Background/Purpose: Sero-positive rheumatoid arthritis (RA) is a chronic autoimmune disease that without effective treatment, leads to joint damage and disability. Not all patients respond to currently available medications. Human mesenchymal stem cells (hMSCs) have the potential to be used therapeutically to attenuate immune-mediated diseases. To understand how to select hMSCs for RA therapy 1) hMSC culture medium was tested for the ability to suppress RA and healthy control allogeneic T-cells; 2) the degree of T-cell suppression was compared using hMSCs from distinct donors; 3) preconditioning of hMSCs with cytokines was tuned for maximal suppressive effect and likely mediators of suppression measured.

Methods: CD4+ T-cells obtained from healthy controls (HC) and RA patients were stimulated for 4 days to proliferate. Medium used in suppression assays were:1) culture conditioned medium (CCM) in which hMSCs were preconditioned by a single cytokine; TNFα, IFNγ, IL-1β or a combination of the three cytokines (all-3) or 2) unconditioned control medium (UC) that was cultured like CCM but was not preconditioned with cytokines. T-cell proliferation was measured using eFluor670 vital dye and proliferation was measured using flow cytometry. IDO mRNA and protein were quantified by qRT-PCR and western blot, respectively. Statistical analyses were performed using two-tailed Student’s t-test and one-way ANOVA was used for multiple comparison tests in GraphPad Prism 8.1.2.

Results: RA and HC CD4+ T-cell proliferation was suppressed by all-3-CCM compared to UC (76%±2 of UC p< 0.0001). IFNγ-CCM was also suppressive (43%±6 of UC p< 0.001). CCM containing all-3 cytokines suppressed HC CD4+ T-cell proliferation best (68%±9 p< 0.05) when hMSCs were preconditioned with cytokines for 48 hours compare to 24 hours (42%±8). CCM from different donors varied in potency of suppression in both RA and HC. Differences between individual hMSC donors were best detected using all-3-CCM and IFNγ-CCM. CD4+ T-cell suppression was positively correlated with increases in IDO mRNA and IDO protein in both RA and HC.

Conclusion: The data demonstrate that hMSC can be tuned using cytokines to maximally suppress HC and RA T-cells.  IDO mediates hMSC effects on T-cells, but hMSC also secrete other molecules that contribute toT-cell suppression. Standardizing cell-based therapy using functional assays ex vivo should facilitate comparing potency of distinct cellular products across disease indications. The suppression assays described directly test an admixture of soluble mediators that arise from hMSC rather than just describing mRNA or protein levels. Whether hMSCs should be cytokine primed prior to therapeutic infusion, is of great interest and has implications for hMSC-therapy in RA.

*This project was supported by the Clinical and Translational Science Collaborative (CTSC) of Cleveland which is funded by the       National Institutes of Health (NIH), National Center for Advancing Translational Science (NCATS), Clinical and Translational       Science Award (CTSA) grant, UL1TR002548. The content is solely the responsibility of the authors and do not necessarily       represent the official views of the NIH. And David and Virginia Baldwin Foundation.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/development-of-functional-assays-to-pre-qualify-human-mesenchymal-stem-cells-for-rheumatoid-arthritis-treatment/