3-D Explant Method Facilitates the Study of Lymphocytes in Synovium and Reveals a Population of Resident Memory-Like T Cells in Rheumatoid Arthritis

Lauren Henderson¹, Deepak Rao², Nikola Teslovich^3,4, Sandra King⁵, Fumitaka Mizoguchi⁶, Sarah Ameri⁶, Allyn Morris⁷, Christopher Elco⁵, Margaret Chang⁸, Anais Levescot⁹, James Lederer¹⁰, Scott Martin¹¹, Barry Simmons¹¹, John Wright¹¹, Michael Brenner², Soumya Raychaudhuri^{12,13,14,15,16}, Robert Fuhlbrigge¹⁷ and Peter Nigrovic^1,18, ¹Division of Immunology, Boston Children's Hospital, Harvard Medical School, Boston, MA, ²Division of Rheumatology, Immunology, and Allergy, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, ³Program in Medical and Population Genetics, Broad Institute of Massachusetts Technical Institute and Harvard University, Cambridge, MD, ⁴Divisions of Genetics and Rheumatology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, ⁵Department of Dermatology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, ⁶Division of Rheumatology, Immunology, and Allergy, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, ⁷Division of Rheumatology, Immunology, and Allergy, Brigham and Women's Hospital, Boston, MA, ⁸Division of Immunology, Boston Children's Hospital, Boston, MA, ⁹Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, Harvard Medical School, boston, MA, ¹⁰Department of Surgery, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, ¹¹Department of Orthopedic Surgery, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, ¹²Divisions of Genetics and Rheumatology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, ¹³Program in Medical and Population Genetics, Broad Institute of Massachusetts Technical Institute, Harvard University, Cambridge, MA, ¹⁴Partners Center for Personalized Genetic Medicine, Boston, MA, ¹⁵Institute of Inflammation and Repair, University of Manchester, Manchester, United Kingdom, ¹⁶Rheumatology Unit, Karolinska Institutet, Karolinska University Hospital Solna, Stockholm, Sweden, ¹⁷Children's Hospital Colorado, Aurora, CO, ¹⁸Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, Harvard Medical School, Boston, MA

Meeting: 2017 Pediatric Rheumatology Symposium

Keywords: CyTOF, T cells and rheumatoid arthritis (RA)

Session Information

Date: Thursday, May 18, 2017

Title: Genetics and Pathogenesis Poster Session

Session Type: Abstract Submissions

Session Time: 5:30PM-7:00PM

Background/Purpose: Tissue resident memory T (T_RM) cells survive indefinitely in barrier tissues and mediate swift immunologic memory responses at sites of microbe entry. T_RM cells have been implicated in recurrent site-specific inflammation in skin, intestine, and lung, but little is known about T_RM cells in synovium. We employed a highly efficient 3-dimensional (3-D) explant culture technique, developed to recover T_RMfrom skin, to investigate synovial infiltrating T cells.

Methods: Subjects with rheumatoid arthritis (RA) were identified by International Classification of Disease (ICD) codes, supported by medical record review by a board-certified rheumatologist. Synovial tissue samples were obtained from RA patients undergoing medically necessary joint surgery. Collagenase digestion and/or 3-D explant culture was used to isolate synovial T cells. In the 3-D explant culture system, 2mm x 2mm pieces of synovial tissue were placed on Cellfoam matrices, and cultured in T cell media enriched with IL-2 and IL-15 for 3 weeks. Multidimensional analysis of surface markers and cytokine production upon stimulation was performed by mass cytometry (CyTOF).

Results: Synovial samples were obtained from 13 women and 2 men with established RA. Treatment regimens varied (methotrexate, n=9; tumor necrosis factor inhibitors, n=6; prednisone, n=5; non-steroidal anti-inflammatory drugs, n=5). 3-D explant culture of RA synovium samples yielded 5-fold more mononuclear cells, on average, than collagenase digestion (mean number mononuclear cells per mg tissue ± SEM: 25,000 ± 17,000 vs. 5,600 ± 4,700). Approximately, 80% of cells collected by the 3-D explant culture method were CD3⁺ T cells, with the majority being CD4⁺ T cells (~80%). The ratio of CD4⁺ to CD8⁺ cells was not significantly different between the recovery methods. Multidimensional mass cytometry analyses demonstrated dramatic differences in phenotype between synovial and blood CD4⁺ memory T cells, with significantly increased CD49d and MHCII and decreased CD27 on synovial T cells. A Th1 phenotype predominated in synovial T cells isolated by explant cultures, with ~35% of CD4⁺ memory T cells expressing IFNγ and Tbet upon stimulation. Notably, memory CD4⁺ T cells with a phenotype consistent with T_RM cells (CD62L^–, CCR7^–, CD69⁺) were identified in all tested synovial samples. While CD69 expression may be induced by recent activation, many of these potential T_RM (CD62L^–, CCR7^–, CD69⁺) cells did not express two other markers of recent activation, CD25 and MHCII.

Conclusion: 3-D explant culture is a novel tool that can be employed to study lymphocytes that are resident in synovium. Memory T cells with T_RM features were identified in synovial samples from RA patients, supporting the hypothesis that this T cell subset may contribute to the persistence and recurrence of inflammatory arthritis. Further study will be needed to define the functional characteristics and pathophysiologic role of these cells.

Disclosure: L. Henderson, None; D. Rao, None; N. Teslovich, None; S. King, None; F. Mizoguchi, None; S. Ameri, None; A. Morris, None; C. Elco, None; M. Chang, None; A. Levescot, None; J. Lederer, None; S. Martin, None; B. Simmons, None; J. Wright, None; M. Brenner, None; S. Raychaudhuri, None; R. Fuhlbrigge, None; P. Nigrovic, None.

To cite this abstract in AMA style:

Henderson L, Rao D, Teslovich N, King S, Mizoguchi F, Ameri S, Morris A, Elco C, Chang M, Levescot A, Lederer J, Martin S, Simmons B, Wright J, Brenner M, Raychaudhuri S, Fuhlbrigge R, Nigrovic P. 3-D Explant Method Facilitates the Study of Lymphocytes in Synovium and Reveals a Population of Resident Memory-Like T Cells in Rheumatoid Arthritis [abstract]. Arthritis Rheumatol. 2017; 69 (suppl 4). https://acrabstracts.org/abstract/3-d-explant-method-facilitates-the-study-of-lymphocytes-in-synovium-and-reveals-a-population-of-resident-memory-like-t-cells-in-rheumatoid-arthritis-2/. Accessed .

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