Biology subjects

Lederer, J. A.

Publications and source records attributed to Lederer, J. A..

5 recordsLinked to original sources

bioRxiv2018

The immune cell landscape in kidneys of lupus nephritis patients

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.

immunology↗

bioRxiv2018

Defining Inflammatory Cell States in Rheumatoid Arthritis Joint Synovial Tissues by Integrating Single-cell Transcriptomics and Mass Cytometry

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 IL1B to pro-inflammatory monocytes. These populations are potentially key mediators of RA pathogenesis.

immunology↗

bioRxiv2018

High dimensional analyses of cells dissociated from cryopreserved synovial tissue

BackgroundDetailed 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.\n\nMethodsMultiple 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.\n\nResultsUpon 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{micro}g/mL of Liberase TL enzyme. 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: 1) diverse fibroblast phenotypes, 2) distinct populations of memory B cells and antibody-secreting cells, and 3) multiple CD4+ and CD8+ T cell activation states. Bulk RNA sequencing 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.\n\nConclusionWe 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.

immunology↗

bioRxiv2018

A protocol for single-cell transcriptomics from cryopreserved renal tissue and urine for the Accelerating Medicine Partnership (AMP) RA/SLE network

OBJECTIVEThere is a critical need to define the cells that mediate tissue damage in lupus nephritis. Here we aimed to establish a protocol to preserve lupus nephritis kidney biopsies and urine cell samples obtained at multiple clinical sites for subsequent isolation and transcriptomic analysis of single cells.\n\nMETHODSFresh and cryopreserved kidney tissue from tumor nephrectomies and lupus nephritis kidney biopsies were disaggregated by enzymatic digestion. Cell yields and cell composition were assessed by flow cytometry. Transcriptomes of leukocytes and epithelial cells were evaluated by low-input and single cell RNA-seq.\n\nRESULTSCryopreserved kidney tissue from tumor nephrectomies and lupus nephritis biopsies can be thawed and dissociated to yield intact, viable leukocytes and epithelial cells. Cryopreservation of intact kidney tissue provides higher epithelial cell yields compared to cryopreservation of single cell suspensions from dissociated kidneys. Cell yields and flow cytometric cell phenotypes are comparable between cryopreserved kidney samples and paired kidney samples shipped overnight on wet ice. High-quality single cell and low-input transcriptomic data were generated from leukocytes from both cryopreserved lupus nephritis kidney biopsies and urine, as well as from a subset of kidney epithelial cells.\n\nCONCLUSIONThe AMP RA/SLE cryopreserved tissue analysis pipeline provides a method for centralized processing of lupus nephritis kidney biopsies and urine samples to generate robust transcriptomic analyses in multi-center studies.

immunology↗

bioRxiv2017

Single Cell Transcriptomics And Flow Cytometry Reveal Disease-Associated Fibroblast Subsets In Rheumatoid Arthritis

Fibroblasts mediate normal tissue matrix remodeling, but they can cause fibrosis or tissue destruction following chronic inflammation. In rheumatoid arthritis (RA), synovial fibroblasts expand, degrade cartilage, and drive joint inflammation. Little is known about fibroblast heterogeneity or if aberrations in fibroblast subsets relate to disease pathology. Here, we used an integrative strategy, including bulk transcriptomics on targeted subpopulations and unbiased single-cell transcriptomics, to analyze fibroblasts from synovial tissues. We identify 7 phenotypic fibroblast subsets with distinct surface protein phenotypes, and these collapsed into 3 subsets based on transcriptomics data. One subset expressing PDPN, THY1, but lacking CD34 was 3-fold expanded in RA relative to osteoarthritis (P=0.007); most of these cells expressed CDH11. The subsets were found to differ in expression of cytokines and matrix metalloproteinases, localization in synovial microanatomy, and in response to TNF. Our approach provides a template to identify pathogenic stromal cellular subsets in complex diseases.

immunology↗