肾移植术后稳定状态受者淋巴细胞亚群的动态变化及其与肾功能的相关性分析

目的  探讨肾移植术后稳定状态受者外周血淋巴细胞亚群的动态变化及其与肾功能的相关性。方法  筛选行首次肾移植且术后半年内移植肾功能稳定的受者45例, 采用流式细胞术(FCM)检测受者术后15 d及1、3、6个月共计180份外周血样本淋巴细胞亚群比例和绝对值。分析淋巴细胞亚群随术后时间延长的动态变化及其与血清肌酐(Scr)和血尿素氮(BUN)的相关性。结果  受者术后4个时间点Scr值比较, 差异均无统计学意义(均为P > 0.05)。术后15 d与术后1个月、术后1个月与术后3个月BUN比较, 差异均有统计学意义(P=0.002、P=0.001);术后15 d与术后1个月比较, CD3⁺CD8⁺T细胞、CD3⁺CD4⁺T细胞、自然杀伤(NK)细胞比例及CD4/CD8比值, 差异均有统计学意义(P=0.009、P=0.004、P < 0.001、P=0.004)。B细胞比例术后15 d与术后1个月比较、术后1个月与术后3个月比较, 差异均有统计学意义(均为P < 0.001)。CD3⁺T细胞、CD3⁺CD8⁺T细胞、CD3⁺CD4⁺T细胞和NK细胞绝对值术后15 d与术后1个月比较, 差异均有统计学意义(P=0.001、P=0.002、P=0.003、P < 0.001)。CD3⁺CD8⁺T细胞绝对值术后3个月和术后6个月比较, 差异有统计学意义(P=0.015)。B细胞绝对值术后1个月与术后3个月比较, 差异有统计学意义(P=0.001)。淋巴细胞亚群比例和绝对值与Scr均不相关(均为P > 0.05), CD3⁺CD8⁺T细胞、NK细胞比例和绝对值与BUN均呈负相关(P < 0.001~0.05), CD3⁺CD4⁺T细胞、B细胞比例与BUN均呈正相关(P < 0.001~0.05), CD3⁺T细胞绝对值与BUN呈负相关(P < 0.05)。结论  肾移植术后稳定状态受者的淋巴细胞亚群中T细胞和NK细胞术后1个月内升高至稳定状态, B细胞术后3个月内降低至稳定状态, 且淋巴细胞亚群的动态变化与BUN相关。     

外周血淋巴细胞亚群绝对值和功能的动态监测在肾移植术后早期病毒感染风险预测中的价值

目的  探讨不同淋巴细胞亚群的绝对值和功能对于评估肾移植受者术后早期发生病毒感染风险的预测和诊断价值。方法  将95例肾移植受者纳入前瞻性观察队列研究,根据术后的免疫状态分为稳定组（77例）和感染组（18例）。分别于术前、术后2周、术后1个月、术后2个月、术后6个月采集外周血样本进行流式细胞检测。比较两组CD4⁺T细胞、CD8⁺T细胞、自然杀伤（NK）细胞绝对值的动态变化,通过检测干扰素（IFN）-γ⁺CD4⁺T细胞、IFN-γ⁺CD8⁺T细胞、IFN-γ⁺NK细胞比例分析两组受者淋巴细胞亚群功能,评估淋巴细胞亚群绝对值和功能在肾移植术后早期对病毒感染的预测和诊断价值。结果  在病毒感染时,感染组的CD4⁺T细胞、CD8⁺T细胞、NK细胞绝对值整体处于相对较低的水平;在术后2个月时,感染组的CD4⁺T细胞、NK细胞绝对值均低于稳定组;在术后6个月时,感染组的CD4⁺T细胞、CD8⁺T细胞绝对值均低于稳定组（均为P < 0.05）。在病毒感染时,感染组的IFN-γ⁺CD4⁺T细胞、IFN-γ⁺CD8⁺T细胞、IFN-γ⁺NK细胞比例均处于相对较低的水平,尤以IFN-γ⁺CD8⁺T细胞比例降低最为显著;在术后2个月,感染组的IFN-γ⁺CD8⁺T细胞、IFN-γ⁺NK细胞比例显著高于稳定组;在术后6个月,感染组的IFN-γ⁺CD4⁺T细胞、IFN-γ⁺CD8⁺T细胞比例均高于稳定组（均为P < 0.05）。logistic回归分析结果显示,术后2个月时,IFN-γ⁺CD8⁺T细胞和IFN-γ⁺NK细胞比例的升高与病毒感染风险增加均相关（均为P < 0.05）。受试者工作特征（ROC）曲线结果表明,淋巴细胞亚群绝对值联合其IFN-γ分泌功能对于免疫状态低下的受者病毒感染的诊断价值显著高于单用淋巴细胞亚群绝对值（P < 0.05）。结论  动态监测淋巴细胞亚群绝对值和功能的变化对病毒感染的预测、诊断及指导用药具有重要参考价值。     

流式细胞术在肾移植术后感染中的诊断价值

目的  探讨流式细胞术在肾移植术后感染中的诊断价值。方法  根据术后影像学和实验室检查结果,将51例首次肾移植受体分为细菌组33例、真菌组9例、BK病毒组9例;另选择肾移植术后稳定的受体28例作为稳定组。采用流式细胞术分析各组受体外周血淋巴细胞亚群比例和绝对计数。比较各组肾移植受体的肾功能、外周血淋巴细胞亚群的比例及绝对计数;采用受试者工作特征（ROC）曲线分析淋巴细胞亚群比例和绝对计数在肾移植术后感染性疾病中的诊断价值。结果  与稳定组相比,细菌组、真菌组和BK病毒组血清肌酐（Scr）水平和血尿素氮（BUN）水平均有不同程度升高,差异均有统计学意义（P=0.035、0.007、0.024;0.037、0.006、0.032）。与稳定组比较,细菌组和真菌组CD16⁺CD56⁺自然杀伤（NK）细胞比例均下降（P=0.036、0.015）;真菌组CD4⁺/CD8⁺T细胞比例明显下降（P=0.004）。与细菌组相比,真菌组和BK病毒组的CD3⁺CD8⁺T细胞比例均升高（P=0.013、0.008）,CD3⁺CD4⁺T细胞比例均降低（P=0.003、0.010）,CD4⁺/CD8⁺T细胞比例均明显下降（P=0.003、0.005）。与稳定组比较,细菌组、真菌组、BK病毒组CD3⁺T细胞数量、CD3⁺CD8⁺T细胞数量、CD16⁺CD56⁺NK细胞数量均明显降低（P=0.025、0.002、0.003;0.015、0.005、0.006;0.001、0.001、0.031）;真菌组和BK病毒组CD3⁺CD4⁺T细胞数量降低（P=0.001、0.003）;BK病毒组CD19⁺B细胞数量明显降低（P=0.019）。与细菌组比较,真菌组CD3⁺CD4⁺T细胞数量明显降低（P=0.023）。ROC曲线分析显示,CD3⁺CD4⁺T细胞和CD16⁺CD56⁺NK细胞数量诊断真菌感染的准确度较高,ROC曲线下面积分别为0.8492和0.8889;CD3⁺T细胞、CD3⁺CD4⁺T细胞和CD19⁺B细胞数量诊断BK病毒感染的准确度较高,ROC曲线下面积分别为0.8472、0.8452和0.8115。结论  采用流式细胞术检测外周血淋巴细胞亚群可以评估患者机体免疫功能状态,绝对计数能够直观地判断免疫程度,两者结合对于肾移植受者感染性疾病的诊断和鉴别诊断具有指导意义。     

淋巴细胞亚群分类对肾移植受者活动性肺结核的诊断价值

目的  探讨淋巴细胞亚群分类在诊断肾移植受者活动性肺结核中的临床价值。方法  回顾性分析52例肾移植术后受者的临床资料。根据影像学检查和病原学检查结果,将52例受者分为稳定组（19例）、结核组（9例）、细菌组（12例）以及真菌组（12例）。比较各组受者的肾功能情况;分析并比较各组受者淋巴细胞亚群的比例和绝对值;分析淋巴细胞亚群分类在肾移植术后活动性肺结核中的诊断价值。结果  与稳定组比较,结核组、细菌组、真菌组的血尿素氮和血清肌酐水平均明显升高（均为P＜0.05）,CD3⁺、CD8⁺、CD4⁺、自然杀伤（NK）细胞和CD19⁺淋巴细胞亚群的比例差异无统计学意义（均为P>0.05）,CD3⁺、CD8⁺、CD4⁺、NK和CD19⁺淋巴细胞亚群的绝对值明显降低（均为P＜0.05）。结核组和真菌组的CD8⁺淋巴细胞亚群比例明显高于细菌组（均为P＜0.05）。CD8⁺淋巴细胞亚群比例在鉴别诊断肾移植受者活动性肺结核和细菌性肺炎中的最佳临界值是33.27%,灵敏度和特异度分别为0.889和0.833,曲线下面积（AUC）为0.880。结论  淋巴细胞亚群分类可为肾移植受者活动性肺结核与细菌性肺炎的鉴别诊断和个体化治疗方案提供辅助诊断依据。     

肝移植术后发生急性排斥反应受者淋巴细胞亚群的变化及意义

目的  探讨肝移植受者术后发生急性排斥反应时淋巴细胞亚群的变化及意义。方法  选取接受肝移植且发生急性排斥反应的受者作为排斥组(17例),利用倾向评分匹配方法按1∶1比例选取肝功能稳定的肝移植受者作为对照组(17例)。分析肝移植术后急性排斥反应的发生情况,对比两组受者他克莫司浓度。比较两组受者外周血淋巴细胞亚群的绝对值和比例,采用受试者工作特征(ROC)曲线分析淋巴细胞亚群对肝移植术后急性排斥反应发生的诊断价值。比较排斥组治疗前后淋巴细胞亚群绝对值和比例的变化。结果  排斥组17例受者中,4例在术后28 d内发生急性排斥反应,13例在术后29~180 d发生急性排斥反应。两组他克莫司谷浓度差异无统计学意义(P=0.295)。与对照组比较,排斥组受者外周血T细胞、CD4⁺T细胞、B细胞和自然杀伤(NK)T细胞的比例均上升(均为P < 0.05)。肝移植术后早期NKT细胞比例升高是肝移植术后发生急性排斥反应的独立危险因素[比值比(OR)1.774,95%可信区间(CI)1.059~2.971,P=0.029]。ROC曲线分析结果提示,CD4⁺T细胞、B细胞和NKT细胞比例的曲线下面积(AUC)分别为0.76、0.73和0.77。联用CD4⁺T细胞、B细胞和NKT细胞比例的AUC为0.89,当临界值为0.69时,灵敏度为0.706,特异度为0.941。排斥组所有受者治疗后均逐渐恢复,最终肝功能正常,治疗后T细胞、CD4⁺T细胞、CD8⁺T细胞和NK细胞比例均降低(均为P < 0.05)。结论  NKT细胞比例升高提示肝移植术后急性排斥反应发生风险增加,联用CD4⁺T细胞、B细胞和NKT细胞比例可早期发现和诊断肝移植术后急性排斥反应。     

以西罗莫司为基础三联抗肿瘤疗法对大鼠肝癌肝移植复发模型T淋巴细胞的影响

目的  探讨以西罗莫司为基础联合槐耳颗粒、胸腺肽α-1的三联抗肿瘤疗法对大鼠肝癌肝移植复发模型T淋巴细胞的影响。方法 72只Sprague-Dawley(SD)大鼠以随机数字法分为三联组、西罗莫司组、槐耳组、胸腺肽组、阳性对照组、空白组, 每组12只。除空白组外, 其余各组均采用化学诱癌法建立模拟肝癌肝移植术后复发的动物模型。模型建立后, 取阳性对照组大鼠鉴定模型是否成功建立。采用流式细胞术分别检测各组大鼠外周血调节性T细胞(Treg)占CD4⁺T淋巴细胞比例(Treg%)、CD4⁺T淋巴细胞占淋巴细胞总数比例(CD4⁺T%)及CD8⁺T淋巴细胞占淋巴细胞总数比例(CD8⁺T%)。采用Spearman秩相关分析Treg%与CD4⁺T%、CD8⁺T%及CD4⁺/CD8⁺T淋巴细胞比值(CD4⁺/CD8⁺)之间的关系。结果 大鼠肝癌组织病理切片提示建模成功。阳性对照组的Treg%高于空白组, 差异有统计意义(P<0.05)。三联组的Treg%明显低于阳性对照组、胸腺肽组和槐耳组, 明显高于空白组(均为P<0.05)。与阳性对照组比较, 三联组、西罗莫司组和胸腺肽组的CD4⁺T%和CD8⁺T%较高, 差异有统计学意义(均为P<0.05)。三联组的CD4⁺T%和CD8⁺T%均高于胸腺肽组、西罗莫司组和槐耳组, 差异有统计学意义(均为P<0.05)。各组大鼠的外周血Treg%与CD4⁺ T%、CD8⁺ T%和CD4⁺/CD8⁺均呈负相关, 且三联抗肿瘤疗法可降低Treg%与CD4⁺/CD8⁺之间的负性相关关系。结论 西罗莫司为基础的三联抗肿瘤疗法可降低大鼠外周血Treg水平, 提高T淋巴细胞数量及CD4⁺/CD8⁺, 发挥抗肿瘤细胞生长和增殖的作用。      

人类白细胞抗原-G阳性的脐带间充质干细胞 体外诱导调节性T细胞的实验研究

目的  探讨人类白细胞抗原（HLA）-G阳性的脐带间充质干细胞在体外诱导调节性T细胞（Treg）产生的效果。方法  从新生儿脐带中分离脐带间充质干细胞,采用脂质体转染的方式将PEGFP-N1-HLA-G质粒转染到脐带间充质干细胞中,设为PEGFP-N1-HLA-G组; 转染空载体PEGFP-N1质粒的脐带间充质干细胞设为PEGFP-N1组; 相同条件下,未加入空载体的脐带间充质干细胞设为空白对照组。采用流式细胞仪检测脐带间充质干细胞标志物; 采用蛋白质免疫印迹法鉴定各组细胞HLA-G蛋白的表达; 各组细胞与健康人外周血中CD4⁺T细胞混合培养24 h和48 h后,采用流式细胞仪检测CD4⁺ CD25⁺ Foxp3⁺Treg占全部T细胞的比例。结果  脐带间充质干细胞CD45、CD34和HLA-DR呈阴性表达,CD29、CD44和CD105呈阳性表达; PEGFP-N1-HLA-G组可以表达HLA-G蛋白,与空白对照组和PEGFP-N1组比较差异均有统计学意义（均为P < 0.01）。PEGFP-N1-HLA-G组细胞在与CD4⁺T细胞混合培养24 h后,CD4⁺ CD25⁺ Foxp3⁺Treg占全部T细胞的（15.3±1.9）%,在培养48 h后,CD4⁺ CD25⁺ Foxp3⁺Treg占全部T细胞的（14.3±2.1）%,与空白对照组和PEGFP-N1组比较,差异均有统计学意义（均为P < 0.05）。结论  HLA-G基因修饰后脐带间充质干细胞能够有效地在体外诱导CD4⁺ CD25⁺ Foxp3⁺Treg的产生。     

西罗莫司促进小鼠心脏移植模型中调节性T细胞的分化和增殖

目的  探讨西罗莫司(SRL)对小鼠异位心脏移植模型的移植物存活时间以及脾脏中调节性T细胞(Treg)分化和增殖的影响。方法  应用Cuff法建立雄性BALB/c→C57BL/6小鼠颈部异位心脏移植模型。术后随机分为3组各10只受体:对照组术后不予特殊药物治疗, SRL组术后1~14 d予SRL 10 mg/(kg·d)灌胃, 环孢素(CsA)组术后1~14 d予CsA 30 mg/(kg·d)灌胃。记录移植心脏存活时间, 于移植心停搏或术后14 d取脾, 分离单个核细胞, 上流式细胞仪检测CD4⁺CD25⁺Treg占CD4⁺T细胞比例(CD4⁺CD25⁺Treg%), 采用逆转录-聚合酶链反应(RT-PCR)法半定量检测Foxp3信使核糖核酸(mRNA)表达水平。结果  与对照组比较, CsA组和SRL组均明显延长小鼠移植心的生存时间(均为P < 0.01), 而两者之间比较差异无统计学意义(P>0.05)。与对照组比较, CsA组脾脏中CD4⁺CD25⁺Treg%明显降低, 而SRL组则明显升高(均为P < 0.01), 后两组比较差异有统计学意义(P < 0.01)。SRL组脾脏T细胞的Foxp3 mRNA表达明显高于对照组和CsA组, 其中对照组亦明显高于CsA组(均为P < 0.01)。结论  在小鼠心脏移植模型中, SRL明显延长移植物的存活期, 促进CD4⁺CD25⁺Treg的增殖与生长, 有利于免疫耐受的形成。     

ImmuKnow检测免疫细胞功能在肾移植术后免疫功能监测中的应用

目的  探讨ImmuKnow检测免疫细胞功能在监测肾移植术后患者免疫功能变化的应用价值。方法  2013年1月至2014年12月在广州医科大学附属第二医院器官移植科实施肾移植手术的106例尿毒症患者, 分别于术前、术后12个月内发生感染或急性排斥反应时抽取血液标本。采用ImmuKnow测定CD4⁺ T细胞内的三磷腺苷(ATP)含量。观察与比较不同临床状态肾移植患者的ATP含量, 包括术前组、稳定组、急性排斥反应组和感染组(含重症肺炎)。检测外周血T细胞亚群CD4⁺T细胞、CD8⁺T细胞及自然杀伤(NK)细胞比例。采用Pearson相关分析法了解ATP值与他克莫司(FK506)和环孢素(CsA)血药谷浓度的关系。结果  感染组患者ATP含量低于术后稳定组患者(P < 0.001), 其中发生重症肺炎患者ATP含量低于发生其他感染的患者(P < 0.05)。感染组患者的CD4⁺T细胞百分比低于稳定组患者(P < 0.05)。ATP含量与移植患者术后FK506和CsA血药谷浓度无相关性。结论  ImmuKnow检测可用于监测肾移植患者术后免疫功能状态。CD4⁺T细胞内ATP含量检测对术后感染, 特别是对重症肺炎有提示和预警作用。     

体外光化学疗法对皮肤移植小鼠产生调节性T细胞的影响

目的  探讨输注体外光化学法处理的脾淋巴细胞对皮肤移植受体小鼠产生调节性T细胞(Treg)及移植物存活时间的影响。方法  以C57BL/6小鼠为供体,BALB/c小鼠为受体,建立小鼠皮肤移植模型。分离C57BL/6和BALB/c小鼠脾淋巴细胞(CSP、BSP),制备经8-甲氧基补骨脂素联合长波紫外线(PUVA)处理的小鼠脾淋巴细胞(PUVA-SP)。根据受者静脉输注的成分将实验动物随机分为5组(每组12只)：PUVA-BSP组、PUVA-CSP组、BSP组、CSP组及磷酸盐缓冲液(PBS)对照组,每组受体分别于术前7 d、手术当日和术后7 d按组别从尾静脉注入PUVA-BSP、PUVA-CSP、BSP、CSP或PBS。观察受体移植物的存活时间,检测受体外周血中CD4⁺CD25⁺Foxp3⁺Treg表达情况。结果  皮肤移植术后,PUVA-BSP组和PUVA-CSP组的受体小鼠外周细胞CD4⁺CD25⁺Foxp3⁺Treg的比例明显高于输注BSP组、CSP组和PBS对照组;PUVA-CSP组高于PUVA-BSP,BSP组和CSP组低于PBS对照组。PUVA-BSP组和PUVA-CSP组的受者小鼠移植皮片存活时间明显长于BSP组、CSP组和PBS对照组(均为P＜0.05)。结论  输注足够数量的PUVA-SP可诱导受体体内产生较多的CD4⁺CD25⁺Foxp3⁺Treg,可以显著延长移植物的存活时间。     

肺移植术后T细胞亚群研究进展

T细胞是肺移植术后免疫应答的主要效应细胞,其细胞亚群水平对肺移植受者机体免疫状态具有重要影响。本文综述CD4^＋ T细胞、CD8^＋ T细胞和调节性T细胞的免疫学机制及其与肺移植术后原发性移植肺功能障碍、排斥反应、免疫耐受和感染等的关系,同时探讨监测肺移植术后T细胞亚群的临床意义。     

CD8+ T cells produce RANTES during acute rejection of murine allogeneic skin grafts

BACKGROUND: Based on their chemoattractant properties, it is likely that chemokines play a role in recruiting alloantigen-primed T cells to allografts and in amplifying inflammation within the graft. The graft-infiltrating leukocytes producing specific chemokines remain largely unknown. METHODS: We tested the intragraft RNA expression of the chemokine RANTES (regulated on activation normal T expressed and secreted) and granzyme B during rejection of full thickness, allogeneic skin grafts by C57BL/6 mice. Grafts with different immunogenetic disparities were chosen to test expression when rejection was mediated by CD4+, CD8+, or both CD4+ and CD8+ T cells. RNA expression was also tested in purified CD4+ and CD8+ T cell populations from skin graft recipients. Immunohistology was performed on graft sections to test colocalization of RANTES protein and graft-infiltrating CD4+ and CD8+ T cells. RESULTS: Intra-allograft RANTES RNA expression was not observed during CD4+ T cell-mediated rejection. Expression of RANTES and granzyme B RNA was observed at low levels in purified populations of CD8+, but not CD4+, T cells from the spleen and lymph nodes of graft recipients beginning at day 7 after transplantation and increased thereafter. Intra-allograft RANTES protein was associated with a small number of graft-infiltrating CD8+ T cells but was also associated with endothelial cells and with many graft-infiltrating CD4+ T cells. CONCLUSIONS: CD8+ T cells produce RANTES during allogeneic skin graft rejection. In the allograft, the chemokine also colocalizes with CD4+ T cells that do not produce RANTES.     

他克莫司对肝移植受者外周血T淋巴细胞亚群及共刺激分子表达的影响

目的 探讨他克莫司（FK506）对肝移植受者外周血T淋巴细胞亚群及其表面共刺激分子表达的影响。方法 采用荧光标记单克隆抗体和流式细胞技术，测定术后采用FK506治疗的肝移植受者（FK506治疗组）在用FK506后1、2、3、4周时的外周血T淋巴细胞亚群及其表面共刺激分子CD28、CD152和ICOS分子的表达情况，以健康志愿者（健康对照组）和患终末期肝脏疾病拟行肝移植者（肝病对照组）为对照。结果 CD3^＋T淋巴细胞在各组间的差异均无统计学意义（P〉0．05）。经FK506治疗后，肝移植患者的CD4^＋T淋巴细胞逐渐减少，CD8^＋T淋巴细胞逐渐增加，并恢复至健康对照组水平（P〉0．05）。FK506治疗组T淋巴细胞亚群表面CD28分子和ICOS分子表达逐渐下降，并明显低于健康对照组（P〈0．05），而CD152分子表达增加，且明显高于健康对照组（P〈0．05）；其ICOS分子表达水平的下降晚于CD28分子，CD4^＋CD28^＋T淋巴细胞、CD8^＋CD28^＋T淋巴细胞和CD4^＋ICOS^＋T淋巴细胞均呈现相近的变化规律。结论 FK506能迅速纠正移植受者T淋巴细胞亚群紊乱，并抑制正性共刺激分子CD28和ICOS的表达，促进负性共刺激分子CD152的表达。     

Rejection in lung transplantation--an immunohistochemical study of transbronchial biopsies.

The number, distribution, and phenotype of mononuclear cells infiltrating the allograft lung transplant were determined immunohistochemically with monoclonal antibodies directed against cellular antigens (CD3, CD4, CD8, CD22, CD25, CD16, CD56, CD68, HLA-DR) on frozen sections of transbronchial biopsies. Seventy-two transbronchial biopsies from 21 patients undergoing lung or heart-lung transplantation were evaluated histologically and immunohistologically in a prospective study. Four major results were obtained in the graft lung parenchyma: (1) whatever the histological grading of rejection, T lymphocytes expressing CD3 were present and in a significantly higher number than in control subjects (P < 0.0005); (2) there was a positive correlation between histological rejection and the number of CD3+, CD8+, CD25+, CD16+ cells (P < 0.01); (3) the CD4/CD8 ratio was inverted (0.52 +/- 0.04), with no correlation with the histological rejection; and (4) the number and location of CD3+, CD25+ cells did not correlate with CMV identification in bronchoalveolar lavage. Immunohistochemical criteria could be used for diagnosis of rejection in the management of heart-lung transplantation.     

CD69 expression on peripheral CD8 T cells correlates with acute rejection in renal transplant recipients

BACKGROUND: The development of a noninvasive method to diagnose renal allograft rejection could prevent the complications associated with graft biopsy and allow more accurate surveillance of allograft function. The present study determines whether expression of CD69 on peripheral T lymphocytes of renal allograft recipients correlates with the presence of acute graft rejection. METHODS: Peripheral blood T lymphocytes from healthy volunteers, renal allograft recipients with elevated creatinine but no evidence of rejection on biopsy, and renal allograft recipients with biopsy-proven rejection were analyzed by flow cytometry for the expression of CD69 and various intracellular cytokines (interleukin-2, interferon-gamma). Results were then compared with the degree of rejection on biopsy. RESULTS: CD69 expression on CD3+, CD4+, and CD8+ T-cell subsets was low in controls and transplant recipients without allograft rejection. In contrast, patients with renal allograft rejection showed significantly elevated percentages of CD69+ cells in the CD3+ (P<0.01) and CD8+ subsets (P<0.01). The fraction of CD69+ and CD8+ T cells was found to be a more clinically useful test based on receiver-operator characteristics. CD69 expression on CD4+ T cells did not correlate with rejection. Significant intracellular cytokine levels were not detected in unstimulated T cells from any of the groups; stimulation with mitogens increased expression equally among the three groups. CONCLUSIONS: We demonstrate that expression of CD69 on CD3+ and CD8+ peripheral blood T cells correlates closely with the presence of acute graft rejection in renal allograft recipients. Measurement of this surface marker may provide a rapid, noninvasive, and accurate means by which graft rejection can be identified.     

Observational support for an immunoregulatory role of CD3+CD4+CD25+IFN-gamma+ blood lymphocytes in kidney transplant recipients with good long-term graft outcome.

There is evidence that interferon-gamma (IFN-gamma)-dependent interactions of dendritic cell (DC), T regulatory (Treg), and T suppressor (Ts) subpopulations contribute to allograft acceptance. We measured DC subsets, CD3+CD4+CD25+ (Treg phenotype) and CD3+CD8+CD28(-) (Ts phenotype) peripheral blood lymphocytes (PBL) expressing Foxp3, Th1 or Th2 cytokines, peripheral T- and B-cell counts, and plasma cytokines in 33 kidney transplant recipients with a serum creatinine of < or =1.8 mg/dl and 32 recipients with a serum creatinine of > or =2.0 mg/dl more than 100 days post-transplant. Cell subsets were measured in whole blood using four-color flow cytometry. Patients with increased creatinine had less frequently detectable CD3+CD4+CD25+IFN-gamma+ PBL than patients with good graft function (P = 0.017). In patients with good graft function, CD3+CD4+CD25+IFN-gamma+ PBL were associated with high Foxp3+, IL-2+, IL-12+, IL-4+, and IL-10+ CD3+CD4+CD25+ T PBL (P < 0.001), low CD3+CD8+CD28(-)Foxp3+ (P = 0.002), CD3+CD4+DR+ (P = 0.002), CD3+CD8+DR+ T (P = 0.005) and CD19+ B PBL (P = 0.005), and low lineage(-)HLA-DR+CD11c+CD123(-) DC1 (P = 0.006). Patients with impaired graft function did not show these associations. Additional flow cytometric analysis confirmed strong co-expression of IFN-gamma and Foxp3 by CD4+CD25+ PBL particularly in patients with good graft function. Our data support an immunoregulatory role of CD3+CD4+CD25+Foxp3+IFN-gamma+ cells in a subgroup of transplant recipients with good graft acceptance.     

Immunomodulatory Effects of Mixed Hematopoietic Chimerism: Immune Tolerance in Canine Model of Lung Transplantation

Long-term survival after lung transplantation is limited by acute and chronic graft rejection. Induction of immune tolerance by first establishing mixed hematopoietic chimerism (MC) is a promising strategy to improve outcomes. In a preclinical canine model, stable MC was established in recipients after reduced-intensity conditioning and hematopoietic cell transplantation from a DLA-identical donor. Delayed lung transplantation was performed from the stem cell donor without pharmacological immunosuppression. Lung graft survival without loss of function was prolonged in chimeric (n = 5) vs. nonchimeric (n = 7) recipients (p ≤ 0.05, Fisher's test). There were histological changes consistent with low-grade rejection in 3/5 of the lung grafts in chimeric recipients at ≥1 year. Chimeric recipients after lung transplantation had a normal immune response to a T-dependent antigen. Compared to normal dogs, there were significant increases of CD4+INFγ+, CD4+IL-4+ and CD8+ INFγ+ T-cell subsets in the blood (p < 0.0001 for each of the three T-cell subsets). Markers for regulatory T-cell subsets including foxP3, IL10 and TGFβ were also increased in CD3+ T cells from the blood and peripheral tissues of chimeric recipients after lung transplantation. Establishing MC is immunomodulatory and observed changes were consistent with activation of both the effector and regulatory immune response.