RhoA、CD4^(+)CD25^(+)调节性T细胞、MYBL2在胃癌患者中的表达及对预后和生存时间的影响

目的:探讨RhoA、CD4^(+)CD25^(+)调节性T细胞、成髓细胞瘤转录因子第2亚型(MYBL2)在胃癌患者中的表达及对预后和生存时间的影响。方法:选取2017年1月至2019年1月于本院就诊的134例胃癌患者术后癌组织标本作为研究对象,另选取其中62例胃癌患者相应的癌旁组织标本及40例同期非胃癌患者正常胃黏膜组织标本进行对照,检测癌组织、癌旁组织、正常胃黏膜组织中RhoA、CD4^(+)CD25^(+)调节性T细胞、MYBL2的表达情况,分析其对胃癌预后及生存时间的影响。结果:RhoA,CD4^(+)CD25^(+)调节性T细胞、MYBL2在癌组织中的阳性率均明显高于癌旁组织和正常胃黏膜组织(P<0.05)。胃癌患者术后平均生存时间为(28.61±1.34)个月,其中RhoA、CD4^(+)CD25^(+)调节性T细胞、MYBL2阳性患者的生存时间均短于阴性患者(P<0.05)。RhoA(+)、CD4^(+)CD25^(+)调节性T细胞(+)、MYBL2(+)是胃癌患者预后的危险因素(P<0.05)。结论:检测RhoA,CD4^(+)CD25^(+)调节性T细胞、MYBL2的表达可作为胃癌病情严重程度、预后及生存时间评估的重要补充手段。

外周血CD4^(+)CD25^(+)、CD8^(+)CD28^(+)调节性T细胞水平对早期宫颈癌患者腹腔镜根治术后预后的预测价值

目的分析外周血CD4^(+)CD25^(+)、CD8^(+)CD28^(+)调节性T细胞水平对早期宫颈癌(CC)患者腹腔镜根治术后预后的预测价值。方法招募2018年9月至2020年9月于儋州市人民医院接受腹腔镜根治术治疗的早期CC患者204例,根据患者术后随访期间预后情况分为预后不良组(43例)和预后良好组(161例)。比较两组临床资料。采用Spearman秩相关分析外周血CD4^(+)CD25^(+)调节性T细胞水平与CD8^(+)CD28^(+)调节性T细胞水平的相关性。采用多因素logistic回归分析早期CC患者腹腔镜根治术后预后不良的影响因素。采用受试者工作特征(ROC)曲线评估外周血CD4^(+)CD25^(+)、CD8^(+)CD28^(+)调节性T细胞水平对早期CC患者腹腔镜根治术后预后不良的预测价值。结果预后不良组CD4^(+)CD25^(+)调节性T细胞、癌胚抗原(CEA)、糖类抗原125(CA125)水平,术后切缘阳性占比以及术中宫旁浸润占比高于预后良好组,CD8^(+)CD28^(+)调节性T细胞水平低于预后良好组,差异有统计学意义(P<0.05)。Spearman秩相关分析结果显示,早期CC患者外周血CD4^(+)CD25^(+)调节性T细胞水平与CD8^(+)CD28^(+)调节性T细胞水平呈负相关(r_(s)=-0.478,P<0.05)。多因素logistic回归分析结果显示,较高的CEA、CA125、CD4^(+)CD25^(+)调节性T细胞水平是促进早期CC患者腹腔镜根治术后预后不良发生的独立危险因素(P<0.05),较高的CD8^(+)CD28^(+)调节性T细胞水平是抑制早期CC患者腹腔镜根治术后预后不良发生的独立保护因素(P<0.05)。ROC曲线分析结果显示,外周血CD4^(+)CD25^(+)、CD8^(+)CD28^(+)调节性T细胞水平能有效预测早期CC患者腹腔镜根治术后预后不良(P<0.05),两项指标联合可进一步提高预测效能[AUC(95%CI)=0.939(0.898~0.979),P<0.001],灵敏度和特异度分别为86.00%、88.20%。结论外周血CD4^(+)CD25^(+)、CD8^(+)CD28^(+)调节性T细胞水平与早期CC患者腹腔镜根治术后预后不良有关,二者能有效预测早期CC患者腹腔镜根治术后预后不良。

口腔扁平苔藓组织中IL-35表达水平与外周血CD4^(+)CD25^(+)Treg的相关性

目的探究口腔扁平苔藓(OLP)组织中白介素-35(IL-35)表达水平与外周血CD4^(+)CD25^(+)调节性T细胞(Treg)的相关性。方法收集2016年9月至2018年9月期间我院口腔科收治的口腔扁平苔藓患者38例,设为OLP组,另选同期来我院体检的健康人群20例作为对照组。取两组外周静脉血,荧光定量PCR检测外周血单个核细胞内IL-35两个亚基EB病毒诱导蛋白3(EBI3)、IL-12 p35和CD4^(+)CD25^(+)Treg标志物叉状头转录因子P3(FOXP3)的表达,酶联免疫吸附法检测血清IL-35、白介素-10(IL-10)、白介素-17(IL-17)和转化生长因子β1(TGF-β1)的含量,流式细胞仪检测外周血CD4^(+)CD25^(+)Treg细胞水平,分析IL-35和CD4^(+)CD25^(+)Treg水平与OLP患者临床病理特征之间的关系,Pearson法分析EBI3、IL-35与CD4^(+)CD25^(+)Treg的相关性。结果与对照组相比,OLP组外周血单个核细胞内EBI3、IL-12 p35、FOXP3的mRNA均显著上调(P<0.05)。与对照组相比,OLP组患者血清IL-35、IL-10、IL17、TGF-β1含量显著均上调,差异有统计学意义(P<0.05);与对照组相比,OLP组患者外周血CD4^(+)T细胞和CD4^(+)CD25^(+)Treg细胞水平均显著下降,差异有统计学意义(P<0.05);IL-35和CD4^(+)CD25^(+)Treg水平与患者基底细胞变性程度有关(P<0.05),而与性别、年龄、病程、疾病类型和淋巴细胞浸润无关(P>0.05)。OLP中IL-35与外周血CD4^(+)CD25^(+)Treg的水平呈正相关(r=0.3644,P<0.05)。结论OLP中IL-35与CD4^(+)CD25^(+)Treg表达均显著升高,且二者具有正相关关系。

外周血CD4^(+)PD-1^(+)Tcells及CD4^(+)T淋巴细胞ATP含量与复发性卵巢癌疗效的相关性分析

目的 探讨外周血CD4^(+)程序性细胞死亡受体-1(PD-1)^(+)T cells及CD4^(+)T淋巴细胞三磷酸腺苷(ATP)含量与复发性卵巢癌疗效的相关性。方法 选取30例复发性卵巢癌患者为复发组,30例未复发卵巢癌患者为非复发组;另选取30名同期体检健康者作为对照组。评估外周血CD4^(+)PD-1^(+)T cells及CD4^(+)T淋巴细胞ATP含量与复发性卵巢癌疗效的相关性。结果 复发组和非复发组的CD4^(+)PD-1^(+)T cells较对照组明显升高(P<0.05)。复发组和非复发组的CD4^(+)T淋巴细胞ATP含量较对照组明显降低(P<0.05)。复发组治疗后CD4^(+)PD-1^(+)Tcells显著低于治疗前(P<0.05),治疗后CD4^(+)T淋巴细胞ATP含量显著高于治疗前(P<0.05)。CD4^(+)PD-1^(+)T cells与复发性卵巢癌疗效成负相关(r=-0.393,P=0.039),CD4^(+)T淋巴细胞ATP含量与复发性卵巢癌疗效成正相关(r=0.449,P=0.031)。结论 复发性卵巢癌患者外周血CD4^(+)PD-1^(+)T cells及CD4^(+)T淋巴细胞ATP含量与疗效密切相关。

Analysis of CD4^+CD25^+ Regulatory T Cells and Foxp3 mRNA in the Peripheral Blood of Patients with Asthma

The changes of CD4^＋CD25^＋ regulatory T cells （CD4^＋CD25^＋ Treg） and Foxp3 mRNA in peripheral blood mononuclear cells （PBMCs） from patients with asthma were investigated in order to elucidate the possible roles of CD4^＋CD25^＋ Treg in the development of asthma. The peripheral blood samples were collected from 29 healthy controls （normal control group） and 78 patients with asthma which included 30 patients in exacerbation group, 25 patients in persistent group, and 23 patients in remission group. By using flow cytometry and RT-PCR, the CD4^＋CD25^＋ Treg ratio and Foxp3 mRNA in PBMCs were detected. The CD4^＋CD25^＋ Treg ratio and Foxp3 mRNA in PBMCs of exacerbation and persistent groups were lower than that of remission and normal control groups （P〈0.05）. Although the CD4^＋CD25^＋ Treg ratio and Foxp3 mRNA of remission group were also lower than that of normal control group, there was no significant difference between them （P〉0.05）. As compared with persistent group, exacerbation group had lower CD4^＋CD25^＋ Treg ratio and Foxp3 mRNA （P〈0.05）. It was indicated that the decrease of CD4^＋CD25^＋ Treg ratio and its function in PBMCs may be responsible for pathogenesis of asthma.

Effects of estrogen on CD4^+ CD25^+ regulatory T cell in peripheral blood during pregnancy

Objective To investigate the effects of estrogen(E_2)level on regulatory T cells(Treg)in peripheral blood during pregnancy.Methods:A total of 30 healthy non-pregnant women were selected as control group,90 pregnant women of early,middle and late pregnancy and 30 postpartum women at 1 month after parturition were selected as experimental groups including early pregnancy group,middle pregnancy group and late pregnancy group;the proportions of CD4^+CD25^+Treg and CD4^+CD25^+CD127^-Treg among CD4 T cells were detected by flow cytometry;the serum estrogen content in peripheral blood was detected by electrochemical immune luminescence method.Results:E_2 level was coincident with the change of Tregs number during pregnancy.The estrogen content in peripheral blood increased gradually from early pregnancy to late pregnancy,then decreased significantly after parturition,and the level at 1 month after parturition down to the level in non-pregnancy group(P>0.05);the level of E_2 in pregnancy groups were significantly higher than those in non-pregnancy group(P<0.01);and there were significant differences among early pregnancy group,middle pregnancy group and late pregnancy group(P<0.05).The proportions of CD4^+CD25^+Treg and CD4^+CD25^+CD127^-Treg in pregnancy groups were significantly higher than those in non-pregnancy group(P<0.05),but decreased significantly after parturition,and there was no significant difference between non-pregnancy group and postpartum women group(P>0.05):the proportions in middle and late pregnancy groups were significantly higher than those in early pregnancy group(P<0.05).but decreased slightly in late pregnancy group,there was no significant difference between late pregnancy group and middle pregnancy group(P>0.05).There was correlation between Tregs number with estrogen level during pregnancy.The proportion of CD4^+CD25^+Treg and CD4^+CD25^+CD 127^-Treg were positively correlated with estrogen level.Conclusions:High proportion of CD4^+CD25^+Trcg and CD4^+CD25^+CD127^-Treg is closely related to the high level of E,during pregnancy.It suggested that high level of estrogen may induce an increase of CD4^+CD25^+Treg in peripheral blood.and then influence the immune function of pregnant women.The results of this experiment might play an important role of estrogen in immune-modulation during pregnancy.

An Association between Immunosenescence and CD4^+CD25^+ Regulatory T Cells: A Systematic Review

Objective Age-related increment of the prevalence of CD4^＋CD25^＋ regulatory T （Treg） cells were described controversially, and whether such changes explain immune dysfunction in the elderly is still unclear. The aim of this systematic review is to evaluate the role of the Tregs in immunosenescence. Methods Medline and manual searches were performed to identify all published epidemiological and animal studies investigating the efficacy of the association between immunosenescence and Treg cells. Results It was founded that the frequency, phenotypic characteristics, and number/function of Tregs were altered significantly with aging. Medical conditions in individuals with advanced ageas well as apoptosis intensity of Treg cells had an impact on the accumulation of Tregs which in turn could deteriorate cytotoxic activity of CD8＋ T and NK cells and production of IL-2. The range of immune cells that could be suppressed by Treg cells was quite wide and covered CD4^＋CD25^＋ T cells, NK cells, dendritic cells and even monocytes. These changes were observed both in humans and experimental animals. Besides, it was believed that frequency of Tregs increased with age and was accompanied by intensified suppressive activity for Tregs in patients, for example, with Alzheimer disease （AD） and Parkinson disease （PD）. The impaired condition of CD4＋ T cells, so-called immunosenescence, rendered transplant recipients less responsive to an allogeneic kidney graft, an effect that was limited to transplant recipients who were aged over 60 years. Conclusions Treg cells are associated with immunosenescence. All these changes contribute to the aging-related decline of immune responses and lead to the higher risk of immune-mediated diseases, cancer or infections in aged individuals.

急性非ST抬高心肌梗死患者外周血CD4^(+)CD25^(+)Foxp3^(+)Treg和IL-27变化的临床意义

目的观察急性非ST抬高心肌梗死患者(Non-STMI)治疗过程中外周血CD4^(+)CD25^(+)T/CD4^(+)T、CD4^(+)CD25highFoxp3^(+)Treg/CD4^(+)CD25high T比例及细胞因子TGF-β1、IL-10、INF-γ、IL-27的浓度变化,阐明Non-STMI患者是否存在调节性T细胞比例及功能异常。方法选取2012年8月至2016年4月入住中山大学附属第一医院急诊病房和CCU的Non-STMI40名,不稳定性心绞痛(UA)患者19名和同龄健康志愿者20名作为对照组(HC)。患者诊断明确后次日清晨和经规范治疗后5~7天,留取空腹外周血标本。流式细胞术检测CD4^(+)CD25^(+)T/CD4^(+)T、CD4^(+)CD25high Foxp3^(+)Treg/CD4^(+)CD25high T细胞。ELISA法检测细胞因子TGF-β1、IL-10、INF-γ和IL-27浓度。结果UA、Non-STMI组CD4^(+)CD25highFoxp3^(+)Treg/CD4^(+)CD25highT比例较HC组减低(P<0.01);Non-STMI明显低于UA组(P<0.05),而治疗后Non-STMI组Foxp3^(+)Treg细胞比例显著升高(P<0.05)。入院时Non-STMI和UA组TGF-β1、IL-10浓度较HC组明显降低(P<0.05),而INF-γ和IL-27浓度明显升高(P<0.05)。治疗后Non-STMI组IL-27迅速下降,而UA组变化不明显。治疗前IL-27浓度与CD4^(+)CD25highFoxp3^(+)Treg/CD4^(+)CD25highT呈线性负相关、与INF-γ浓度呈线性正相关。治疗后只有IL-27和CD4^(+)CD25high Foxp3^(+)Treg/CD4^(+)CD25highT呈线性负相关。结论Non-STMI患者CD4^(+)CD25highFoxp3^(+)Treg比例及功能异常,细胞因子IL-27有助于判断患者的临床疗效。

Changes of CD4^+CD25^+ Regulatory T Cells in Patients with Acute Coronary Syndrome and the Effects of Atorvastatin

The function of CD4＋CD25＋ regulatory T lymphocytes （Treg） in patients with acute coronary syndrome （ACS） and the effects of atorvastatin were investigated. Forty-eight patients with ACS were randomly divided into two groups： group C receiving conventional therapy （n=24）, and group C＋A receiving conventional therapy＋atorvastatin （10 mg/day, n=24）. T lymphocytes from ACS patients （before and 2 weeks after the treatment） or 18 healthy subjects were separated and the flow cytometry was used to measure the percentage of Treg. The inhibitory ability of Treg on effector T cells was determined by mixed lymphocyte reaction （MLR）. ELISA was used to measure the serum levels of cytokines （IL-10, TGF-β1 and IFN-γ） before and after treatment. The results showed that as compared with normal control group, Treg percentage was decreased significantly （P〈0.01）, the inhibitory ability of Treg on the T lymphocytes proliferation was reduced （P〈0.01）, IFN-γ levels were increased and IL-10 and TGF-β1 levels were lowered in ACS patients. After treatment with atorvastatin, Treg percentage and the inhibitory ability of Treg on T lymphocytes proliferation were significantly increased in ACS patients. Serum IFN-γ was decreased significantly, while IL-10 and TGF-β1 were elevated significantly as compared with the non-atorvastatin group. The number of Treg was positively correlated with serum TGF-β1, but negatively with serum IFN-γ and CRP. It was concluded that ACS was associated with decreased number and defected function of Treg, which may play an important role in initiating immune-inflammatory response in ACS. The inhibitory effects of atorvastatin on inflammation in ACS may be due to its beneficial effects on Treg and restoration of immune homeostasis.

Influence of Danshen Injection on airway inflammation and CD4^+ CD25^+ regulatory T cells of asthmatic rats

Objective： To investigate the influence of Danshen Injection on airway inflammation and CD4^＋CD25^＋ regulatory T cells（CD4^＋CD25^＋ Tr） of asthmatic rats, and elucidate the possible mechanism of Danshen Injection in treatment of asthma. Methods： 30 Wister rats were randomly divided into control group, asthma group and Danshen Injection treated group. Bronchoalveolar lavage fluids （BALF） were collected, and cytology studies were conducted. Lung tissues were obtained and pathologic analyses were done with hematoxylin and eosin stain （HE）. Flow cytometry was used to detect the CD4^＋CD25^＋ Tr ratio in peripheral blood mononuclear cells （PBMCs）. Results： Total cell, the percentage of lymphocytes, neutrophils and eosinophils （Eos） in BALF of Danshen Injection-treated group were lower than that in asthma group （P〈0.05, P〈0.01）. Compared with asthma group, less infiltration of inflammatory cells in lung tissues was observed in Danshen Injection-treated group. CD4^＋CD25^＋ Tr of asthma group was lower than that of control and Danshen Injection treated group （P〈0.05）. Conclusion： Danshen Injection can suppress airway inflammation of asthmatic rats, probably by increasing the number of CD4^＋CD25^＋ Tr.

25-羟基胆固醇通过调控干扰素调节因子4抑制CD4^(+)T细胞IL-17的表达

目的探讨25-羟基胆固醇(25-hydroxycholesterol,25-HC)对人外周血单个核细胞(PBMCs)产生IL-17的抑制作用及其机制。方法用抗人CD3抗体联合抗CD28抗体刺激培养外周血单个核细胞(PBMCs)和纯化CD4^(+)T细胞,加或不加25-HC进行培养,ELISA、ELISPOT和PCR检测IL-17和IFN-γ的表达;流式细胞术检测T细胞转录因子(RORγt、RUNX1和IRF4)的表达、细胞表面活化分子的表达以及T细胞分裂增殖情况。通过电转染过表达IRF4,检测其对IL-17表达的影响。结果25-HC呈时间和剂量依赖的方式抑制T细胞产生IL-17,且主要抑制CD4^(+)T细胞中IL-17的表达。进一步研究发现,25-HC抑制T细胞晚期活化分子的表达和细胞分裂增殖,抑制IL-17相关转录因子IRF4的表达;过表达IRF4后,25-HC对IL-17表达的抑制作用被恢复。结论25-HC抑制Th17细胞的活化和增殖,并通过下调IRF4的表达抑制CD4^(+)T细胞产生IL-17。25-HC可能是IL-17相关炎症性疾病的一个具有前景的调控靶点。

类风湿性关节炎患者抗环瓜氨酸肽抗体、CD4^(+)CD25^(+)调节性T细胞与类风湿因子的检验价值分析

目的探讨抗环瓜氨酸肽抗体(Anti-CCP)、CD4^(+)CD25^(+)调节性T细胞与类风湿因子(RF)在类风湿性关节炎患者中的检出结果及意义。方法选取2019年1月至2021年5月的昆明市延安医院的130例疑似类风湿性关节炎患者作为研究对象,进行Anti-CCP、CD4^(+)CD25^(+)调节性T细胞与RF水平进行检测,以晨僵持续1 h以上、3组关节肿胀与特异性抗体检测阳性作为金标准,分析上述指标的诊断效能,并对比类风湿性关节炎患者和非类风湿性关节炎患者的上述指标水平、不同活动度的类风湿性关节炎患者的指标水平。结果RF、Anti-CCP与CD4^(+)CD25^(+)调节性T细胞检测对类风湿性关节炎的诊断准确率为97.69%、特异度为98.11%、敏感度为97.70%、阳性预测值为98.84%、阴性预测值为96.30%。类风湿性关节炎患者的RF平均值、Anti-CCP平均值指标高于非类风湿性关节炎患者,且CD4^(+)CD25^(+)调节性T细胞低于非类风湿性关节炎患者,差异有统计学意义(P<0.05)。高度活动组患者RF平均值、Anti-CCP平均值高于低中度活动组,且CD4^(+)CD25^(+)调节性T细胞低于低中度活动组,差异有统计学意义(P<0.05)。结论Anti-CCP、CD4^(+)CD25^(+)调节性T细胞与RF指标检测有助于对类风湿性关节炎患者进行鉴别,并判断患者的疾病活动情况。

Isolation and identification of CD4^+CD25^+ regulatory T cells in rat

Objective： To establish a stable and high efficient method for collection of CD4^＋CD25^＋ regulatory T cells from rats in vitro. Methods： CD4^＋CD25^＋ regulatory T cells were isolated from the rat splenic cells through two steps by magic cell sorting （MACS） system. The first step was negative selection of CD4^＋T cells by cocktail antibodies and anti-IgG magic microbeads, and the second step was positive selection of CD25^＋T cells by anti-CD25 PE and anti-PE magic microbeads. The purity and viability of separated cells were measured by flow cytometry （FACS） and Trypan blue staining. The suppressive ability of seperated cells on the proliferation of CD4^＋CD25^- T cells was assessed by cell proliferation assay. Results： The purity of negatively enriched CD4^＋ T cells was 79%-87% （83.6%±2.5% ） , and the purity of positively enriched CD4^＋CD25^＋ T cells was 86%- 93% （ 90.2±1.8% ） with the viability of 92%～95% （92.8% ± 3.4% ）. The enriched cells significantly suppressed the proliferation of CD4^＋CD25^- T cells in mixed lymphocyte culture （P 〈 0.05）. Conclusion： An effective method can be established for enrichment of CD4^＋CD25^＋ regulatory T cells in two steps by MACS, with satisfied cell purity, viability and function.

冷冻消融联合养肺方治疗对Lewis肺癌CD4^(+)CD25^(+)Foxp3^(+)Treg的影响及机制

目的 探讨冷冻消融联合养肺方治疗对Lewis肺癌CD4^(+)CD25^(+)Foxp3^(+)Treg细胞影响及机制。方法 构建C57BL/6小鼠肺癌皮下移植瘤模型,随机分为模型组、冷冻消融组、冷冻消融联合养肺方低、中、高剂量组,每组5只。模型组行假手术,在小鼠移植瘤部位切开缝合,其余各组均行双循环冷冻消融术。术后冷冻消融联合养肺方低、中、高剂量组分别给予1.64、3.28、6.56 g/kg养肺方灌胃,模型组、冷冻消融组分别给予等体积的生理盐水灌胃,均1次/d,连续给药14 d。给药期间观察并记录各组小鼠肿瘤体积,末次给药后剥离脾脏和移植瘤,称取瘤体质量并计算抑瘤率;流式细胞术检测脾脏CD4^(+)T、CD4^(+)CD25^(+)Foxp3^(+)T细胞比例;qRT-PCR和Western blot法检测瘤组织中Foxp3表达。结果 各组小鼠肿瘤体积均逐渐增长,增长速度为模型组>冷冻消融组>冷冻消融联合养肺方低剂量组>冷冻消融联合养肺方中剂量组>冷冻消融联合养肺方高剂量组。手术联合药物干预14 d后,与模型组比较,各治疗组肿瘤体积小(P<0.05);与冷冻消融组比较,冷冻消融联合养肺方低、中、高剂量组肿瘤体积小(P<0.01);冷冻消融联合养肺方高剂量组肿瘤体积小于低剂量组(P<0.01)。与模型组比较,冷冻消融联合养肺方低、中、高剂量组瘤体质量小(P<0.05);与冷冻消融组比较,冷冻消融联合养肺方低、中、高剂量组瘤体质量小(P<0.05)。冷冻消融组、冷冻消融联合养肺方低、中、高剂量组抑瘤率逐渐升高。与模型组比较,各治疗组CD4^(+)T细胞比例低(P<0.05),冷冻消融联合养肺方低、中、高剂量组的CD4^(+)CD25^(+)Foxp3^(+)T%细胞比例低(P<0.05);与冷冻消融组比较,冷冻消融联合养肺方高剂量组CD4^(+)T、CD4^(+)CD25^(+)Foxp3^(+)T细胞比例低(P<0.05);冷冻消融联合养肺方低、中、高剂量组CD4^(+)T、CD4^(+)CD25^(+)Foxp3^(+)T细胞比例逐渐降低。与模型组比较,冷冻消融联合养肺方低、中、高剂量组肿瘤组织中Foxp3 mRNA相对表达量和Foxp3蛋白表达均降低(P<0.01);冷冻消融联合养肺方中、高剂量组Foxp3 mRNA相对表达量和Foxp3蛋白表达均低于冷冻消融组(P<0.01)和冷冻消融联合养肺方低剂量组(P<0.05)。结论冷冻消融联合养肺方通过减少Lewis肺癌小鼠脾脏CD4^(+)CD25^(+)Foxp3^(+)Treg细胞比例,下调肿瘤组织中Foxp3表达,发挥抑制Lewis肺癌增殖作用,其机制可能与增强机体抗肿瘤免疫应答,改善肿瘤免疫抑制有关。

MBD2 promotes Th2 differentiation in ovalbumin-induced CD4+T cells

Introduction:Allergen-specific CD4+T cells play a central role in autoimmune disorders,allergies and asthma,with Th2-type immunity being the typical functional response of CD4+T cells.This study aimed to investigate the role of MBD2 in regulating Th2 cell differentiation.Methods:Splenic mononuclear cells were extracted from C57BL/6 mice,and CD4+T cells were isolated using magnetic beads and confirmed through flow cytometry.Lentivirus was employed to construct MBD2-silenced CD4+T cells.In vitro experiments were performed to treat splenogenic mononuclear cells and CD4+T cells with Ovalbumin(OVA),and Th2 cell ratios and IL-4 levels were assessed using flow cytometry and ELISA.Results:The purity of the isolated CD4+T cells was 95.73%,confirming successful isolation of primary CD4+T cells.Compared to the control group,the Th2 cell ratio exhibited an increase in the Th2-induced group.Treatment with 5-Aza(concentrations,1-100μM)promoted Th2 cell differentiation and increased IL-4 levels.Notably,when combined with Th2 induction and 10μM 5-Aza treatment,silencing MBD2 further amplified Th2 cell ratios and elevated IL-4 levels in cell supernatants.Furthermore,OVA(concentration,200μg/mL)induced the differentiation of CD4+T cells into Th2 cells and increased IL-4 secretion.Interestingly,silencing MBD2 significantly increased the Th2 cell ratio and IL-4 levels in OVA-treated CD4+T cells.Conclusion:In summary,OVA promoted CD4+T cell differentiation into Th2 cells and enhanced IL-4 levels.MBD2 was identified as a mediator of Th2 cell differentiation in splenic-derived CD4+T cells,influenced by OVA or 5-Aza treatment.

Peripheral CD4^(+)CD8^(+) double positive T cells:A potential marker to evaluate renal impairment susceptibility during systemic lupus erythematosus

Lupus nephritis(LN) has a high incidence in systemic lupus erythematosus(SLE) patients, but there is a lack of sensitive predictive markers. The purpose of the study was to investigate the association between the CD4^(+)CD8^(+)double positive T(DPT) lymphocytes and LN. The study included patients with SLE without renal impairment(SLE-NRI), LN, nephritic syndrome(NS), or nephritis. Peripheral blood lymphocyte subsets were analyzed by flow cytometry. Biochemical measurements were performed with peripheral blood in accordance with the recommendations proposed by the National Center for Clinical Laboratories. The proportions of DPT cells in the LN group were significantly higher than that in the SLE-NRI group(t=4.012, P<0.001), NS group(t=3.240,P=0.001), and nephritis group(t=2.57, P=0.011). In the LN group, the risk of renal impairment increased significantly in a DPT cells proportion-dependent manner. The risk of LN was 5.136 times(95% confidence interval, 2.115–12.473) higher in cases with a high proportion of DPT cells than those whose proportion of DPT cells within the normal range. These findings indicated that the proportion of DPT cells could be a potential marker to evaluate LN susceptibility, and the interference of NS and nephritis could be effectively excluded when assessing the risk of renal impairment during SLE with DPT cell proportion.

Combined TIM-3 and PD-1 blockade restrains hepatocellular carcinoma development by facilitating CD4+ and CD8+T cellmediated antitumor immune responses

BACKGROUND Immune checkpoint inhibitors(ICIs)targeting programmed cell death protein 1(PD-1)and T cell immunoglobulin and mucin domain-containing protein 3(TIM-3)are beneficial to the resumption of anti-tumor immunity response and hold extreme potential as efficient therapies for certain malignancies.However,ICIs with a single target exhibit poor overall response rate in hepatocellular carcinoma(HCC)patients due to the complex pathological mechanisms of HCC.AIM To investigate the effects of combined TIM-3 and PD-1 blockade on tumor development in an HCC mouse model,aiming to identify more effective immunotherapies and provide more treatment options for HCC patients.METHODS The levels of PD-1 and TIM-3 on CD4+and CD8+T cells from tumor tissues,ascites,and matched adjacent tissues from HCC patients were determined with flow cytometry.An HCC xenograft mouse model was established and treated with anti-TIM-3 monoclonal antibody(mAb)and/or anti-PD-1 mAb.Tumor growth in each group was measured.Hematoxylin and eosin staining and immunohistochemical staining were used to evaluate T cell infiltration in tumors.The percentage of CD4+and CD8+T cells in tissue samples from mice was tested with flow cytometry.The percentages of PD-1+CD8+,TIM-3+CD8+,and PD-1+TIM-3+CD8+T cells was accessed by flow cytometry.The levels of the cytokines including tumor necrosis factor alpha(TNF-α),interferon-γ(IFN-γ),interleukin(IL)-6,and IL-10 in tumor tissues were gauged with enzyme-linked immunosorbent assay kits.RESULTS We confirmed that PD-1 and TIM-3 expression was substantially upregulated in CD4+and CD8+T cells isolated from tumor tissues and ascites of HCC patients.TIM-3 mAb and PD-1 mAb treatment both reduced tumor volume and weight,while combined blockade had more substantial anti-tumor effects than individual treatment.Then we showed that combined therapy increased T cell infiltration into tumor tissues,and downregulated PD-1 and TIM-3 expression on CD8+T cells in tumor tissues.Moreover,combined treatment facilitated the production of T cell effector cytokines TNF-α and IFN-γ,and reduced the production of immunosuppressive cytokines IL-10 and IL-6 in tumor tissues.Thus,we implicated that combined blockade could ameliorate T cell exhaustion in HCC mouse model.CONCLUSION Combined TIM-3 and PD-1 blockade restrains HCC development by facilitating CD4+ and CD8+T cell-mediated antitumor immune responses.

Effect of CD4^+CD25^+ regulatory T cells in the development of anterior chamber-associated immune deviation

AIM: To investigate whether CD4(+)CD25(+) regulatory T (Treg) cells play a role in the development of anterior chamber-associated immune deviation (ACAID). METHODS: The dynamic changes in the frequency of CD4(+)D25(+) T cells, CD4(+)D25(+) FoxP3(+) T cells and CD4(+)CD25(+) PD-1(+) T cells from spleens of mice with ACAID were analyzed by flow cytometry. Foxp3 mRNA expression in purified CD4(+)CD25(+) T cells was analyzed using real-time PCR. The suppressive effect of purified CD4(+)CD25(+) T cells on the proliferation of CD4(+)CD25(-) T cells was evaluated by [H-3] thymidine incorporation. A blocking experiment was performed to further address the role of CD4(+)CD25(+) T cells in ACAID. The expression of IL-10 in purified CD4(+)CD25(+) T cells was evaluated by ELISA. RESULTS: Increased frequencies of CD4(+)CD25(+) T cells, CD4(+)CD25(+) Foxp3(+) T cells and CD4(+)CD25(+) PD-1(+) T cells were observed in ACAID. The CD4(+)CD25(+) T cells from mice with ACAID showed enhanced suppressive effect on the proliferation of CD4(+)CD25(-) T cells. Treatment of BALB/c mice with anti-CD25 antibody after injection of OVA into the anterior chamber significantly inhibited the induction of ACAID. Furthermore, purified CD4(+)CD25(+) T cells from ACAID mice secreted IL-10. CONCLUSION: Our results demonstrate that Treg cells are induced in the mice undergoing ACAID. These Treg cells may play a role in the development of ACAID.

CD4^+CD25^(high) Regulatory Cells in Peripheral Blood of NSCLC Patients

The proportion and changes of CD4^＋CD25^high regulatory T cells （Trs） in peripheral blood of non-small cell lung cancer （NSCLC） patients were analyzed and their clinical significance explored. The peripheral blood was collected from 61 patients with NSCLC and 15 healthy controls. By using monoclonal antibodies, the blood samples were evaluated with the flow cytometry for lymphocyte subsets （CD3^＋, CD4^＋ and CD8^＋） and CD4^＋CD25^high Tr cells. The results showed that the proportion of CD4^＋CD25^high Tr cells in NSCLC group was significantly higher than in control group [（4.36 ±2.07） % vs （2.04±1.03） %, P〈0.01]. The proportion of CD4^＋CD25^ high Tr cells in late stage was higher than that in early stage [stages Ⅰ ＋Ⅱ （2.264±0.6） %; stage Ⅲ（3.284± 1.38） %; stage IV （6.06 4±4.08） %] （P〈0.05）. Kaplan-Meier survival analysis revealed that the prognosis of the patients who had higher proportion of CD4^＋CD25^high Tr cells in peripheral blood was worse （P=0.0026）. In conclusion, the relative increase in CD4^＋CD25^high Tr cells in peripheral blood may be related to im- munosuppression and tumor progression in patients with NSCLC. This finding suggests that CD4^＋CD25^high Tr cells in peripheral blood of NSCLC may be positive for prognosis analysis. The use of depletion of the CD4^＋CD25^high Tr cell therapy to treat NSCLC patients may be an effective strategy.

Immunotherapy of rat glioma without accumulation of CD4^+CD25^+FOXP3^+ regulatory T cells

Immunotherapy may be used for the treatment of glioblastoma multiforme; however, the induced immune response is inadequate when either T cells or dendritic cells are used alone. In this study we established a novel vaccine procedure in rats, using dendritic cells pulsed with C6 tumor cell lysates in combination with adoptive transfer of T lymphocytes from syngenic donors. On day 21 after tumor inoculation, all the rats were sacrificed, the brains were harvested for calculation of glioma volume, cytolytic T lymphocyte responses were measured by cytotoxic assay, and the frequency of regulatory T lymphocytes （CD4＋CD25~FOXP3~） in the peripheral blood was investigated by flow cytometric analysis. The survival rate of rats bearing C6 glioma was observed. Results showed that the co-immunization strategy had significant anti-tumor potential against the pre-established C6 glioma, and induced a strong cytolytic T lymphocyte response in rats. The frequency of peripheral blood CD4＊CD25＊FOXP3＊ regulatory T lymphocytes was significantly decreased following the combination therapy, and the rats survived for a longer period. Experimental findings indicate that the combined immunotherapy of glioma cell lysate-pulsed dendritic cell vaccination following adoptive transfer of T cells can effectively inhibit the growth of gliomas in rats, boost anti-tumor immunity and produce a sustained immune response while avoiding the accumulation of CD4＋CD25＋FOXP3＋ regulatory r lymphocytes.