CD4~+ CD25~+调节性T细胞与肿瘤免疫的研究进展

CD4+CD25+调节性T细胞是CD4+T细胞的一个重要亚群,具有免疫抑制和免疫无能两大功能。CD4+CD25+T细胞与自身免疫性疾病的发生、移植耐受具有密切关联。近几年的研究表明,CD4+CD25+T细胞与肿瘤的发生发展和转归亦有着密切联系。本文就调节性T细胞的作用机制及特点与肿瘤免疫的关系作一综述。     

喘可治注射液对小鼠体外CD4+CD25+T细胞的影响

利用荧光抗体标记和流式细胞术检测喘可治对刀豆蛋白A(ConA)诱导的T细胞CD69和CD25表达的影响,研究喘可治是否具有促进CD4 CD25 调节性T细胞升高的作用.结果发现喘可治对ConA诱导的T细胞活化标志分子CD69的表达具有抑制作用,但对CD25的表达具有促进作用.说明喘可治对T细胞活化具有抑制作用,CD25表达的上调并不是由活化引起的,而很可能是CD4 CD25 Tr水平升高的标志.     

CD4+CD25+调节性T细胞作用机制的双模式

CD4 CD25 调节性T细胞是具有免疫抑制功能的细胞群,在多种生理病理过程中发挥了重要作用。它们的作用机制主要包括细胞-细胞接触依赖和可溶性细胞因子介导两种抑制模式。由于CD4 CD25 调节性T细胞的抑制机制复杂,争议较大,进一步阐明它们的作用机制将有利于多种免疫相关疾病的防治。     

非霍奇金淋巴瘤患者外周血中CD4+CD25+ 调节性T 细胞 亚群变化初探

目的:检测非霍奇金淋巴瘤(non-Hodgkin’s lymphoma,NHL)患者外周血中CD4+CD25+调节性T细胞(CD4+CD25+regulatoryT cell,Treg)的改变,探讨Treg与NHL的相关性。方法:病例组(n=60)为本院收治的初诊NHL患者,对照组(n=60)为本院健康体检者,用流式细胞技术联合标记CD4、CD25检测对照组及病例组化疗前、化疗后的外周血中CD4+CD25+调节性T细胞的分布特点。结果:(1)病例组化疗前外周血中CD4+细胞比例显著低于对照组(P<0.05),CD4+CD25+调节性T细胞比例显著高于对照组(P<0.05);(2)病例组化疗后,CD4+细胞比例明显高于化疗前(P<0.05),CD4+CD25+调节性T细胞比例明显低于化疗前(P<0.05);(3)病例组化疗后CD4+细胞比例与对照组无显著差异(P>0.05),而CD4+CD25+调节性T细胞比例显著高于对照组(P<0.05)。结论:非霍奇金淋巴瘤患者外周血中CD4+CD25+调节性T细胞比例升高,存在机体免疫抑制,化疗可降低CD4+CD25+调节性T细胞比例。     

香菇多糖对急性弓形虫感染过程中CD4+CD25+Foxp3+调节性T细胞的调节作用研究

目的 探讨香菇多糖(Lentinan,Lent)对急性弓形虫感染BALB/c小鼠CD4+ CD25+ Foxp3+调节性T细胞(Tregs)数量和功能的调节作用.方法 对RH强毒株感染的BALB/c小鼠进行不同时间点的Lent预处理,动态观察用药后各组感染小鼠的生存率;在感染后第0、3、5、8和10天提取小鼠的脾细胞,FACS检测Tregs细胞数量的动态变化,ELISA法检测脾细胞培养上清中IL-10的分泌水平.结果 感染前6 d 1 mg/kg Lent用药组与药物未处理组相比显著提高了弓形虫感染小鼠的生存率;具有免疫抑制功能的Tregs数量于感染后8d达峰值,同时免疫应答中关键细胞因子IL-10的分泌水平也于感染后8d和10d显著增加.结论 对急性弓形虫感染的BALB/c小鼠采用Lent预处理之后能有效的调节Tregs的数量和功能,从而调控Th1/Th2之间的动态平衡达到治疗弓形虫的作用,为弓形虫病的临床治疗提供的新的理论依据.     

小鼠沙眼衣原体肺感染过程中Treg细胞与Th17反应关系

[目的]对沙眼衣原体在BALB/c小鼠肺部感染过程中CD4+ CD25+Foxp3+调节性T细胞(regulatory T cells,Treg)与Th17反应关系进行初步探讨.[方法]取6-8周龄的BALB/c小鼠,鼻腔吸入25 μL含5×103 IFU的沙眼衣原体鼠肺炎菌株(Chlamydia muridarum,Cm),建立沙眼衣原体小鼠肺感染模型.监测感染后不同时期小鼠体重变化;检测肺组织衣原体包涵体形成单位( IFU)及肺组织病理改变;利用流式细胞术检测Cm感染后小鼠体内Treg细胞百分率;ELISA检测肺组织上清液IL-6、TGF-β、IL-17、IL-2细胞因子的的表达;qRT-PCR检测KC( keratinocyte derived chemokine) mRNA和MIP-2( macrophage inflammatory protein-2)mRNA的表达差异.[结果]用5xl03 IFU Cm经鼻腔吸人感染后小鼠发生沙眼衣原体肺炎,表现为体重下降、肺组织大量炎症细胞浸润并可检测到衣原体繁殖.Cm感染后第3天,小鼠体内Treg细胞占CD4 +T细胞的百分比明显降,随后开始恢复,第7天恢复原来水平,一直持续到衣原体清除.TGF-β、IL-2的表达与Treg细胞动态变化一致.与Th17相关细胞因子IL-6、IL-17和Th17相关趋化因子KC、MIP-2的表达于第3天开始升高,至第7天达到最高水平,随后逐渐减少.[结论]在衣原体感染BALB/c小鼠过程中,Treg可能通过提供TGF-β并在IL-6帮助下促进Th17应答产生.     

CD4+CD25+调节性T细胞在造血干细胞移植后诱导免疫耐受的作用

异基因造血干细胞移植是治疗良恶性血液病的有效手段,尤其单倍型移植是具有中国特色的移植体系。然而,移植物抗宿主病(graft-versus-host disease, GVHD)的发生是移植后患者死亡的最重要原因。造血干细胞移植的成功实施需要安全有效的诱导免疫耐受,其中T淋巴细胞在GVHD发生中起到最重要的作用。CD4~+CD25~+调节性T细胞(Treg)是近年来发现的一类能够抑制免疫应答的重要调节性免疫细胞,本文综述了Treg在造血干细胞移植后诱导免疫耐受的作用。     

CD4+CD25+FoxP3+ 调节性T 细胞与黑龙江省HIV/AIDS 患者病情 相关性的研究

目的:比较黑龙江省HIV/AIDS患者与健康对照者(healthy controls,HCs)外周血CD4+CD25+FoxP3+调节性T细胞数量、免疫抑制功能的变化,探讨CD4+CD25+FoxP3+调节性T细胞在HIV/AIDS感染过程中的作用。方法:采用流式细胞仪检测21例HIV/AIDS患者及20例健康对照组的外周血CD4+CD25+FoxP3+调节性T细胞数量的百分比及绝对数量;采用共同培养方法检测HIV/AIDS患者外周血CD4+CD25+FoxP3+调节性T细胞免疫抑制功能的变化;实时荧光定量聚合酶链反应(RT-FQ-PCR)检测HIV/AIDS患者外周血CD4+CD25+FoxP3+调节性T细胞中FoxP3mRNA的表达。结果:黑龙江省HIV/AIDS患者外周血CD4+CD25+FoxP3+调节性T细胞比率明显高于HCs(P<0.01),而CD4+CD25+FoxP3+调节性T细胞的绝对计数显著下降,且与CD4+T细胞绝对计数成反比;混合淋巴细胞共同培养结果显示,HIV/AIDS患者外周血CD4+CD25+FoxP3+调节性T细胞的抑制功能无明显变化;HIV/AIDS患者外周血CD4+CD25+FoxP3+调节性T细胞的FoxP3 mRNA相对表达量无显著变化。结论:黑龙江省HIV/AIDS患者CD4+CD25+FoxP3+调节性T细胞的数量变化与病情相关。     

牙龈卟啉单胞菌脂多糖对CD4^＋CD25^＋调节性T细胞免疫抑制功能的影响

目的探讨牙龈卟啉单胞菌（Porphyromonas gingivalis,Pg）对CD4^＋CD25^＋调节性T细胞（regulatory T cells,Tregs）免疫抑制功能的影响。方法采用酚水法提取Pg ATCC 33277株脂多糖（lipopolysaccharide,LPS）。免疫磁珠法分离BALB／c小鼠脾脏CD4^＋CD25^＋Tregs并进行体外培养,同时给予不同剂量（0～500ng／ml）Pg—LPS干预,培养48h后收集细胞及上清液。Real-TimePCR法测定培养细胞Foxp3mRNA的表达,ELISA法分别测定细胞上清液中IL-10、TGF-β水平;采用体外淋巴细胞混合培养法对Pg-LPS干预后的CD4^＋CD25^＋Tregs进行功能抑制试验。结果Pg-LPS干预不影响CD4^＋CD25^＋Tregs分泌IL-10和TGF-β,但是能够显著上调CD4^＋CD25^＋TregsFoxp3mRNA的表达,增强其免疫抑制作用;当Ps—LPS浓度低于300ng／m1时,CD4^＋CD25^＋TregsFoxp3mRNA表达以及免疫抑制作用的增强与Ps—LPS浓度之间呈剂量-效应关系。结论Pg-LPS能够增强CD4^＋CD25^＋Tregs的免疫抑制作用,这种免疫抑制增强效应可能与CD4^＋CD25^＋Tregs Foxp3基因表达的上调有关,并且不具有抑制性细胞因子依赖性。     

CD4^＋CD25^＋Foxp3^＋调节性T细胞的体外扩增及其在移植物抗宿主病中的应用

同种异基因造血干细胞移植是急、慢性白血病及其他恶性血液病重要的治疗方法,但急慢性移植物抗宿主病（graft—versus-host disease,GVHD）作为异基因造血干细胞移植的主要并发症严重影响移植患者的存活率,阻碍移植的临床推广。很多研究发现,高表达Foxp3的CD4^＋CD25^＋调节性T细胞（regulatory T cells,Treg）不仅能控制急慢性GVHD的发生,而且不影响移植物抗白血病效应（graft-versusleukemia,GVL）,在急慢性GVHD发生发展及治疗方面有重要的作用。但Treg细胞在体内的数量很少,不能满足临床应用需求。目前应用外源的IL-2联合TCR、CD28信号通路共同刺激以及运用树突状细胞（dendritic cell,DC）刺激均能达到体外有效扩增Treg细胞的目的。这些扩增的Treg细胞在控制造血干细胞移植过程中急慢性GVHD的发生及防治自身免疫性疾病和移植排斥等方面具有明显作用,在疾病控制和临床应用中具有广阔前景。     

HIV/HCV共感染者抗逆转录病毒治疗后免疫恢复情况

由于具有相同的传播途径,人类免疫缺陷病毒(Human immunodeficiency virus,HIV)和丙型肝炎病毒(Hepatitis C virus,HCV)共感染非常普遍,但是关于合并感染的程度,两种病毒之间的相互关系,在艾滋病抗逆转录病毒治疗(Antiretroviral therapy,ART)前后,HCV合并感染对HIV患者免疫细胞恢复的影响仍不明确。为了通过分析CD4⁺和CD8⁺T淋巴细胞数的变化,以了解辽宁省HIV/HCV共感染者ART后免疫恢复的情况,本研究从辽宁省艾滋病抗病毒治疗数据库中筛选符合要求的HIV感染者和HIV/HCV共感染者,收集感染者基本人口学资料及HCV抗体检测结果、HIV/HCV共感染途径等资料。采用t检验或卡方检验进行组间比较,采用Kaplan-Meier乘积极限法绘制生存分析函数图。结果显示,本研究共纳入HIV感染者12742人,HIV/HCV共感染者340人。HIV感染者和HIV/HCV共感染者的不同人口学特征均差异显著(P<0.001)。HIV感染和HIV/HCV共感染者ART治疗后CD4⁺细胞数和CD4⁺/CD8⁺比值显著升高(P<0.05),CD8⁺细胞数比ART前显著下降(P<0.05)。HIV/HCV共感染者随着ART时长,CD4⁺T淋巴细胞数恢复情况始终显著低于HIV感染者(P<0.05)。生存分析曲线表明,HCV/HIV共感染者从艾滋病诊断开始随着ART的治疗CD4⁺细胞恢复情况显著低于HIV感染者,Log-Rank检验统计量为4.483(P=0.034)。本研究揭示,HCV感染对ART患者CD4⁺和CD8⁺T淋巴细胞的恢复有影响。ART后HIV/HCV共感染者中CD4⁺T淋巴细胞计数的改善低于HIV单一感染者,并且单一感染患者对ART的反应比合并感染患者更好。因此,建议在启动ART之前,对每个感染HIV的患者进行HCV抗体筛查。     

口服卡介菌诱导免疫耐受的CD4+CD25+调节性T 细胞机制研究

目的:研究口服卡介菌诱导免疫耐受对CD4+CD25+调节性T细胞的影响。方法:采用口服MPB制备EAE大鼠模型,随机分为BCG组(0.5mg/kg)和EAE模型组(PBS),每组各15只,连续经口灌服给药14d,同时选取15只健康大鼠作为对照组。分别于免疫后15d、27d流式细胞术检测外周血、胸腺及脾脏中CD4+CD25+T淋巴细胞百分率,ELISA检测血清IL-6、TGF-β、IgE、IgG含量。结果:与EAE模型组相比,免疫后BCG组大鼠外周血、胸腺及脾脏中CD4+CD25+T淋巴细胞百分率增加,血清IL-6、TGF-β含量上升,血清IgE、IgG抗体水平下降。结论:口服BCG通过上调淋巴器官中CD4+CD25+T淋巴细胞比例,抑制效应性T细胞活性,发挥免疫耐受作用。     

Decreased regulatory T‐cells and CD4+/CD8+ ratio correlate with disease onset and progression in patients with generalized vitiligo

The aim of present study was to evaluate CD4⁺/CD8⁺ ratio and CD4⁺CD25^hiFoxP3⁺ Tregs in GV patients with reference to their effect on disease onset and progression. Flow cytometry was used for determination of CD4⁺/CD8⁺ ratio and Tregs in 82 patients and 50 controls. CD8⁺ T‐cell counts were significantly higher in GV patients as compared with controls (p = 0.003). Active GV patients showed higher CD8⁺ T‐cell counts compared with stable GV patients (p = 0.001). The CD4⁺/CD8⁺ ratio decreased significantly in patients as compared with controls (p = 0.001). Moreover, the ratio in active GV patients significantly lowered as compared with stable GV patients (p = 0.002). Significant decrease in Treg cell percentage and counts in GV patients was observed compared with controls (p = 0.009, p = 0.008) with significant reduction in FoxP3 expression (p = 0.024). Treg cell percentage and counts were significantly decreased in active GV patients compared with stable GV patients (p = 0.007, p = 0.002). Our results suggest that an imbalance of CD4⁺/CD8⁺ ratio and natural Tregs in frequency and function might be involved in the T‐cell mediated pathogenesis of GV and its progression.     

Immune responses to p53 in patients with cancer:enrichment in tetramer+ p53 peptide-specific T cells and regulatory T cells at tumor sites

Objective: A majority of human cancers, including head and neck cancer (HNC), overexpress p53. Although T cells specific for wild-type (wt) sequence p53 peptides are detectable in the peripheral blood of patients with HNC, it is unknown whether such T cells accumulate in tumor-involved tissues. Also, the localization of regulatory T cells (Treg) to tumor sites in HNC has not been investigated to date. Methods: Tumor infiltrating lymphocytes (TIL), tumor-involved or non-involved lymph node lymphocytes (LNL) and peripheral blood mononuclear cells (PBMC) were obtained from 24 HLA-A2.1+ patients with HNC. Using tetramers and four-color flow cytometry, the frequency of Treg and CD3+CD8+ T cells specific for wt p53 epitopes as well as their functional attributes were determined. Results: The CD3+CD8+ tetramer+ cell frequency was significantly higher (P<0.001) in TIL than autologous PBMC as was the percentage of CD4+CD25+ T cells (P<0.003). TIL were enriched in FOXp3+, GITR+ and CTLA-4+ Treg. CD8+ TIL had low expression and produced little IFN- after ex vivo stimulation relative to autologous PBMC or PBMC from NC. Conclusions: Anti-wt p53 epitope-specific T cells and Treg preferentially localize to tumor sites in patients with HNC. However, despite enrichment in tumor peptide-specific T cells, the effector cell population (CD3+CD8+) in TIL or PBMC was unresponsive to activation in the tumor microenvironment enriched in Treg.     

Dynamic demethylation of the IL2RA promoter during in vitro CD4+ T cell activation in association with IL2RA expression

IL2RA, a subunit of the high affinity receptor for interleukin-2 (IL2), plays a crucial role in immune homeostasis. Notably, IL2RA expression is induced in CD4+ T cells in response to various stimuli and is constitutive in regulatory T cells (Tregs). We selected for our study 18 CpGs located within cognate regulatory regions of the IL2RA locus and characterized their methylation in naive, regulatory, and memory CD4+ T cells. We found that 5/18 CpGs (notably CpG + 3502) show dynamic, active demethylation during the in vitro activation of naive CD4+ T cells. Demethylation of these CpGs correlates with appearance of IL2RA protein at the cell surface. We found no influence of cis located SNP alleles upon CpG methylation. Treg cells show constitutive demethylation at all studied CpGs. Methylation of 9/18 CpGs, including CpG +3502, decreases with age. Our data thus identify CpG +3502 and a few other CpGs at the IL2RA locus as coordinated epigenetic regulators of IL2RA expression in CD4+ T cells. This may contribute to unravel how the IL2RA locus can be involved in immune physiology and pathology.     

调节性T 细胞在发热结缔组织病患者外周血中的表达及临床意义

目的:研究Treg细胞在发热CTD患者外周血表达对结核感染的诊断价值。方法:对103例发热CTD患者进行T-SPOT.TB试验,将39例阳性者设为实验组-1,进行抗结核治疗,将64例阳性者设为实验组-2,另选取40例健康者作为对照组,检测三组外周血CD4+CD25+Treg细胞、Foxp3基因、IL-10、TGF-β的表达。结果:实验组CD4+CD25+Foxp3 Treg细胞占CD4+T比例高于对照组(P0.05),实验组-1治疗前外周血CD4+CD25+Foxp3 Treg细胞占CD4+T比例高于实验组-1治疗后、实验组-2(P0.05);实验组TGF-β表达量低于对照组(P0.05),实验组-1治疗前低于实验组-1治疗后及实验组-2(P0.05);实验组-1治疗前IL-10表达量低于实验组-1治疗后、实验组-2及对照组(P0.05)。结论:CD4+CD25+Foxp3 Treg细胞在发热CTD伴有结核感染患者外周血中的表达升高,其变化可作为结核感染诊断的辅助性指标。     

Reversal of new-onset type 1 diabetes in mice by syngeneic bone marrow transplantation

Autologous hematopoietic stem cell transplantation (HSCT) has recently been performed as a novel strategy to treat patients with new-onset type 1 diabetes (T1D). However, the mechanism of autologous HSCT-induced remission of diabetes remains unknown. In order to help clarify the mechanism of remission-induction following autologous HSCT in patients with T1D, mice treated with multiple low doses of streptozotocin to induce diabetes were used as both donors (n = 20) and recipients (n = 20). Compared to streptozocin-treated mice not receiving transplantation, syngeneic bone marrow transplantation (syn-BMT) from a streptozocin-treated diabetic donor, if applied during new-onset T1D (day 10 after diabetes onset), can reverse hyperglycemia without relapse (P < 0.001), maintain normal blood insulin levels (P < 0.001), and preserve islet cell mass. Compared to diabetic mice not undergoing HSCT, syn-BMT, results in restoration of Tregs in spleens (P < 0.01), increased Foxp3 mRNA expression (P < 0.01) and increased Foxp3 protein expression (P < 0.05). This diabetic-remission-inducing effect occurred in mice receiving bone marrow from either streptozocin-treated diabetic or non-diabetic normal donors. We conclude that autologous HSCT remission of diabetes is more than transient immune suppression, and is capable of prolonged remission-induction via regeneration of CD4+CD25+FoxP3+ Tregs.