IL-7通过调节CD127表达调控脓毒症患者CD8 +T细胞的体外活性

IL-7 regulates in vitro activity of CD8+T cells in patients with sepsis through modulation of CD127 expression

目的:观察脓毒症患者中膜型CD127(membrane-bound CD127,mCD127)和可溶型CD127(soluble CD127,sCD127)的表达,探讨IL-7调控脓毒症患者CD8 +T细胞活性的机制。 方法:采用前瞻性研究队列,入组47例脓毒症患者和18例健康对照者,分离血清和外周血单个核细胞(peripheral blood mononuclear cells,PBMCs),纯化CD8 +T细胞,酶联免疫吸附试验检测血清中IL-7和sCD127,流式细胞术检测CD8 +T细胞表面mCD127表达,实时定量PCR法检测CD8 +T细胞中总CD127 mRNA和sCD127 mRNA相对表达量。使用重组人IL-7刺激CD8 +T细胞并抑制信号转导和转录激活因子5(signal transducer and activator of transcription 5,STAT5)、磷脂酰肌醇-3-激酶(phosphatidylinositol 3-kinase,PI3K)通路,检测CD8 +T细胞表面mCD127表达、总CD127 mRNA和sCD127 mRNA相对表达量的变化,利用CD8 +T细胞与MCF-7细胞共培养系统评估IL-7刺激后CD8 +T细胞杀伤功能的变化。采用 t检验或LSD- t检验进行统计分析。 结果:脓毒症患者血清中IL-7和sCD127水平均低于对照组[(101.82±12.58) pg/ml vs (111.07±11.10) pg/ml, P<0.01;(278.58±62.31) pg/ml vs (334.62±70.55) pg/ml, P<0.01],且两者水平呈正相关( r=0.46, P<0.01)。脓毒症患者CD8 +T细胞中mCD127 +CD8 +T细胞比例和mCD127平均荧光强度均高于对照组( P<0.05)。脓毒症患者CD8 +T细胞中sCD127 mRNA相对表达量低于对照组(1.34±0.33 vs 1.80±0.60, P<0.001)。重组人IL-7刺激后CD8 +T细胞sCD127分泌水平、总CD127和sCD127 mRNA相对表达量升高( P<0.05),抑制STAT5后IL-7诱导的sCD127分泌水平、总CD127和sCD127 mRNA相对表达量降低( P<0.05),但抑制PI3K后IL-7诱导的sCD127分泌水平、总CD127和sCD127 mRNA相对表达量变化无统计学意义( P>0.05)。脓毒症患者CD8 +T细胞诱导靶细胞死亡的比例低于对照组[(12.49±2.12)% vs (23.83±3.76)%, P<0.001],重组人IL-7刺激后CD8 +T细胞诱导靶细胞死亡的比例升高( P<0.05),细胞因子和毒性颗粒分泌增加( P<0.05),抑制STAT5后IL-7诱导的CD8 +T细胞杀伤功能减弱( P<0.05),但抑制PI3K后IL-7诱导的CD8 +T细胞杀伤功能变化无统计学意义( P>0.05)。 结论:IL-7可增加sCD127分泌,通过STAT5信号通路增强脓毒症患者CD8 +T细胞的体外杀伤功能。

Objective To investigate the expression of membrane-bound CD127(mCD127)and soluble CD127(sCD127)in patients with sepsis,and to assess the mechanism of IL-7 in regulating CD8+T cell activity in these patients.Methods A prospective cohort study was conducted on 47 patients with sepsis(sepsis group)and 18 healthy controls(control group).Serum samples and peripheral blood mononuclear cells(PBMCs)were isolated.CD8+T cells were purified.IL-7 and sCD127 levels in serum were measured by enzyme-linked immunosorbent assay.Expression of mCD127 on CD8+T cells was measured by flow cytometry.Total CD127 and sCD127 expression at mRNA level in CD8+T cells was semi-quantified by real-time PCR.CD8+T cells were stimulated with recombinant human IL-7,along with signal transducer and activator of transcription 5(STAT5)inhibitor or phosphatidylinositol 3-kinase(PI3K)inhibitor.Changes in mCD127 expression on CD8+T cells and the expression of total CD127 and sCD127 at mRNA level were then measured.The cytotoxicity of CD8+T cells in response to IL-7 stimulation was assessed using a co-culture system with CD8+T cells and MCF-7 cells.Student′s t test and LSD-t test were used for statistical analysis.Results Serum IL-7 and sCD127 were lower in sepsis group than in control group[(101.82±12.58)pg/ml vs(111.07±11.10)pg/ml,P<0.01;(278.58±62.31)pg/ml vs(334.62±70.55)pg/ml,P<0.01].Serum IL-7 was positively correlated with serum sCD127 in patients with sepsis(r=0.46,P<0.01).The percentage of mCD127+CD8+T cells in CD8+T cells and the mean fluorescence intensity of mCD127 in sepsis group were higher than those in control group(P<0.05).The expression of sCD127 at mRNA level in CD8+T cells was lower in sepsis group than in control group(1.34±0.33 vs 1.80±0.60,P<0.001).Stimulation with recombinant human IL-7 promoted sCD127 secretion and total CD127 and sCD127 expression at mRNA level in CD8+T cells(P<0.05).Inhibition of STAT5 suppressed the IL-7-induced sCD127 secretion and total CD127 and sCD127 expression at mRNA level(P<0.05).However,inhibition of PI3K could not achieve those effects(P>0.05).CD8+T cells-induced target cell death was inhibited in sepsis group as compared with that in control group[(12.49±2.12)%vs(23.83±3.76)%,P<0.001].Recombinant human IL-7 promoted the CD8+T cell-induced target cell death(P<0.05)and increased the secretion of cytokines and cytotoxic granule proteins(P<0.05).Inhibition of STAT5 suppressed IL-7-mediated CD8+T cell cytotoxicity(P<0.05).However,inhibition of PI3K did not affect IL-7-mediated CD8+T cell cytotoxicity(P>0.05).Conclusions IL-7 promoted sCD127 secretion and enhanced the in vitro cytotoxicity of CD8+T cells in patients with sepsis through STAT5 signal pathway.