目的:通过观察进展期食管胃交界部腺癌(AEGJ)患者拟行术前新辅助放化疗术后化疗联合腹腔热灌注化疗(HIPEC)后血免疫细胞及癌组织中PD-L1的表达,证实PD-L1对AEGJ患者预后的影响,探讨HIPEC治疗后免疫细胞水平与PD-L1表达的关系。方法:选取...目的:通过观察进展期食管胃交界部腺癌(AEGJ)患者拟行术前新辅助放化疗术后化疗联合腹腔热灌注化疗(HIPEC)后血免疫细胞及癌组织中PD-L1的表达,证实PD-L1对AEGJ患者预后的影响,探讨HIPEC治疗后免疫细胞水平与PD-L1表达的关系。方法:选取2018年1月至2019年6月河北北方学院附属第一医院收治的进展期AEGJ术前新辅助放化疗术后辅助化疗联合HIPEC患者46例。采用转录组测序技术检测AEGJ术后辅助HIPEC患者的血免疫细胞水平,免疫组化检测AEGJ组织中PD-L1表达,应用相应的统计学方法观察免疫细胞与PD-L1水平对AEGJ患者预后的预测情况,以及二者的相关性。结果:截至2022年6月30日,患者肿瘤特异生存率(CSS)为28.26%,无疾病复发率(RFS)为45.65%,中位CSS和RFS分别为31个月和24个月。PD-L1阳性率为52.17%,PD-L1阳性和PD-L1阴性患者的RFS率和CSS率分别为16.67%、77.27%和54.17%、90.91%,前者的中位RPS和CSS明显短于后者(12个月vs 37个月)和(24个月vs 37个月)。本研究共检测出22种免疫细胞,与CPS<1组相比,CPS≥1组中CD8^(+)T细胞、活化树突状细胞和静息NK细胞水平明显降低(均P<0.05),而M2型巨噬细胞在前者中显著低于后者(0.013±0.012 vs 0.033±0.003,P<0.001)。ROC曲线结果显示,CD8^(+)T细胞和M2型巨噬细胞均可预测AEGJ患者治疗的预后(AUC分别为0.690和0.698)。COX多因素回归分析提示CD8^(+)T细胞可能是AEGJ患者治疗后的保护因子[HR=0.590,95%CI(0.248~0.708)],而M2型巨噬细胞可能作为AEGJ患者治疗后的风险因素[HR=6.448,95%CI(1.440~9.165)]。PD-L1的表达与M2型巨噬细胞水平呈正相关(R=0.44,P<0.01),与CD8^(+)T细胞呈负相关(R=-0.47,P<0.05)。结论:HIPEC辅助治疗可能调控M2型巨噬细胞、CD8^(+)T细胞的功能,其中M2型巨噬细胞水平与PD-L1表达呈正相关,这种免疫细胞的增多可能是影响AEGJ术后辅助HIPEC预后的因素。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
OBJECTIVE Programmed death ligand-1(PD-L1)and indoleamine 2,3-dioxygenase 1(IDO1)are immune checkpoints which can be induced by interferon-γ(IFN-γ)in the tumor microenvironment,leading to immune escape of tumors.Myr...OBJECTIVE Programmed death ligand-1(PD-L1)and indoleamine 2,3-dioxygenase 1(IDO1)are immune checkpoints which can be induced by interferon-γ(IFN-γ)in the tumor microenvironment,leading to immune escape of tumors.Myricetin(MY)is a flavonoid distributed in many edible and medicinal plants.The aim of this study is to clarify the effect and the mechanism of MY on inhibiting IFN-γ-induced PD-L1 and IDO1 in lung cancer cells.METHODS Expressions of PD-L1 and major histocompatibility complex-I(MHC-I)were evaluated by flow cytometry and Western blotting,and the expression of IDO1 was measured by Western blotting.qRT-PCR was used to detect their mRNA levels.The function of T cells was evaluated using a co-culture system consist of lung cancer cells and the Jurkat-PD-1 T cell line that overexpressing PD-1.Molecular docking analysis,Western blotting and immunofluorescence were used for mechanism study.RESULTS MY potently inhibited IFN-γ-induced PD-L1 and IDO1 expression in human lung cancer cells,while didn't show obvious effect on the expression of MHC-I.In addition,MY restored the survival,proliferation,CD69 expression and interleukin-2(IL-2)secretion of Jurkat-PD-1 T cells suppressed by IFN-γ-treated lung cancer cells in the co-culture system.Mechanistically,IFN-γup-regulated PD-L1 and IDO1 at the transcriptional level through the JAK-STAT-IRF1 axis,which was targeted and inhibited by MY.CONCLUSION Our research revealed a new insight into the anti-tumor effects of MY which inhibited IFN-γ-induced PD-L1 and IDO1 expression,supporting the potential of MY in anti-tumor immunotherapy.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
文摘目的:通过观察进展期食管胃交界部腺癌(AEGJ)患者拟行术前新辅助放化疗术后化疗联合腹腔热灌注化疗(HIPEC)后血免疫细胞及癌组织中PD-L1的表达,证实PD-L1对AEGJ患者预后的影响,探讨HIPEC治疗后免疫细胞水平与PD-L1表达的关系。方法:选取2018年1月至2019年6月河北北方学院附属第一医院收治的进展期AEGJ术前新辅助放化疗术后辅助化疗联合HIPEC患者46例。采用转录组测序技术检测AEGJ术后辅助HIPEC患者的血免疫细胞水平,免疫组化检测AEGJ组织中PD-L1表达,应用相应的统计学方法观察免疫细胞与PD-L1水平对AEGJ患者预后的预测情况,以及二者的相关性。结果:截至2022年6月30日,患者肿瘤特异生存率(CSS)为28.26%,无疾病复发率(RFS)为45.65%,中位CSS和RFS分别为31个月和24个月。PD-L1阳性率为52.17%,PD-L1阳性和PD-L1阴性患者的RFS率和CSS率分别为16.67%、77.27%和54.17%、90.91%,前者的中位RPS和CSS明显短于后者(12个月vs 37个月)和(24个月vs 37个月)。本研究共检测出22种免疫细胞,与CPS<1组相比,CPS≥1组中CD8^(+)T细胞、活化树突状细胞和静息NK细胞水平明显降低(均P<0.05),而M2型巨噬细胞在前者中显著低于后者(0.013±0.012 vs 0.033±0.003,P<0.001)。ROC曲线结果显示,CD8^(+)T细胞和M2型巨噬细胞均可预测AEGJ患者治疗的预后(AUC分别为0.690和0.698)。COX多因素回归分析提示CD8^(+)T细胞可能是AEGJ患者治疗后的保护因子[HR=0.590,95%CI(0.248~0.708)],而M2型巨噬细胞可能作为AEGJ患者治疗后的风险因素[HR=6.448,95%CI(1.440~9.165)]。PD-L1的表达与M2型巨噬细胞水平呈正相关(R=0.44,P<0.01),与CD8^(+)T细胞呈负相关(R=-0.47,P<0.05)。结论:HIPEC辅助治疗可能调控M2型巨噬细胞、CD8^(+)T细胞的功能,其中M2型巨噬细胞水平与PD-L1表达呈正相关,这种免疫细胞的增多可能是影响AEGJ术后辅助HIPEC预后的因素。
基金Science and Technology Development Fund,Macao SAR(0129/2019/A3176/2017/A3)and University of Macao(MYRG2018-00165-ICMS)。
文摘OBJECTIVE Programmed death ligand-1(PD-L1)and indoleamine 2,3-dioxygenase 1(IDO1)are immune checkpoints which can be induced by interferon-γ(IFN-γ)in the tumor microenvironment,leading to immune escape of tumors.Myricetin(MY)is a flavonoid distributed in many edible and medicinal plants.The aim of this study is to clarify the effect and the mechanism of MY on inhibiting IFN-γ-induced PD-L1 and IDO1 in lung cancer cells.METHODS Expressions of PD-L1 and major histocompatibility complex-I(MHC-I)were evaluated by flow cytometry and Western blotting,and the expression of IDO1 was measured by Western blotting.qRT-PCR was used to detect their mRNA levels.The function of T cells was evaluated using a co-culture system consist of lung cancer cells and the Jurkat-PD-1 T cell line that overexpressing PD-1.Molecular docking analysis,Western blotting and immunofluorescence were used for mechanism study.RESULTS MY potently inhibited IFN-γ-induced PD-L1 and IDO1 expression in human lung cancer cells,while didn't show obvious effect on the expression of MHC-I.In addition,MY restored the survival,proliferation,CD69 expression and interleukin-2(IL-2)secretion of Jurkat-PD-1 T cells suppressed by IFN-γ-treated lung cancer cells in the co-culture system.Mechanistically,IFN-γup-regulated PD-L1 and IDO1 at the transcriptional level through the JAK-STAT-IRF1 axis,which was targeted and inhibited by MY.CONCLUSION Our research revealed a new insight into the anti-tumor effects of MY which inhibited IFN-γ-induced PD-L1 and IDO1 expression,supporting the potential of MY in anti-tumor immunotherapy.
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。
