The inflamed joint is a complex place. Many different types of cells must interact with each other to initiate and sustain inflammation and tissue injury. These include both those resident in the joint before inflammation started and cells that were recruited or developed later. To get a handle on this complexity, investigators need methods that can provide highly detailed portraits of individual cells. A major goal of the Joint Biology Consortium’s Cellular Systems Core, directed by Dr. Jim Lederer, is to accelerate research by JBC members through two such methods: cytometry by time of flight (CyTOF) mass cytometry and RNA sequencing (RNA-seq).
- CyTOF is conceptually similar to flow cytometry. A pool of cells is tagged with labeled antibodies that are specific for either surface or intracellular features, and then cells are evaluated one by one to quantitate how much of each label is present. What makes CyTOF different from flow cytometry is the way antibodies are detected. Flow cytometry uses fluorescence, a powerful method but one that is constrained by two limitations: 1) The spectra of available fluorescent labels often overlap. 2) Cells exhibit intrinsic fluorescence (termed autofluorescence) that results in variable background signals, especially if cells are large or come from inflamed and damaged tissue. In CyTOF, antibodies are labeled with metals, which can be characterized very specifically when individual cells are vaporized and subjected to mass spectrometry. As a result, instead of a practical limit of 12-15 labels by flow cytometry, CyTOF can easily evaluate 40 or more markers per cell. Using this technology, JBC Young Investigator Dr. Deepak Rao of BWH and colleagues recently identified a new population of T cells in seropositive rheumatoid joints, termed T peripheral helper (TPH) cells.1 By providing labeled antibodies as well as associated expertise, the JBC CyTOF Core is making this technology broadly available, resulting in current application to a range of lineages, including other lymphocytes, neutrophils, macrophages and fibroblasts, in joint tissue as well as in blood and even in lung tissue.
- RNA sequencing is a high-fidelity way to look at the genes that are being actively expressed in cells (i.e. their messenger RNA, mRNA). Though not completely synonymous with the set of proteins that a cell possesses, this mRNA signature provides key insight into what role a cell is playing within a tissue. Similarly, cell types can be classified into groups based on their gene expression profiles. RNA-seq comes in many variants, from general bulk sequencing, to low-input sequencing of small cell populations, to RNA-seq of individual cells. These tools have helped JBC investigators not only to understand TPH cells better, complementing the CyTOF studies detailed earlier, but also to define new subsets of fibroblasts within the joint. In a study recently published in Nature Communications, Dr. Fumitaka Mizoguchi, bioinformatics graduate student Kamil Slowikowski, and their colleagues at BWH and elsewhere used techniques including low-input RNA-seq to define three distinct subsets of fibroblasts within the joint. One of these exhibited more pro-inflammatory cytokine production and more aggressive invasive behavior than the other and was correspondingly increased in rheumatoid joints compared to osteoarthritis joints, suggesting a new mechanism by which non-immune cells can also contribute to inflammation and injury within the joint.2
The JBC Cellular Systems Core helps to make the latest in RNA-seq technology accessible to investigators, both through the Broad Institute in Cambridge, MA and at the new 10X Single Cell RNAseq Core operated by the BWH Human Immunology Center. Through JBC microgrants and other funding mechanisms, JBC investigators are using these tools to understand how each cell type, and indeed each cell individually, contributes to disease in rheumatoid arthritis and other diseases.
- Rao DA, Gurish MF, Marshall JL, Slowikowski K, Fonseka CY, Liu Y, Donlin LT, Henderson LA, Wei K, Mizoguchi F, Teslovich NC, Weinblatt ME, Massarotti EM, Coblyn JS, Helfgott SM, Lee YC, Todd DJ, Bykerk VP, Goodman SM, Pernis AB, Ivashkiv LB, Karlson EW, Nigrovic PA, Filer A, Buckley CD, Lederer JA, Raychaudhuri S, Brenner MB. Pathologically expanded peripheral T helper cell subset drives B cells in rheumatoid arthritis. Nature. Feb 01 2017;542(7639):110-114.
- Mizoguchi F, Slowikowski K, Wei K, Marshall JL, Rao DA, Chang SK, Nguyen HN, Noss EH, Turner JD, Earp BE, Blazar PE, Wright J, Simmons BP, Donlin LT, Kalliolias GD, Goodman SM, Bykerk VP, Ivashkiv LB, Lederer JA, Hacohen N, Nigrovic PA, Filer A, Buckley CD, Raychaudhuri S, Brenner MB. Functionally distinct disease-associated fibroblast subsets in rheumatoid arthritis. Nature Communications. Feb 23 2018;9(1):789.