Identification of prognostic molecular subtypes and model based on CD8+ T cells for lung adenocarcinoma

Background:Cytotoxic T lymphocytes(CD8+T)cells function critically in mediating anti-tumor immune response in cancer patients.Characterizing the specific functions of CD8+T cells in lung adenocarcinoma(LUAD)could help better understand local anti-tumor immune responses and estimate the effect of immunotherapy.Methods:Gens related to CD8+T cells were identified by cluster analysis based on the single-cell sequencing data of three LUAD tissues and their paired normal tissues.Weighted gene co-expression network analysis(WGCNA),consensus clustering,differential expression analysis,least absolute shrinkage and selection operator(LASSO)and Cox regression analysis were conducted to classify molecular subtypes for LUAD and to develop a risk model using prognostic genes related to CD8+T cells.Expression of the genes in the prognostic model,their effects on tumor cell invasion,and interactions with CD8+T cells were verified by cell experiments.Results:This study defined two LUAD clusters(CD8+0 and CD8+1)based on CD8+T cells,with cluster CD8+0 being significantly associated with the prognosis of LUAD.Three heterogeneous subtypes(clusters 1,2,and 3)differing in prognosis,genome mutation events,and immune status were categorized using 42 prognostic genes.A prognostic model created based on 11 significant genes(including CD200R1,CLEC17A,ZC3H12D,GNG7,SNX30,CDCP1,NEIL3,IGF2BP1,RHOV,ABCC2,and KRT81)was able to independently estimate the death risk for patients in different LUAD cohorts.Moreover,the model also showed general applicability in external validation cohorts.Low-risk patients could benefit more from taking immunotherapy and were significantly related to the resistance to anticancer drugs.The results from cell experiments demonstrated that the expression of CD200R1,CLEC17A,ZC3H12D,GNG7,and SNX30 was significantly downregulated,while that of CDCP1,NEIL3,IGF2BP1,RHOV,ABCC2 and KRT81 was upregulated in LUAD cells.Inhibition of CD200R1 greatly increased the invasiveness of the LUAD cells,but inhibiting CDCP1 expression weakened the invasion ability of LUAD cells.Conclusion:This study defined two prognostic CD8+T cell clusters and classified three heterogeneous molecular subtypes for LUAD.A prognostic model predictive of the potential effects of immunotherapy on LUAD patients was developed.

肿瘤微环境中与CD8^(+)T细胞耗竭相关的分化及代谢重编程的研究进展

CD8^(+)T细胞耗竭是导致机体免疫应答减弱及恶性肿瘤复发转移的核心原因。T细胞不仅在迁移进入肿瘤微环境(tumor microenvironment,TME)时需要突破重重屏障,而且在浸润至肿瘤微环境的T细胞的分化及代谢过程依然受到多因素的调控,往往造成CD8+T细胞抗肿瘤活性受到抑制,进一步形成耗竭性T细胞,导致免疫逃逸。因此,研究导致T细胞耗竭的相关因素及发生机制是逆转机体低免疫应答的关键,为抗肿瘤免疫治疗提供坚实的基础及依据。该文综述了近年来关于CD8^(+)T细胞在肿瘤微环境中的浸润分化、代谢重编程及耗竭性T细胞形成的相关过程,以期为改善T细胞抗肿瘤疗效提供潜在策略。

LncRNA LINC01137通过诱导CD8^(+)T细胞耗竭促进非小细胞肺癌进展的机制研究

目的研究长链非编码RNA(long non-coding RNA,LncRNA)LINC01137在非小细胞肺癌(nonsmall cell lung cancer,NSCLC)免疫逃逸中的生物学功能及其潜在的调节机制。方法采集24例健康志愿者和24例NSCLC患者血液样本,并收集NSCLC肿瘤组织和癌旁组织检测LINC01137水平。利用Starbase数据库预测LINC01137与miR-22-3p的结合位点,荧光素酶报告基因分析进行验证。采用A549细胞来源的外泌体和/或sh-LINC01137干扰序列转染A549细胞,检测细胞增殖和侵袭能力;收集转染后的细胞上清液培养CD8^(+)T细胞,检测CD8^(+)T细胞耗竭标志物干扰素-γ(interfereron-γ,IFN-γ)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、颗粒霉素B(granzyme B)和白细胞介素-2(interleukin-2,IL-2)水平,以及PD-1+Tim3^(+)CD8^(+)T细胞百分比。采用外泌体和/或miR-22-3p模拟物(miR-22-3p mimic)转染CD8^(+)T细胞,检测PD-1蛋白水平。结果与癌旁组织相比,NSCLC肿瘤组织中LINC01137表达(3.357±0.548 vs 1.011±0.371)明显升高;与健康志愿者相比,NSCLC患者外周血LINC01137表达(3.216±0.342 vs 1.007±0.313)亦明显升高,差异具有统计学意义(t=-17.367,-17.147,均P<0.001)。肿瘤组织LINC01137表达与外周血中LINC01137表达呈正相关(r=0.755,P<0.05)。在A549细胞来源的外泌体中LINC01137显著富集。与Exo+sh-NC组相比,Exo+sh-LINC01137组细胞活力(65.852%±4.715%vs 100.153%±11.934%)及细胞侵袭(21.464%±3.481%vs 43.126%±1.447%)能力显著降低,差异具有统计学意义(t=4.630,9.953,均P<0.01)。NSCLC患者外周血中LINC01137表达和CD8^(+)T细胞百分比呈负相关(r=-0.520,P<0.05)。与Exo+sh-NC组相比,Exo+sh-LINC01137组IFN-γ(3865.314±543.852 pg/ml vs 1786.971±105.982 pg/ml),TNF-α(4631.930±510.715pg/ml vs 1973.242±379.623pg/ml),Granzyme B(3876.496±312.438pg/ml vs 1879.439±287.584pg/ml)和IL-2 mRNA水平(3.286±0.437 vs 1.015±0.314)升高,PD-1+Tim3^(+)CD8^(+)T细胞百分比(7.680%±2.185%vs 18.952%±3.216%)降低,差异具有统计学意义(t=-6.497,-7.237,-8.146,-7.310,5.021,均P<0.01)。miR-22-3p是LINC01137的靶基因。与Exo+NC mimic组相比,Exo+miR-22-3p组PD-1蛋白水平(0.384±0.087 vs 1.003±0.147)显著降低,差异有统计学意义(t=6.277,P<0.01)。结论NSCLC患者肿瘤组织及外周血中LINC01137表达显著上调;NSCLC细胞来源的外泌体中LINC01137通过靶向CD8^(+)T细胞中miR-22-3p并抑制其表达,诱导CD8^(+)T细胞耗竭,促进NSCLC细胞免疫逃逸。

抑制细胞毒性CD8^(+)T细胞治疗白癜风研究进展

白癜风是一种色素脱失性皮肤病,易诊治难根治,致病机制可能涉及遗传、自身免疫、氧化应激、黑色素细胞破坏等方面,最终致病环节为细胞毒性CD8^(+)T细胞异常增殖靶向损伤黑素细胞。综述了阻断细胞毒性CD8^(+)T细胞应激损伤黑素细胞途径治疗白癜风研究进展。

Glutamine deprivation impairs function of infiltrating CD8^(+)T cells in hepatocellular carcinoma by inducing mitochondrial damage and apoptosis

BACKGROUND The functions of infiltrating CD8^(+)T cells are often impaired due to tumor cells causing nutrient deprivation in the tumor microenvironment.Thus,the mechanisms of CD8^(+)T cell dysfunction have become a hot research topic,and there is increased interest on how changes in metabolomics correlate with CD8^(+)T cell dysfunction.AIM To investigate whether and how glutamine metabolism affects the function of infiltrating CD8^(+)T cells in hepatocellular carcinoma.METHODS Immunohistochemical staining and immunofluorescence were performed on surgically resected liver tissues from patients.Differentially expressed genes in infiltrating CD8^(+)T cells in hepatocellular carcinoma were detected using RNA sequencing.Activated CD8^(+)T cells were co-cultured with Huh-7 cells for 3 d.The function and mitochondrial status of CD8^(+)T cells were analyzed by flow cytometry,quantitative real-time polymerase chain reaction,and transmission electron microscopy.Next,CD8^(+)T cells were treated with the mitochondrial protective and damaging agents.Functional alterations in CD8^(+)T cells were detected by flow cytometry.Then,complete medium without glutamine was used to culture cells and their functional changes and mitochondrial status were detected.RESULTS There were a large number of infiltrating PD-1+CD8^(+)T cells in liver cancer tissues.Next,we cocultured CD8^(+)T cells and Huh-7 cells to explore the regulatory effect of hepatoma cells on CD8^(+)T cells.Flow cytometry results revealed increased PD-1 expression and decreased secretion of perforin(PRF1)and granzyme B(GZMB)by CD8^(+)T cells in the co-culture group.Meanwhile,JC-1 staining was decreased and the levels of reactive oxygen species and apoptosis were increased in CD8^(+)T cells of the co-culture group;additionally,the mitochondria of these cells were swollen.When CD8^(+)T cells were treated with the mitochondrial protective and damaging agents,their function was restored and inhibited,respectively,through the mitochondrial damage and apoptotic pathways.Subsequently,complete medium without glutamine was used to culture cells.As expected,CD8^(+)T cells showed functional downregulation,mitochondrial damage,and apoptosis.CONCLUSION Glutamine deprivation impairs the function of infiltrating CD8^(+)T cells in hepatocellular carcinoma through the mitochondrial damage and apoptotic pathways.

Neutrophils inhibit CD8^(+)T cells immune response by arginase-1 signaling in patients with sepsis

BACKGROUND:Patients with sepsis often exhibit an acute inflammatory response,followed by an immunosuppressive phase with a poor immune response.However,the underlying mechanisms have not been fully elucidated.METHODS:We sought to comprehensively characterize the transcriptional changes in neutrophils of patients with sepsis by transcriptome sequencing.Additionally,we conducted a series of experiments,including real-time quantitative polymerase chain reaction(RT-qPCR)and flow cytometry to investigate the role of arginase-1 signaling in sepsis.RESULTS:Through the analysis of gene expression profiles,we identified that the negative regulation of T cell activation signaling was enriched,and the expression of arginase-1 was high in neutrophils from patients with sepsis.Furthermore,we conducted flow cytometry and found that the function of CD8^(+)T cells in septic patients was impaired.Moreover,neutrophils from septic patients inhibited the percentage of polyfunctional effector CD8^(+)T cells through arginase-1.Additionally,the proportions of granzyme B^(+)IFN^(-)γ^(+)CD8^(+)T and TNF^(-)α^(+)IFN^(-)γ^(+)CD8^(+)T cells increased after inhibition of arginase-1 signaling.CONCLUSION:The impaired effector function of CD8^(+)T cells could be restored by blocking arginase-1 signaling in patients with sepsis.

雌性和雄性非肥胖糖尿病小鼠中CD8^(+)T细胞分化亚群差异研究

目的以雌性和雄性非肥胖糖尿病(nonobese diabetic,NOD)小鼠以及健康对照癌症研究所(institute for cancer research,ICR)小鼠为研究对象,比较分析初始、效应、记忆、耗竭以及调节性CD8^(+)T细胞分化亚群差异,探讨1型糖尿病(type 1 diabetes,T1D)背景下的性别因素对CD8^(+)T细胞分化命运的影响。方法采用流式细胞术检测雌雄NOD小鼠脾脏、胰腺引流淋巴结(pancreatic draining lymph nodes,pLN)、胰腺浸润淋巴细胞(pancreas-infiltrating lymphocytes,PIL)、初始T细胞(naive T cells,T_(N))、中枢记忆T细胞(central memory T cells,T_(CM))、效应T细胞(effector T cells,T_(EFF))、效应前体样T细胞(effector precursor T cells,T_(EP))、耗竭T细胞(exhausted T cells,T_(EX))、耗竭前体T细胞(precursor exhausted T cells,T_(PEX))以及调节性T细胞(regulatory T cells,Tregs)等CD8^(+)T细胞分化亚群的频率和表型差异。结果与雄性NOD小鼠比较,雌性NOD小鼠pLN及PIL中IFN-γ^(+)、CD107a^(+)和CCL5^(+)CD8^(+)T_(EFF)频率显著升高(P<0.01,P<0.05,P<0.05),同时脾脏中CD8^(+)T_(N)、CD8^(+)T_(CM)、CD8^(+)T_(EX)、CD8^(+)T_(PEX)和CD122^(+)CD8^(+)Tregs亚群的频率均显著降低(P<0.01,P<0.01,P<0.01,P<0.01,P<0.001);而雌性和雄性ICR小鼠体内除CD122^(+)CD8^(+)Tregs差异变化与NOD小鼠一致(P<0.05),其余上述各CD8^(+)T细胞分化亚群无显著差异。结论雄激素可能通过抑制记忆T细胞向效应T细胞分化,同时促进效应T细胞功能耗竭,导致雌雄发病率差异。

富含萝卜硫素的西兰花提取物对小鼠外周血CD8^(+)T淋巴细胞活化及肠道菌群的影响

目的:探讨富含萝卜硫素(SFN)的西兰花提取物对小鼠肠道菌群及外周血CD8^(+)T淋巴细胞活化的影响。方法:将15只C57BL/6N小鼠随机分为空白对照(CON)组、阴性对照(NC)组和SFN组,每组5只小鼠,分别给予蒸馏水、不含SFN的西兰花提取物、含0.3 g/L SFN的西兰花提取物125μL灌胃,连续4周。每2 d测量1次体重。实验结束前采集小鼠外周血检测活化的CD8^(+)T淋巴细胞百分比以及CD8^(+)T淋巴细胞中IL-2、TNF-α的含量。收集小鼠粪便,进行肠道菌群分析。结果:3组小鼠不同时间点体重差异无统计学意义(P>0.05)。SNF组小鼠外周血活化的CD8^(+)T淋巴细胞百分比、CD8^(+)T淋巴细胞中IL-2和TNF-α含量高于CON组和NC组(P<0.05)。3组小鼠肠道菌群组成存在差异,SFN组小鼠肠道中疣微菌门及阿克曼菌属的相对丰度高于CON组和NC组(P<0.05)。肠道中阿克曼菌属的相对丰度与CD8^(+)T淋巴细胞中IL-2和TNF-α含量呈正相关(r=0.934和0.664,P<0.05)。结论:长期连续服用富含SFN的西兰花提取物能增加小鼠肠道中阿克曼菌属等有益菌的丰度,同时可增强外周血CD8^(+)T淋巴细胞的功能。

阴道灌洗液中炎症因子与CD4^(+)和CD8^(+)T细胞在高级别宫颈鳞状上皮内病变与早期宫颈癌发病中的作用研究

目的:探讨阴道灌洗液中CD4^(+)与CD8^(+)T细胞亚群分布及局部炎症因子水平在高级别宫颈鳞状上皮内病变(high-grade cervical squamous intraepithelial lesions,HSIL)与早期宫颈鳞癌发病中的作用。方法:收集桂林市人民医院高危型人类乳头状瘤病毒(high-risk human papilloma virus,HR-HPV)感染的HSIL患者120例和早期宫颈癌(cervical cancer,CC)患者144例的临床资料。并随机选取未感染HR-HPV的健康女性60例作为对照组。比较3组患者的临床资料、血清和宫颈灌洗液炎症因子(IL-2、INF-γ、IL-10、TNF-α)、CD4^(+)与CD8^(+)T细胞分布与比值。多因素逻辑回归分析HSIL进展至早期CC的影响因素,绘制ROC及校准图对模型评估。结果:对照组、HSIL组与早期CC组患者血IL-2、INF-γ、IL-10、TNF-α水平、CD4^(+)细胞分布、CD8^(+)T细胞分布与CD4^(+)/CD8^(+)比值比较,差异均无统计学意义(均P>0.05);3组患者阴道灌洗液中IL-2、INF-γ、IL-10、TNF-α水平、CD4^(+)细胞分布、CD8^(+)T细胞分布与CD4^(+)/CD8^(+)比值比较,差异均具有统计学意义(均P<0.05),且早期CC组高于HSIL组及对照组(均P<0.05),HSIL组高于对照组(P<0.05)。逻辑回归分析显示,模型2中产次>2次(OR=3.119,95%CI:4.353~6.737)与阴道灌洗液CD4^(+)T细胞百分比(OR=0.327,95%CI:0.188~0.478)(P<0.001,AUC=0.908),模型3中CD4^(+)/CD8^(+)(OR=0.809,95%CI:0.356~1.868)(P<0.001,AUC=0.873)对HSIL发展至早期CC有独立影响;两模型ROC曲线无明显差异(Z=1.550 4,P=0.121)。模型3中仅用CD4^(+)/CD8^(+)就能良好预测,该模型为优,该模型校准曲线与标准曲线接近。结论:HR-HPV感染后,宫颈局部免疫状态参与HSIL及HSIL进展至早期CC,且CD4^(+)/CD8^(+)T细胞比值可作为其独立保护因素。

LAG3对多房棘球蚴感染小鼠模型CD8^(+)T细胞免疫功能调节作用的研究

目的研究淋巴细胞激活基因3(Lymphocyte activation gene 3,LAG3)对多房棘球蚴慢性感染小鼠CD8^(+)T细胞免疫功能的调节作用。方法取C57BL/6野生型(Wild-type,WT)和LAG3缺陷型(Knock-out,KO)小鼠各10只,每只小鼠经肝门静脉接种3000个多房棘球蚴原头节建立多房棘球蚴感染模型。感染12周后,分别取两组小鼠肝脏和脾脏组织,采用苏木精-伊红(Hematoxylin-eosin,HE)染色观察肝脏病灶周围炎性细胞浸润和病理表现,通过免疫组织化学观察肝脏病灶周围“炎症微环境”与脾脏中CD8^(+)T的比例。收集两组小鼠肝脏与脾脏淋巴细胞,采用流式细胞术筛选不同CD8^(+)T细胞亚群,检测CD8^(+)T细胞、效应记忆CD8^(+)T细胞(Effector memory CD8^(+)T cell,CD8^(+)Tem)、中心记忆CD8^(+)T细胞(Central memory CD8^(+)T cell,CD8^(+)Tcm)、初始CD8^(+)T细胞(Naive CD8^(+)T cell,CD8^(+)Tn)比例与绝对数,以及分泌细胞因子γ干扰素(IFN-γ)、肿瘤坏死因子α(TNF-α)、白细胞介素10(IL-10)和白细胞介素17A(IL-17A)的比例。通过多房棘球蚴虫体蛋白体外刺激两组小鼠肝脏淋巴细胞,采用流式细胞术检测CD8^(+)T细胞分泌细胞因子IFN-γ、IL-10和IL-17A的比例。结果HE染色结果显示,与野生型小鼠比较,LAG3缺陷型小鼠肝脏形成的炎性病灶数量增多,差异有统计学意义(P<0.05)。免疫组织化学结果显示,与野生型小鼠比较,LAG3缺陷型小鼠肝脏病灶周围CD8^(+)T细胞募集升高,差异有统计学意义(P<0.05);LAG3缺陷型小鼠脾脏CD8^(+)T细胞募集有降低的趋势,差异无统计学意义(P>0.05)。流式细胞术结果显示,与野生型小鼠比较,LAG3缺陷型小鼠肝脏效应记忆型CD8^(+)T细胞比例有升高的趋势,差异无统计学意义(P>0.05);脾脏CD8^(+)T细胞比例降低,脾脏CD8^(+)Tem表型比例升高,差异均有统计学意义(P<0.01)。LAG3缺陷型小鼠肝脏和脾脏CD8^(+)T细胞分泌TNF-α和IL-10能力均增强,差异均有统计学意义(P<0.05)。多房棘球蚴的虫体蛋白体外刺激肝脏淋巴细胞实验结果显示,与野生型小鼠比较,LAG3缺陷型小鼠CD8^(+)T细胞分泌IFN-γ、IL-10比例升高,差异均有统计学意义(P<0.05);LAG3缺陷型小鼠CD8^(+)T细胞分泌IL-17A的能力有升高趋势(P>0.05)。结论在小鼠多房棘球蚴慢性感染中,LAG3可抑制肝脏和脾脏CD8^(+)T细胞免疫应答能力,调节炎症反应。

构建胃癌中CD8^(+)T细胞相关预后模型并筛选治疗药物

目的:胃癌是高度异质性的恶性肿瘤,其病死率高,患者就诊时多已处于晚期且预后差。CD8^(+)T细胞可识别和消灭肿瘤细胞,对癌症的进展有重要影响。本研究旨在构建CD8^(+)T细胞浸润相关预后模型,为胃癌患者预后评估提供新的生物标志物及其药物治疗提供客观依据。方法:从癌症基因组图谱(The Cancer Genome Atlas,TCGA)数据库中下载371例胃癌样本的数据为训练集。从基因表达综合(Gene Expression Omnibus,GEO)数据库中下载433例胃癌样本的数据为测试集。计算训练集胃癌样本中CD8^(+)T细胞相对含量,将样本分为CD8^(+)T细胞高含量组和CD8^(+)T细胞低含量组,采用Kaplan-Meier法分析和比较2组之间的生存关系;采用加权基因共表达网络(weighted gene co-expression network analysis,WGCNA)分析训练集数据,筛选出与CD8^(+)T细胞最具相关性的基因模块;对该模块基因进行单因素Cox回归分析、最小绝对收缩和选择算子(least absolute shrinkage and selection operator,LASSO)回归分析及多因素Cox回归分析筛选出与预后相关的基因,构建预后风险评分模型。应用模型计算训练集和测试集患者的风险评分并分别将患者分为高风险组和低风险组,并对模型进行内部及外部测试。应用肿瘤免疫单细胞枢纽2(tumor immune single-cell hub 2,TISCH2)数据库及人类蛋白质图谱(The Human Protein Atlas,HPA)数据库对模型基因进行验证。结合模型进行临床病理特征分析、功能富集分析、免疫相关性分析及化学治疗药物敏感性分析。结果:生存分析显示CD8^(+)T细胞低含量组的预后更差。WGCNA分析筛选出与CD8^(+)T细胞最具相关性的基因模块为Ebisque4。单因素Cox回归分析、LASSO逻辑回归分析及多因素Cox回归分析最终筛选出9个独立预后基因并构建预后风险评分模型。模型内部及外部测试结果均表明该模型具有较强的预测能力。TISCH2数据库验证表明模型基因在肿瘤微环境中具有不同的表达模式;HPA数据库验证表明模型基因的表达情况与预后模型较一致。临床病理特征分析表明风险评分与组织浸润深度相关;功能富集分析表明高风险组富集于分子间相互作用的通路,而低风险组则富集于代谢相关通路;免疫相关性分析表明高、低风险组之间的多个免疫细胞、免疫功能和免疫检查点均有差异;化学治疗药物敏感性分析表明高风险组对帕唑帕尼的敏感性更高,低风险组对西妥昔单抗的敏感性更高。结论:肿瘤微环境中的CD8^(+)T细胞浸润对胃癌患者的预后有显著的影响。基于CD8^(+)T细胞相关基因构建的预后模型具有普适性和临床适用性,对胃癌患者的药物治疗有重要的提示作用。

杀伤细胞凝集素样受体B1对类风湿关节炎外周血CD8^(+)T细胞功能的影响

目的研究杀伤细胞凝集素样受体B1(CD161)对类风湿关节炎(RA)病人外周血CD8^(+)T细胞及其亚群功能的影响。方法自2023年5―8月,整合基因组学数据库(GEO)不同数据集中对照组及RA组病人的外周血单细胞数据、Bulk转录组测序数据及临床相关数据,观察CD161在对照组及RA组中的表达差异情况。通过相关性分析指出CD161与RA病人临床炎症指标及治疗反应预后的相关性。运用单细胞无监督聚类、细胞注释方法观察CD161主要表达的细胞亚群及不同细胞亚群在对照组及RA组中的变化趋势。利用单细胞生物学评分功能计算不同亚群细胞的生物学功能评分,结合基因差异表达分析和功能富集分析,探索RA病人CD8^(+)T细胞及其亚群关键生物学功能与CD161基因表达的潜在关系。结果在人体外周全血中,与对照组相比,RA组中CD161高表达(8.779±0.657)(P<0.05),且CD161的表达与RA病人炎症因子C反应蛋白(CRP,r=−0.21)及血沉(ESR,r=−0.25)呈负相关(P<0.05)。CD161主要表达于NK细胞1.35±0.87、CD4^(+)T细胞0.62±0.82及CD8^(+)T细胞0.85±1.01,但在CD8^(+)T细胞中表达异质性最显著(标准差1.01大于平均数0.85),且CD161在CD8^(+)记忆T细胞中表达丰度最高1.38±0.34。与对照组相比,RA组中CD8^(+)T及CD8^(+)记忆T细胞频率均下降。与对照组相比,在RA病人外周血CD8^(+)记忆T细胞中,CD161表达降低(1.46±0.33比0.97±0.37,P<0.05),CD161与CD8^(+)记忆T细胞迁移(0.24±0.12比0.16±0.11)、细胞毒作用(0.52±0.27比0.45±0.38)呈正相关(均P<0.05),与趋化及促炎能力(0.15±0.04比0.17±0.02)呈负相关(P<0.05),与细胞增殖(0.02±0.04比0.03±0.04)或凋亡(0.12±0.22比0.18±0.18)相关性差异无统计学意义(P>0.05)。结论CD161介导CD8^(+)记忆T细胞细胞毒作用及促炎功能改变促进RA发生、发展。

慢阻肺小鼠肺组织CD8^(+)T细胞功能耗竭及抑制性受体阻断剂干预研究

目的 初步探讨慢性阻塞性肺疾病小鼠CD8^(+)T细胞功能耗竭状态及抑制性受体抗体干预后变化。方法 将28只小鼠分为正常对照组、COPD对照组、PD-1抗体干预治疗组(COPD+Anti-PD-1组),以烟熏及LPS吸入方式建立COPD模型,PD-1抗体干预治疗组在造模12周后腹腔注射PD-1干预,16周后用小动物肺功能仪检测各组小鼠肺功能,流式细胞仪检测各组小鼠肺组织CD8^(+)T细胞PD-1表达水平及分泌细胞因子功能。留取小鼠部分肺组织行病理检查评估肺气肿程度。结果 COPD对照组CD8^(+)T细胞数量较正常对照组增多(P<0.01),COPD对照组表达PD-1的CD8^(+)T细胞较正常对照组增多(P<0.05),PD-1抗体干预治疗组CD8^(+)T细胞表达TNF-α比例较COPD对照组增多(P<0.01)。PD-1抗体干预治疗组小鼠气道阻力较COPD对照组小鼠下降(P<0.05)。PD-1抗体干预治疗组平均线性截距较COPD对照组下降(P<0.05),PD-1抗体干预治疗组平均肺泡数较COPD对照组增加(P<0.01)。结论 慢性阻塞性肺疾病小鼠肺组织CD8^(+)T细胞较正常对照组表达抑制性受体PD-1,存在功能耗竭,应用PD-1抗体治疗后可部分恢复CD8^(+)T细胞功能及改善肺功能。

外周血CD8^(+)T淋巴细胞低表达与肝细胞肝癌的临床相关性分析

1目的分析肝细胞肝癌(HCC)患者外周血中CD8^(+)T淋巴细胞低表达的危险因素并构建临床预测模型。方法收集2018年8月至2021年12月安徽医科大学第二附属医院收治143例肝细胞肝癌患者为研究对象,应用流式细胞仪检测患者外周血淋巴细胞,对比正常人群外周血CD8^(+)T淋巴细胞计数范围将143例患者分为CD8^(+)T淋巴细胞低表达组及CD8^(+)T淋巴细胞正常表达组,采用单因素分析分析肝癌患者外周血CD8^(+)T淋巴细胞表达下降的危险因素,多因素logistic二元回归分析D8^(+)T淋巴细胞表达下调的危险因素。应用R4.2.1建立Nomogram预测模型图,绘制ROC曲线计算一致性指数,绘制校准曲线内部验证进行决策曲线分析(DCA)并进行比较。结果143例HCC患者45例(31.5%)患者CD8^(+)T细胞表达下降,98例(68.5%)患者CD8^(+)T细胞表达正常或增高。两组患者在甲胎蛋白(AFP)、血小板(PLT)、谷草转氨酶(AST)、总胆红素(TBIL)、白蛋白(ALB)、凝血酶原时间(PT)、国际标准值(INR)、K值、R15有显著性统计学差异,P<0.05。多因素分析发现AFP>20ng/L,(0R=0.22,95%CI:0.06-0.86,P<0.05)、Ks值,(0R=0.22,95%CI:0.06-0.86,P<0.05)、肝硬化是肝细胞肝癌患者D8^(+)T细胞低表达的独立危险因素。

PRKCH的表达对CD8^(+)T细胞功能及黑色素瘤免疫治疗的影响

目的 探讨PRKCH表达对CD8^(+)T细胞功能的影响及其在黑色素瘤免疫治疗效果预测中的作用。方法 运用Kaplan-Meier法分析黑色素瘤免疫治疗队列以及肿瘤基因组图谱(TCGA)数据中PRKCH在黑色素瘤中的表达及其与总生存期(overall survival, OS)的相关性。利用单细胞RNA测序(single-cell RNA-sequencing, scRNA-seq)数据分析PRKCH的细胞表达群体,利用蛋白质互作网络(protein-protein interaction networks, PPI)和基因集富集分析(gene set enrichment analysis, GSEA)评估PRKCH诱导抗肿瘤免疫应答的机制。利用逆转录病毒过表达和敲减PRKCH,检测小鼠CD8^(+)T细胞功能。利用B16-OVA小鼠黑色素瘤模型验证PRKCH过表达的OT-1 CD8^(+)T细胞对肿瘤生长的影响。结果 在黑色素瘤免疫治疗队列和TCGA数据中,高水平的PRKCH显著延长患者OS(P<0.05)。scRNA-seq分析显示PRKCH可以在T细胞表达。PPI、GSEA和scRNA-seq分析显示PRKCH的高表达与更多的细胞毒性和记忆性T细胞形成相关。过表达和敲减PRKCH分别增强和减少CD8^(+)T细胞的增殖及IFN-γ、Granzyme B的表达。过表达PRKCH增强OT-1 CD8^(+)T细胞的肿瘤抑制能力。结论 PRKCH增强CD8^(+)T细胞抑制肿瘤进展的能力,并可以用于预测黑色素瘤免疫治疗效果。

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.

癸酸能够活化CD8^(+)T细胞并提高其抗肿瘤免疫反应能力

目的:探究中链脂肪酸癸酸对CD8^(+)T细胞活化的影响,及其对CD8^(+)T细胞介导的抗肿瘤免疫反应的作用和机制。方法:建立C57BL/6小鼠黑色素瘤B16F10皮下荷瘤模型,随机分为癸酸组(10 mg/kg癸酸灌胃)和对照组(等量溶剂灌胃),观察癸酸对小鼠肿瘤生长以及生存率的影响,采用流式细胞术检测肿瘤微环境中浸润CD8^(+)T细胞的活化水平。建立B16F10-OVA和OT-I T细胞共培养体系,采用流式细胞术检测癸酸对CD8^(+)T细胞的肿瘤细胞杀伤能力的影响。采用α-CD8抗体清除B16F10荷瘤小鼠体内CD8^(+)T细胞,观察对小鼠肿瘤体积的影响。小鼠原代CD8^(+)T细胞经癸酸处理后,采用WB、ELISA及qPCR、流式细胞术检测T细胞受体(TCR)活化、效应细胞因子产生以及增殖和代谢水平。在B16F10荷瘤小鼠模型中,观察α-PD-1抗体联合癸酸给药对小鼠肿瘤生长以及生存率的影响。结果:在小鼠黑色素瘤荷瘤模型中,与对照组相比,癸酸组小鼠移植瘤体积显著降低且生存率显著提高(均P<0.05),肿瘤浸润CD8^(+)T细胞IFN-γ和TNF-α的表达水平显著升高(P<0.01)。经癸酸处理的OT-I T细胞对B16F10-OVA细胞的杀伤水平显著升高(P<0.01)。在荷瘤小鼠模型中用α-CD8抗体清除CD8^(+)T细胞后,癸酸对移植瘤的抑制作用显著降低(P<0.0001)。小鼠原代CD8^(+)T细胞经癸酸处理后,TCR活化水平显著升高、细胞因子IL-2和IFN-γ的产生增多、线粒体代谢水平显著上调(均P<0.05)。在黑色素瘤荷瘤小鼠模型中,癸酸与α-PD-1抗体联用,能够显著抑制小鼠移植瘤生长并提高其生存率(均P<0.05)。结论:癸酸能够促进CD8^(+)T细胞活化、增强其抗肿瘤免疫反应能力。

艾滋病合并结核病对CD8^(+)T淋巴细胞的影响

目的探讨艾滋病(AIDS)合并结核病(TB)对CD8^(+)T淋巴细胞的影响。方法将单纯人类免疫缺陷病毒(HIV)感染者、单纯TB患者、HIV感染合并TB者各200例分别作为HIV组、TB组、HIV/TB组,分析各组血清CD8^(+)T细胞及CD4^(+)T细胞变化。结果三组CD8^(+)T细胞水平比较呈现HIV组>HIV/TB组>TB组,且组间差异有统计学意义(χ2=128.779,P<0.001)。HIV/TB组、HIV组CD8^(+)T细胞水平较正常水平升高(ZHIV/TB=8.343,PHIV/TB<0.001;ZHIV=7.988,PHIV<0.001),而TB组CD8^(+)T细胞较正常水平明显下降(ZTB=8.682,PTB<0.001)。三组CD4^(+)T细胞水平均较正常下降(ZHIV=11.088,PHIV<0.001;ZTB=5.562,PTB<0.001;ZHIV/TB=12.077,PHIV/TB<0.001)。按照CD4^(+)T细胞计数进行分层分析发现,当CD4^(+)T细胞计数≤100 cells/μL时,三组CD8^(+)T细胞水平比较:HIV组>TB组,HIV组>HIV/TB组,且HIV/TB组CD8^(+)T细胞水平较正常显著下降(Z0-100=1.604,P0-100=0.109);当CD4^(+)T细胞≥101 cells/μL时,CD8^(+)T细胞三组比较:HIV/TB组>TB组,HIV组>TB组;当CD4^(+)T细胞>500 cells/μL,三组CD8^(+)T细胞水平均在正常范围内。三组CD8^(+)T细胞与CD4^(+)T细胞变化趋势的差异分析结果提示,以HIV组为参照,随着CD4^(+)T细胞计数的下降,TB组、HIV/TB组的CD8^(+)T细胞均比HIV组下降趋势更明显,差异有统计学意义(斜率分别为0.344和0.216,P<0.001)。结论随着CD4^(+)T细胞的下降,HIV感染合并TB和TB均可导致CD8^(+)T细胞下降,且TB的CD8^(+)T细胞下降更加显著。

白驳丸及其拆方对白癜风小鼠CD8^(+)T细胞及黑素细胞的影响

目的探讨白驳丸及其拆方对白癜风小鼠CD8^(+)T细胞及黑素细胞的影响。方法正常C57BL/6小鼠作为空白组,同品系小鼠随机分为模型组、阳性药组、白驳丸及其拆方组。除空白组外,其余各组均构建内源性自身反应性CD8^(+)T细胞靶向皮肤黑素细胞的白癜风动物模型。造模成功后,分别予以托法替尼、白驳丸及其拆方水溶液灌胃,空白组及模型组等量纯水灌胃,各组小鼠均灌胃10周。采用小型摄影棚及数码相机拍摄小鼠尾部皮肤,Image J图像处理后观察皮损脱色情况;采用全组织包埋免疫荧光观察皮损CD8^(+)T细胞及黑素细胞数量及分布;采用流式细胞术观察皮损CD8^(+)T细胞及黑素细胞比例及功能。结果与空白组比较,模型组尾部皮损脱色面积增大,CD8^(+)T细胞浸润及黑素细胞缺失增多,CD3^(+)CD8^(+)T、CD8^(+)CD69^(+)T、CD8^(+)CXCR3^(+)T细胞比例增加,CD45^(-)CD117^(+)黑素细胞比例下降。与模型组比较,白驳丸及其拆方可不同程度改善白癜风小鼠尾部皮损脱色情况,减少CD8^(+)T细胞浸润及黑素细胞缺失,降低CD3^(+)CD8^(+)T、CD8^(+)CD69^(+)T、CD8^(+)CXCR3^(+)T细胞比例,增加CD45^(-)CD117^(+)黑素细胞比例;其中以白驳丸全方组效果最为显著,全方减养血活血通络药组效果最不明显。结论白驳丸及其拆方可不同程度地抑制CD8^(+)T细胞数量及功能,保护黑素细胞,改善白癜风小鼠尾部皮损脱色情况,其中养血活血通络药可能发挥核心作用。

Are CD45RO^(+)and CD45RA^(-)genuine markers for bovine memory T cells?

Effective vaccination in duces memory T cells,which protect the host against pathogen re-infecti ons.Therefore,detection of memory T cells is essential for evaluating vaccine efficacy,which was originally dependent on cytokine induction assays.Currently,two isoforms of CD45 tyrosine phosphatase,CD45RO expression and CD45RA exclusion(CD45RO^(+)/CD45RA^(-)) are used extensively for detecting memory T cells in cattle.The CD45RO^(+)/CD45RA^(-) markers were first established in humans around three decades ago,and were adopted in cattle soon after.However,in the last two decades,some published data in humans have challenged the initial paradigm,and required multiple markers for identifying memoryT cells.On the contrary,memoryT cell detection in cattle still mostly relies on CD45RO^(+)/CD45RA^(-)despite some con troversial evidence.In this review,we summarized the current literature to exami ne if CD45RO^(+)/CD45RA^(-)are valid markers for detecting memoryT cells in cattle.It seems CD45RA and CD45RO(CD45RA/RO)as markers for identifyi ng bovine memoryT cells are questi on able.