Low-Dose Decitabine Inhibits Cytotoxic T Lymphocytes-Mediated Platelet Destruction via Modulating PD-1 Methylation in Immune Thrombocytopenia

Abstract

Cytotoxic T lymphocytes (CTLs)-mediated platelet destruction plays an important role in the pathogenesis of primary immune thrombocytopenia (ITP). The programmed cell death protein 1 (PD-1) signaling can turn off autoreactive T cells and induce peripheral tolerance. Herein, we found that the expression of PD-1 and its ligand PD-L1 on CD8+ T cells from ITP patients was decreased. Activating PD-1 pathway by PD-L1-Fc fusion protein inhibited CTLs-mediated platelet destruction in ITP in vitro. PD-1 promoter hypermethylation in CD8+ T cells was found in ITP patients, resulting in decreased PD-1 expression. The demethylating agent decitabine at a low dose was proved to restore the methylation level and expression of PD-1 on CD8+ T cells and reduce the cytotoxicity of CTLs of ITP patients. The phosphorylation levels of phosphatidylinositol 3-kinase (PI3K) and AKT in CD8+ T cells were significantly downregulated by low-dose decitabine. Furthermore, blocking PD-1 could counteract the effect of low-dose decitabine on CTLs from ITP patients. Therefore, our data suggest that the aberrant PD-1/PD-L1 pathway is involved in the pathophysiology of ITP and enhancing PD-1/PD-L1 signaling is a promising therapeutic approach for ITP management. Our results reveal the immunomodulatory mechanism of low-dose decitabine in ITP by inhibiting CTLs cytotoxicity to autologous platelets through PD-1 pathway.

Keywords: PD-1; PD-L1; cytotoxic T lymphocytes; decitabine; immune thrombocytopenia.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2021 Han, Yu, Hou, Zhao, Liu, Sun, Wang, Xu, Li, Sun, Hu, Liu, Li, Peng, Zhou and Hou.

Figures

Figure 1

The expression of programed cell death protein 1 (PD-1) and PD-1 ligand (PD-L1) in immune thrombocytopenia (ITP) patients was lower than that of healthy controls. (A) The mRNA expression level of PD-1 in PBMCs from ITP patients had no difference with that of healthy controls (0.51 × 10−3 ± 0.39 × 10−3 vs. 0.37 × 10−3 ± 0.31 × 10−3, n = 13, P = 0.37, Unpaired t-test). (B,C) Significantly decreased PD-1 expression on CD8+ T cells (B, median; 2.70, IQR: 2.36–3.21, n = 35, vs. median: 2.56 IQR: 2.24–2.82, n = 40, P = 0.023, Mann Whitney test) and CD4+ T cells (C, 3.35 ± 0.53, n = 35 vs. 3.10 ± 0.45, n = 40, P = 0.032, Unpaired t-test) of ITP patients compared with healthy controls were observed. (D,E) Decreased PD-L1 expression on CD8+ T cells (D, 5.98 ± 1.35, n = 35 vs. 5.00 ± 1.77, n = 40, P = 0.010, Unpaired t-test) and CD4+ T cells (E, median: 5.58, IQR: 4.50–7.33, n = 35, vs. median: 2.25, IQR: 1.77–3.54, n = 40, P < 0.0001, Mann Whitney test) of ITP patients compared with healthy controls were observed. (F) The expression level of PD-L1 on platelet from ITP patients had no difference with that of healthy controls (median: 5.90, IQR: 5.42–6.31, n = 18, vs. median: 5.74, IQR: 5.34–6.61, n = 18, P = 0.894, Mann Whitney test). (G,H) Linear regression analysis of the PD-1 (G) and PD-L1 (H) expression on CD8+ T cells and platelet counts in ITP patients. *P < 0.05, **P < 0.01, ****P < 0.0001.

Figure 2

PD-L1-Fc reduced CTLs-mediated platelet apoptosis in ITP. (A) Platelets were gated by CD61 positive. (B) The gated dot-plots presented the apoptosis of platelets treated with PBS or PD-L1-Fc and cultured with CTLs of ITP patients treated with PBS or PD-L1-Fc. (C) A significant reduction was observed in CTLs-induced platelet apoptosis from ITP patients cultured with PD-L1-Fc in vitro after 72 h (9.76 ± 4.45 vs. 8.69 ± 4.29, n = 18, P = 0.006, Paired t-test). **P < 0.01.

Figure 3

Increased PD-1 methylation in ITP patients. (A) Bisulfite sequencing PCR analysis of PD-1 methylation. The 340-bp region (−975 to −636) containing 10 CpG residues in CpG rich area upstream of transcriptional start site (TSS) was amplified and sequenced. (B) The percentage of methylated residues in the PD-1 promoter of CD8+ T cells in ITP patients was significantly higher compared with healthy controls (position 1, 18.00 ± 14.83% vs. 58.00 ± 13.04%, P = 0.006; position 2, 30.00 ± 10.00% vs. 78.00 ± 13.04%, P = 0.002; position 3, 30.00 ± 10.00% vs. 72.00 ± 10.95%, P = 0.002; position 4, 34.00 ± 11.40% vs. 78.00 ± 13.04%, P = 0.002; position 5, 34.00 ± 16.73% vs. 74.00 ± 11.40%, P = 0.006; position 6, 18.00 ± 13.04% vs. 44.00 ± 15.17%, P = 0.02; position 7, 32.00 ± 8.37% vs. 66.00 ± 5.48%, P = 0.0006; position 8, 34.00 ± 5.48% vs. 70.00 ± 15.81%, P = 0.005; position 9, 34.00 ± 5.58% vs. 76.00 ± 13.42%, P = 0.002; position 10, 8.00 ± 4.47% vs. 30.00 ± 7.07%, P = 0.002, n = 5, Multiple t-tests). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

Low-dose decitabine (Dec) increased PD-1 expression on CD8+ T cells and ameliorated thrombocytopenia in ITP. (A,B) Low-dose decitabine significantly increased PD-1 expression on CD8+ T cells in vitro (B, median: 2.33, IQR: 1.98–3.54 vs. median: 2.61, IQR: 2.09–4.18, n = 16, P = 0.001, Wilcoxon test). (C) Platelet counts in severe combined immunodeficiency (SCID) mice that received 5 × 104 splenocytes from either wild type C57 mice (♦, n = 10) treated with an equal volume of PBS, non-immunized CD61-knockout (KO) mice (▾, n = 10) treated with an equal volume of PBS, or platelet-immunized CD61-KO mice treated with an equal volume of PBS (•, n = 10) or decitabine (▴, n = 10). On day 21 and 28, a significantly higher platelet count was observed in the decitabine-treated group compared with PBS (day 21, 360.0 × 109 ± 27.04 × 109/L vs. 643.0 × 109 ± 45.97 × 109/L; P = 0.0005; day 28, 401.0 × 109 ± 23.87 × 109/L vs. 713.0 × 109 ± 52.35 × 109/L; P = 0.001). The horizontally dotted lines represent the normal mean platelet count ± SEM (dotted lines) from 40 SCID mice. Significance among groups was determined by ANOVA. (D) The expression of PD-1 on CD8+ T cells were upregulated in the spleen in decitabine treated ITP mice (D, 2.10 ± 0.65 vs. 3.18 ± 0.92, n = 6, P = 0.039, Unpaired t-test). (E) Increased platelet count was observed after low-dose decitabine treatment in ITP patients (17.08 × 109 ± 8.53 × 109/L vs. 70.75 × 109 ± 47.79 × 109/L, n = 12, P = 0.004, Paired t-test). (F,G) Elevated PD-1 expression on CD8+ T cells was observed after low-dose decitabine treatment in ITP patients (G, 1.24 ± 1.00 vs. 3.62 ± 2.28, n = 12, P = 0.018, Paired t-test). (H) Linear regression analysis of PD-1 expression and platelet counts in active ITP mice on day 28 after decitabine therapy (PBS group and decitabine group). (I) Linear regression analysis of the PD-1 expression on CD8+ T cells and platelet counts in ITP patients before and after decitabine treatment. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

Low-dose decitabine (Dec) decreased PD-1 promoter methylation in ITP in vitro. (A) The representative plots of bisulfite sequencing analysis of PD-1 methylation. (B) Low-dose decitabine significantly decreased the percentage of methylated CpG residues of PD-1 in CD8+ T cells (position 2, 14.00 ± 5.48% vs. 4.00 ± 5.48%, P = 0.020; position 3, 22.00 ± 8.37% vs. 10.00 ± 7.07%, P = 0.040, position 3, 26.00 ± 8.94% vs. 12.00 ± 8.47%, P = 0.034, n = 5, Multiple t-test). *P < 0.05.

Figure 6

Low-dose decitabine (Dec) inhibited CTLs-mediated platelet apoptosis in ITP. (A) The representative dot-plots characterized the apoptosis of platelets treated with PBS or 100 nM decitabine and cultured with CTLs of ITP patients treated with PBS or 100 nM decitabine. (B) A significant reduction in CTLs induced apoptosis of platelets cultured with low-dose decitabine was observed (15.40 ± 8.23 vs. 14.05 ± 7.32, n = 15, P = 0.018, Paired t-test). (C–E) Platelet spontaneous apoptosis and platelet apoptosis cultured with CTLs was measured before (C) and after (D) 12-week decitabine treatment for ITP patients. (E) CTLs-induced platelet apoptosis was significantly inhibited after 12-week decitabine administration (before: median: 4.98, IQR: 2.66–13.95 vs. after: median: 0.61, IQR: 0.37–5.34, n = 6, P = 0.031, Wilcoxon test). (F) Isolated CD8+ T cells were treated with PBS control or decitabine. Western blotting was performed on cell lysates. (G,H) Graphs present densitometry data of relative phosphorylated (p-) protein to total protein after PBS or 100 nM decitabine treatment (G, p-PI3K, 0.82 ± 0.095 vs. 0.53 ± 0.03, P = 0.017; H, p-AKT, 0.75 ± 0.035 vs. 0.50 ± 0.036, n = 3, P = 0.035, Paired t-test). *P < 0.05.

Figure 7

Blocking PD-1 can partly counteract the therapeutic effect of low-dose decitabine in inhibiting CTLs-mediated platelet apoptosis in ITP in vitro and in vivo. (A) Anti-human PD-1 antibody (Anti-hPD-1) partly counteracted decitabine effect in inhibiting CTLs-induced platelets apoptosis in ITP in vitro (13.63 ± 8.05 at Anti-hPD-1 vs. 13.12 ± 6.53 at Dec + Anti-hPD-1, n = 16, P = 0.961; 13.63 ± 8.05 at Anti-hPD-1 vs. 11.86 ± 6.48 at Dec, n = 16, P = 0.043, ANOVA). (B) Anti-mouse PD-1 antibody (Anti-mPD-1) offset the low-dose decitabine treatment in active ITP mice on days 21 and 28 (day 21, 595.0 × 109 ± 55.42 × 109/L at Dec vs. 396.7 × 109 ± 42.40 × 109/L at Dec + Anti-mPD-1, P = 0.026; day 28, 685.0 × 109 ± 51.03 × 109/L at Dec vs. 476.7 × 109 ± 27.16 × 109/L at Dec + Anti-mPD-1, n = 6, P = 0.008, ANOVA). *P < 0.05, **P < 0.01.