Glucocorticoid hormone differentially modulates the in vitro expansion and cytokine profile of thymic and splenic Treg cells

Objective: Functional disturbances in regulatory T cells (Treg) have been described in autoimmune diseases, and their potential therapeutic use is intensively studied. Our goal was to investigate the influence of glucocorticoid hormone on the in vitro differentiation of Treg cells from thymic and splenic CD4(+) T cells under different conditions to establish methods for generating stable and functionally suppressive iTregs for future use in adoptive transfer experiments. Methods: Thymic and splenic CD4(+) T lymphocytes were isolated from 3 to 4 week-old control and in vivo dexamethasone (DX) pretreated BALB/c mice using magnetic bead negative selection, followed by CD25 positive selection. The cells were cultured with anti-CD3/CD28 beads and IL-2 in the presence or absence of TGF beta and/or DX for 3-6 days. Multiparametric flow cytometry was performed using CD4, CD25, CD8, TGF beta (LAP) cell surface and Foxp3, IL-4, IL-10, IL-17 and IFN gamma intracellular staining. Quantitative RT-PCR was performed to measure IL-10, TGF beta cytokine and Foxp3 mRNA levels. Results: Differentiation of thymus-derived CD4(+) cells in vitro into iTreg cells was most effective (24-25%) when anti-CD3/CD28 beads, IL-2, and TGF beta were present. Splenic CD4(+) T cell expansion under same conditions resulted in a higher (44-45%) iTreg cell ratio that further increased (up to 50% Treg) in the presence of DX. Elevated immunosuppressive cytokine (IL-10 and TGF beta) production by iTregs could be measured both at protein and mRNA levels without elevation of Th1/Th2 or Th17 cytokine production. We got the highest iTreg ratio (74%) and TGF beta production when CD4(+) CD25(+) splenic T cells were stimulated in the presence of TGF beta. In vivo 4 days DX pretreatment resulted in enhanced in vitro expansion and Foxp3 expression of thymus-derived iTregs and decreased differentiation of spleen-derived iTreg cells. In these Tregs the relative expression of IL-10 mRNA significantly decreased under all in vitro stimulation conditions, while TGFP mRNA level did not change. Conclusion: DX promotes the expansion of thymic and splenic Treg cells, and enhances Foxp3(+) expression and the production of immunosuppressive cytokines IL-10 and TGF beta in vitro. In vivo pretreatment of mice with DX inhibited the immunosuppressive cytokine production of in vitro differentiated Treg cells. We hypothesize that patients receiving GC therapy may need special attention prior to in vitro expansion and transplantation of Treg cells.