儿童急性B淋巴细胞白血病CD4＋CD25highFoxp3＋调节性T细胞亚群改变及意义

Changes of CD4 ＋CD25highFoxp3 ＋regulatory T cells and their significance in childhood B-cell acute lymphocytic leukemia

目的：探讨急性B淋巴细胞白血病（B-ALL）患儿CD4＋CD25highFoxp3＋调节性T细胞（ Treg）亚群改变及其在B-ALL免疫逃逸机制中的作用。方法急性期B-ALL患儿42例，正常同年龄对照组28例，分别于化疗前直接取血备检。采用流式细胞检测外周血CD4＋CD25 high Foxp3＋、CD4＋CD25high ICOS＋Foxp3＋、CD4＋CD25high ICOS-Foxp3＋细胞比例及IL-10、TGF-β、IL-35、TGF-βRII、ICOS、CD28蛋白表达水平；荧光定量PCR （ real-time PCR）检测CD4＋T细胞Smad3/4、TIEG1、Itch等mRNA表达；ELISA检测血浆中TGF-β浓度。结果（1）急性B-ALL患儿CD4＋CD25high Foxp3＋Treg细胞比例显著升高（P＜0．05），其中ICOS＋Foxp3＋和ICOS-Foxp3＋细胞比例均明显高于对照组（P＜0．05）， ICOS＋Foxp3＋/ICOS -Foxp3＋比值低于对照组（0．73±0．21 vs 1．87±0．59，P＜0．05）；（2）急性期B-ALL患儿ICOS＋Foxp3＋细胞转录因子Foxp3及抑制性细胞因子IL-10、IL-35和TGF-β表达水平显著上调（P＜0．05），ICOS-Foxp3＋细胞Foxp3表达略有增加，但差异无统计学意义（P＞0．05），其mTGF-β表达明显高于对照组（ P＜0．05）；（3）急性B-ALL患儿血浆TGF-β浓度明显高于对照组[（25.83±12．65） ng/ml vs （8．59±5．73） ng/ml， P＜0．05]，CD4＋T细胞表面TGF-βRII及下游信号分子Smad3/4、TIEG1、Itch表达水平显著上调（P＜0．05），CD4＋CD25highFoxp3＋Treg细胞表面ICOS、CD28表达亦明显增高（P＜0．05）。结论 TGF-β、ICOS、CD28信号过度活化可能是导致急性B-ALL患儿CD4＋CD25highFoxp3＋Treg细胞异常及其亚群比例失调的重要因素。

Objective To investigate the changes of CD4 ＋CD25highFoxp3 ＋regulatory T （Treg） cells and their significance in immune escape of childhood B-cell acute lymphocytic leukemia （ B-ALL ） . Methods Forty-two children with B-ALL and twenty-eight age-matched healthy children were enrolled in this study.Flow cytometry analysis was performed to evaluate the proportion of CD 4 ＋CD25high Foxp3 ＋Treg cells as well as CD4 ＋CD25high ICOS＋Foxp3 ＋and CD4 ＋CD25high ICOS-Foxp3 ＋subsets in peripheral blood samples.The expression of associated molecules including IL-10, TGF-β, IL-35, TGF-βRII, ICOS and CD28 at protein level were also measured by flow cytometry analysis .The transcription level of Smad3/4, TIEG1 and Itch by CD4 ＋T cells were determined by quantitative real-time PCR.The concentration of TGF-βin plasma was detected by enzyme-linked immunosorbent assay.Results （1）The proportion of CD4 ＋CD25highFoxp3 ＋Treg cells in children with B-ALL were significantly higher than those of health subjects （P〈0.05）.The proportion of both ICOS ＋Foxp3 ＋and ICOS -Foxp3 ＋subsets were increased in comparison with those of control group （P〈0.05）, while the ratio of ICOS ＋Foxp3 ＋to ICOS-Foxp3 ＋was decreased （0.73 ±0.21 vs 1.87 ±0.59, P〈0.05）.（2） The expression of Foxp3, TGF-β, IL-10 and IL-35 by ICOS＋Foxp3 ＋Treg cells and the expression of membrane bound TGF-βby ICOS -Foxp3 ＋Treg cells were significantly increased in children with B-ALL （P〈0.05）.However, the expression of Foxp3 by ICOS -Foxp3 ＋Treg cells showed no significant difference between the two groups （P〉0.05）.（3）The concentra-tion of TGF-βin plasma from children with B-ALL were higher than those from control group [ （ 25 .83 ±12.65） ng/ml vs （8.59 ±5.73） ng/ml, P〈0.05].The expression of TGF-βRII and its associated mole-cules （Smad3/4, TIEG1 and Itch） by CD4 ＋T cells were significantly up-regulated.Moreover, an increased expression of ICOS and CD28 by CD4 ＋CD25highFoxp3 ＋Treg cells were also observed in children with B-ALL （P〈0.05）.Conclusion The hyper-activity of TGF-β, ICOS and CD28 signaling might be closely associ-ated with the increased proportion of CD4 ＋CD25high Foxp3 ＋Treg cells and the imbalance of its subsets in children with B-ALL.