OBJECTIVE To evaluate whether the IDO1 inhibitor 1-methyl-L-tryptophan(1-MT)combine calcium influx inhibitor carboxyamidotriazole(CAI)could further enhance the suppression of programmed death 1(PD-1)in CD8^+T cells an...OBJECTIVE To evaluate whether the IDO1 inhibitor 1-methyl-L-tryptophan(1-MT)combine calcium influx inhibitor carboxyamidotriazole(CAI)could further enhance the suppression of programmed death 1(PD-1)in CD8^+T cells and investigate the curative effect of the combined use.METHODS CD8^+T cells were isolated from normal mice spleen by negative selection using magnetic cell separation.The isolated CD8^+T cells were cultured in RPMI 1640 medium containing 10%FBS and 100 U·mL^(-1)IL-2 and activated by the addition of anti-CD3 and anti-CD28(1 g·L^(-1) each mabs).CD8^+T cells were pretreated for 48 h with drug and the fluo-3 as a marker of intracellular calcium concentration was detected by flow cytometry.The calcineurin(Ca N)levels were assayed with ELISA in CD8^+T cells after 48 h incubation with 10μm CAI.The nuclear translocations of NFAT and AHR were detected by immunofluorescent staining after 48 h of drug treatment.The expression of PD-1 in CD8^+T cells was analyzed by flow cytometry.RESULTS Intracellular fluorescent intensity was markedly debase due to CAI treatment(P<0.01).Meanwhile,the changes of CaN content had a resembled correlation(P<0.01).Immunofluorescence experiment showed that after combination therapy the transfer of NFAT and AHR in nuclear substantially reduced.Flow cytometry revealed that after the combination caused a significant decrease in PD-1 expression in CD8^+T cells.CONCLUSION CAI and 1-MT could inhibit markedly the expression of PD-1 in CD8^+T cells by inhibiting the nuclear translocation of NFAT and AHR,respectively and the combination of them has synergetic effect.展开更多
程序性细胞死亡受体1(programmed cell death 1,PD-1)单抗治疗肺腺癌可能引起罕见而严重的血液学不良反应,可出现重症贫血等表现。尽管糖皮质激素被推荐用于免疫相关不良事件的管理,但针对PD-1单抗诱发的重症贫血的治疗经验仍十分有限,...程序性细胞死亡受体1(programmed cell death 1,PD-1)单抗治疗肺腺癌可能引起罕见而严重的血液学不良反应,可出现重症贫血等表现。尽管糖皮质激素被推荐用于免疫相关不良事件的管理,但针对PD-1单抗诱发的重症贫血的治疗经验仍十分有限,其疗效和安全性尚未充分验证。本文报道了1例化疗联合PD-1单抗治疗后发生重症贫血的晚期肺腺癌患者,经过系列检查,考虑诊断炎症性贫血,经足量糖皮质激素治疗后,血红蛋白显著回升,以期为临床中这类罕见血液学毒性的识别和治疗提供新的见解。展开更多
程序性死亡因子-1(programmed cell death protein 1,PD-1)和细胞程序性死亡-配体1(programmed death-ligand 1,PD-L1)作为重要的免疫检查点,在肿瘤免疫治疗领域具有重要意义。目前,晚期消化系统肿瘤如胃癌、结直肠癌、肝癌的治疗手段有...程序性死亡因子-1(programmed cell death protein 1,PD-1)和细胞程序性死亡-配体1(programmed death-ligand 1,PD-L1)作为重要的免疫检查点,在肿瘤免疫治疗领域具有重要意义。目前,晚期消化系统肿瘤如胃癌、结直肠癌、肝癌的治疗手段有限,PD-1/PD-L1免疫检查点抑制剂作为后线的新兴治疗选择之一,仍面临有相当数量患者治疗效果不理想的情况。非编码RNA(non-coding RNA,ncRNA)如微小RNA(microRNA,miRNA)、长链非编码RNA(long non-coding RNA,lncRNA)和环状RNA(circular RNA,circRNA)已被证实能够影响PD-1/PD-L1的表达及功能,深入研究ncRNA对PD-1/PD-L1的作用有助于为癌症中免疫检查点抑制剂的应用提供新的思路,从而提高临床免疫治疗的有效性。本文就ncRNA干预PD-1/PD-L1调控消化系统肿瘤的研究进展进行综述。展开更多
程序性死亡受体1(programmed cell death protein 1,PD-1)和程序性死亡配体1(programmed cell death 1 ligand 1,PD-L1)是一对经典免疫检查点,其相互作用在肿瘤免疫逃逸中发挥重要作用。但由于PD-1和PD-L1存在复杂的糖基化修饰,且真核...程序性死亡受体1(programmed cell death protein 1,PD-1)和程序性死亡配体1(programmed cell death 1 ligand 1,PD-L1)是一对经典免疫检查点,其相互作用在肿瘤免疫逃逸中发挥重要作用。但由于PD-1和PD-L1存在复杂的糖基化修饰,且真核细胞中的PD-1和PD-L1在蛋白质水平的表达量低,限制了靶向二者相互作用的药物筛选及抑制剂的开发。本研究针对二者相互作用的关键结构域,利用金门桥组装(golden gate assembly)无缝克隆技术构建了表达功能性截短体蛋白质PD-1^(33-150)和PD-L1^(19-239)的真核载体,并利用HEK-293T细胞系验证了其高效表达。经亲和层析纯化,每升细胞培养基可获得蛋白质纯度>95%的PD-1^(33-150)和PD-L1^(19-239)蛋白分别为5 mg和3 mg。利用生物膜干涉技术(biolayer interferometry,BLI)和流式细胞术(flow cytometry,FCM),我们检测了纯化后PD-1^(33-150)和PD-L1^(19-239)的结合动力学参数、平衡常数以及其细胞结合活性。与昆虫细胞和大肠杆菌在蛋白质水平表达的PD-1胞外域相比,哺乳细胞表达的PD-1^(33-150)对PD-L1的亲和力提高了近24和50倍,同时使结合的解离速率降低至原来的1/400以下,这可能是由于不同的糖基化修饰对PD-1和PD-L1蛋白的相互作用具有重要影响。综上,本研究建立了人源PD-1^(33-150)/PD-L1^(19-239)功能性截短体在蛋白质水平的高效表达与纯化平台,为PD-1/PD-L1抗体药物筛选及小分子免疫检查点抑制剂的开发提供了高质量的分子工具。展开更多
目的探究脓毒症中免疫耗竭的特征并分析其与肠道菌群异位的相关性。方法采用随机数字表法将130只小鼠分为盲肠穿刺结扎(cecal ligation and puncture,CLP)组(n=100)和假手术(sham)组(n=30),通过CLP建立小鼠脓毒症模型;流式细胞术检测分...目的探究脓毒症中免疫耗竭的特征并分析其与肠道菌群异位的相关性。方法采用随机数字表法将130只小鼠分为盲肠穿刺结扎(cecal ligation and puncture,CLP)组(n=100)和假手术(sham)组(n=30),通过CLP建立小鼠脓毒症模型;流式细胞术检测分析小鼠外周血CD4^(+)T、CD8^(+)T和PD-1+T细胞亚群比例变化;心、肝、肾等脏器匀浆涂板检测细菌定植量变化;HE染色分析免疫器官的病理改变;免疫组化染色检测免疫器官内CD4、CD8和PD-1阳性细胞表达及定位,使用Image J软件对阳性细胞数变化进行定量。结果HE染色提示各级免疫器官随病程进展出现不同程度病理损伤;与Sham组小鼠相比,CLP组小鼠各脏器及外周血细菌定植量均显著增高(P<0.05),以肝脏感染最为严重;CLP组小鼠在术后第1、3、5天时外周血CD4^(+)T淋巴细胞比例较Sham组分别下降56%、70.57%和87.42%(P<0.001),CD8^(+)T淋巴细胞比例只在第5天时下降48.33%(P<0.001),而表达PD-1的CD4^(+)T细胞亚群比例分别升高为Sham组的673.08倍、423.08倍和600倍(P<0.001);免疫组化结果显示,CLP术后胸腺、脾脏和肠系膜淋巴结中CD4阳性细胞比例在早期最高时分别上升为Sham组的7.65倍、2.66倍和3.7倍(P<0.001),后期最低时分别较早期下降82.8%(P<0.001)、41.9%(P<0.01)和60.15%(P<0.001);CD8阳性细胞比例在早期升高后下降不显著;PD-1阳性细胞占比持续升高,最高时分别上升为Sham组的6.24倍、13.9倍和20.96倍(P<0.001),且其表达区域与CD4阳性细胞大致相同。结论脓毒症时炎症反应可引起免疫器官严重损伤,CD4^(+)T淋巴细胞持续耗竭,机体预防感染的能力下降,可能是造成肠道菌群异位加重,导致全身感染的重要原因。展开更多
基金supported by National Natural Science Foundation of China(81402943)CAMS Major Collaborative Innovation Project(2016-I2M-1-011)PUMC Youth Fund(3332015168)
文摘OBJECTIVE To evaluate whether the IDO1 inhibitor 1-methyl-L-tryptophan(1-MT)combine calcium influx inhibitor carboxyamidotriazole(CAI)could further enhance the suppression of programmed death 1(PD-1)in CD8^+T cells and investigate the curative effect of the combined use.METHODS CD8^+T cells were isolated from normal mice spleen by negative selection using magnetic cell separation.The isolated CD8^+T cells were cultured in RPMI 1640 medium containing 10%FBS and 100 U·mL^(-1)IL-2 and activated by the addition of anti-CD3 and anti-CD28(1 g·L^(-1) each mabs).CD8^+T cells were pretreated for 48 h with drug and the fluo-3 as a marker of intracellular calcium concentration was detected by flow cytometry.The calcineurin(Ca N)levels were assayed with ELISA in CD8^+T cells after 48 h incubation with 10μm CAI.The nuclear translocations of NFAT and AHR were detected by immunofluorescent staining after 48 h of drug treatment.The expression of PD-1 in CD8^+T cells was analyzed by flow cytometry.RESULTS Intracellular fluorescent intensity was markedly debase due to CAI treatment(P<0.01).Meanwhile,the changes of CaN content had a resembled correlation(P<0.01).Immunofluorescence experiment showed that after combination therapy the transfer of NFAT and AHR in nuclear substantially reduced.Flow cytometry revealed that after the combination caused a significant decrease in PD-1 expression in CD8^+T cells.CONCLUSION CAI and 1-MT could inhibit markedly the expression of PD-1 in CD8^+T cells by inhibiting the nuclear translocation of NFAT and AHR,respectively and the combination of them has synergetic effect.
文摘程序性细胞死亡受体1(programmed cell death 1,PD-1)单抗治疗肺腺癌可能引起罕见而严重的血液学不良反应,可出现重症贫血等表现。尽管糖皮质激素被推荐用于免疫相关不良事件的管理,但针对PD-1单抗诱发的重症贫血的治疗经验仍十分有限,其疗效和安全性尚未充分验证。本文报道了1例化疗联合PD-1单抗治疗后发生重症贫血的晚期肺腺癌患者,经过系列检查,考虑诊断炎症性贫血,经足量糖皮质激素治疗后,血红蛋白显著回升,以期为临床中这类罕见血液学毒性的识别和治疗提供新的见解。
文摘目的探究脓毒症中免疫耗竭的特征并分析其与肠道菌群异位的相关性。方法采用随机数字表法将130只小鼠分为盲肠穿刺结扎(cecal ligation and puncture,CLP)组(n=100)和假手术(sham)组(n=30),通过CLP建立小鼠脓毒症模型;流式细胞术检测分析小鼠外周血CD4^(+)T、CD8^(+)T和PD-1+T细胞亚群比例变化;心、肝、肾等脏器匀浆涂板检测细菌定植量变化;HE染色分析免疫器官的病理改变;免疫组化染色检测免疫器官内CD4、CD8和PD-1阳性细胞表达及定位,使用Image J软件对阳性细胞数变化进行定量。结果HE染色提示各级免疫器官随病程进展出现不同程度病理损伤;与Sham组小鼠相比,CLP组小鼠各脏器及外周血细菌定植量均显著增高(P<0.05),以肝脏感染最为严重;CLP组小鼠在术后第1、3、5天时外周血CD4^(+)T淋巴细胞比例较Sham组分别下降56%、70.57%和87.42%(P<0.001),CD8^(+)T淋巴细胞比例只在第5天时下降48.33%(P<0.001),而表达PD-1的CD4^(+)T细胞亚群比例分别升高为Sham组的673.08倍、423.08倍和600倍(P<0.001);免疫组化结果显示,CLP术后胸腺、脾脏和肠系膜淋巴结中CD4阳性细胞比例在早期最高时分别上升为Sham组的7.65倍、2.66倍和3.7倍(P<0.001),后期最低时分别较早期下降82.8%(P<0.001)、41.9%(P<0.01)和60.15%(P<0.001);CD8阳性细胞比例在早期升高后下降不显著;PD-1阳性细胞占比持续升高,最高时分别上升为Sham组的6.24倍、13.9倍和20.96倍(P<0.001),且其表达区域与CD4阳性细胞大致相同。结论脓毒症时炎症反应可引起免疫器官严重损伤,CD4^(+)T淋巴细胞持续耗竭,机体预防感染的能力下降,可能是造成肠道菌群异位加重,导致全身感染的重要原因。
