Background/Purpose: Recent studies have demonstrated that naturally occurring CD4⁺Foxp3⁺regulatory T cells (nTregs) are unstable and dysfunctional in the presence of pro-inflammatory cytokines. All-trans RA (atRA), the active derivative of vitamin A, has been demonstrated to regulate Treg differentiation and stabilize nTreg in the healthy subjects. We here plan to determine whether atRA can similarly stabilize nTreg cells isolated from patients with active systemic lupus erythematosus (SLE) under inflammatory conditions.

Methods: CD4⁺CD25⁺CD127^-/low human nTregs were sorted from PBMCs in patients with SLE or healthy controls, and then expanded in the presence or absence of atRA for 7 days. These cells were extensively washed and restimulated with or without IL-1β/IL-6 for additional 3 days. Cell suspensions were harvested for cytokine assay with Elisa and cells were stained with flow cytometry analysis. In vitro, suppression assay was performed, CD3⁺CD25^–T cells labeled with carboxyfluorescein succinimidyl ester (CFSE) were stimulated with anti-CD3 in the presence of APC with or without different populations of nTreg cells. The CFSE dilution was examined by flow cytometry, and the suppression ratio was then calculated. Xenograft-vs-host diseases (xGVHD) models were conducted for determining the suppressive activities of Treg cells in vivo. NSG mice after γ-irradiation irradiation were injected with human CD25-depleted PBMCs with or without expanded SLE nTregs pretreated with atRA, rapamycin, DMSO control, and nTreg that had been exposed to IL-1β/IL-6. Mouse survival was monitored twice per week, and survival curves were analyzed by the log-rank test. Pathology in organ organs, human CD3+ engraftment and IgG were analyzed accordingly. The statistical comparisons were performed by the Student t test by using Prism software (GraphPad).

Results: In the presence of IL-1β/IL-6, atRA also prevents nTregs isolated from active SLE patients from converting to Th1 and/or Th17 cells and sustains their Foxp3 expression and suppressive function in vitro. atRA also diminished IL-1β/IL-6 signaling events by reducing STAT1, STAT3 activation. Adoptive transfer of SLE nTregs pretreated with atRA enhanced their suppressive function on xGVHDs, and only atRA primed nTregs sustained the suppressive effect on xGVHD after stimulation with IL-1β/IL-6.

Conclusion: Our results have demonstrated that atRA also stabilizes the Foxp3 expression and Treg function of patients with SLE, particularly on the inflammatory condition. Thus, a new protocol has been developed that usage of autologous atRA-programed Treg cells likely treats patients with SLE and other autoimmune diseases.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/all-trans-retinoic-acid-stabilizes-natural-t-regulatory-cells-isolated-from-patients-with-systemic-lupus-erythematosus-under-inflammatory-conditions/