肺鳞状细胞癌和腺癌PD-L1蛋白及相关miRNA表达差异的研究

摘要 目的：探讨肺鳞状细胞癌（鳞癌）和腺癌PD-L1蛋白及相关miRNA表达的差异。方法：2019年5月至2020年11月来我院就诊的非小细胞肺癌初治患者纳入本项研究；按照病理类型，将患者分为腺癌组和鳞癌组；H&E染色检测免疫细胞数量；免疫组化检测PD-L1、ki-67、PD-1、CTLA-4和LAG-3的表达；miRNA测序筛选鳞癌和腺癌间差异表达的miRNA。结果：H&E染色结果显示鳞癌组微环境中免疫细胞的数量为86.86±8.96个/高倍视野（HPF），腺癌组的数量为26.29±3.99个/HPF（t=6.173，P＜0.001）；肺鳞癌组微环境免疫细胞PD-1、CTLA-4和LAG-3阳性表达的比例分别为53.71±6.88%、35.29±3.25%和34.43±3.29%，腺癌组阳性表达的比例分别为22.29±3.80%、13.43±2.32%和24.00±1.98%（t=3.997，P=0.002；t=5.476，P＜0.001；t=2.719，P=0.019）；肺鳞癌组患者PD-L1蛋白阳性表达的比例为76.67%，腺癌组的比例为36.67%（P=0.001）；肺鳞癌PD-L1（miR-135、miR-24和miR-30b等）和PD-1（miR-802、miR-155和miR-3127-5p等）相关miRNA的表达均显著高于腺癌。结论：肺鳞癌PD-L1蛋白及相关miRNA的表达、微环境免疫细胞PD-1、CTLA-4和LAG-3阳性比例均显著高于腺癌。     

CTLA-4在弥漫大B细胞淋巴瘤患者外泌体的表达及初步机制研究

摘要 目的：探讨细胞毒性T淋巴细胞相关抗原4（CTLA-4）在弥漫大B细胞淋巴瘤（DLBCL）患者外泌体的表达及初步机制。方法：2019年6月至2020年11月就诊于本院的DLBCL患者纳入本项研究,分为缓解组和复发组;选取来医院体检的健康志愿者做为对照组;采用试剂盒分离外泌体,CD63抗体包被磁珠结合后,流式细胞术检测CTLA-4+外泌体的比例;流式细胞术检测CD4⁺T细胞、CD8⁺T细胞和调节性T细胞（Treg细胞）的比例。结果：相对于对照组,缓解组DLBCL患者CTLA-4+外泌体的比例升高了37.42%;复发组DLBCL患者CTLA-4+外泌体的比例为6.04%,相对于缓解组,差异具有显著的统计学意义;复发组DLBCL患者CD4/CD8⁺T细胞比值和Treg细胞比例分别为0.85和0.44%,相对于缓解组,差异均具有显著的统计学意义;相关性分析结果显示CTLA-4+外泌体比例与CD4/CD8⁺T细胞比值呈负相关,而与Treg细胞比例呈正相关。结论：CTLA-4+外泌体比例在DLBCL患者显著升高,且与淋巴瘤的治疗效果和抗肿瘤免疫反应均具有紧密的相关性。     

长期肾移植受者外周血B淋巴细胞亚群分布特征

摘要 目的：观察移植肾功能稳定的长期受者（>10年）外周血B细胞亚群分布特征及其相关因素。方法：54名肾移植受者接受流式细胞仪检查,测算外周血总B细胞、未转化记忆B细胞、转化记忆B细胞、双阴性B细胞（CD19⁺CD27^-IgD^-）比例及数量（个/微升）。患者均服用包括环孢霉素的免疫抑制治疗。术后时间16.33±5.98年,GFR：91.63±11.28 mL/min/1.73 m²。结果：1长期肾移植患者外周血B细胞中幼稚B细胞最多（37.92% ± 22.06%）,未转化记忆B细胞最少(16.23% ± 11.10%)。B细胞亚群数量与白细胞总数、中性粒细胞比例等相关。2 以上述条件为控制因素行相关分析,转化记忆B细胞比例和GFR相关（r=-0.279,P=0.045）,双阴性B细胞数量和环孢霉素浓度相关（r=-0.300,P=0.029）。线性回归显示双阴性B细胞数目与环孢霉素浓度相关（R²=0.123,P=0.049）。3按GFR将患者分为肾功能减退组（GFR<90 mL/min/1.73 m²,n=19）和肾功能正常组（GFR≥90 mL/min/1.73 m²,n=35）。前者转化记忆B细胞比例显著升高（23.61% ± 10.96% vs.17.48%±8.91%,P=0.030）。按环孢霉素谷浓度将患者分为低浓度组（<64 mmol/L,n=28）和高浓度组（≥64 mmol/L,n=26）,前者双阴性B细胞数量显著升高（13.74±10.70 vs. 8.14±6.72/μL,P=0.027）。转化记忆B细胞比例与GFR分组相关（r=-0.326,P=0.018）,双阴性B细胞数量和环孢霉素浓度分组相关（r=-0.350,P=0.01）。结论：移植肾功能稳定的长期存活受者（>10年）外周血幼稚B细胞较多。转化记忆B细胞增多与移植肾功能减退相关,增多的双阴性B细胞和低孢霉素浓度治疗相关。     

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。     

调强放射治疗对食管癌患者外周血Treg细胞、血清肿瘤标志物及应激激素水平的影响

摘要 目的：观察调强放射（IMRT）治疗对食管癌患者外周血调节性T细胞（Treg）细胞、血清肿瘤标志物及应激激素水平的影响。方法：选取2019年9月~2021年3月期间自贡市第一人民医院收治的食管癌患者80例,按照随机数字表法将患者分为对照组（普通适形放疗,40例）和研究组（IMRT治疗,40例）。对比两组临床总有效率、外周血Treg细胞比例、血清肿瘤标志物及应激激素水平,观察放疗期间出现的不良反应。结果：研究组的临床总有效率高于对照组（P<0.05）。两组放疗后癌胚抗原（CEA）、糖类抗原 199（CA199）、细胞角蛋白19片段（CYFRA21-1）均下降,且研究组低于对照组（P<0.05）。两组放疗后生长激素（GH）、催乳素（PRL）均下降,且研究组低于对照组（P<0.05）。两组放疗后外周血Treg细胞占CD4⁺T细胞的比例下降,且研究组较对照组低（P<0.05）。不良反应发生率两组组间对比,未见显著性差异（P>0.05）。结论：食管癌患者采用IMRT治疗,可有效降低外周血Treg细胞比例、血清肿瘤标志物及应激激素水平,疗效较好,具有一定的临床应用价值。     

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。     

miR-125a-5p转染对肝癌细胞增殖、侵袭、迁移的影响及相关机制研究

摘要 目的：探究miR-125a-5p转染对肝癌细胞增殖、侵袭、迁移的影响及相关机制。方法：将肝癌细胞分为对照组、下调组和上调组,并通过细胞转染建立稳定转染的下调组和上调组。MMT法检测细胞增殖能力,流式细胞仪检测细胞凋亡能力,Transwell小室实验检测细胞侵袭能力,细胞划痕实验检测细胞迁移能力,Western blot法检测P13K/Akt通路中AKT、Bax、Bcl-2、P13K、P-AKT蛋白表达量。结果：与上调组相比,下调组24、48、72 h细胞增殖率,细胞侵袭、迁移细胞数,AKT、Bcl-2、P13K、P-AKT蛋白表达量显著降低,具有统计学差异（29.67±9.87 vs 17.34±5.71,t=5.192,P＜0.05、34.75±11.56 vs 15.17±5.04,t=7.365,P＜0.05、38.48±12.81 vs 12.51 ±4.13,t=9.153,P＜0.05,72.53±24.17 vs 36.28±12.07,t=6.365,P＜0.05、86.51±28.75 vs 46.28±15.32,t=5.858,P＜0.05,1.26±0.41 vs 0.81±0.26,t=4.397,P＜0.05、1.35±0.44 vs 0.76±0.24,t=5.584,P＜0.05、1.48±0.46 vs 0.79±0.26,t=6.194,P＜0.05、1.22±0.39 vs 0.73±0.24,t=5.584,P＜0.05）;与上调组相比,下调组24、48、72h细胞凋亡率,Bax蛋白表达量显著升高,具有统计学差异（17.62±5.84 vs 29.31±9.75,t=4.879,P＜0.05、14.97±4.65 vs 34.19±11.36,t=7.427,P＜0.05、11.26±3.74 vs 38.62±12.86,t=9.690,P＜0.05,0.75±0.24 vs 1.33±0.43,t=5.587,P＜0.05）。结论：下调miR-125a-5p的表达,可通过作用于P13K/Akt通路,调控AKT、Bax、Bcl-2、P13K、P-AKT蛋白表达量,进而起到抑制肝癌细胞增殖、促进肝癌细胞凋亡以及抑制肝癌细胞的侵袭、迁移能力。     

研究报告: 基于AS1411适配体的DNA-RNA杂交载体的抗肿瘤研究

目的 研究连接适配体的DNA-RNA分子作为杂交载体靶向肿瘤细胞并导入功能性RNA分子进入细胞的有效性，以及对肿瘤细胞的影响。方法 设计合成短的互补DNA、RNA分子，组装成DNA-RNA杂合链；连接AS1411适配体为靶向分子，再分别连接p21 saRNA和TIGIT siRNA作为药物分子，记为P21 saRNA和TIGIT siRNA，构成杂交载体，通用结构式为AS1411-DNA/RNA-sxRNA；检测AS1411-DNA/RNA-sxRNA能否靶向结合并进入肿瘤细胞及其对瘤细胞生存、迁移、侵袭和凋亡的影响。结果 将设计的杂交载体各部分等摩尔加入杂交缓冲体系并于特定温度条件下孵育，TBM聚丙烯酰胺凝胶电泳检测到AS1411-DNA/RNA-sxRNA成功组装；AS1411-DNA/RNA-sxRNA杂交载体在10%血清条件下也显示出良好的抗降解稳定性；荧光显微镜和激光共聚焦显微镜下观察，SKOV3细胞表面及胞内存在绿色大量荧光信号，杂交载体成功进入肿瘤细胞。杂交载体孵育后：在mRNA水平上，p21基因表达（2.14±0.25）是对照组（1.02±0.10）2倍以上，P<0.05；TIGIT基因表达（0.63±0.09）低于对照组（1.09±0.15），P<0.05；在蛋白质水平上，p21基因表达（1.57±0.16）是对照组（1.10±0.09）1.5倍以上，P<0.05；TIGIT基因表达（0.61±0.12）低于对照组（1.01±0.07），P<0.05。CCK-8实验显示，P21 saRNA（3.10±0.13）和TIGIT siRNA（2.91±0.13）杂交载体孵育组与空白对照组（3.67±0.15）相比，卵巢癌细胞增殖能力显著下降（P<0.05）；划痕实验结果显示，P21 saRNA孵育组愈合率（42.53±2.90）%、TIGIT siRNA孵育组愈合率（36.23±3.43）%，明显低于空白对照组（76.47±3.64）%，P<0.05；Transwell检测迁移能力发现：P21 saRNA孵育组（128.25±5.36）、TIGIT siRNA孵育组（119.50±8.79）低于对照组（186.5±8.56）；侵袭能力：P21 saRNA孵育组（145.5±9.45）、TIGIT siRNA孵育组（112.25±5.63）也显著低于对照组（202.50±10.12），P<0.05；细胞凋亡率：P21 saRNA孵育组（11.74%±2.47%）、TIGIT siRNA孵育组（17.12%±2.04%）明显高于对照组（5.66%±1.44%），P<0.05。结论 所制备的AS1411-DNA/RNA-sxRNA杂交载体能够有效靶向肿瘤细胞，携带功能性小RNA靶向导入肿瘤细胞并调控目的基因表达，使肿瘤细胞的增殖、侵袭和迁移能力受到抑制；该结果为利用DNA-RNA偶联AS1411适配体作为靶向工具的杂交载体，靶向杀伤表面表达NCL蛋白的肿瘤细胞提供了实验基础。     

慢性牙周炎患者血清CGRP、PGE2、CCL20与牙周临床指标和Th17/Treg失衡的相关性分析

摘要 目的：探讨慢性牙周炎（CP）患者血清降钙素基因相关肽（CGRP）、前列腺素E₂（PGE₂）、C-C基序趋化因子配体20（CCL20）与牙周临床指标和辅助性T细胞17/调节性T细胞（Th17/Treg）失衡的相关性。方法：选取2018年3月～2022年1月本院收治的92例CP患者,根据病情严重程度分为轻度组28例、中度组33例、重度组31例。检查患者牙周临床指标,采用酶联免疫吸附法检测血清CGRP、PGE₂、CCL20水平,流式细胞术检测外周血Th17、Treg百分比并计算Th17/Treg。比较3组血清CGRP、PGE₂、CCL20水平和牙周临床指标及外周血Th17、Treg、Th17/Treg,采用Pearson相关系数分析CP患者血清CGRP、PGE₂、CCL20水平与牙周临床指标和Th17/Treg的相关性。结果：轻度组、中度组、重度组血清CGRP水平和外周血Treg比例依次降低,血清PGE₂、CCL20水平和探诊深度（PD）、出血指数（BI）、附着丧失（AL）、菌斑指数（PLI）及外周血Th17比例、Th17/Treg依次升高（P＜0.05）。Pearson相关系数显示,CP患者血清CGRP水平与PD、BI、AL、PLI、Th17/Treg呈负相关,PGE₂、CCL20水平与PD、BI、AL、PLI、Th17/Treg呈正相关（P＜0.05）。结论：血清CGRP、PGE₂、CCL20水平与CP患者牙周临床指标和Th17/Treg失衡密切相关,可能是CP发生发展的关键因子。     

龈沟液Chemerin、APN、25(OH)D3与伴2型糖尿病的慢性牙周炎患者牙周指标和Th17/Treg失衡的关系

摘要 目的：探讨龈沟液趋化素（Chemerin）、脂联素（APN）、25-羟维生素D3 [25(OH)D3]与伴2型糖尿病（T2DM）的慢性牙周炎（CP）患者牙周指标和Th17/Treg失衡的关系。方法：选择2021年1月至2022年1月我院口腔科门诊接诊的125例伴T2DM的CP患者,根据CP病情严重程度分为轻度组（42例）、中度组（53例）和重度组（30例）。检测龈沟液中Chemerin、APN、25(OH)D₃水平以及外周血中Th17细胞占比、Treg细胞占比,计算Th17/Treg比值,记录出血指数（SBI）、菌斑指数（PLI）、附着丧失（AL）、牙周袋探诊深度（PD）。分析龈沟液中Chemerin、APN、25(OH)D₃与牙周指标、外周血Th17/Treg的相关性。结果：重度组龈沟液 Chemerin水平和外周血中Th17细胞占比、Th17/Treg比值、PLI、SBI、AL、PD高于中度组和轻度组（P＜0.05）,龈沟液APN、25(OH)D₃水平,外周血Treg细胞占比低于中度组和轻度组（P＜0.05）。龈沟液 Chemerin与PLI、SBI、AL、PD、外周血Th17细胞占比、Th17/Treg比值呈正相关（P＜0.05）,与Treg细胞占比呈负相关（P＜0.05）;龈沟液APN、25(OH)D₃与PLI、SBI、AL、PD、外周血中Th17细胞占比、Th17/Treg比值呈负相关（P＜0.05）,与Treg细胞占比呈正相关（P＜0.05）。结论：伴T2DM 的CP患者龈沟液中Chemerin水平增高,APN、25(OH)D₃水平降低,且与牙周指标和Th17/Treg失衡有关。     

Graves病患者外周血中CD4+CD25+Treg细胞上的PD-1/PD-L1的表达及意义

目的:探究Graves病患者外周血中CD4+CD25+Treg细胞上的PD-1/P-L1的表达及意义。方法:收集2017年6月至2017年12月就诊于西南医科大学附属医院内分泌科的Graves病、Graves眼病及体检中心的健康者的外周血进行流式检测。结果:与正常对照组相比,Graves病及Graves眼病组CD4+CD25+Treg细胞比例明显降低(P0.05);PD-1+Treg、PD-L1+Treg、PD-1+/PD-L1+Treg细胞比例均较正常组明显降低(P0.05);此外,Graves眼病组的结果较Graves病组更低(P0.05)。结论:CD4+CD25+Treg的降低会导致Graves病和Graves眼病患者的免疫状态活化,甲状腺自身抗体的增加和活化,促进疾病的发生。此外于PD-1和PD-L1阳性细胞比例的减少对于其对免疫的负向调节有所减弱,从而导致患者体内免疫耐受的降低和免疫稳态的打破,可能导致机体对甲状腺抗原的耐受消失,导致GD及眼病的发生发展。     

山萘酚通过增强Treg细胞免疫抑制功能延长移植物生存时间

目的:探讨应用山萘酚增强Treg细胞免疫抑制功能,从而抑制大鼠移植物排斥反应并改善移植物生存的作用和机制。方法:以Wister大鼠和SD大鼠分别为供、受体,建立同种异体皮肤移植排斥反应动物模型。观察受体老鼠皮肤移植物的情况,记录移植物失功时间(移植物皮片80%面积发生排斥)。RT-PCR检测移植7天后脾细胞、淋巴细胞FOXP3、CTLA-4和IL-10的mRNA水平,用HE染色组织病理学观察术后7天移植皮片的淋巴细胞浸润程度。体外实验T细胞增殖抑制试验加入山萘酚作为对照,观察Treg功能情况。结果:1.山萘酚能增强移植后同种异体移植物的生存时间(DMSO组6.3±0.3天,山萘酚组13.7±0.39天,P<0.01);2.RT-PCR显示山萘酚可增强细胞CTLA-4(对照组9.24±0.17,山萘酚组12.48±0.145,P<0.05)、FOXP3(对照组0.96±0.07,山萘酚组1.41±0.07,P<0.01)和IL-10(对照组0.95±0.12,山萘酚组1.50±0.16,P<0.05)的mRNA水平;3.体外T细胞增殖抑制实验中,山萘酚可增强Treg细胞的免疫抑制功能。结论:在大鼠皮肤移植模型中,山萘酚可延长皮肤移植物的生存时间,提高Treg细胞相关IL-10、FOXP3和CTLA-4的mRNA水平;体外实验中,能抑制效应T细胞的增殖,表明山萘酚在提高移植物生存方面存在一定的价值。     

PD-1、PD-L1的表达与肺癌临床病理特征及预后的相关性分析

目的:分析程序性死亡因子-1(PD-1)、程序性死亡1-配体(PD-L1)的表达与肺癌临床病理特征及预后的相关性。方法:回顾性分析我院2015年3月～2016年6月收治的73例肺癌患者的临床资料,取距离切除肿瘤边缘3cm内的非癌组织作为癌旁组织。比较两组PD-1、PD-L1的表达,分析其和肺癌患者临床病理特征和预后的关系,采用COX比例回归分析肺癌患者预后的影响因素。结果:肺癌组织PD-1、PD-L1阳性表达率均显著高于癌旁组织(P0.05)。不同性别、年龄、病理类型、吸烟情况、EGFR表达、肿瘤大小肺癌患者PD-1、PD-L1的阳性表达率比较差异无统计学意义(P0.05);低分化程度、临床分期Ⅲ及Ⅳ期、有淋巴结转移肺癌患者PD-1、PD-L1阳性表达率分别高于中分化程度、临床分期Ⅲ期、无淋巴结转移患者,差异有统计学意义(P0.05)。PD-1、PD-L1阳性表达及阴性表达组无疾病进展生存期比较均有统计学差异(P0.05)。COX比例风险回归模型显示分化程度、临床分期、淋巴结转移、PD-1、PD-L1的表达是影响肺癌患者预后的危险因素(P0.05)。结论:肺癌组织PD-1、PD-L1呈高表达,可能参与肺癌的发生发展,有助于病情严重程度的评价和预后预测。     

PD-L1 Blockade Differentially Impacts Regulatory T Cells from HIV-Infected Individuals Depending on Plasma Viremia

Blocking the PD-1/PD-L1 pathway has emerged as a potential therapy to restore impaired immune responses in human immunodeficiency virus (HIV)-infected individuals. Most reports have studied the impact of the PD-L1 blockade on effector cells and neglected possible effects on regulatory T cells (Treg cells), which play an essential role in balancing immunopathology and antiviral effector responses. The aim of this study was to define the consequences of ex vivo PD-L1 blockade on Treg cells from HIV-infected individuals. We observed that HIV infection led to an increase in PD-1+ and PD-L1+ Treg cells. This upregulation correlated with disease progression and decreased under antiretroviral treatment. Treg cells from viremic individuals had a particularly high PD-1 expression and impaired proliferative capacity in comparison with Treg cells from individuals under antiretroviral treatment. PD-L1 blockade restored the proliferative capacity of Treg cells from viremic individuals but had no effect on its suppressive capacity. Moreover, it increased the viral production in cell cultures from viremic individuals. This increase in viral production correlated with an increase in Treg cell percentage and a reduction in the CD4/Treg and CD8/Treg cell ratios. In contrast to the effect of the PD-L1 blockade on Treg cells from viremic individuals, we did not observe a significant effect on the proliferative capacity of Treg cells from individuals in whom viremia was controlled (either spontaneously or by antiretroviral treatment). However, PD-L1 blockade resulted in an increased proliferative capacity of HIV-specific-CD8 T cells in all subjects. Taken together, our findings suggest that manipulating PD-L1 in vivo can be expected to influence the net gain of effector function depending on the subject’s plasma viremia.     

肾透明细胞癌中免疫检查点相关的免疫逃逸机制的研究进展

摘要：近年来,免疫治疗在晚期肾透明细胞癌的治疗中异军突起,使人们对于肾癌治疗有了全新的认识。肿瘤免疫治疗药物是通过抑制免疫检查点从而抑制肿瘤细胞免疫逃逸,使免疫细胞可以杀伤肿瘤细胞来发挥治疗作用。因此,了解肾透明细胞癌中免疫检查点相关免疫逃逸机制对于制定有效的治疗策略以及开发新的免疫治疗药物至关重要。本文对目前肾透明细胞癌中主要的免疫检查点（PD-1/PD-L1、CTLA-4、B7-H4、LAG-3、TIM-3和HLA-G）相关的免疫逃逸机制进行综述。     

Cytotoxic activity of anti-mucin 1 chimeric antigen receptor T cells expressing PD-1-CD28 switch receptor against cholangiocarcinoma cells

Background aimsCholangiocarcinoma (CCA) is a lethal bile-duct cancer that is difficult to treat by current standard procedures. This drawback has prompted us to develop adoptive T-cell therapy for CCA, which requires an appropriate target antigen for binding of chimeric antigen receptor (CAR) T cells. Mucin 1 (MUC1), an overexpressed protein in CCA cells, is a potential target antigen for the CAR T-cell development. However, MUC1 overexpression also is associated with the upregulation of programmed death-ligand 1 (PD-L1), an immune checkpoint protein that prohibits anti-tumor functions of T cells, probably causing poor overall survival of patients with CCA.MethodsTo overcome this problem, we developed anti-MUC1-CAR T cells containing PD-1-CD28 switch receptor (SR), namely αM.CAR/SR T cells, to target MUC1 and switch on the inhibitory signal of PD-1/PD-L1 interaction to activate CD28 signaling. Our lentiviral construct contains the sequences that encode anti-MUC1-single chain variable fragment, CD137 and CD3ζ, linked with P2A, PD-1 and CD28.ResultsInitially, the upregulations of MUC1 and PD-L1 proteins were confirmed in CCA cell lines. αM.CAR and SR were co-expressed in 53.53 ± 13.89% of transduced T cells, mainly CD8⁺ T cells (85.7 ± 0.75%, P<0.0001) with the effector memory phenotype (59.22 ± 16.31%, P < 0.01). αM.CAR/SR T cells produced high levels of intracellular tumor necrosis factor-α and interferon-γ in response to the activation by CCA cells expressing MUC1, including KKU-055 (27.18 ± 4.38% and 27.33 ± 5.55%, respectively, P < 0.05) and KKU-213A (47.37 ± 12.67% and 54.55 ± 8.66%, respectively, P < 0.01). Remarkably, the cytotoxic function of αM.CAR/SR T cells against KKU-213A cells expressing PD-L1 was significantly enhanced compared with the αM.CAR T cells (70.69 ± 14.38% versus 47.15 ± 8.413%, respectively; P = 0.0301), correlated with increased granzyme B production (60.6 ± 9.89% versus 43.2 ± 8.95%, respectively; P = 0.0402). Moreover, the significantly enhanced disruption of KKU-213A spheroids by αM.CAR/SR T cells (P = 0.0027), compared with αM.CAR T cells, was also observed.ConclusionTaken together, the cytotoxic function of αM.CAR/SR T cells was enhanced over the αM.CAR T cells, which are potential to be further tested for CCA treatment.     

The CTLA-4 and PD-1/PD-L1 Inhibitory Pathways Independently Regulate Host Resistance to Plasmodium-induced Acute Immune Pathology

The balance between pro-inflammatory and regulatory immune responses in determining optimal T cell activation is vital for the successful resolution of microbial infections. This balance is maintained in part by the negative regulators of T cell activation, CTLA-4 and PD-1/PD-L, which dampen effector responses during chronic infections. However, their role in acute infections, such as malaria, remains less clear. In this study, we determined the contribution of CTLA-4 and PD-1/PD-L to the regulation of T cell responses during Plasmodium berghei ANKA (PbA)-induced experimental cerebral malaria (ECM) in susceptible (C57BL/6) and resistant (BALB/c) mice. We found that the expression of CTLA-4 and PD-1 on T cells correlates with the extent of pro-inflammatory responses induced during PbA infection, being higher in C57BL/6 than in BALB/c mice. Thus, ECM develops despite high levels of expression of these inhibitory receptors. However, antibody-mediated blockade of either the CTLA-4 or PD-1/PD-L1, but not the PD-1/PD-L2, pathways during PbA-infection in ECM-resistant BALB/c mice resulted in higher levels of T cell activation, enhanced IFN-γ production, increased intravascular arrest of both parasitised erythrocytes and CD8⁺ T cells to the brain, and augmented incidence of ECM. Thus, in ECM-resistant BALB/c mice, CTLA-4 and PD-1/PD-L1 represent essential, independent and non-redundant pathways for maintaining T cell homeostasis during a virulent malaria infection. Moreover, neutralisation of IFN-γ or depletion of CD8⁺ T cells during PbA infection was shown to reverse the pathologic effects of regulatory pathway blockade, highlighting that the aetiology of ECM in the BALB/c mice is similar to that in C57BL/6 mice. In summary, our results underscore the differential and complex regulation that governs immune responses to malaria parasites.     

Tumour-draining lymph nodes in head and neck cancer are characterized by accumulation of CTLA-4 and PD-1 expressing Treg cells

IntroductionHigh Tregs infiltration within the tumour microenvironment (TME) of various cancers shows a positive correlation with poor prognosis. Despite the fact that tumour draining lymph nodes (TDLNs) are recognized as key organs playing a crucial role in response to immunotherapy and modulating anti-cancer immunity, the distribution of Tregs and their role in TDLNs remain uncertain thus far. The purpose of this project is to investigate the density of Tregs in TDLNs and non-TDLNs and their expression of immune checkpoint molecules – PD-1 and CTLA-4.MethodsSamples including TDLNs, non-TDLNs and metastatic lymph nodes (LNs) from 23 patients with oral squamous cell carcinoma (OSCC) were analyzed by multicolour flow cytometry with a focus on Tregs population and expression of CTLA-4 and PD-1.ResultsTDLNs and metastatic LNs were characterized by a significantly higher infiltration of Tregs defined as CD4+FoxP3+CD25^highCD127^low cells and significantly higher expression of CTLA-4 and PD-1 on Tregs compared with non-TDLNs. Tregs in TDLNs and metastatic LNs co-expressed CTLA-4 and PD-1 abundantly. High expression of these immune check-point molecules correlated with positive N-stage but not with T-stage.ConclusionTDLNs and metastatic LNs are characterized by a high accumulation of Tregs expressing high levels of CTLA-4 and PD-1. High infiltration of Tregs can be a potential driver of an immunosuppressive milieu in TDLNs that can, in turn, favour cancer progression. High accumulation of Tregs expressing CTLA-4 and PD-1 in TDLNs is associated with lymph node involvement, but not with the size of the primary tumour.