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
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
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。
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.
