CyTOF analyses in rheumatoid arthritis. Deepak Rao, MD PhD Rheumatology, Immunology, Allergy, BWH
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1 CyTOF analyses in rheumatoid arthritis Deepak Rao, MD PhD Rheumatology, Immunology, Allergy, BWH
2 Agenda: CyTOF analyses Analysis of synovial tissue Panel for analysis of synovial cells Comparison of CyTOF and flow data Traditional gating and visne visualization Analysis of synovial T cell function Cells stimulated in vitro then analyzed for activation markers and cytokines Broad phenotyping of circulating T cells (PBMC) RA, GCA, PMR
3 Synovial workflow Tissue disaggregation Arthroplasty Synovial Biopsy gentlemacs Single cell suspension Kevin Wei, AMP Trituration Single cell suspension
4 Synovial workflow Step 1. Determine analytical methods Single cell suspension Assess cell yield and cell viability 1. < 100,000 cells 2. <500,000 cells 3. >500,000 cells RNAseq only RNAseq + Cytof (50%) (50%) RNAseq + Cytof (limit CyTOF to 200,000 cells) Kevin Wei, AMP
5 Synovial workflow Cell sorting Step 2. Collect 1000 cells for low input RNAseq Step 3. collect remaining cells into FACS buffer Step 4. Sort single cells into 384 well plate T B M F Single cell suspension Single cell suspension Lysis buffer FACS buffer T B M F Once 1000 cells collected, Exchange collection tube (step 3) and continue collection for each subset Kevin Wei, AMP T B M F Collect cells until sample is depleted Collect up to 144 cells for each subtype
6 t-sne1 AMP Synovial CyTOF: RA synovial biopsy t-sne2 visne analysis CyTOF Panel CD45 VE-Cadherin CD66b VCAM-1 CD14 Podoplanin CD64 Cadherin-11 CD16 FAP CD11c CD146 CCR2 CD90 CD3 CD34 CD4 CD19 CD8 CD20 CD69 CD38 CD45RO CD138 CXCR5 IgD PD-1 IgM ICOS IgA HLA-DR FcRL4 TCRgd RANKL FoxP3 Live/dead
7 Parallel analysis of the same synovial sample by flow and mass cytometry RA Synovial Tissue: Flow cytometry analysis RA Synovial Tissue: CyTOF analysis
8 Podoplanin CD14 CD8 Synovial CyTOF: RA Synovial Biopsy Fibroblasts Monocytes/Macs CD8+ T cells Leukocytes T cells CD4+ T cells CD45 CD3 CD4 Biaxial gating of dot plots AMP
9 CD90 Podoplanin CD14 CD8 Synovial CyTOF: RA Synovial Biopsy Fibroblasts Monocytes/Macs CD8+ T cells Leukocytes T cells CD4+ T cells CD45 CD3 CD4 CD34
10 CD90 CCR2 Podoplanin CD14 CD8 Synovial CyTOF: RA Synovial Biopsy Fibroblasts Monocytes/Macs CD8+ T cells Leukocytes T cells CD4+ T cells CD45 CD3 CD4 CD34 CD11c
11 CD90 CCR2 CD19 Podoplanin CD14 CD8 Synovial CyTOF: RA Synovial Biopsy Fibroblasts Monocytes/Macs CD8+ T cells Leukocytes T cells CD4+ T cells CD45 CD3 CD4 CD34 CD11c CD38
12 CD90 CCR2 CD19 PD-1 Podoplanin CD14 CD8 Synovial CyTOF: RA Synovial Biopsy Fibroblasts Monocytes/Macs CD8+ T cells Leukocytes T cells CD4+ T cells CD45 CD3 CD4 CD34 CD11c CD38 ICOS
13 t-sne1 AMP Synovial CyTOF: RA synovial biopsy t-sne2 visne analysis CyTOF Panel CD45 VE-Cadherin CD66b VCAM-1 CD14 Podoplanin CD64 Cadherin-11 CD16 FAP CD11c CD146 CCR2 CD90 CD3 CD34 CD4 CD19 CD8 CD20 CD69 CD38 CD45RO CD138 CXCR5 IgD PD-1 IgM ICOS IgA HLA-DR FcRL4 TCRgd RANKL FoxP3 Live/dead
14 CyTOF: RA synovium CD4+ T cells in a Seropositive RA synovial tissue
15 CyTOF: RA synovium CD4+ T cells in a Seropositive RA synovial tissue
16 Pathologically expanded peripheral B cell-helper T cells in RA Cytokine production after in vitro stimulation RNAseq transcriptomics In vitro functional assays: B cell help Immunofluorescence microscopy PD-1 CD20 CD3
17 Analysis of synovial T cells: 3-D culture In vitro stimulation CyTOF Lauren Henderson
18 Analysis of synovial T cells: 3-D culture In vitro stimulation Lauren Henderson
19 Immunophenotyping of PBMCs by CyTOF Identify immune cell populations with altered frequencies in specific autoimmune diseases Analytical methods in the Raychaudhuri lab Chamith Fonseka Determine expression of many different proteins on specific immune cell populations of interest Data generated so far: RA vs controls (n=7 each) RA vs controls w/ and w/o stimulation (Raychaudhuri lab, n=~30 each) RA, OA, SLE, controls (AMP, Lederer lab, in progress) GCA, PMR, seronegative RA, controls (n=4 each)
20 CyTOF: Memory CD4+ T cells from blood
21 CyTOF: Memory CD4+ T cells from blood
22 CyTOF for analysis of rheumatic disease samples Powerful, accessible discovery tool to measure 35+ markers on cell samples Staining protocols have undergone substantial maturation and now regularly utilized (Raychaudhuri, Lederer, Brenner labs) Panels developed for broad analysis of synovial cells Panels developed to phenotype T cells, B cells, myeloid cells in detail Simple data analysis strategies that we can teach you (e.g. visne) New, powerful analysis methods under development in the Raychaudhuri lab
Methods for high-dimensional analysis of cells dissociated from cryopreserved synovial tissue
Donlin et al. Arthritis Research & Therapy (218) 2:139 https://doi.org/1.1186/s1375-18-1631-y RESEARCH ARTICLE Open Access Methods for high-dimensional analysis of cells dissociated from cryopreserved
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