外周血T细胞CD38和HLA-DR的表达水平在儿童传染性单核细胞增多症中的临床意义

摘要 目的：探讨传染性单核细胞增多症（IM）患儿外周血T细胞活化分子CD38和人类白细胞抗原DR（HLA-DR）表达水平的临床意义。方法：采用流式细胞术分别检测45例IM患儿急性期和恢复期的活化分子CD38和HLA-DR在T细胞的表达水平,并与30例健康体检儿童进行对比。分析IM患儿急性期CD38和HLA-DR在T细胞的表达水平与EB病毒载量、肝功能指标、外周血异型淋巴细胞比例、淋巴细胞计数的相关性,并采用ROC曲线分析CD8⁺CD38⁺T和CD8⁺HLA-DR+T细胞百分比的诊断效能。结果：与对照组比较,IM急性期患儿的CD38和HLA-DR在T细胞的表达水平显著升高（P＜0.05）。CD8⁺CD38⁺T、CD8⁺HLA-DR+T细胞百分比分别与EBV-DNA、ALT、AST、LDH、异型淋巴细胞百分比、淋巴细胞计数呈正相关（P＜0.05）,与白蛋白（ALB）呈负相关（P＜0.05）;CD4⁺CD38⁺T、CD4⁺HLA-DR+T细胞百分比与上述指标无显著相关性（P均＞0.05）。IM恢复期CD38和HLA-DR在T细胞的表达水平较急性期明显降低（P＜0.05）。ROC曲线分析CD8⁺CD38⁺T、CD8⁺HLA-DR+T细胞百分比显示诊断儿童IM的AUC值分别为0.931和0.993,特异度均为100%,灵敏度分别为88.89 %和93.33 %。结论：流式法检测CD38和HLA-DR在T细胞的变化有助于判断病情变化。外周血CD8⁺CD38⁺T、CD8⁺HLA-DR+T细胞百分比不仅能反映出IM急性期肝功能损伤严重程度,还可作为儿童IM的流式诊断指标。     

外周血Treg细胞、T淋巴细胞及其亚群与早期宫颈癌的关系及对淋巴结转移的预测价值研究

摘要 目的：分析外周血Treg细胞、T淋巴细胞及其亚群与早期宫颈癌的关系及对淋巴结转移的预测价值。方法：选择我院自2017年1月至2020年12月接诊的60例接受子宫颈癌根治术及盆腔淋巴清扫术的早期宫颈癌患者作为观察组,另选同期的60例健康体检者作为对照组。比较两组外周血Treg细胞、T淋巴细胞及其亚群水平,使用受试者工作特征曲线（ROC）下面积（AUC）评价外周血Treg细胞、T淋巴细胞及其亚群对淋巴结转移的预测效能。结果：观察组外周血Treg细胞、CD8⁺T细胞水平高于对照组,CD3⁺T细胞、CD4⁺T细胞、CD4⁺/CD8⁺比值均低于对照组（P＜0.05）;观察组术后外周血Treg细胞、CD8⁺T细胞水平较术前降低,CD3⁺T细胞、CD4⁺T细胞、CD4⁺/CD8⁺比值均较术前升高（P＜0.05）;在60例早期宫颈癌患者中,发生淋巴结转移12例;淋巴结转移组术前外周血Treg细胞水平、CD8⁺T细胞高于非淋巴结转移组,CD3⁺T细胞、CD4⁺T细胞、CD4⁺/CD8⁺比值均低于非淋巴结转移组（P＜0.05）;经多因素Logistic回归分析,外周血Treg细胞、CD3⁺T细胞、CD4⁺/CD8⁺比值均是早期宫颈癌患者发生淋巴结转移的独立预测因素（P＜0.05）;经ROC曲线分析,外周血Treg细胞、CD3⁺T细胞联合CD4⁺/CD8⁺比值预测早期宫颈癌患者发生淋巴结转移的AUC为0.910。结论：外周血Treg细胞、T淋巴细胞及其亚群水平与早期宫颈癌的病情演变有关,其中外周血Treg细胞、CD3⁺T细胞联合CD4⁺/CD8⁺比值预测淋巴结转移的效能较好,值得进一步研究应用。     

脓毒症患者外周血T淋巴细胞PD-1表达变化及胸腺肽α-1的免疫调理作用研究

摘要 目的：探讨脓毒症患者外周血T淋巴细胞程序性细胞死亡受体1（PD-1）表达特点,分析胸腺肽?琢-1治疗对患者免疫功能的影响。方法：选择2018年3月至2020年6月我院重症医学科收治的140例脓毒症患者（脓毒症组）和同期于我院进行体检的95例健康志愿者（对照组）,根据急性生理与慢性健康评估Ⅱ（APACHE Ⅱ）、序贯器官衰竭评估（SOFA）评分结果将脓毒症患者分为APACHE Ⅱ 0~10分组（51例）、11~20分组（62例）和＞20分组（27例）;SOFA评分0~5分组（48例）、6~10分组（60例）和＞10分组（32例）。检测外周血CD4⁺T细胞上PD-1表达、CD8⁺T细胞上PD-1表达,比较组间差异性。Pearson秩相关性分析外周血CD4⁺T细胞上PD-1表达、CD8⁺T细胞上PD-1表达与APACHE Ⅱ、SOFA评分相关性。根据治疗方法将脓毒症患者分为A组（60例）和B组（80例）,A组给予常规综合治疗和乌司他丁治疗,B组在A组的基础上联合胸腺肽α-1治疗,比较两组治疗前后外周血T淋巴细胞（CD3⁺、CD4⁺、CD8⁺）、NK细胞（CD3-CD16⁺CD56⁺）差异。结果：脓毒症组外周血CD4⁺T细胞上PD-1表达、CD8⁺T细胞上PD-1表达高于对照组（P＜0.001）,外周血CD4⁺T细胞上PD-1表达、CD8⁺T细胞上PD-1表达随APACHE Ⅱ、SOFA评分的增加而增高,各组间差异显著（P＜0.05）。Pearson秩相关分析结果显示外周血CD4⁺T细胞上PD-1表达、CD8+T细胞上PD-1表达与APACHE Ⅱ评分、SOFA评分呈正相关（r=0.569、0.475;0.653、0.509,P均＜0.05）。B组治疗后CD3⁺、CD4⁺、CD3-CD16⁺CD56⁺高于A组（P＜0.05）,CD8⁺低于A组（P＜0.05）。结论：脓毒症患者外周血CD4⁺、CD8⁺T细胞上PD-1表达均增高,其表达与病情严重程度密切相关。给予胸腺肽α-1治疗可改善患者免疫功能。     

CD39的临床意义及其与肝癌浸润T细胞耗竭的相关性分析

摘要 目的：探索CD39分子（编码基因ENTPD1）在原发性肝细胞肝癌（Hepatocellular carcinoma, HCC）中的表达、临床意义及其与HCC中免疫浸润和T细胞耗竭的相关性。方法：TIMER、GEPIA、Kaplan-Meier Plotter、TCGA等数据库分析CD39在肝癌中的差异性表达、与免疫浸润的关系、相关基因的表达及其与肝癌患者预后的关系。免疫荧光染色、细胞测序和流式检测验证临床HCC患者的癌及癌旁组织中CD39的差异性表达及其和CD8⁺T细胞耗竭特性的相关性。结果：（1）生物信息学分析结果显示：CD39在多种肿瘤组织中表达上调（包括HCC）（P＜0.05）；且其表达水平与HCC的临床预后等显著相关（P＜0.05）；与CD39低表达组相比，HCC中CD39高表达组患者无复发生存期（relapse-free survival, RFS）（P=0.025）和无进展生存期（progression-free survival, PFS）（P=0.026）较短；CD39与HCC肿瘤微环境中CD8⁺T、CD4⁺T、巨噬细胞等免疫细胞浸润水平均有明显正相关。（2）临床HCC样本验证：免疫荧光和流式结果显示CD39在癌组织中的表达水平高于癌旁正常组织，与耗竭相关分子LAYN、TIM3、CTLA4等共表达，且在耗竭CD8⁺T细胞中表达比例显著高于非耗竭细胞。结论：CD39在HCC肿瘤组织及浸润的CD8⁺T细胞中高表达，与HCC的RFS、PFS、免疫细胞浸润等紧密相关，且参与CD8⁺T细胞耗竭。     

CD38基因敲除的淋巴瘤细胞株构建

摘要 目的：构建Luc⁺CD38^-的Raji细胞株,并进行功能的初步验证,为后期探索淋巴瘤细胞CD38位点免疫逃逸现象奠定基础。方法：通过CRISPR-cas9技术和PiggyBac(PB)转座子系统,对Luc⁺Raji细胞的CD38基因位点进行敲除,构建Luc⁺CD38^-Raji细胞株,使用流式细胞术检测与Luc⁺CD38^-Raji细胞株以1：1的比例共孵育CD19 CAR-T和CD38 CAR-T以及未转导的原始T细胞表面活化因子CD69的表达水平,荧光素酶检测法检测上述几组效应细胞对Luc⁺CD38^-Raji细胞株的杀伤效率。结果：成功构建Luc⁺CD38^-Raji细胞,激活实验结果显示,CD19 CAR-T与CD38 CAR-T均可以被Luc⁺Raji细胞激活。而Luc⁺CD38^-Raji19号单克隆细胞由于缺失CD38的表达,仅能够激活CD19 CAR-T。杀伤实验结果显示,两种CAR-T细胞均能够对Luc⁺Raji细胞进行杀伤,而CD38 CAR-T对Luc⁺CD38^-Raji19号单克隆细胞的杀伤效率与原始的T细胞相似。结论：成功构建了Luc⁺CD38^-Raji细胞株,为后期探索淋巴瘤CD38位点免疫逃逸现象奠定基础。     

扶正方对Lewis肺癌小鼠免疫功能、PI3K/AKT信号通路和外周血IL-2、IL-6、INF-γ的影响

摘要 目的：观察扶正方对Lewis肺癌小鼠免疫功能、磷脂酰肌醇3激酶（PI3K）/蛋白激酶B（AKT）信号通路和外周血白细胞介素（IL）-2、IL-6、γ干扰素（INF-γ）的影响。方法：将40只Lewis肺癌小鼠随机分为模型组（M组）、扶正方低剂量组（A组）、扶正方高剂量组（B组）、顺铂组（S组）,每组10只,A组、B组分别给予扶正方0.4 mL/20 g、0.8 mL/20 g灌胃,M组给予生理盐水0.4 mL/20 g灌胃,S组给予顺铂1 mg/mL,0.4 mL灌胃,连续14d,比较各组小鼠一般情况、肿瘤重量,胸腺指数、脾脏指数、脾脏CD3⁺细胞、CD4⁺细胞、CD8⁺细胞比例细胞百分比,鼠肿瘤组织PI3K/AKT信号通路蛋白表达水平及外周血IL-2、IL-6、INF-γ水平。结果：A组、B组、S组小鼠肿瘤重量低于M组,S组小鼠肿瘤重量低于A组、B组（P<0.05）,治疗前各组小鼠体重比较无统计学差异（P>0.05）,治疗后A组、B组小鼠体重高于S组、M组（P<0.05）。A组、B组小鼠胸腺指数显著高于M组、S组（P<0.05）。A组、B组CD3⁺、CD4⁺、CD4⁺/CD8⁺显著高于M组、S组,CD8⁺显著低于M组、S组（P<0.05）,B组CD3⁺、CD4⁺、CD4⁺/CD8⁺显著高于A组,CD8⁺低于A组（P<0.05）。A组、B组、S组小鼠肿瘤组织PI3K蛋白、AKT蛋白表达水平显著低于M组（P<0.05）。A组、B组、S组小鼠外周血IL-2、INF-γ水平显著高于M组,IL-6水平显著低于M组（P<0.05）。结论：扶正方可以提升Lewis肺癌小鼠免疫功能,调节IL-2、IL-6、INF-γ细胞因子水平,抑制PI3K/AKT信号通路起到抗肺癌的作用。     

手足口病合并脑炎患儿外周血T淋巴细胞亚群、血清VCAM-1及CRP的表达水平及其检测价值分析

摘要 目的：探讨与分析手足口病（HFMD）合并脑炎患儿外周血T淋巴细胞亚群、血清VCAM-1及CRP的表达水平及其检测价值。方法：2017年4月到2020年10月选择在本院诊治的手足口病合并脑炎患儿42例作为合并组,同期选择手足口病不合并脑炎患儿68例作为对照组,检测两组外周血T淋巴细胞亚群、血清血管细胞粘附分子-1（VCAM-）及C-反应蛋白（CRP）表达水平,并判断检测价值与进行相关性分析。结果：合并组的CD4⁺、CD8⁺T淋巴细胞相对比例都明显少于对照组（P<0.05）。合并组的血清VCAM-1及CRP含量明显高于对照组（P<0.05）。在80例患儿中,Spearsman分析显示CD4⁺、CD8⁺T淋巴细胞相对比例和血清VCAM-1、CRP含量都与手足口病合并脑炎的发生存在相关性（P<0.05）。二分类Logistic回归分析显示CD4⁺、CD8⁺T淋巴细胞相对比例和血清VCAM-1、CRP含量都为导致手足口病合并脑炎发生的重要因素（P<0.05）。结论：手足口病合并脑炎患儿多伴随有外周血T淋巴细胞亚群异常与血清VCAM-1、CRP的高表达,CD4⁺、CD8⁺T淋巴细胞相对比例、血清VCAM-1、CRP含量都为导致手足口病合并脑炎发生的重要因素。     

组蛋白去乙酰化酶3对外周CD4+ T细胞分化的影响

目的 探讨组蛋白去乙酰化酶3（HDAC3）对外周CD4⁺ T细胞分化及功能的调控作用。方法 采用CD4cre酶介导Hdac3杂合基因缺失小鼠（Hdac3^fl/flCD4^cre+/-）及其野生型正常对照小鼠（Hdac3^fl/fl，WT），流式细胞术检测HDAC3缺失对外周CD4⁺和CD8⁺ T细胞比例和数量的影响；在体外佛波酯（PMA）和离子霉素（Ionomycin）刺激条件下，流式细胞术检测HDAC3缺失对CD4⁺ T细胞中IFN-γ、IL-4和IL-17A的表达以及Tfh细胞产生的影响；采用ELISA检测HDAC3缺失对小鼠血清IFN-γ、IL-4和IL-17表达的影响；分选Hdac3^fl/flCD4^cre+/-和WT小鼠外周初始CD4⁺ T细胞，分别在Th1和Th2分化条件下培养，细胞内染色检测HDAC3缺失对Th1、Th2以及Th17相关细胞因子及其特异转录因子表达的影响；采用Microarray检测HDAC3缺失对CD4⁺ T细胞分化亚群相关基因表达的影响；采用链脲佐菌素（STZ）处理小鼠构建I型糖尿病（TIDM）疾病模型，检测HDAC3缺失对T1DM发病的影响。结果 与WT小鼠相比，Hdac3^fl/flCD4^cre+/-小鼠外周CD4⁺和CD8⁺ T细胞的比例和数量显著降低。Hdac3^fl/flCD4^cre+/-小鼠CD4⁺ T细胞及血清中IFN-γ的表达显著降低，而IL-4和IL-17A的表达显著增加，Tfh细胞比例也显著增加；HDAC3缺失抑制体外培养CD4⁺ T细胞向Th1分化但促进其向Th2分化；Microarray检测发现HDAC3缺失导致Th1型细胞谱系基因表达降低，而Th2、Th17以及Tfh细胞谱系基因表达增加；在STZ诱导条件下，HDAC3缺失抑制小鼠T1DM的发生和CD4⁺ T细胞向Th1分化。结论 HDAC3促进外周CD4⁺ T细胞向Th1细胞分化并加重T1DM的发生。     

T淋巴细胞亚群、血红蛋白及血小板在类风湿关节炎患者中的表达及临床意义分析

摘要 目的：探讨T淋巴细胞亚群、血红蛋白及血小板在类风湿关节炎患者中的表达及临床意义。方法：选取我院2020年1月到2023年1月收治的100例类风湿关节炎患者作为研究对象,依照患者病情活动性进行分组,将活动期类风湿关节炎的35例患者分为活动期组,将65例缓解期类风湿关节炎患者分为缓解期组,另选取同期体检的50名健康志愿者作为对照组,对比三组患者CD3⁺、CD4⁺、CD8⁺以及CD4⁺/CD8⁺比值,并对比三组受检者血红蛋白及血小板表达水平。应用Spearman相关分析分析T淋巴细胞亚群、血红蛋白及血小板与类风湿关节炎活动程度的相关性,并应用logistic回归分析分析T淋巴细胞亚群、血红蛋白及血小板对类风湿关节炎活动期的独立预测价值。结果：三组受检者T淋巴细胞亚群表达水平对比有差异,且活动期组CD3⁺、CD4⁺、CD4⁺/CD8⁺水平较缓解期组和对照组低,CD8⁺水平较高（P＜0.05）;三组受检者血红蛋白及血小板表达水平对比差异显著,且活动期组血红蛋白水平较缓解期组和对照组低,血小板水平较高（P＜0.05）;Spearman相关分析结果显示：CD3⁺、CD4⁺、CD4⁺/CD8⁺、血红蛋白与类风湿关节炎病情活动程度呈负相关,CD8⁺、血小板与类风湿关节炎病情活动程度呈正相关（P＜0.05）;logistic回归分析结果表明：CD4⁺/CD8⁺升高、血红蛋白升高及血小板降低为类风湿关节炎活动期的独立影响因素（P＜0.05）。结论：类风湿关节炎患者在疾病活动期T淋巴细胞亚群相关细胞比例、血红蛋白及血小板表达水平会出现明显变化,且与其活动程度具有明显相关性。以CD4⁺/CD8⁺升高、血红蛋白升高及血小板降低情况可独立判定类风湿关节炎活动期,因此临床上对于上述指标升高的类风湿关节炎患者需及时改善治疗措施,改善患者预后水平。     

慢阻肺患者运动负荷气道反应性与T细胞亚群的关系

摘要 目的：探讨与分析慢性阻塞性肺疾病（COPD）患者运动负荷气道反应性与T细胞亚群的关系。方法：2020年1月到2022年4月选择在本院诊治的慢阻肺患者88例作为慢阻肺组,同期选择在本院进行健康体检者88例作为健康组,检测两组T细胞亚群含量,判定两组的运动负荷气道反应性情况并进行相关性分析。结果：慢阻肺组的CD8⁺T淋巴细胞比例明显高于健康组,CD3⁺T淋巴细胞、CD4⁺T淋巴细胞比例明显低于健康组（P<0.05）。慢阻肺组的运动负荷气道反应性发生率为20.9 %,明显高于健康组的1.2 %（P<0.05）。在慢阻肺中,Spearsman分析显示运动负荷气道反应性发生率与CD3+T淋巴细胞、CD4⁺T淋巴细胞、CD8⁺T淋巴细胞比例存在相关性（P<0.05）。logistic回归分析显示CD3⁺T淋巴细胞、CD4⁺T淋巴细胞、CD8⁺T淋巴细胞比例都为影响运动负荷气道反应性发生的重要危险因素（P<0.05）。结论：慢阻肺患者多伴随有T细胞亚群异常,也多伴随有运动负荷气道反应性,运动负荷气道反应性与T细胞亚群存在相关性,也表明T细胞亚群紊乱是导致运动负荷气道反应性发生的重要因素。     

结核分枝杆菌核糖体蛋白丙氨酸乙酰转移酶过表达菌株的构建及分析

rimI基因编码的核糖体蛋白丙氨酸乙酰转移酶(ribosomal-protein-alanine acetyltransferase,RimI)为结核分枝杆菌GCN5相关N-乙酰转移酶家族成员,其在结核分枝杆菌中的生物学功能尚不十分清楚。为探索RimI的生物学特性及其对结核分枝杆菌致病性的影响,本研究以耻垢分枝杆菌为模式菌,构建过表达结核分枝杆菌rimI基因的重组菌株Msm∷pMV261-rimI。分别培养 Msm∷pMV261-rimI菌株和对照Msm∷pMV261菌株,分析两者生长速率、菌落形态和生物膜形成的差异,以及耐受低氧、低pH值、H₂O₂、二硫苏糖醇(dithiothreitol,DTT)和0.05%～1%十二烷基硫酸钠(sodium dodecyl sulfate,SDS)等逆环境的能力;并将两种菌株分别接种于鼠巨噬细胞RAW264.7,观察两者在巨噬细胞内的存活能力。结果表明,相较于对照菌株,过表达rimI的菌株在生长前中期速率降低,生物膜早期成膜变缓,但不影响生物膜的后期成熟。同时,过表达rimI的菌株抵抗低氧、低pH值、H₂O₂等逆环境的能力增强,在巨噬细胞内的存活能力增强。结果提示,rimI基因对分枝杆菌的生物膜形成、抗逆性及细胞内生存具有重要作用,可能与结核分枝杆菌的毒力密切相关。     

Mycobacterium tuberculosis Multidrug Resistant Strain M Induces an Altered Activation of Cytotoxic CD8+ T Cells

In human tuberculosis (TB), CD8⁺ T cells contribute to host defense by the release of Th1 cytokines and the direct killing of Mycobacterium tuberculosis (Mtb)-infected macrophages via granule exocytosis pathway or the engagement of receptors on target cells. Previously we demonstrated that strain M, the most prevalent multidrug-resistant (MDR) Mtb strain in Argentine, is a weak inducer of IFN-γ and elicits a remarkably low CD8-dependent cytotoxic T cell activity (CTL). In contrast, the closely related strain 410, which caused a unique case of MDR-TB, elicits a CTL response similar to H37Rv. In this work we extend our previous study investigating some parameters that can account for this discrepancy. We evaluated the expressions of the lytic molecules perforin, granzyme B and granulysin and the chemokine CCL5 in CD8⁺ T cells as well as activation markers CD69 and CD25 and IL-2 expression in CD4⁺ and CD8⁺ T cells stimulated with strains H37Rv, M and 410. Our results demonstrate that M-stimulated CD8⁺ T cells from purified protein derivative positive healthy donors show low intracellular expression of perforin, granzyme B, granulysin and CCL5 together with an impaired ability to form conjugates with autologous M-pulsed macrophages. Besides, M induces low CD69 and IL-2 expression in CD4⁺ and CD8⁺ T cells, being CD69 and IL-2 expression closely associated. Furthermore, IL-2 addition enhanced perforin and granulysin expression as well as the degranulation marker CD107 in M-stimulated CD8⁺ T cells, making no differences with cells stimulated with strains H37Rv or 410. Thus, our results highlight the role of IL-2 in M-induced CTL activity that drives the proper activation of CD8⁺ T cells as well as CD4⁺ T cells collaboration.     

Histone deacetylase 9 deficiency exaggerates uterine M2 macrophage polarization

The maternal-foetal interface is an immune-privileged site where the semi-allogeneic embryo is protected from attacks by the maternal immune system. Uterine macrophages are key players in establishing and maintaining pregnancy, and the dysregulation of the M1-M2 subpopulation balance causes abortion. We separated two distinct mouse uterine macrophage subpopulations during early pregnancy, CD45⁺F4/80⁺CD206⁻ M1-like (M1) and CD45⁺F4/80⁺CD206⁺ M2-like (M2) cells. The M1 preponderance was significantly exaggerated at 6 hours after lipopolysaccharide (LPS) treatment, and adoptive transfer of M2 macrophages partially rescued LPS-induced abortion. RNA sequencing analysis of mouse uterine M2 versus M1 revealed 1837 differentially expressed genes (DEGs), among which 629 was up-regulated and 1208 was down-regulated. Histone deacetylase 9 (Hdac9) was one of the DEGs and validated to be significantly up-regulated in uterine M2 as compared with M1. Remarkably, this differential expression profile between M1 and M2 was also evident in primary splenic macrophages and in vitro polarized murine peritoneal, bone marrow–derived and RAW 264.7 macrophages. In Hdac9/HDAC9 knockout RAW 264.7 and human THP-1–derived macrophages, the expression of M1 differentiation markers was unchanged or decreased whereas M2 markers were increased compared with the wild-type cells, and these effects were unrelated to compromised proliferation. Furthermore, Hdac9/HDAC9 ablation significantly enhanced the phagocytosis of fluorescent microspheres in M2 Raw 264.7 cells yet decreased the capacity of THP-1-derived M1 macrophages. The above results demonstrate that Hdac9/HDAC9 deficiency exaggerates M2 macrophage polarization in mouse and human macrophages, which may provide clues for our understanding of the epigenetic regulation on macrophage M1/M2 polarization in maternal-foetal tolerance.     

Identification and Enhancement of HLA-A2.1-Restricted CTL Epitopes in a New Human Cancer Antigen-POTE

Identification of CD8⁺ T cell epitopes that can induce T cells to kill tumor cells is a fundamental step for development of a peptide cancer vaccine. POTE protein is a newly identified cancer antigen that was found to be expressed in a wide variety of human cancers, including prostate, colon, lung, breast, ovary and pancreas. Here, we determined HLA-A2.1-restricted cytotoxic T lymphocyte (CTL) epitopes in the POTE protein, and also designed enhanced epitopes by amino acid (AA) substitutions. Five 9-mer peptides were first selected and their binding affinity to HLA-A2 molecules was measured by the T2 binding assay. POTE 272–280 and POTE 323–331 showed the strongest HLA-A2 binding affinity. AA substituted peptides POTE 252-9V (with valine at position 9), POTE 553-1Y (with tyrosine at position 1) and POTE 323-3F (with phenylalanine at position 3) conferred higher affinity for HLA-A2, and induced CTL responses cross-reactive with wild type antigens. While POTE 252-9V was the strongest in this respect, POTE 323-3F had the greatest increase in immunogenicity compared to wild type. Importantly, two modified epitopes (POTE-553-1Y and POTE-323-3F) induced CTLs that killed NCI-H522, a POTE-expressing HLA-A2⁺ human non-small cell lung cancer cell line, indicating natural endogenous processing of these epitopes. In conclusion, the immunogenicity of POTE epitopes can be enhanced by peptide modification to induce T cells that kill human cancer cells. A combination of POTE 553-1Y and POTE 323-3F epitopes might be an attractive vaccine strategy for HLA-A2 cancer patients to overcome tolerance induced by tumors and prevent escape.     

Co-ordinated isolation of CD8+ and CD4+ T cells recognizing a broad repertoire of cytomegalovirus pp65 and IE1 epitopes for highly specific adoptive immunotherapy

Background aimsAdoptive transfer of cytomegalovirus (CMV)-specific memory T cells can be used for treatment of CMV reactivation after allogeneic stem cell transplantation. As co-ordinated CD8⁺ and CD4⁺ T cells specific for a broad repertoire of CMV epitopes may be most effective for adoptive immunotherapy, the aim of this study was to isolate these cells from peripheral blood of CMV seropositive donors, irrespective of their HLA type.MethodsActivation of CMV-specific CD8⁺ and CD4⁺ T cells was compared after stimulation of donor peripheral blood with minimal epitope peptides, pools of overlapping 15-mer peptides or full-length protein. Furthermore, the kinetics of interferon (IFN)-γ production after stimulation was analyzed to determine the optimal time-point for IFN-γ-based isolation of CMV-specific T cells. The specificity, phenotype and functionality of generated T-cell lines were analyzed.ResultsCMV protein-spanning 15-mer peptide pools induced simultaneous activation of both CD8⁺ and CD4⁺ CMV-specific T cells, while full-length CMV protein only efficiently activated CD4⁺ CMV-specific T cells. Isolation of IFN-γ-secreting cells at the peak of the IFN-γ response after 4-h stimulation with CMV pp65 and IE1 peptide pools resulted in efficient enrichment of CMV-specific T cells. The T-cell lines contained high frequencies of CD8⁺ and CD4⁺ T cells recognizing multiple CMV pp65 and IE1 epitopes, and produced IFN-γ and tumor necrosis factor (TNF)-α upon specific restimulation.ConclusionsThis study provides a feasible strategy for the rapid generation of clinical-grade CD8⁺ and CD4⁺ T-cell lines with high specificity for multiple CMV pp65 and IE1 epitopes, which may be used for effective adoptive immunotherapy.     

Positioning of APOBEC3G/F Mutational Hotspots in the Human Immunodeficiency Virus Genome Favors Reduced Recognition by CD8+ T Cells

Due to constitutive expression in cells targeted by human immunodeficiency virus (HIV), and immediate mode of viral restriction upon HIV entry into the host cell, APOBEC3G (A3G) and APOBEC3F (A3F) have been considered primarily as agents of innate immunity. Recent bioinformatic and mouse model studies hint at the possibility that mutation of the HIV genome by these enzymes may also affect adaptive immunity but whether this occurs in HIV-infected individuals has not been examined. We evaluated whether APOBEC-mediated mutations within common HIV CD8⁺ T cell epitopes can potentially enhance or diminish activation of HIV-specific CD8⁺ T cells from infected individuals. We compared ex vivo activation of CD8⁺ T lymphocytes from HIV-infected individuals by wild type HIV peptide epitopes and synthetic variants bearing simulated A3G/F-induced mutations by measuring interferon-γ (IFN-γ) production. We found that A3G/F-induced mutations consistently diminished HIV-specific CD8⁺ T cell responses against the common epitopes we tested. If this reflects a significant trend in vivo, then adaptation by HIV to enrich sequences that are favored for mutation by A3G/F (A3G/F hotspots) in portions of its genome that encode immunogenic CD8⁺ T cell epitopes would favor CTL escape. Indeed, we found the most frequently mutated A3G motif (CCC) is enriched up to 6-fold within viral genomic sequences encoding immunodominant CD8⁺ T cell epitopes in Gag, Pol and Nef. Within each gene, A3G/F hotspots are more abundant in sequences encoding epitopes that are commonly recognized due to their HLA restriction. Thus, in our system, mutations of the HIV genome, mimicking A3G/F activity, appeared to abrogate or severely reduce CTL recognition. We suggest that the physiological significance of this potential effect in facilitating CTL escape is echoed in the adaptation of the HIV genome to enrich A3G/F hotspots in sequences encoding CTL epitopes that are more immunogenic at the population level.     

Imaging effector functions of human cytotoxic CD4+ T cells specific for Plasmodium falciparum circumsporozoite protein

Malaria vaccines, comprised of irradiated Plasmodium falciparum sporozoites or a synthetic peptide containing T and B cell epitopes of the circumsporozoite protein (CSP), elicit multifunctional cytotoxic and non-cytotoxic CD4⁺ T cells in immunised volunteers. Both lytic and non-lytic CD4⁺T cell clones recognised a series of overlapping epitopes within a ‘universal’ T cell epitope EYLNKIQNSLSTEWSPCSVT of CSP (NF54 isolate) that was presented in the context of multiple DR molecules. Lytic activity directly correlated with T cell receptor (TCR) functional avidity as measured by stimulation indices and recognition of naturally occurring variant peptides. CD4⁺ T cell-mediated cytotoxicity was contact-dependent and did not require de novo synthesis of cytotoxic mediators, suggesting a granule-mediated mechanism. Live cell imaging of the interaction of effector and target cells demonstrated that CD4⁺ cytotoxic T cells recognise target cells with their leading edge, reorient their cytotoxic granules towards the zone of contact, and form a stable immunological synapse. CTL attacks induced chromatin condensation, nuclear fragmentation and formation of apoptotic bodies in target cells. Together, these findings suggest that CD4⁺ CTLs trigger target cell apoptosis via classical perforin/granzyme-mediated cytotoxicity, similar to CD8⁺ CTLs, and these multifunctional sporozoite- and peptide-induced CD4⁺ T cells have the potential to play a direct role as effector cells in targeting the exoerythrocytic forms within the liver.     

Full length antigen priming enhances the CTL epitope-based DNA vaccine efficacy

Although CD8⁺ cytotoxic T lymphocyte (CTL) epitope-based DNA vaccination is valuable experience on vaccine research but many attempts are still continued to achieve acceptable protective response. To study the role of full length antigen in CTL epitope immunization, we evaluated cellular immunity of diverse patterns of complete Herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) and the immunodominant CTL epitope (498–505) DNA injection in C57BL/6 mice. Optimal immune response was observed in the group immunized with the full length of gB in the first injection and CTL epitope in the second and third vaccination as assessed by lymphocyte proliferation assay (MTT), cytokine assay (ELISA) and CTL assay. B cell and spatially CD4⁺ T cell epitopes in full length protein might be important for appropriate priming of CTL immune response. These findings may have important implication for the improvement of CTL epitope based DNA vaccine against HSV and other pathogens.     

NLRC5 potentiates anti-tumor CD8+ T cells responses by activating interferon-β in endometrial cancer

ObjectivesNLR family CARD domain containing 5 (NLRC5) could promote major histocompatibility complex class I (MHC-I)-dependent CD8⁺ T cell-mediated anticancer immunity. In this study, the immunosurveillance role and underlying mechanisms of NLRC5 in endometrial cancer (EC) were characterized.MethodsCD8⁺ T cells were separated from healthy women's peripheral blood by using magnetic beads. The effect of NLRC5 and interferon-β (IFN-β) on immunosurveillance of EC were examined through a mouse tumor model and a CD8⁺ T cell-EC cell coculture system after NLRC5 overexpression and IFN-β overexpression or depletion. The effect of NLRC5 on IFN-β expression was examined with gain- and loss-of-function experiments.ResultsNLRC5 overexpression in the EC cell and CD8⁺ T cell coculture system inhibited EC cell proliferation and migration and promoted EC cell apoptosis and CD8⁺ T cell proliferation. In vivo, NLRC5 overexpression increased the proportion of CD8⁺ T cells and inhibited EC progression. Furthermore, IFN-β overexpression in the EC cell and CD8⁺ T cell coculture system activated CD8⁺ T cell proliferation; however, genetic depletion of IFN-β exerted the opposite effects. In addition, NLRC5 could negatively regulate IFN-β expression in EC cells. Mechanistically, NLRC5 potentiated the antitumor responses of CD8⁺ T cells to EC by activating IFN-β.ConclusionsTaken together, our findings demonstrated that NLRC5 potentiates anti-tumor CD8⁺ T cells responses by activating interferon-β in EC, suggesting that genetically escalated NLRC5 and IFN-β may act as potential candidates for the clinical translation of adjuvant immunotherapies to patients with EC.