B cells and antibody-secreting cells, and multiple CD4+ and CD8+ T-cell activation states. Bulk RNA-seq of sorted cell populations demonstrated robust separation of synovial lymphocytes, fibroblasts, and macrophages. Single-cell RNA-seq produced transcriptomes of over 1000 genes/cell, including transcripts encoding characteristic lineage markers identified. Conclusions: We have established a robust protocol to acquire viable cells from cryopreserved synovial tissue with intact transcriptomes and cell surface phenotypes. A centralized pipeline to generate multiple high-dimensional analyses of synovial tissue samples collected across a collaborative network was developed. Integrated analysis of such datasets from large patient cohorts may help define molecular heterogeneity within RA pathology and identify new therapeutic targets and biomarkers

Effets du belimumab sur la fonction rénale, le contrôle global et les biomarqueurs du lupus systémique

Le Belimumab (BEL) a montré son efficacité dans les manifestations extra-rénales du lupus systémique (LS) dans 4 études pivotales. Dans cette étude ont été évaluées l’efficacité et la tolérance du BEL (IV)+associé au traitement standard (TS) au cours de la glomérulonéphrite lupique active (GNL). Étude de phase 3, en double aveugle (NCT01639339), randomisée (1 :1) incluant des adultes avec un LS et une GNL confirmée par biopsie (classe III, IV et/ou V) qui ont reçu chaque mois du BEL 10mg/kg IV ou placebo (PBO), en sus du TS. La randomisation a été stratifiée sur l’origine ethnique et sur le traitement (corticoïdes+soit cyclophosphamide IV en induction suivi par azathioprine en maintien, ou mycophénolate mofétil (MMF) en induction et maintien). Critère principal : Réponse rénale primaire (RRP); à la semaine (S) 104 (basée sur ratio protéinurie/créatininurie (RPCU) ≤0,7g/g ; baisse du débit de filtration estimé (DFG) <20 % de la valeur avant poussée ou ≥60ml/min/1,73m2 ; pas de traitement de secours). Critères secondaires principaux : réponse rénale complète (RRC) à S104 (RPCU <0,5 ; baisse du DFG <10 % de la valeur avant poussée ou ≥90ml/min/1,73m2 ; pas de traitement de secours) ; RRP à S52 ; délai jusqu’aux événements rénaux (premier événement survenu : insuffisance rénale terminale, doublement de la créatininémie, aggravation rénale, échec du traitement de la maladie rénale ou décès). Analyses supplémentaires : poussée sévère (SFI) ; proportion de pts avec un SLEDAI-S2K <4 ; proportion de pts avec dose prednisone ≤7,5 ou 5mg/jour ; changement vs baseline des biomarqueurs (anti-ADNdb, anti-C1q, C3, C4) ; tolérance. Résultats 448 pts randomisés (efficacité : 223/groupe ; tolérance : 224/groupe). À S104, RRP : 96 (43,0 %) pts BEL vs 72 (32,3 %) pts PBO (OR [IC95 %] 1,6 [1,0 ; 2,3] ; p=0,031) ; RRC : 67 (30,0 %) pts BEL vs 44 (19,7 %) pts PBO (OR [IC95 %] 1,7 [1,1 ; 2,7] ; p=0,017). À S52, plus de pts BEL vs PBO ont atteint une RRP (104 [46,6 %] vs 79 [35,4 %] ; OR [IC95 %] 1,6 [1,1 ; 2,4] ; p=0,025). Le risque d’événements rénaux ou décès était plus faible dans le bras BEL (35 (15,7 %)) comparé au bras PBO (63 évènements (28,3 %) ; HR [IC95 %] 0,5 [0,3 ; 0,8] ; p=0,001). Des poussées sévères selon le SFI ont été rapportées chez 42 (18,8 %) pts BEL vs 70 (31,4 %) pts PBO (HR [IC95 %] 0,6 [0,4 ; 0,8] ; p=0,004). À S104, plus de pts BEL vs PBO ont atteint un score SLEDAI-S2K <4 (62 [27,8 %] vs 41 [18,4 %] ; OR [IC95 %] 1,8 [1,1 ; 2,8] ; p=0,016). À S104, 36,8 % (n=82) des pts BEL vs 27,8 % (n=62) des pts PBO recevaient des doses moyennes en équivalent prednisone ≤5mg/j (OR [IC95 %] 1,5 [1,0 ; 2,3] ; p=0,044) ; 40,8 % (n=91) des pts BEL et 29,6 % (n=66) des pts PBO recevaient des doses ≤7,5mg/j (OR [IC95 %] 1,7 [1,1 ; 2,5) ; (p=0,014). Chez les patients avec des autoanticorps à baseline, la diminution médiane exprimée en pourcentage (IQR) (BEL vs PBO) était de : −74,2 % (−85,1 ; −49,5) vs −36,6 (−69,7 ; 28,6) pour les anti-ADNdb et de −73,2 (−84,1 ; −59,0) vs −57,9 (−76,1 ; −33,2) pour les anti-C1q à S104. Chez les patients avec une consommation du complément à baseline, l’augmentation médiane exprimée en pourcentage (IQR) (BEL vs PBO) était de+43,8 % (17,1 ; 88,9) vs 30,0 (13,5 ; 59,8) pour le C3 et de+115,5 % (60,0 ; 177,8) vs 66,7 (22,2 ; 166,7) pour le C4. Tolérance : des événements indésirables (EI≥1) ont été rapportés par 95,5 % des pts BEL et 94,2 % des pts PBO ; 12,9 % des pts de chaque groupe avaient au moins un EI entraînant l’arrêt du traitement ; 25,9 % des pts BEL et 29,9 % des pts PBO avaient ≥1 EI grave, le plus fréquent étant les infections (13,8 % des pts BEL vs 17,0 % des pts PBO) ; 1,8 % des pts BEL et 1,3 % des pts PBO ont eu un EI fatal durant le traitement (principalement lié à une infection). Dans cette étude à 2 ans, BEL+TS a amélioré la fonction rénale, l’activité globale du LS, les taux de biomarqueurs, tout en réduisant l’utilisation des corticoïdes, avec un profil de sécurité favorable vs TS seul [1]

Use of consensus methodology to determine candidate items for systemic lupus erythematosus classification criteria

Objective. Given the complexity and heterogeneity of systemic lupus erythematosus (SLE), high-performing classification criteria are critical to advancing research and clinical care. A collaborative effort by the European League Against Rheumatism and the American College of Rheumatology was undertaken to generate candidate criteria, and then to reduce them to a smaller set. The objective of the current study was to select a set of criteria that maximizes the likelihood of accurate classification of SLE, particularly early disease. Methods. An independent panel of international SLE experts and the SLE classification criteria steering committee (conducting SLE research in Canada, Mexico, United States, Austria, Germany, Greece, France, Italy, and Spain) ranked 43 candidate criteria. A consensus meeting using nominal group technique (NGT) was conducted to reduce the list of criteria for consideration. Results. The expert panel NGT exercise reduced the candidate criteria for SLE classification from 43 to 21. The panel distinguished potential &quot;entry criteria,&quot; which would be required for classification, from potential &quot;additive criteria.&quot; Potential entry criteria were antinuclear antibody (ANA) = 1:80 (HEp-2 immunofluorescence), and low C3 and/or low C4. The use of low complement as an entry criterion was considered potentially useful in cases with negative ANA. Potential additive criteria included lupus nephritis by renal biopsy, autoantibodies, cytopenias, acute and chronic cutaneous lupus, alopecia, arthritis, serositis, oral mucosal lesions, central nervous system manifestations, and fever. Conclusion. The NGT exercise resulted in 21 candidate SLE classification criteria. The next phases of SLE classification criteria development will require refinement of criteria definitions, evaluation of the ability to cluster criteria into domains, and evaluation of weighting of criteria. Copyright © 2019. All rights reserved

Phenotypic associations of genetic susceptibility loci in systemic lupus erythematosus

Objective: Systemic lupus erythematosus is a clinically heterogeneous autoimmune disease. A number of genetic loci that increase lupus susceptibility have been established. This study examines if these genetic loci also contribute to the clinical heterogeneity in lupus. Materials and methods: 4001 European-derived, 547 Hispanic, 1590 African-American and 1191 Asian lupus patients were genotyped for 16 confirmed lupus susceptibility loci. Ancestry informative markers were genotyped to calculate and adjust for admixture. The association between the risk allele in each locus was determined and compared in patients with and without the various clinical manifestations included in the ACR criteria. Results: Renal disorder was significantly correlated with the lupus risk allele in ITGAM (p=5.0 × 10-6, OR 1.25, 95% CI 1.12 to 1.35) and in TNFSF4 (p=0.0013, OR 1.14, 95% CI 1.07 to 1.25). Other significant findings include the association between risk alleles in FCGR2A and malar rash (p=0.0031, OR 1.11, 95% CI 1.17 to 1.33), ITGAM and discoid rash (p=0.0020, OR 1.20, 95% CI 1.06 to 1.33), STAT4 and protection from oral ulcers (p=0.0027, OR 0.89, 95% CI 0.83 to 0.96) and IL21 and haematological disorder (p=0.0027, OR 1.13, 95% CI 1.04 to 1.22). All these associations are significant with a false discovery rate of and lt;0.05 and pass the significance threshold using Bonferroni correction for multiple testing. Conclusion: Significant associations were found between lupus clinical manifestations and the FCGR2A, ITGAM, STAT4, TNSF4 and IL21 genes. The findings suggest that genetic profiling might be a useful tool to predict disease manifestations in lupus patients in the future

Phenotypic associations of genetic susceptibility loci in systemic lupus erythematosus

Objective: Systemic lupus erythematosus is a clinically heterogeneous autoimmune disease. A number of genetic loci that increase lupus susceptibility have been established. This study examines if these genetic loci also contribute to the clinical heterogeneity in lupus. Materials and methods: 4001 European-derived, 547 Hispanic, 1590 African-American and 1191 Asian lupus patients were genotyped for 16 confirmed lupus susceptibility loci. Ancestry informative markers were genotyped to calculate and adjust for admixture. The association between the risk allele in each locus was determined and compared in patients with and without the various clinical manifestations included in the ACR criteria. Results: Renal disorder was significantly correlated with the lupus risk allele in ITGAM (p=5.0 × 10-6, OR 1.25, 95% CI 1.12 to 1.35) and in TNFSF4 (p=0.0013, OR 1.14, 95% CI 1.07 to 1.25). Other significant findings include the association between risk alleles in FCGR2A and malar rash (p=0.0031, OR 1.11, 95% CI 1.17 to 1.33), ITGAM and discoid rash (p=0.0020, OR 1.20, 95% CI 1.06 to 1.33), STAT4 and protection from oral ulcers (p=0.0027, OR 0.89, 95% CI 0.83 to 0.96) and IL21 and haematological disorder (p=0.0027, OR 1.13, 95% CI 1.04 to 1.22). All these associations are significant with a false discovery rate of and lt;0.05 and pass the significance threshold using Bonferroni correction for multiple testing. Conclusion: Significant associations were found between lupus clinical manifestations and the FCGR2A, ITGAM, STAT4, TNSF4 and IL21 genes. The findings suggest that genetic profiling might be a useful tool to predict disease manifestations in lupus patients in the future