氧化低密度脂蛋白干预记忆性CD8^+T细胞亚群的分化

Oxidized low-density lipoprotein modulates differentiation of murine memory CD8^+T cell subpopulations

目的探讨氧化低密度脂蛋白(ox-LDL)对CD8+记忆性T细胞亚群分化的影响及其机制。方法将雌性10周龄未发生糖尿病NOD小鼠随机分为2组(n=5):ox-LDL组及对照组。断颈处死各组小鼠磁珠分离脾脏CD8+T细胞体外培养(IL-2),ox-LDL组加入ox-LDL 30μg/m L体外刺激24 h,ox-LDL未处理组作为对照组。24 h后分离细胞。体外实验利用流式细胞仪检测各组以下指标:胞内CD8+IFN-γ+确定胰岛β细胞特异性杀伤性CD8+T细胞的比例;细胞表面分子CD8、CD44、CD62L确定CD8+记忆性T细胞的不同亚群分化;T细胞相关转录因子TCF-1及STAT-3的活化确定关键性转录因子的作用。体内实验中,将各组CD8+记忆性T细胞通过尾静脉注射的方法移植给未发病NOD小鼠,同时设立未移植的模型对照组,每5 d检测血糖1次,监测小鼠的血糖改变及生存率。结果相比对照组,ox-LDL(30μg/m L)作用24 h后,胰岛β细胞特异性杀伤性CD8+T细胞活化减少[(0.7±0.03)%vs(2.7±0.14)%,P<0.01],记忆性T细胞中的效应记忆性T细胞形成数目减少[(10.3±0.71)%vs(30.3±1.36)%,P<0.01],干细胞样记忆性T细胞增加[(72.3±3.8)%vs(55.1±2.61)%,P<0.05]。T细胞转录因子TCF-1及p-STAT-3活化的也受到抑制[TCF-1:(14.5±0.82)%vs(34.2±1.23)%,磷酸化STAT-3:(3.3±0.12)%vs(22.1±1.1)%,P<0.01]。T细胞移植给未发病NOD小鼠后,ox-LDL处理组小鼠血糖升高明显低于对照组(P<0.05),且生存率提高(P<0.05)。结论 ox-LDL干扰记忆性CD8+T细胞核转录因子TCF-1活化及STAT-3的磷酸化,抑制长效杀伤作用的效应性和记忆性CD8+T细胞形成,诱导干细胞样记忆性CD8+T细胞形成,从而阻止胰岛β细胞自身免疫损伤及Ⅱ型糖尿病的发生。

Objective To investigate effect of oxidized low-density lipoprotein （ox-LDL） on memory CD8＋T cell subpopulation differentiation in mice with autoimmune diabetes. Methods Cultured splenic CD8 ＋ T cells from pre-diabetic NOD mice isolated with magnetic beads were treated with 30 μg/mL ox-LDL and 10 U/mL interleukin-2 （IL-2） for 24 h and the control cells were treated with IL-2 only. Flow cytometry was used to determine the percentage of splenic CD8 ＋IFN-γ ＋ T cells, expressions of CD8, CD44 and CD62L on the T cells, and the activation of T cell factor-1 （TCF-1） and STAT-3. The CD127＋memory T cells were purified and transplanted into the pre-diabetic NOD mice via the tail vein, and the blood glucose was recorded weekly and survival time of the mice was monitored. Results Treatment with ox-LDL significantly reduced islet βcell-specific cytotoxic CD8＋T cells as compared with the control group[（0.7＆#177;0.03）%vs （2.7＆#177;0.14）%, P〈0.01]. The percentage of effector memory CD8＋T cells （Tem） in the total memory CD8＋T cells was reduced[（10.3 ＆#177; 0.71）%vs （30.3 ＆#177; 1.36）%, P〈0.01]and that of stem cell-like memory T cells was significantly increased [（72.3 ＆#177; 3.8）% vs （55.1 ＆#177; 2.61）%, P〈0.05] following ox-LDL treatment, which also resulted in significantly decreased activation of TCF-1 [（14.5 ＆#177; 0.82）% vs （34.2 ＆#177; 1.23）%, P〈0.01] and pSTAT-3[（3.3 ＆#177; 0.12）% vs （22.1 ＆#177; 1.1）%, P〈0.01]. Transplantation of ox-LDL-treated memory T cells in pre-diabetic NOD mice obviously inhibited the increase of blood glucose and prolonged the survival time of the mice （P〈0.05）. Conclusion Ox-LDL decreases the activation of transcriptional factors TCF-1 and phosphorylation of STAT-3, inhibits the formation of effector memory CD8 ＋ T cells with long-term cytotoxicity, but promote the generation of stem cell-like memory CD8＋ T cells, which result in suppression of islet β cell-specific effector cytotoxic CD8 ＋ T cell differentiation to lessen autoimmune injury to the isletβcells.