Showing papers by "Chad Nusbaum published in 2018"
Hospital for Special Surgery1, Cornell University2, Brigham and Women's Hospital3, Harvard University4, University of Pittsburgh5, University of Birmingham6, University of Rochester Medical Center7, University of California, San Diego8, Barts Health NHS Trust9, Stanford University10, University of Massachusetts Medical School11, Queen Mary University of London12, The Feinstein Institute for Medical Research13, University of Colorado Boulder14
TL;DR: A robust protocol to acquire viable cells from cryopreserved synovial tissue with intact transcriptomes and cell surface phenotypes is established and integrated analysis of large patient cohorts may help define molecular heterogeneity within RA pathology and identify new therapeutic targets and biomarkers.
Abstract: 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. 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. 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. 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.
85 citations
Brigham and Women's Hospital1, Barts Health NHS Trust2, Hospital for Special Surgery3, University of Rochester Medical Center4, University of Alabama at Birmingham5, University of Massachusetts Medical School6, Harvard University7, University of California, San Diego8, Northwestern University9, Cornell University10, The Feinstein Institute for Medical Research11, University of Pittsburgh12, Queen Mary University of London13, University of Birmingham14, University of Colorado Denver15
TL;DR: Using bulk and single-cell data, inflammatory mediators to source cell populations are mapped, for example attributing IL6 production to THY1+HLAhigh fibroblasts and naïve B cells, and IL1B to pro-inflammatory monocytes, which are potentially key mediators of RA pathogenesis.
Abstract: To define the cell populations in rheumatoid arthritis (RA) driving joint inflammation, we applied single-cell RNA-seq (scRNA-seq), mass cytometry, bulk RNA-seq, and flow cytometry to sorted T cells, B cells, monocytes, and fibroblasts from 51 synovial tissue RA and osteoarthritis (OA) patient samples. Utilizing an integrated computational strategy based on canonical correlation analysis to 5,452 scRNA-seq profiles, we identified 18 unique cell populations. Combining mass cytometry and transcriptomics together revealed cell states expanded in RA synovia: THY1+HLAhigh sublining fibroblasts (OR=33.8), IL1B+ pro-inflammatory monocytes (OR=7.8), CD11c+T-bet+ autoimmune-associated B cells (OR=5.7), and PD-1+ Tph/Tfh (OR=3.0). We also defined CD8+ T cell subsets characterized by GZMK+, GZMB+, and GNLY+ expression. Using bulk and single-cell data, we mapped inflammatory mediators to source cell populations, for example attributing IL6 production to THY1+HLAhigh fibroblasts and naive B cells, and ILB to pro-inflammatory monocytes. These populations are potentially key mediators of RA pathogenesis.
48 citations
Broad Institute1, Brigham and Women's Hospital2, University of Michigan3, The Feinstein Institute for Medical Research4, University of California, San Francisco5, University of California, Berkeley6, Medical University of South Carolina7, University of North Carolina at Chapel Hill8, New York University9, Johns Hopkins University10, Albert Einstein College of Medicine11, University of California, San Diego12, University of Cincinnati Academic Health Center13, University of Cincinnati14, Cincinnati Children's Hospital Medical Center15, University of Rochester Medical Center16
TL;DR: Analysis of kidney samples from lupus nephritis patients and healthy controls revealed 21 subsets of leukocytes active in disease, including multiple populations of myeloid, T, NK and B cells, demonstrating both pro-inflammatory and resolving responses.
Abstract: Lupus nephritis is a potentially fatal autoimmune disease, whose current treatment is ineffective and often toxic. To gain insights into disease mechanisms, we analyzed kidney samples from lupus nephritis patients and healthy controls using single-cell RNA-seq. Our analysis revealed 21 subsets of leukocytes active in disease, including multiple populations of myeloid, T, NK and B cells, demonstrating both pro-inflammatory and resolving responses. We found evidence of local activation of B cells correlated with an age-associated B cell signature, and of progressive stages of monocyte differentiation within the kidney. A clear interferon response was observed in most cells. Two chemokine receptors, CXCR4 and CX3CR1, were broadly expressed, pointing to potential therapeutic targets. Gene expression of immune cells in urine and kidney was highly correlated, suggesting urine may be a surrogate for kidney biopsies. Our results provide a first comprehensive view of the complex network of leukocytes active in lupus nephritis kidneys.
11 citations
Cornell University1, Brigham and Women's Hospital2, University of Pittsburgh3, University of Birmingham4, University of Rochester Medical Center5, Harvard University6, University of California, San Diego7, Hospital for Special Surgery8, Barts Health NHS Trust9, Stanford University10, University of Massachusetts Amherst11, Queen Mary University of London12, The Feinstein Institute for Medical Research13, University of Colorado Boulder14
TL;DR: A robust protocol to acquire viable cells from cryopreserved synovial tissue with intact transcriptomes and cell surface phenotypes is established and integrated analysis of such datasets from large patient cohorts may help define molecular heterogeneity within RA pathology and identify new therapeutic targets and biomarkers.
Abstract: 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 (AMP) 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.
5 citations