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_RM from 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.

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ACR Meeting Abstracts - 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/