The sphingosine-1-phosphate receptor agonist FTY720 and its phosphorylated form affect the function of CD4+CD25+ T cells in vitro

Abstract

Abstract. The sphingosine-1-phosphate receptor agonist FTY720 and FTY720-P have a wide variety of fundamental functions. Many studies have demonstrated that CD4 + CD25 + regulatory T (Treg) cells engage in the maintenance of immunological self-tolerance by actively suppressing self-reactive lymphocytes. Although FTY720 has also recently shown to possess an additional effect that increases the functional activity of Treg cells, the mechanism leading to the enhanced Treg activity after FTY720 treatment is still not clear. We isolated Treg cells, which were co-cultured with FTY720 or FTY720-P. The proliferation of co-cultured Treg cells was detected by the cell counting kit-8. The changes of the phenotype CD25 + and forkhead box P3 (Foxp3) + of co-cultured Treg cells were measured by flow cytometry. The levels of IL-10 and TGF-β1 in the supernatants were detected by ELISA. Cytokine mRNA expressions in co-cultured Treg cells were analyzed by real-time quantitative PCR. Mixed lymphocyte reaction assay examined the suppressive function. We found that neither FTY720 nor FTY720-P affected the proliferation of co-cultured Treg cells. The percentages of CD25 + and Foxp3 + were enhanced in the high-dose FTY720-P group. The levels of TGF-β1 in the supernatants were enhanced in the high-dose FTY720 group. Medium and high-dose FTY720-P also enhanced the levels of TGF-β1. TGF-β1 and Foxp3 mRNA expression were upregulated in the high-dose FTY720-P group. The proliferation of effector T (Teff) cells was suppressed significantly in the medium and high-dose FTY720-P group at a Treg/Teff cell ratio of 1:1. At a ratio of 1:1, the proliferation of Teff cells was also suppressed in the high-dose FTY720 group. It can be concluded that high-dose FTY720-P can enhance the immune function of co-cultured Treg cells, and that medium-dose FTY720-P and high-dose FTY720 could partly enhance the function. The reason may be attributed to enhanced levels of TGF-β1 and Foxp3. IntroductionAs a synthetic structural analog of myriocin, the sphingosine-1-phosphate (S1P) receptor agonist FTY720 can regulate a wide variety of fundamental functions including cell survival, cytoskeletal rearrangements, and cell motility (1,2). It has been shown to control some autoimmunities and allergic diseases as well as to suppress transplant rejection and graft vs. host disease (3). The drug has shown efficacy in advanced clinical trials for the treatment of multiple sclerosis (4). Once FTY720 is phosphorylated in vivo by sphingosine kinase 2 (SphK2), the phosphorylated compound (FTY720-P) acts as an agonist on 4 of the 5 known S1P receptors (S1P1, S1P3, S1P4 and S1P5) (5). In contrast to classical immunosuppressants, such as cyclosporine A or FK506, FTY720 does not impair antigen-driven T-cell activation and does not interfere with T-cell proliferation at therapeutically relevant concentrations, but induces a severe deprivation in lymphocytes in the blood due to modification of S1P signaling (6-8).Regulatory T (Treg) cells have been demonstrated to engage in the main...

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“…Activation of S1P1 is linked to inhibition of Treg cell differentiation [ 142 ]; however, FTY720 and phosphorylated FTY720 increase Treg levels and activity in vivo and in vitro [ 143 – 146 ]. The exact mechanism is unclear but may require higher doses [ 144 ]. The inhibitory effects of atorvastatin on inflammation in acute coronary syndrome (ACS) may be due to its beneficial effects on natural Tregs [ 147 ].…”

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“…Importantly modulation of SPNS2 could be a more favourable approach than the S1P-blocking antibody Sphingomab 27,28 or the prodrug FTY720 (ref. 24) (which is phosphorylated in vivo to a functional antagonist of S1PR1) as these interventions increase regulatory T cell activity, suppress proliferation of effector T cells 29,30 and increase vascular permeability 18 . Furthermore, as lymphatic endothelial cell-specific deletion of Spns2 is sufficient to regulate lymphocyte circulation to allow a higher percentage of effector T cells and NK cells in the lung (and liver) and more tumour cell killing, targeting SPNS2 is potentially a more promising option for regulating metastatic colonization than existing S1P pathway modulators.…”

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“…In vivo experiments show that FTY720 causes differentiation and an increase in T reg numbers in the spleen [ 414 ], but FTY720 does not affect the proliferation of these cells [ 414 , 415 ]. Wolf et al have shown that FTY720 does disrupt T reg proliferation by inhibiting IL-2-dependent STAT5 phosphorylation [ 416 ].…”

“…Research on FTY720 show that this drug supports the functions of these cells. The active form of FTY720-P induces an increased expression of TGF-β1 and FoxP3 marker in T reg cells [ 415 , 421 ]. On the other hand it does not cause a significant increase in IL-10 production in these cells.…”