Low-dose decitabine modulates T-cell homeostasis and restores immune tolerance in immune thrombocytopenia

Abstract

Our previous clinical study showed that low-dose decitabine exhibited sustained responses in nearly half of patients with refractory immune thrombocytopenia (ITP). The long-term efficacy of decitabine in ITP is not likely due to its simple role in increasing platelet production. Whether decitabine has the potential to restore immune tolerance in ITP is unknown. In this study, we analyzed the effect of decitabine on T-cell subpopulations in ITP in vitro and in vivo. We found that low-dose decitabine promoted the generation and differentiation of regulatory T (Treg) cells and augmented their immunosuppressive function. Splenocytes from CD61 knockout mice immunized with CD61+ platelets were transferred into severe combined immunodeficient mouse recipients to induce a murine model of ITP. Low-dose decitabine alleviated thrombocytopenia and restored the balance between Treg and helper T (Th) cells in active ITP mice. Treg deletion and depletion offset the effect of decitabine in restoring CD4+ T-cell subpopulations in ITP mice. For patients who received low-dose decitabine, the quantity and function of Treg cells were substantially improved, whereas Th1 and Th17 cells were suppressed compared with the pretreatment levels. Next-generation RNA-sequencing and cytokine analysis showed that low-dose decitabine rebalanced T-cell homeostasis, decreased proinflammatory cytokines, and downregulated phosphorylated STAT3 in patients with ITP. STAT3 inhibition analysis suggested that low-dose decitabine might restore Treg cells by inhibiting STAT3 activation. In conclusion, our data indicate that the immunomodulatory effect of decitabine provides one possible mechanistic explanation for the sustained response achieved by low-dose decitabine in ITP.

© 2021 by The American Society of Hematology.

Figures

Graphical abstract

Figure 1.

Low-dose decitabine had no influence on the apoptosis of PBMCs from patients with ITP or healthy control subjects, but significantly elevated the percentage of Treg cells in patients with ITP without changing the percentage of CD4+ T cells in PBMCs. (A) Representative dot plots of flow cytometry analysis of apoptosis in PBMCs from patients with ITP. The percentage of Annexin-positive and propidium iodide (PI)-negative cells in PBMCs showed the cell apoptosis rate. (B,C) Decitabine induced apoptosis of PBMCs from healthy control subjects and patients with ITP at doses of 1 μM and 10 μM (n = 6). (D-F) Percentages of Treg cells in CD4+ T cells were significantly increased by decitabine at the doses of 10 nM and 100 nM in patients with ITP (n = 11). (G-I) In cultured isolated CD4+ T cells, low-dose decitabine (100 nM) significantly increased the percentage of Treg cells in patients with ITP (I; n = 7), but not in healthy control subjects (H; n = 8). *P < .05, **P < .01. Dec, decitabine; HC, healthy controls; ns, not significant.

Figure 2.

The inhibitory function of Treg cells was enhanced by low-dose decitabine in ITP in vitro. Teff cells (2 × 105 cells per well) were seeded in a 96-well plate with or without Treg cells (5 × 104 cells per well) in the presence of decitabine 100 nM. The cell division index was calculated based on the dilution of CFSE fluorescence measured by flow cytometry and represents the average number of cell divisions that Teff cells in the original population have undergone [division index = sum (i × N(i)/2i)/sum (N(i)/2i), “i” is division number (undivided = 0), and “N(i)” is the number of cells in division “i”]. (A,C) Representative histograms of CD4+CFSE+ Teff cells from one healthy control subject (A) and patient with ITP (C). (B,D) Treg cells suppressed the proliferation of Teff cells. Decitabine (100 nM) enhanced the inhibitory function of Treg cells in ITP (D; n = 24), but not in healthy control subjects (B; n = 13). *P < .05, **P < .01, ***P < .001. Dec, decitabine; HC, healthy control subjects; ns, not significant.

Figure 3.

Low-dose decitabine ameliorated thrombocytopenia in active ITP murine models via Treg cells. ITP models were established in irradiated SCID mice with engraftment of 2 × 104 splenocytes from CD61 knockout mice immunized against wild-type C57 mice platelets; platelet counts were then monitored every week for 4 weeks (mean ± standard error of the mean). (A) From day 7, decitabine (0, 0.01 mg/kg, 0.03 mg/kg) was administered. The horizontally dotted lines represent the baseline platelet counts of SCID mice (mean ± standard error of the mean). On days 21 and 28, significantly higher platelet counts were observed in the group administered the 0.03-mg/kg dose of decitabine compared with control group. (B) Decitabine-treated ITP mice had a significantly higher percentage of splenic Treg cells in CD4+ T cells compared with control mice (n = 11 in the control group and n = 13 in the decitabine group, respectively). The percentage of Th1 (C) and Th17 (D) in CD4+ T cells were decreased in decitabine-treated spleens compared with controls. No significance was found in Th22 cells (E) in splenic CD4+ T cells (n = 9 in the control group and n = 10 in the decitabine group). Treg deletion (F) by magnetic beads and depletion (K) by anti-CD25 antibody partly offset the effect of decitabine on increasing platelet counts of ITP mice (n = 6). Treg-deleted splenocyte-transferred mice (G) and anti-CD25 antibody-treated mice (L) were significantly depleted of Treg cells in the spleens on day 28 (n = 6). (H-J) After Treg deletion, decitabine had no significant effect on splenic Th1, Th17, or Th22 cells on day 28 in ITP mice (n = 6). (M-O) With Treg depletion by anti-CD25 antibody, decitabine had no significant effect on splenic Th1, Th17, or Th22 cells on day 28 in ITP mice (n = 6). *P < .05, **P < .01, ***P < .001. Dec, decitabine; ns, not significant.

Figure 4.

Immunologic responses to low-dose decitabine in patients with ITP. (A-E) Percentages of Treg, Th1, Th17, Th2, and Th22 cells in peripheral blood were analyzed by using flow cytometry. Treg cells (n = 22), Th1 cells (n = 17), Th17 cells (n = 15), Th2 cells (n = 8), and Th22 cells (n = 16) in CD4+ T cells before and after decitabine treatment are shown. (F) Ex vivo suppression assays were performed to examine the suppressive activity of Treg cells obtained from patients with ITP before and after decitabine treatment. CFSE-labeled CD4+CD25– Teff cells were stimulated and cocultured with Treg cells from each patient. Representative histograms show proliferation of CD4+ Teff cells with or without Treg cells before (above) or after (below) decitabine treatment. (G-H) Treg cell–mediated suppression of CD4+ Teff cell proliferation was measured using division index before and after decitabine treatment (n = 14). (I) Decitabine enhanced immunosuppressive function of Treg cells. *P < .05, **P < .01, ****P < .0001. NR, nonresponders; R, responders.

Figure 5.

Profiles and enriched pathway analysis of decitabine-induced genes. (A) Venn diagram of overlapping genes of decitabine-altered genes in 4 responders. (B) Heat map of decitabine-altered genes in 4 responders with unsupervised clustering. Red to green colors represent relatively high to low gene expression, respectively, and gray indicates below the detectable level. (C) Venn diagram of overlapping genes of decitabine-altered genes in 3 nonresponders. (D) No overlap of decitabine-altered genes was observed between responders and nonresponders. (E-G) Pathways were predicted by Gene Ontology (E), KEGG (F), and Reactome (G) databases based on the expression changes of decitabine altered genes in 4 responders. AF, after; BE, before; NR, nonresponders; R, responders.

Figure 6.

Low-dose decitabine inhibited the STAT3 signaling pathway. (A) Representative western blots of phosphorylated-STAT3 (p-STAT3), total STAT, phosphorylated-AKT (p-AKT), total AKT, and GAPDH in PBMCs from patients with ITP before and after decitabine treatment. (B-C) Graphs present densitometry data of relative phosphorylated protein to total protein (n = 3). Treg cells in CD4+ T cells from healthy control subjects (D; n = 10) and patients with ITP (E; n = 8) in the STAT3 inhibitor plus decitabine group were no different from those in the STAT3 inhibitor group in vitro. In the presence of AKT inhibitor, decitabine significantly increased the percentage of Treg cells from patients with ITP in vitro (G; n = 10), but not in the healthy control subjects (F; n = 8). (H) The platelet counts in the STAT3 inhibitor plus decitabine group were not significantly different from those in mice receiving STAT3 inhibitor (n = 6). (I) AKT inhibitor plus decitabine increased platelet counts in mice compared with AKT inhibitor on day 21 (n = 6). *P < .05. Dec, decitabine; ns, not significant.