Low-Dose Decitabine Restores Immune Tolerance in ITP By Modulating Regulatory T Cells

Immune thrombocytopenia (ITP) is an aquired autoimmune disease triggered by increased platelet destruction and impaired platelet production. Loss of immune tolerance is considered to be the pivotal mechanism of ITP. Decitabine, a hypomethylating agent promoting cell differentiation at low dose, has been applied in the treatment of myelodysplastic syndrome (MDS) with a considerable platelet response. Our previous and current studies demonstrated that low-dose decitabine could significantly increase the number of mature polyploidy megakaryocytes and had a long-term efficacy that could not be explained by its role on promoting platelet production in ITP patients. Recent reports have demonstrated that decitabine could decrease the production of interferon-γ (IFN-γ) and tumor necrosis factor-a (TNF-a), and increase immune suppressive regulatory T cells. Thus, low-dose decitabine might correct the loss of immune tolerance to induce a more durable response in patients with ITP.

CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs), which play an important role in maintenance of peripheral tolerance, were reported to be decreased in number and function in ITP (Stasi R et. al. Blood 2008). In the in vitro study, peripheral blood mononuclear cells (PBMCs) were isolated and cultured with 100nM decitabine or PBS for 72h. We performed flow cytometry to determine the percentage of Tregs. It was shown that the percentage of Tregs in PBMCs and CD4⁺ T cells were significantly elevated after decitabine treatment. We also sorted the CD4⁺CD25⁺CD127^- Tregs from ITP patients and co-cultured them with CFSE-labeled CD4⁺CD25^-T cells to evaluate the suppressive activity of Tregs. The results indicated that the inhibitory function of Tregs after decitbine treatments was significantly elevated compared with controls.

As compared to healthy individuals, patients with ITP also had increased number of T_H1, T_H17, T_H22, Bregs and CD16+ monocytes. We subsequently investigated the effects of low-dose decitabine on those cells in patients with active ITP. Three cycles of decitabine were administered intravenously at a dose of at 3.5 mg/m² for 3 days per cycle with a 4-week interval between cycles. After decitabine treatment, there was a significant increase in the relative number of CD25⁺Foxp3⁺ Tregs in the CD4⁺ T cell population. We also observed an increase in Tregs function after the decitabine treatment. In contrast, decitabine could significantly decrease the percentage of CD4⁺IFN-γ⁺T_H1 in the CD4⁺T cell population and the percentage of CD16⁺monoyctes. However, we have not observed the effect of decitabine on the CD4⁺IFN-γ^-CD17⁺T_H17, CD4⁺IFN-γ^-CD22⁺T_H22, and CD19⁺CD24⁺CD38⁺Bregs by now.

We further established the murine ITP model by transferring the splenocytes of C57BL/6 CD61 knockout mice, which were immunized against platelets from wild-type syngeneic C57BL/6 mice into severe combined immunodeficient (SCID) mice. We treated the ITP mice model with a low-dose range of decitabine (0.01, 0.03 or 0.10 mg/kg) intravenously three times a week. Our data showed a significant effect on PLT increase and the expansion of the Tregs population and the suppression of TH1 cells and CD16⁺ monocytes .

In addition, programmed death-1 (PD-1) is a co-receptor that is expressed predominantly by T cells as well as B cells, monocytes and was found to be lower on the PBMCs and in serum of ITP patients (Zhong J et. al. Hematology. 2016). And research has reported that PD-1 signaling pathway is important in the pathogenesis of autoimmune diseases by inhibition of self-reactive T cells and increasing the development and functions of Tregs. Furthermore, it was reported that the expression of PD-1 is enhanced by decitabine in MDS (Yang H et. al. Leukemia 2014). In our current studies, decreased PD-1 expression on CD4⁺T cells of ITP patients was observed and decitabine could elevate the PD-1 expression on CD4⁺T cells after the decitabine treatment in ITP patients. Therefore, decitabine may correct the loss of immune tolerance by upregulating the Expression of PD-1 and be a promising therapeutic strategy for the management in ITP patients.

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