Figure 2.
Decitabine enhanced metabolic and immunosuppressive function in MDSCs. (A) The scatter plots showing apoptosis in adherent cells. The percentage of Annexin V–positive and PI-negative cells represented the cell apoptosis rate. (B-C) Decitabine induced significantly higher percentage of Annexin V–positive and PI-negative cells at the higher doses of 1 μM and 10 μM (healthy control, HC: ordinary one-way ANOVA, ∗∗∗∗P < .0001, multiple comparisons: P0 nM vs 100 nM = .8105, ∗∗∗∗P0 nM vs 1 μM < .0001, ∗∗∗∗P0 nM vs 10 μM < .0001; ITP patients: ordinary one-way ANOVA, ∗∗∗∗P < .0001, multiple comparisons: P0 nM vs 100 nM = .3248, ∗∗∗∗P0 nM vs 1 μM < .0001, ∗∗∗∗P0 nM vs 10 μM < .0001). ns, Not significant. (D) There was no significant difference between the control group and the decitabine-treated group in healthy controls (one-way ANOVA, n = 7, P = .0696; multiple comparisons: P0 nM vs 10 nM = .8793, P0 nM vs 50 nM = .4241, P0 nM vs 100 nM = .0521, P0 nM vs 1 μM = .2371, P0 nM vs 10 μM = .9251). (E) After 7 d of culture with decitabine (50 nmol/L, 100 nmol/L, or 1 μmol/L), PBMCs from patients with ITP had significantly increased MDSCs than before decitabine treatment (one-way ANOVA, n = 5, ∗P < .0001; multiple comparisons: P0 nM vs 10 nM = .0772, ∗∗∗P0 nM vs 50 nM = .0010, ∗∗∗∗P0 nM vs 100 nM < .0001, ∗∗P0 nM vs 1 μM = .0027). (F) MDSCs from ITP patients and healthy controls were treated with PBS and decitabine (100 nmol/L), respectively. OCR was measured following injections of oligomycin (1 mol/L), carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP) (1 mol/L), and rotenone/antimycin A (0.5 mol/L). (G) Respective mitochondrial parameters basal respiration (unpaired t tests, ∗∗PCtrl vs ITP = .0087, ∗PITP vs ITP+Dec = .0484), ATP production (unpaired t tests, ∗PCtrl vs ITP = .0106, ∗PITP vs ITP+Dec = .0199) and maximal respiration (unpaired t tests, ∗∗PCtrl vs ITP = .0036, ∗∗PITP vs ITP+Dec = .0052) calculated via ATP-linked respiration/basal respiration. (H) Increased intracellular ATP levels in MDSCs after decitabine treatment (paired t tests, P = .0575). (I) Representative histogram of the proliferation of CD4+ Teffs (Ⅰ) Ctrl-PBS, (II) Ctrl-Dec, (III) ITP-PBS, (IV) ITP-Dec. (J) MDSC-mediated suppression of CD4+ Teff proliferation was measured using division index. MDSC inhibition in healthy controls was significantly higher than that in ITP patients (unpaired t tests, ∗∗PCtrl vs ITP = .0073). (K) Compared with PBS treatment, decitabine significantly enhanced MDSC inhibitory function in ITP (paired t tests, ∗∗PITP vs ITP+Dec = .0089). (L) The gated dot plots represent CTL-induced platelet apoptosis after coculture with PBS- or decitabine-treated MDSCs in ITP. (Ⅰ) Platelets only. (II) Platelets + CTLs. (III) Platelets + CTLs + PBS-MDSCs. (IV) Platelets + CTLs + Dec-MDSCs. (M-N) CTL-induced platelet apoptosis was significantly lower following coculture with decitabine-treated MDSCs (paired t tests, PLT + CTLs vs PLT + CTLs + PBS-MDSC, ∗∗∗P = .0004; PLT + CTLs + PBS-MDSC vs PLT + CTLs + Dec-MDSC, ∗∗∗P = .0010).

Decitabine enhanced metabolic and immunosuppressive function in MDSCs. (A) The scatter plots showing apoptosis in adherent cells. The percentage of Annexin V–positive and PI-negative cells represented the cell apoptosis rate. (B-C) Decitabine induced significantly higher percentage of Annexin V–positive and PI-negative cells at the higher doses of 1 μM and 10 μM (healthy control, HC: ordinary one-way ANOVA, ∗∗∗∗P < .0001, multiple comparisons: P0 nM vs 100 nM = .8105, ∗∗∗∗P0 nM vs 1 μM < .0001, ∗∗∗∗P0 nM vs 10 μM < .0001; ITP patients: ordinary one-way ANOVA, ∗∗∗∗P < .0001, multiple comparisons: P0 nM vs 100 nM = .3248, ∗∗∗∗P0 nM vs 1 μM < .0001, ∗∗∗∗P0 nM vs 10 μM < .0001). ns, Not significant. (D) There was no significant difference between the control group and the decitabine-treated group in healthy controls (one-way ANOVA, n = 7, P = .0696; multiple comparisons: P0 nM vs 10 nM = .8793, P0 nM vs 50 nM = .4241, P0 nM vs 100 nM = .0521, P0 nM vs 1 μM = .2371, P0 nM vs 10 μM = .9251). (E) After 7 d of culture with decitabine (50 nmol/L, 100 nmol/L, or 1 μmol/L), PBMCs from patients with ITP had significantly increased MDSCs than before decitabine treatment (one-way ANOVA, n = 5, ∗P < .0001; multiple comparisons: P0 nM vs 10 nM = .0772, ∗∗∗P0 nM vs 50 nM = .0010, ∗∗∗∗P0 nM vs 100 nM < .0001, ∗∗P0 nM vs 1 μM = .0027). (F) MDSCs from ITP patients and healthy controls were treated with PBS and decitabine (100 nmol/L), respectively. OCR was measured following injections of oligomycin (1 mol/L), carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP) (1 mol/L), and rotenone/antimycin A (0.5 mol/L). (G) Respective mitochondrial parameters basal respiration (unpaired t tests, ∗∗PCtrl vs ITP = .0087, ∗PITP vs ITP+Dec = .0484), ATP production (unpaired t tests, ∗PCtrl vs ITP = .0106, ∗PITP vs ITP+Dec = .0199) and maximal respiration (unpaired t tests, ∗∗PCtrl vs ITP = .0036, ∗∗PITP vs ITP+Dec = .0052) calculated via ATP-linked respiration/basal respiration. (H) Increased intracellular ATP levels in MDSCs after decitabine treatment (paired t tests, P = .0575). (I) Representative histogram of the proliferation of CD4+ Teffs (Ⅰ) Ctrl-PBS, (II) Ctrl-Dec, (III) ITP-PBS, (IV) ITP-Dec. (J) MDSC-mediated suppression of CD4+ Teff proliferation was measured using division index. MDSC inhibition in healthy controls was significantly higher than that in ITP patients (unpaired t tests, ∗∗PCtrl vs ITP = .0073). (K) Compared with PBS treatment, decitabine significantly enhanced MDSC inhibitory function in ITP (paired t tests, ∗∗PITP vs ITP+Dec = .0089). (L) The gated dot plots represent CTL-induced platelet apoptosis after coculture with PBS- or decitabine-treated MDSCs in ITP. (Ⅰ) Platelets only. (II) Platelets + CTLs. (III) Platelets + CTLs + PBS-MDSCs. (IV) Platelets + CTLs + Dec-MDSCs. (M-N) CTL-induced platelet apoptosis was significantly lower following coculture with decitabine-treated MDSCs (paired t tests, PLT + CTLs vs PLT + CTLs + PBS-MDSC, ∗∗∗P = .0004; PLT + CTLs + PBS-MDSC vs PLT + CTLs + Dec-MDSC, ∗∗∗P = .0010).

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