乳腺癌患者外周血CD4+CD25+Foxp3+调节性T细胞水平的检测及意义

目的：探讨乳腺癌患者外周血CD4+CD25+Foxp3+调节性T细胞（Treg）水平检测的意义。 方法：流式细胞术检测74例乳腺癌患者与30例健康对照者外周血CD4+CD25+Foxp3+Treg占CD4+T细胞百分比,分析CD4+CD25+Foxp3+Treg细胞水平与乳腺癌患者临床病理特征及相关免疫组化指标的关系。 结果：乳腺癌患者外周血CD4+CD25+Foxp3+Treg占CD4+T细胞的百分比高于健康对照者[（9.15± 2.24）% vs.（2.29±1.36）%],差异有统计学意义（P<0.05）。统计分析显示,乳腺癌患者外周血CD4+CD25+Foxp3+Treg细胞水平与肿瘤组织学分级、淋巴结转移、pTNM分期以及HER-2、pS2、nm23的表达有关（均P<0.05）,而与肿瘤大小、病理类型以及雌激素受体（ER）、孕激素受体（PR）、p53、Ki-67表达无关（均P>0.05）。进一步相关性分析显示,CD4+CD25+Foxp3+Treg细胞水平与肿瘤组织学分级、淋巴结转移数、pTNM分期、HER-2的表达呈正相关（r=0.583,r=0.333,r=0.919,r=0.604,均P<0.05）而与pS2、nm23表达呈负相关（r=-0.229,r=-0.401,均P<0.05）。 结论：乳腺癌患者外周血CD4+CD25+Foxp3+Treg细胞水平升高,并与与乳腺癌的进展、转移密切相关,对其检测可能有助于患者预后及治疗效果的评估。

     

肝癌患者CD8+CD28-Foxp3+调节性T细胞的变化及意义

目的:探讨肝癌患者外周血和癌组织中CD8~+CD28~-Foxp3~+调节性T细胞(CD8~+CD28-Foxp3~+Tregs)的改变及意义。方法:采用流式细胞仪检测72例肝癌患者和22例健康对照人群外周血CD8~+CD28~-Foxp3~+T细与CD8~+T细胞比值(CD8~+CD28-Foxp3~+Tregs/CD8~+T),分析CD8~+CD28~-Foxp3~+Tregs/CD8~+T与肝癌患者临床病理因素的关系;分别用免疫组化和Western blot检测肝癌患者癌组织及癌旁组织中Foxp3阳性细胞与Foxp3蛋白表达水平。结果:与健康对照人群比较,肝癌患者外周血CD8~+CD28~-Foxp3~+Tregs/CD8~+T明显升高(P0.05);外周血CD8~+CD28~-Foxp3~+Tregs/CD8~+T与患者TNM分期、淋巴结转移、分化程度有关(均P0.05);肝癌组织中平均Foxp3阳性细胞数与Foxp3蛋白表达量均明显高于癌旁组织(均P0.05);外周血CD8~+CD28~-Foxp3~+Tregs/CD8~+T,肝癌组织Foxp3阳性细胞数与Foxp3蛋白表达量在高、中、低分化的肝癌中均呈依次升高趋势,但差异均无统计学意义(均P0.05)。结论:CD8~+CD28~-Foxp3~+Tregs在肝癌患者外周血及癌组织中增多,可能与肿瘤免疫抑制作用有关,其检测对肝癌患者病情有一定评估价值。     

乳腺癌微环境中调节性T细胞表达及其意义研究

目的探讨CD4+CD25+Foxp3+调节性T细胞(Treg)在诱导乳腺癌免疫耐受中的作用和机制。方法选取河北医科大学第二医院腺体外科乳腺癌标本40例(淋巴结无转移20例,转移20例)、乳腺纤维腺瘤标本20例,分别应用流式细胞术(FCM)和免疫组化检测CD4+CD25+Foxp3+Treg、TGF-β和IL-10在乳腺癌原发灶、癌周组织、腋窝淋巴结以及乳腺纤维腺瘤中的表达。结果乳腺癌原发灶中三指标表达程度,均较乳腺纤维腺瘤中高(P<0.05);原发灶中表达程度,均高于癌周组织和腋窝淋巴结(P<0.05);淋巴结转移组中癌周组织三项指标表达低于腋窝淋巴结(P<0.05),而淋巴结未转移组中,癌周组织和腋窝淋巴结表达无差异(P>0.05)。结论 CD4+CD25+Foxp3+Treg在乳腺癌微环境中富集可能是导致乳腺癌免疫逃逸的重要因素;CD4+CD25+Foxp3+Treg与TGF-β和IL-10具有一定的协同作用。     

Foxp3+调节性T细胞与乳腺癌淋巴结转移及增殖的关系

目的:探讨Foxp3~+调节性T细胞(Tregs)与乳腺癌淋巴结转移及增殖的关系。方法:用免疫组化法检测168例女性乳腺癌患者癌组织中CD4和Foxp3(调节性T细胞标志)的表达,以42例女性乳腺良性病变乳腺组织为对照,分析Tregs与乳腺癌淋巴结转移及乳腺癌组织细胞核增殖相关抗原Ki-67表达的关系。结果:乳腺癌组织中CD4~+T细胞与Foxp3~+T细胞(Tregs)的数量均高于乳腺良性病变组织,差异均有统计学意义(均P0.05);有淋巴结转移的乳腺癌组织中CD4~+T细胞与Foxp3~+T细胞数均高于无淋巴结转移的乳腺癌组织,但仅后者差异有统计学意义(P0.05)。乳腺癌组织中Tregs的浸润数量与Ki-67表达之间无关(P0.05)。结论:乳腺癌组织微环境存在免疫抑制,Tregs浸润数量与淋巴结转移关系密切,但与肿瘤增殖无关,提示Tregs可作为判断乳腺癌患者有无淋巴结转移的新指标。     

精索静脉曲张不育患者CD_4~+ CD_(25)~+ Foxp3~+调节性T细胞的检测及临床意义

目的:探讨精索静脉曲张(VC)不育症患者外周血CD4+CD25+Foxp3+调节性T细胞(Treg)的变化及其临床意义。方法:72例VC不育症患者(VC组)作为研究对象,以30例非VC且正常生育者为对照(对照组)。流式细胞术检测外周血CD4+CD25+Foxp3+调节性T细胞的比例,ELISA法检测血清抗精子抗体(AsAb)水平,计算机辅助精液分析系统检测精液参数。结果:VC组的精子浓度、活率及活力均低于对照组(P0.05),精子畸形率及AsAb阳性率高于对照组(P0.05);Ⅲ度VC组CD4+CD25+Foxp3+调节性T细胞在总CD4+T细胞所占比例低于正常生育组(P0.05)。AsAb阳性VC组的精子活率、精子活力及CD4+CD25+Foxp3+调节性T细胞占总CD4+T细胞比例均低于AsAb阴性VC组(P0.05),相关分析提示AsAb的存在与Treg/CD4+T比例、精子活率及精子活力负相关(r=-0.245,P0.05;r=-0.314,P0.05;r=-0.263,P0.05)。结论:精索静脉曲张不育患者外周血CD4+CD25+Foxp3+调节性T细胞的水平降低,且AsAb阳性组患者更低,这群调节性T细胞的异常可能是导致精索静脉曲张免疫性不育的重要因素之一。     

CD8+T细胞和调节性T细胞在食管癌中表达及与预后的关系

目的 探讨食管癌组织中CD8+T细胞、Foxp3阳性调节性T细胞(regulatory T cells,Treg)表达及对预后的影响.方法 应用免疫组织化学SP法检测CD8、Foxp3在90例食管癌组织间质、癌巢中的表达,计数阳性细胞,分析阳性细胞数目与预后的关系.结果 间质中CD8+T细胞的数量与浸润深度、分化程度呈负相关(P<0.05);Foxp3+Treg细胞数量与淋巴结转移、病变长度呈正相关(P<0.05).本组病例3年总生存率66.67%(60/90).单因素生存分析显示,无论癌巢或间质中,CD8+T细胞计数高者的总体生存曲线优于计数低者,差异有统计学意义(P<0.05);roxp3+Treg细胞计数高者的累计生存情况较计数低者差(P<0.05).多因素分析显示,间质浸润的CD8+T、Foxp3+Treg细胞数量和病变长度是影响生存期的独立因素(P<0.05).结论 间质中浸润的CD8+T细胞数量、Foxp3+Treg细胞数量是影响食管癌患者预后的独立因素,Foxp3+Treg细胞数量增多预后不良,CD8+T细胞数量增多则预后良好.     

不同分期前列腺癌患者外周血CD4~+ CD25~+ Foxp3~+调节性T细胞的变化及与胰岛素抵抗的相关性

目的 :观察不同分期前列腺癌患者外周血单个核细胞CD4+CD25+Foxp3+调节性T细胞的变化及与胰岛素抵抗的关系。方法:采用流式细胞术检测62例前列腺癌患者(患者组,临床TNM分期Ⅰ期5例、Ⅱ期16例、Ⅲ期21例、Ⅳ期20例)外周血单个核细胞(PBMC)中CD4+CD25+Foxp3+调节性T细胞数目,计算CD4+CD25+Foxp3+调节性T细胞占CD4+T淋巴细胞的百分率;并检测其空腹胰岛素及空腹血糖水平,计算胰岛素抵抗指数(HOMA-IR);采用ELISA法测定外周血胰岛素样生长因子1(IGF-1)水平,分析CD4+CD25+Foxp3+调节性T细胞与胰岛素抵抗的相关性,并与42例健康体检者进行对照。结果:与健康对照组相比,前列腺癌患者HOMAIR明显升高(6.68±1.66 vs 3.68±1.42),IGF-1水平明显下降[(96.39±21.21)ng/ml vs(164.56±30.58)ng/ml],PBMC CD4+CD25+Foxp3+Treg占CD4+T淋巴细胞的百分率[(13.88±0.96)%vs(5.33±0.65)%]及CD4+CD25+Foxp3+Treg绝对值[(3.55±0.29)×107vs(1.99±0.78)×107]明显升高(P0.05,P0.01)。患者PBMC CD4+CD25+Foxp3+Treg占CD4+T淋巴细胞的百分率及CD4+CD25+Foxp3+Treg绝对数﹑HOMA-IR均随TNM分期逐渐加重而增加,IGF-1逐渐下降;相关性分析表明:CD4+CD25+Foxp3+Treg/CD4+T及CD4+CD25+Foxp3+Treg绝对数均与HOMA-IR呈明显正相关(r分别为0.689、0.722,P0.01),与IGF-1呈明显负相关(r分别为-0.896、-0.747,P0.01)。结论:前列腺癌患者存在不同程度的胰岛素抵抗,且随着疾病程度的加重,外周血CD4+CD25+Foxp3+调节性T细胞数目和比例及胰岛素抵抗逐渐加重;CD4+CD25+Foxp3+调节性T细胞可能通过调节胰岛素抵抗参与其形成和发展。     

Foxp3~+调节性T细胞与肝癌肝移植患者术后肿瘤复发关系的研究

目的探讨Foxp3+调节性T细胞(Treg)与超加利福尼亚大学洛杉矶分校(UCSF)标准肝细胞癌(肝癌)肝移植患者术后肿瘤复发的关系。方法回顾性分析2010年1月至2013年12月在解放军第309医院全军器官移植研究所接受肝移植的24例肝癌患者的临床资料。在随访期内肿瘤复发4例(肿瘤复发组),余20例无复发(肿瘤未复发组);以同期健康人的血液标本为对照组。比较肿瘤复发组和肿瘤未复发组术前、术后不同时点甲胎蛋白(AFP)水平;比较肿瘤复发组和肿瘤未复发组术前、术后不同时点及对照组的Foxp3+Treg(Foxp3+Treg%)水平;对术前与术后Foxp3+Treg表达与AFP、CD3+和CD8+T水平的关系进行相关分析。结果与术前及正常水平相比,肿瘤未复发组患者术后Foxp3+Treg表达经历先降低后逐渐升高最终稳定于较低水平。与肿瘤未复发组患者比较,肿瘤复发组患者的AFP和Foxp3+Treg显著升高,明显高于术前及正常水平(均为P0.01),而且在肿瘤复发组患者中Foxp3+Treg早期异常性升高先于AFP。相关性分析提示,Foxp3+Treg与AFP变化一致,呈正相关(P0.01);而与效应性T细胞(CD3+T细胞、CD8+T细胞)表达变化相反,呈负相关(P0.05~0.01)。结论 Foxp3+Treg与肝癌肝移植术后肿瘤复发的关系密切。超UCSF标准的肝癌肝移植患者术后Foxp3+Treg越高,提示复发风险越大,联合检测AFP有助于及早发现肿瘤复发。     

复发性流产患者主动免疫治疗后淋巴细胞免疫表型的变化

目的探讨原因不明复发性流产(URSA)患者行淋巴细胞主动免疫治疗(LIT)后淋巴细胞免疫表型的变化对治疗效果的评估价值。方法采用流式细胞术分析URSA患者LIT前后外周血T淋巴细胞、B淋巴细胞、NK细胞和调节性T细胞免疫表型的变化(P0.05)。结果 25例URSA患者经LIT后成功妊娠16例,治疗后所有URSA患者(n=25)和妊娠成功组(n=16)外周血CD3+T细胞、CD4+HLA-DR+T细胞比例较治疗前均明显增加,CD4+T和CD3-CD56+NK细胞比例明显降低(P0.05);而治疗前后B细胞和Treg细胞、CD56bright CD16-NK、CD56dimCD16+NK、CD3+CD56+NKT及CD69+NK细胞比例则无明显变化(P0.05)。结论 LIT后外周血T细胞、NK细胞的比例发生了明显变化,CD4+T细胞、CD3-CD56+NK细胞比例降低和CD3+T细胞、活化CD4+T细胞增加也许有利于维持妊娠,T细胞和NK细胞的免疫表型有望作为LIT疗效评估的一个重要指标。     

不同免疫抑制方案对肾移植术受者CD4~+ Foxp3~+ 调节性T细胞的影响

目的 探讨不同免疫抑制剂方案对肾移植术受者外周血CD4~+ Foxp3~+调节性T细胞(regulatory T cells,Treg)表达水平的影响.方法 定群研究了2006年1月至2008年1月在本移植中心接受初次移植50例随访满1年肾移植受者,分为钙调神经蛋白抑制组(钙调神经蛋白抑制剂+吗替麦考酚酯+强的松)19例,其中环孢素组10例,他克莫司组9例;雷帕霉素组(雷帕霉素+吗替麦考酚酯+强的松)31例.另取20例行规律血液透析终末期肾病患者为对照组.采用流式细胞仪的方法检测3组外周血CD4~+ Foxp3~+ Treg占CD4~+ T细胞的比例,比较各组间表达水平与不同免疫抑制方案的关系.结果 钙调神经蛋白抑制剂组、雷帕霉素组和终末期肾病组3组年龄、性别比无统计学差异(P>0.05).钙调神经蛋白抑制剂组、雷帕霉素组2组冷缺血时间、HLA错配率、群体反应性抗体(PRA)和急性排斥反应发生率无统计学差异(P>0.05).雷帕霉素组和终末期肾病组CD4~+ Foxp3~+ T细胞占CD4~+ T细胞的比例均明显高于钙调神经蛋白抑制组,差异有统计学意义(P<0.01).使用环孢素患者和他克莫司患者外周血中CD4~+ Foxp3~+ T细胞占CD4~+ T细胞的比例之间无显著性差异(P>0.05).结论 肾移植术后服用雷帕霉素组患者外周血CD4~+ Foxp3~+ Treg占CD4~+ T细胞的比例显著高于服用钙调神经蛋白抑制组患者,提示雷帕霉素有助于诱导宿主对移植肾免疫耐受.     

Acceleration of apoptosis in CD4+CD8+ thymocytes by rapamycin accompanied by increased CD4+CD25+ T cells in the periphery

BACKGROUND: Rapamycin (Rapa) is an immunosuppressant that is used in patients and animal models to control allograft rejection. Its mechanisms of action are not fully understood. In this article, the authors have investigated the effects of therapeutic doses of Rapa on both thymic and peripheral T-cell populations in the adult rat. METHODS: The therapeutic dosage of Rapa was optimized using cardiac transplantation between LEW and DA rats. Thymic morphology was assessed by hematoxylin-eosin staining. Flow cytometric analysis was performed to analyze T-cell phenotype and apoptosis. T-cell receptor (TCR)-mediated T-cell responsiveness was evaluated by 3[H]-thymidine deoxyribose incorporation. RESULTS: Rapa induced atrophy in the thymus but not in peripheral lymphoid organs. Moreover, fibrosis occurred in thymus that was long-lasting after Rapa withdrawal. In animals treated with Rapa, there was a significant reduction in CD4+CD8+ thymocytes caused by accelerated apoptosis, whereas CD4-CD8-, CD4+CD8-, and CD8+CD4- populations remained unaffected. In contrast, the cellularity of the periphery lymphoid organs was not altered. Within the CD4+ thymocyte population, CD4+CD25+ thymocytes were resistant to Rapa-accelerated apoptosis, and in the periphery, the ratio of CD4+CD25+ to CD4+CD25- T cells was increased. Notably, the peripheral CD4+CD25+ T cells were hyporesponsive to TCR-mediated activation. CONCLUSIONS: The resistance of the peripheral CD4+CD25+ T cells to Rapa treatment might contribute to its immunosuppressive action. The long-term effects of Rapa on thymus atrophy and thymocyte development requires consideration with respect to its clinical application.     

CD19+CD5+ B cells in primary IgA nephropathy

The source of IgA and the mechanism for deposition of IgA in the mesangium remain unknown for primary IgA nephropathy. Because CD19(+)CD5(+) B cells are important producers of IgA and contribute to several autoimmune diseases, they may play an important role in IgA nephropathy. In this study, flow cytometry, quantitative PCR, and confocal microscopy were used to assess the frequency, distribution, Ig production, CD phenotypes, cytokine production, and sensitivity to apoptosis of CD19(+)CD5(+) B cells in the peripheral blood, peritoneal fluid, and kidney biopsies of 36 patients with primary IgA nephropathy. All patients with IgA nephropathy were significantly more likely to have CD19(+)CD5(+) B cells in the peripheral blood, peritoneal fluid, and kidney biopsies than were five control subjects and 10 patients with active systemic lupus erythematosus. The 33 patients who had IgA nephropathy and responded to treatment demonstrated a significant decrease in CD19(+)CD5(+) B cells in the peripheral blood, peritoneal fluid, and kidney (all P < 0.01). In the three patients who had IgA nephropathy and did not respond to treatment, the frequency of CD19(+)CD5(+) B cells did not change. CD19(+)CD5(+) B cells isolated from patients with untreated IgA nephropathy expressed higher levels of IgA, produced more IFN-gamma, and were more resistant to CD95L-induced apoptosis than cells isolated from control subjects and patients with lupus; these properties reversed with effective treatment of IgA nephropathy. In conclusion, these results strongly suggest that CD19(+)CD5(+) B cells play a prominent role in the pathogenesis of primary IgA nephropathy.     

CD4+CD25+ cells regulate CD8 cell anergy in neonatal tolerant mice

BACKGROUND: Injection of neonatal BALB/c mice with semi-allogeneic splenocytes leads to antigen-specific tolerance lasting into adulthood. Tolerant mice accept A/J skin grafts and fail to generate CD8 cytotoxic T lymphocyte (CTL) activity against A/J targets. Anergic CD8 T cells are present in tolerant mice, and CD4 regulatory cells function to maintain CD8 cell anergy. METHODS: Neonatal BALB/c mice were injected with 108 live CAF, splenocytes, and mice were deemed tolerant by accepting A/J grafts over 40 days. CD8 cell proliferation was measured by in vitro incorporation of bromodeoxyuridine coupled with fluorescence-activated cell sorter analysis. Alloantigen-specific cytotoxicity was tested using 51Cr release assays of A/J or third-party targets. RESULTS: We demonstrate that A/J-specific anergic CD8 cells are present in neonatal primed mice that develop tolerance but not in neonatal primed mice that reject A/J skin grafts. Anergic CD8 cells show decreased proliferation and no CTL activity against A/J targets. Addition of interleukin-2 (IL-2) to unfractionated cultures fails to restore CTL activity against A/J targets. However, addition of IL-2 to CD4-depleted cultures restores A/J-specific CD8 CTL activity. Removal of CD4+/CD25+ cells, but not CD4+/CD25- cells, also restores CD8 CTL activity against A/J in the presence, but not the absence, of IL-2. Moreover, when added back into cultures, purified CD4+/CD25+ cells from tolerant mice inhibit the generation of CD8 CTL against A/J targets. CONCLUSION: These data indicate that CD8 anergy is associated with the state of tolerance, and that CD4+CD25+ cells from tolerant mice function to maintain A/J-specific CD8 cell anergy in vitro.     

肝癌、肝硬化组织中CD3、CD57、CD20细胞数量与临床病理表现及预后的关系

目的探讨CD3、CD57、CD20细胞在原发性肝细胞癌(HCC)、癌旁、肝硬化及正常肝组织中的数量及意义.方法HCC 60例,单纯性肝硬化62例,正常肝组织23例,以免疫组化SP法进行CD3、CD57、CD20染色,对阳性细胞数进行定量分析并与临床资料进行相关探讨.结果(1)各组CD3+细胞平均数从高到低为癌旁组织、癌组织、肝硬化组织、正常肝组织(P＜0.05);各组CD57+细胞平均数从高到低为癌组织、癌旁组织、正常肝组织、肝硬化组织(P ＜0.05);各组CD20+细胞平均数从高到低为癌组织、癌旁组织、肝硬化组织、正常肝组织(P ＜0.01).(2)HCC中CD3+细胞、CD57+细胞、CD20+细胞与组织学分级均无明显关系.(3)HCC中CD57+细胞和CD20+细胞随着临床分期的发展有下降的趋势(P ＜0.05);HCC中CD3+细胞平均数与临床TNM分期无关.(4)HCC中15月内有转移组的CD57+、CD3+细胞数均少于无转移组(P＜0.01).HCC患者15月内有无转移与HCC和癌旁组织中的B细胞分布均无关.结论临床上,随着HCC患者的病情恶化,CD3+、CD57+、CD20+细胞逐渐减少.CD3+、CD57+、CD20+细胞可成为反映机体抗肿瘤特异性细胞免疫状态和生物学行为及判断患者预后的重要指标.     

血清微环境对小鼠T细胞衰老的调节作用

目的：探讨血清微环境对小鼠T细胞衰老的调节作用。方法：分别取年老（12～14月龄）及年轻（1.5～2月龄）小鼠各10只,提取其脾脏淋巴细胞及血清,实验分4组。组I为年老鼠T淋巴细胞＋10%年轻鼠血清;组II为年老鼠T淋巴细胞＋10%年老鼠血清;组III为年轻鼠T淋巴细胞＋10%年轻鼠血清;组IV为年轻鼠T淋巴细胞＋10%年老鼠血清。培养48h后,经流式细胞术研究CD8＋CD28＋共表达率差异。结果：组I和组II T细胞表面的CD8＋CD28＋共表达率分别是（10.84±0.6841）%和（3.18±0.1789）%,组III和组IV T细胞表面的CD8＋CD28＋共表达分别是（12.5±0.9445）%和（8.36±0.2074）%。各组间对比有统计学差异（P〈0.05）结论：血清微环境具有调节小鼠T细胞衰老的作用,年轻鼠血清能使年老鼠的T细胞表面的CD8＋CD28＋共表达率提高,年老鼠的血清能使年轻鼠的T细胞表面的CD8＋CD28＋共表达率降低。     

CD4+CD25+ regulatory T cells mediate acquired transplant tolerance

The Holy Grail of clinical organ transplantation is the safe induction of allograft tolerance. Transplant tolerance has been successfully induced in animal models. Since T cells play a pivotal role in graft rejection, modulating T cell function has been the primary focus of studies aimed at inducing transplant tolerance. Rodent models of transplant tolerance induction include central deletion and peripheral mechanisms involving activation-induced cell death (AICD), anergy, immune deviation, and production of regulatory T cells. These mechanisms are not mutually exclusive. Although clonal deletion and anergy limit self-reactive T cells in the thymus, these mechanisms alone are not sufficient for controlling self-reactive T cells in the periphery. There is now evidence that the adult animal harbors two functionally distinct populations of CD4(+) T cells; one mediates autoimmune disease and the other dominantly inhibits it. The latter cells express CD4, CD25 and CTLA-4. These thymus-derived T cells have recently been shown to mediate the induction and maintenance of transplant tolerance. These CD4(+)CD25(+) T cells are similar in origin, phenotype, and function to those that maintain natural self-tolerance and T cell homeostasis in the periphery. Against this background, is it possible that alloantigen specific regulatory T cells might be generated and expanded ex vivo before organ transplantation and then infused to induce long-term tolerance, perhaps without the need for chronic immunosuppression?