Figure 5.
LKB1 shRNA interference offset the altered metabolic activity and suppressed function augmentation of MDSC induced by decitabine in vitro. (A) LKB1 shRNA transfection for 3 days successfully silenced LKB1 in the MDSCs of ITP patients, achieving ≥ 69.26% LKB1 knockdown, determined via RT-PCR (n = 7, paired t tests, ∗∗P = .0091). (B) The MDSCs of patients with ITP were treated with NC shRNA, NC shRNA + Dec, LKB1 shRNA, or LKB1 shRNA + Dec, during in vitro culture, and the OCR was measured. (C-E) Respective mitochondrial parameters including basal respiration (paired t tests, ∗∗∗∗PNC shRNA vs LKB1 shRNA < .0001, PLKB1 shRNA vs LKB1 shRNA+Dec = .0909), ATP production (paired t tests, ∗∗PNC shRNA vs LKB1 shRNA = .0036, PLKB1 shRNA vs LKB1 shRNA+Dec = .2269), and maximal respiration (paired t tests, ∗∗PNC shRNA vs LKB1 shRNA = .0035, PLKB1 shRNA vs LKB1 shRNA+Dec = .0561). (F) In ITP patients, intercellular ATP level was lower in LKB1 shRNA-MDSCs than in NC shRNA-MDSCs, with no significant improvement after decitabine treatment (paired t tests, ∗PNC shRNA vs LKB1 shRNA= .0436, PLKB1 shRNA vs LKB1 shRNA+Dec = .1181). (G) Representative histograms of CD4+CFSE+ effector T-cell proliferation. The effector T-cell division index reflects cell proliferation after 5 days of coculture with MDSCs. (H) The inhibitory function of effector T cells cocultured with LKB1 shRNA-MDSCs was lower than that cocultured with NC shRNA-MDSCs (paired t tests, ∗∗∗∗PNC shRNA vs LKB1 shRNA < .0001); there was no significant statistical difference in the immunosuppression of MDSCs transfected with LKB1 shRNA whether treated with decitabine or not (paired t tests, PLKB1 shRNA vs LKB1 shRNA+Dec = .0729); after treatment with decitabine, the immunosuppression of MDSCs transfected with NC shRNA was significantly increased (paired t-test, ∗P NC shRNA vs NC shRNA+Dec =.0113). (I) The gated dot plots present CTL-induced platelet apoptosis after 3 days of coculture with NC shRNA or LKB1 shRNA MDSCs in ITP. (a) Platelets only. (b) Platelets + CTLs. (c) Platelets + CTLs + NC shRNA-MDSC(PBS). (d) Platelets + CTLs + NC shRNA-MDSC(Dec). (e) Platelets + CTLs + LKB1 shRNA-MDSC(PBS). (f) Platelets + CTLs + LKB1 shRNA-MDSC(Dec). (J) LKB1 blockade resulted in significantly weaker immunosuppressive capacity of MDSCs on the cytotoxicity of CTLs, compared with controls (paired t test, ∗∗PPLT+CTL+NC shRNA-MDSC(PBS) vs PLT+CTL+LKB1 shRNA-MDSC(PBS) = .0022). Moreover, LKB1 shRNA interference masked the effect of decitabine to enhance MDSC function (∗∗∗∗PPLT+CTL vs PLT+CTL+NC shRNA-MDSC(Dec) < .0001, paired t test, PPLT+CTL+LKB1 shRNA-MDSC(PBS) vs PLT+CTL+LKB1 shRNA-MDSC(Dec) = .3121, ∗∗∗PPLT+CTL+NC shRNA-MDSC(PBS) vs PLT+CTL+NC shRNA-MDSC(Dec) = .0007).

LKB1 shRNA interference offset the altered metabolic activity and suppressed function augmentation of MDSC induced by decitabine in vitro. (A) LKB1 shRNA transfection for 3 days successfully silenced LKB1 in the MDSCs of ITP patients, achieving ≥ 69.26% LKB1 knockdown, determined via RT-PCR (n = 7, paired t tests, ∗∗P = .0091). (B) The MDSCs of patients with ITP were treated with NC shRNA, NC shRNA + Dec, LKB1 shRNA, or LKB1 shRNA + Dec, during in vitro culture, and the OCR was measured. (C-E) Respective mitochondrial parameters including basal respiration (paired t tests, ∗∗∗∗PNC shRNA vs LKB1 shRNA < .0001, PLKB1 shRNA vs LKB1 shRNA+Dec = .0909), ATP production (paired t tests, ∗∗PNC shRNA vs LKB1 shRNA = .0036, PLKB1 shRNA vs LKB1 shRNA+Dec = .2269), and maximal respiration (paired t tests, ∗∗PNC shRNA vs LKB1 shRNA = .0035, PLKB1 shRNA vs LKB1 shRNA+Dec = .0561). (F) In ITP patients, intercellular ATP level was lower in LKB1 shRNA-MDSCs than in NC shRNA-MDSCs, with no significant improvement after decitabine treatment (paired t tests, ∗PNC shRNA vs LKB1 shRNA= .0436, PLKB1 shRNA vs LKB1 shRNA+Dec = .1181). (G) Representative histograms of CD4+CFSE+ effector T-cell proliferation. The effector T-cell division index reflects cell proliferation after 5 days of coculture with MDSCs. (H) The inhibitory function of effector T cells cocultured with LKB1 shRNA-MDSCs was lower than that cocultured with NC shRNA-MDSCs (paired t tests, ∗∗∗∗PNC shRNA vs LKB1 shRNA < .0001); there was no significant statistical difference in the immunosuppression of MDSCs transfected with LKB1 shRNA whether treated with decitabine or not (paired t tests, PLKB1 shRNA vs LKB1 shRNA+Dec = .0729); after treatment with decitabine, the immunosuppression of MDSCs transfected with NC shRNA was significantly increased (paired t-test, ∗P NC shRNA vs NC shRNA+Dec =.0113). (I) The gated dot plots present CTL-induced platelet apoptosis after 3 days of coculture with NC shRNA or LKB1 shRNA MDSCs in ITP. (a) Platelets only. (b) Platelets + CTLs. (c) Platelets + CTLs + NC shRNA-MDSC(PBS). (d) Platelets + CTLs + NC shRNA-MDSC(Dec). (e) Platelets + CTLs + LKB1 shRNA-MDSC(PBS). (f) Platelets + CTLs + LKB1 shRNA-MDSC(Dec). (J) LKB1 blockade resulted in significantly weaker immunosuppressive capacity of MDSCs on the cytotoxicity of CTLs, compared with controls (paired t test, ∗∗PPLT+CTL+NC shRNA-MDSC(PBS) vs PLT+CTL+LKB1 shRNA-MDSC(PBS) = .0022). Moreover, LKB1 shRNA interference masked the effect of decitabine to enhance MDSC function (∗∗∗∗PPLT+CTL vs PLT+CTL+NC shRNA-MDSC(Dec) < .0001, paired t test, PPLT+CTL+LKB1 shRNA-MDSC(PBS) vs PLT+CTL+LKB1 shRNA-MDSC(Dec) = .3121, ∗∗∗PPLT+CTL+NC shRNA-MDSC(PBS) vs PLT+CTL+NC shRNA-MDSC(Dec) = .0007).

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