ACR Meeting Abstracts

ACR Meeting Abstracts

Abstract Number: 1406

Methods for Generating Multiple High-Dimensional Analyses of Cryopreserved Synovial Tissue Developed By the Accelerating Medicines Partnership RA/SLE Network

Deepak Rao1, Laura T. Donlin2, Kevin Wei3, Nida Meednu4, Jason Turner5, Mandy J. McGeachy6, Fumitaka Mizoguchi7, Joshua Keegan8, James Lederer9, Maria Gutierrez-Arcelus10, Kamil Slowikowski11, Kaylin Muskat12, Joshua Hillman12, Cristina Rozo13, Edd Ricker14, Thomas Eisenhaure15, David Lieb15, Shuqiang Li15, Edward Browne15, Chad Nusbaum15, William H. Robinson16, Stephen Kelly17, Alessandra B. Pernis18, Lionel Ivashkiv19, Susan M. Goodman20, Ellen M. Gravallese21, Michael Holers22, Nir Hacohen23, Costantino Pitzalis17, Peter Gregersen24, Vivian P. Bykerk25, Larry W. Moreland26, Gary Firestein27, Soumya Raychaudhuri28, Andrew Filer29, David L. Boyle30, Michael Brenner10 and Jennifer H. Anolik4, 1Division of Rheumatology, Immunology, and Allergy, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, 2Arthritis and Tissue Degeneration Program and the David Z. Rosensweig Genomics Research Center, Hospital for Special Surgery, New York, NY, 3Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, 4Medicine- Allergy, Immunology and Rheumatology, University of Rochester Medical Center, Rochester, NY, 5Institute of Inflammation and Ageing, University of Birmingham, Birmingham, United Kingdom, 6Medicine, University of Pittsburgh, Pittsburgh, PA, 7Department of Rheumatology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), Tokyo, Japan, 8Brigham and Women's Hospital, Boston, MA, 9Department of Surgery, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, 10Brigham and Women's Hospital and Harvard Medical School, Boston, MA, 11Division of Medicine and Rheumatology, Brigham and Women's Hospital, Harvard Medical Schoo, Boston, MA, 12University of California, San Diego, San Diego, CA, 13Hospital for Special Surgery, New York, NY, 14Weill Cornell Graduate School of Medical Sciences, New York, NY, 15Broad Institute, Cambridge, MA, 16Stanford University School of Medicine, Stanford, CA, 17Centre for Experimental Medicine and Rheumatology, William Harvey Research Institute, Barts and The London, School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom, 18David Z. Rosensweig Genomics Research Center, Hospital for Special Surgery, New York, NY, 19Medicine, Hospital for Special Surgery, New York, NY, 20Medicine, Hospital for Special Surgery/Weill Cornell Medicine, New York, NY, 21Lazare Research Bldg, University of Massachusetts Medical School, Worcester, MA, 22Medicine, Division of Rheumatology, University of Colorado Denver, Aurora, CO, 23Harvard Medical School, Boston, MA, 24The Feinstein Institute for Medical Research, Northwell Health, Manhasset, NY, 252-005, Mt Sinai Hospital, Toronto, ON, Canada, 26Rheumatology & Clinical Immunology, University of Pittsburgh, Pittsburgh, PA, 27EGG, St Cloud, France, 28Division of Medicine and Rheumatology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, 29Institute of Inflammation and Ageing (IIA), University of Birmingham, Birmingham, United Kingdom, 30University of California San Diego, La Jolla, CA

Meeting: 2017 ACR/ARHP Annual Meeting

Date of first publication: September 18, 2017

Keywords: , , ,

Session Information

Date: Monday, November 6, 2017

Title: Rheumatoid Arthritis – Human Etiology and Pathogenesis Poster II

Session Type: ACR Poster Session B

Session Time: 9:00AM-11:00AM

Background/Purpose: Detailed analyses of cells from rheumatoid arthritis (RA) synovium may identify cell phenotypes and functions that drive tissue pathology and joint damage. The AMP RA/SLE network aims to deconstruct RA pathology using multiple high-dimensional analyses of cells obtained from rheumatoid synovium. These studies hold the potential to identify novel therapeutic strategies to counteract pathologic inflammatory responses in RA.

Methods: We developed a method to cryopreserve intact synovial tissue for downstream analyses of viable cells. Synovial tissue fragments were cryopreserved in a 10% DMSO-containing solution, then thawed and disaggregated. Synovial cells were analyzed using a 35-marker mass cytometry panel. In parallel, synovial cell populations, including fibroblasts, T cells, B cells, and macrophages, were flow-cytometrically sorted for low-input and plate-based single cell RNAseq transcriptomics.

Results: Intact cryopreserved synovial tissue yielded high numbers of viable cells after a thawing protocol followed by tissue dissociation. Cells isolated from cryopreserved tissue demonstrated intact expression of lineage markers by flow cytometry, comparable to freshly processed tissues. Optimization of synovial tissue dissociation performed across 6 sites, utilizing >30 arthroplasty and >20 synovial biopsy samples, yielded a consensus dissociation protocol of tissue digestion with 100ug/mL of liberase TL enzyme, which provided high cell yields, preserved surface markers, and minimized variability in RNAseq transcriptomes across replicates.

Arthroplasty samples were mechanically dissociated after enzymatic digestion by gentleMACS. To reduce cell losses when using smaller synovial biopsies, biopsy samples were mechanically dissociated by rapid, continuous magnetic stirring during enzymatic digestion and passage through an 18-gauge syringe. Cryopreserved tissue dissociated by these methods could be analyzed by multiple high-dimensional analyses. Mass cytometry revealed 1) diverse fibroblast phenotypes, 2) clear separation of memory B cells from antibody-secreting cells, and 3) multiple phenotypes of activated CD4+ and CD8+ T cells. To complement mass cytometry analysis, a flow cytometric sorting strategy was developed to collect fibroblasts, macrophages, T cells, B cells for bulk and single cell RNAseq transcriptomics. RNAseq of total synovial cells revealed a transcriptomic effect of tissue dissociation, compared to intact synovial tissue. Nonetheless, RNAseq of sorted cell populations demonstrated robust separation of synovial lymphocytes, fibroblasts, and macrophages. Single cell RNAseq by CelSeq2 yielded transcriptomes of over 1000 genes/cell and demonstrated characteristic lineage markers in the expected cell populations.

Conclusion: We propose that this method of acquisition of viable cells from cryopreserved tissue can serve as a model for centralized generation of multiple, robust high-dimensional analyses of synovial samples acquired in multi-site studies. Integrated analysis of these datasets in a large patient cohort may help define the molecular heterogeneity of RA pathology and identify new therapeutic targets and biomarkers.

Disclosure: D. Rao, None; L. T. Donlin, None; K. Wei, None; N. Meednu, None; J. Turner, None; M. J. McGeachy, None; F. Mizoguchi, None; J. Keegan, None; J. Lederer, None; M. Gutierrez-Arcelus, None; K. Slowikowski, None; K. Muskat, None; J. Hillman, None; C. Rozo, None; E. Ricker, None; T. Eisenhaure, None; D. Lieb, None; S. Li, None; E. Browne, None; C. Nusbaum, None; W. H. Robinson, None; S. Kelly, None; A. B. Pernis, Kadmon coporation, 2; L. Ivashkiv, None; S. M. Goodman, None; E. M. Gravallese, Abbott Immunology Pharmaceuticals, 2,Lilly, Inc, 2,New England Journal of Medicine, 3,Up to Date, 7,Lilly Inc., 5,Sanofi/Genzyme, 5; M. Holers, None; N. Hacohen, None; C. Pitzalis, None; P. Gregersen, None; V. P. Bykerk, None; L. W. Moreland, None; G. Firestein, None; S. Raychaudhuri, Pfizer Inc, 2,Roche Pharmaceuticals, 2; A. Filer, None; D. L. Boyle, None; M. Brenner, None; J. H. Anolik, None.

To cite this abstract in AMA style:

Rao D, Donlin LT, Wei K, Meednu N, Turner J, McGeachy MJ, Mizoguchi F, Keegan J, Lederer J, Gutierrez-Arcelus M, Slowikowski K, Muskat K, Hillman J, Rozo C, Ricker E, Eisenhaure T, Lieb D, Li S, Browne E, Nusbaum C, Robinson WH, Kelly S, Pernis AB, Ivashkiv L, Goodman SM, Gravallese EM, Holers M, Hacohen N, Pitzalis C, Gregersen P, Bykerk VP, Moreland LW, Firestein G, Raychaudhuri S, Filer A, Boyle DL, Brenner M, Anolik JH. Methods for Generating Multiple High-Dimensional Analyses of Cryopreserved Synovial Tissue Developed By the Accelerating Medicines Partnership RA/SLE Network [abstract]. Arthritis Rheumatol. 2017; 69 (suppl 10). https://acrabstracts.org/abstract/methods-for-generating-multiple-high-dimensional-analyses-of-cryopreserved-synovial-tissue-developed-by-the-accelerating-medicines-partnership-rasle-network/. Accessed .

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