肾癌细胞PD-L1表达及与肿瘤微环境中T淋巴细胞功能间相互调控关系的研究
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摘要
目的探讨肾癌中程序性死亡配体1(PD-L1)的表达及与肿瘤微环境中T淋巴细胞功能之间的相互调控作用。方法通过分离人外周血单个核细胞,刺激培养单个CD3~+T淋巴细胞;检测786-O、OSRC细胞系PDL1基因及蛋白表达水平的差异性;ELISA检测外周血T淋巴细胞与786-O、OSRC共培养后γ-干扰素(IFN-γ)的产生水平;通过小干扰RNA下调PD-L1相对高表达的肾癌细胞系与T淋巴细胞共培养后,检测IFN-γ水平的变化;检测786-O、OSRC与T淋巴细胞共培养后PD-L1表达的变化,以及不同浓度IFN-γ对786-O、OSRC中PD-L1表达的影响,分析肾癌细胞系786-O及OSRC中PD-L1的表达与IFN-γ之间的相互调控作用。结果 PD-L1在786-O中的表达水平明显高于OSRC;与单独培养T淋巴细胞相比,与786-O共培养时T淋巴细胞IFN-γ的产生量无明显差异,但与OSRC共培养时T淋巴细胞IFN-γ的产生量明显升高;瞬时干扰降低786-O中PD-L1的表达水平后,与T淋巴细胞共培养,发现T淋巴细胞IFN-γ的产生量较正常共培养组明显升高;共培养体系中T淋巴细胞被激活后两种肿瘤细胞PD-L1的表达明显增高,IFN-γ刺激实验得到相同的结果,且呈剂量依赖性。结论PD-L1高表达的肾癌细胞系可有效抑制外周血T淋巴细胞分泌IFN-γ,导致肿瘤细胞发生免疫逃逸,同时T淋巴细胞分泌的IFN-γ也可以上调肾癌细胞PD-L1的表达,肿瘤微环境中两者存在相互调节作用。
Objective To investigate the mutual regulatory interactions between programmed death-ligand 1(PD-L1) expression in renal carcinoma and T lymphocyte function in tumor microenvironment. Methods Human peripheral blood mononucleated cells were isolated and stimulated to obtain single CD3~+T lymphocytes.The mRNA and protein expression of PD-L1 in786-O and OSRC cell lines were measured.ELISA was used to measure the level of interferon-γ(IFN-γ)after peripheral blood T lymphocytes were co-cultured with 786-O or OSRC cells,and the change in IFN-γlevel was measured after T lymphocytes were cocultured with renal carcinoma cells with relatively high expression of PD-L1 downregulated by small interfering RNA.The change in PD-L1 expression was measured after 786-O and OSRC cells were co-cultured with T lymphocytes,and the influence of different concentrations of IFN-γon the expression of PD-L1 in 786-O and OSRC cells was analyzed to investigate the mutual regulatory interactions between PD-L1 expression and IFN-γin renal carcinoma 786-O and OSRC cells. Results The expression of PD-L1 in 786-O cells was significantly higher than that in OSRC cells.There was no significant difference in the level of IFN-γbetween T lymphocytes cultured alone and those co-cultured with 786-O cells,but T lymphocytes co-cultured with OSRC cells had a significantly higher level of IFN-γthan those cultured alone.After the expression of PD-L1 in 786-O cells was reduced by transient interference,T lymphocytes co-cultured with such 786-O cells had a significant increase in the level of IFN-γcompared with normally co-cultured T lymphocytes.There was a significant increase in the expression of PD-L1 in these two tumor cell lines after T lymphocytes were activated in the co-culture system,which was consistent with the results of stimulation experiment,and the expression of PD-L1 increased in a dose-dependent manner. Conclusion Renal carcinoma cell lines with high PD-L1 expression can effectively inhibit IFN-γsecretion by peripheral blood T lymphocytes and thus lead to immune escape of tumor cells.Meanwhile,IFN-γsecreted by T lymphocytes can upregulate the expression of PD-L1 in renal carcinoma cells,indicating the presence of mutual regulatory interactions between PD-L1 expression and IFN-γsecretion by T lymphocytes in tumor microenvironment.
Objective To investigate the mutual regulatory interactions between programmed death-ligand 1(PD-L1) expression in renal carcinoma and T lymphocyte function in tumor microenvironment. Methods Human peripheral blood mononucleated cells were isolated and stimulated to obtain single CD3~+T lymphocytes.The mRNA and protein expression of PD-L1 in786-O and OSRC cell lines were measured.ELISA was used to measure the level of interferon-γ(IFN-γ)after peripheral blood T lymphocytes were co-cultured with 786-O or OSRC cells,and the change in IFN-γlevel was measured after T lymphocytes were cocultured with renal carcinoma cells with relatively high expression of PD-L1 downregulated by small interfering RNA.The change in PD-L1 expression was measured after 786-O and OSRC cells were co-cultured with T lymphocytes,and the influence of different concentrations of IFN-γon the expression of PD-L1 in 786-O and OSRC cells was analyzed to investigate the mutual regulatory interactions between PD-L1 expression and IFN-γin renal carcinoma 786-O and OSRC cells. Results The expression of PD-L1 in 786-O cells was significantly higher than that in OSRC cells.There was no significant difference in the level of IFN-γbetween T lymphocytes cultured alone and those co-cultured with 786-O cells,but T lymphocytes co-cultured with OSRC cells had a significantly higher level of IFN-γthan those cultured alone.After the expression of PD-L1 in 786-O cells was reduced by transient interference,T lymphocytes co-cultured with such 786-O cells had a significant increase in the level of IFN-γcompared with normally co-cultured T lymphocytes.There was a significant increase in the expression of PD-L1 in these two tumor cell lines after T lymphocytes were activated in the co-culture system,which was consistent with the results of stimulation experiment,and the expression of PD-L1 increased in a dose-dependent manner. Conclusion Renal carcinoma cell lines with high PD-L1 expression can effectively inhibit IFN-γsecretion by peripheral blood T lymphocytes and thus lead to immune escape of tumor cells.Meanwhile,IFN-γsecreted by T lymphocytes can upregulate the expression of PD-L1 in renal carcinoma cells,indicating the presence of mutual regulatory interactions between PD-L1 expression and IFN-γsecretion by T lymphocytes in tumor microenvironment.
引文
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[11]徐光旭,徐曼,耿卫朴,等.PD-L1^+HeLa细胞对人外周血活化T细胞的免疫调节作用[J].第三军医大学学报,2011,33(16):1710-1713.
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[19]姜玉勃,刘昱辰,黄勇,等.PD-1/PD-L1免疫检查点抑制剂抗肿瘤治疗现状及其疗效预测标志物的研究进展[J].癌症进展,2018,16(11):1319-1327.
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[21]ABIKO K,MATSUMURA N,HAMANISHI J,et al.IFN-γfrom lymphocytes induces PD-L1expression and promotes progression of ovarian cancer[J].Br J Cancer,2015,112(9):1501-1509.
[22]DILLINGER B,AHMADI-ERBER S,LAU M,et al.IFN-gamma and tumor gangliosides:Implications for the tumor microenvironment[J].Cell Immunol,2018,325:33-40.
[23]PARRY R V,CHEMNITZ J M,FRAUWIRTH K A,et al.CTLA-4and PD-1receptors inhibit T-Cell activation by distinct mechanisms[J].Mol Cell Biol,2005,25(21):9543-9553.
[24]TAUBE J M,KLEIN A,BRAHMER J R,et al.Association of PD-1,PD-1ligands,and other features of the tumor immune microenvironment with response to anti-PD-1therapy[J].Clin Cancer Res,2014,20(19):5064-5074.
[25]SERRA P,PETAT A,MAURY J M,et al.Programmed cell death-ligand 1(PD-L1)expression is associated with RAS/TP53mutations in lung adenocarcinoma[J].Lung Cancer,2018,118(4):62-68.
[26]JI W M,KONG S K,KIM B S,et al.IFNγinduces PD-L1overexpression by JAK2/STAT1/IRF-1signaling in EBV-positive gastric carcinoma[J].Sci Rep,2017,7(1):17810-17819.
[27]LI L,YOKOYAMA S,NA H,et al.Lac water extract inhibits IFN-γsignaling through JAK2-STAT1-IRF1axis in human melanoma[J].RSC Adv,2018,8(38):21534-21540.
[28]NA L,FORMISANO L,GONZALEZERICSSON P I,et al.Melanoma response to anti-PD-L1immunotherapy requires JAK1signaling,but not JAK2[J].Oncoimmunology,2018,7(6):e1438106.
[29]GOWRISHANKAR K,GUNATILAKE D,GALLAGHER SJ,et al.Inducible but not constitutive expression of PD-L1in human melanoma cells is dependent on activation of NF-κB[J].PLoS One,2015,10(4):e0123410.
[30]FILIPPAKOPOULOS P,KNAPP S.Targetingbromodomains:Epigenetic readers of lysine acetylation[J].Nat Rev Drug Discov,2014,13(5):337-356.
[2]BOCKORNY B,DASANU C A.Intrinsic immune alterations in renal cell carcinoma and emerging immunotherapeutic approaches[J].Expert Opin Biol Ther,2013,13(6):911-925.
[3]OHAEGBULAM K C,ASSAL A,LAZARMOLNAR E,et al.Human cancer immunotherapy with antibodies to the PD-1and PD-L1pathway[J].Trends Mol Med,2015,21(1):24-33.
[4]YE Y,ZHOU L,XIE X,et al.Interaction of B7-H1on intrahepatic cholangiocarcinoma cells with PD-1on tumor-infiltrating T cells as a mechanism of immune evasion[J].J Surg Oncol,2010,100(6):500-504.
[5]田坤,何雯婷,李玉民.PD-1/PD-L1通路在肿瘤免疫逃逸中的研究进展[J].现代肿瘤医学,2016,24(21):3513-3516.
[6]ZOU W,CHEN L.Inhibitory B7-family molecules in the tumor microenvironment[J].Nat Rev Immunol,2008,8(6):467-477.
[7]李巧转,李娴.PD-L1在人胶质瘤中的表达及其对肿瘤内浸润性T细胞凋亡的影响[J].重庆医学,2018,47(1):37-39.
[8]FREEMAN G J,LONG A J,IWAI Y,et al.Engagement of the PD-1immunoinhibitory receptor by a novel B7family member leads to negative regulation of lymphocyte activation[J].J Exp Med,2000,192(7):1027-1034.
[9]邱志敏,王玲,傅颖媛.免疫检查点PD-1/PD-L1抑制剂在恶性肿瘤中的治疗进展[J].实用癌症杂志,2018,33(5):176-178.
[10]陈成,穆传勇,瞿秋霞,等.PD-L1分子在肺癌细胞株上的表达及其生物学意义[J].中国肺癌杂志,2009,12(8):859-863.
[11]徐光旭,徐曼,耿卫朴,等.PD-L1^+HeLa细胞对人外周血活化T细胞的免疫调节作用[J].第三军医大学学报,2011,33(16):1710-1713.
[12]KEIR M E,BUTTE M J,FREEMAN G J,et al.PD-1and its ligands in tolerance and immunity[J].Annu Rev Immunol,2007,26(1):677-704.
[13]SUN C,MEZZADRA R,SCHUMACHER T N.Regulation and function of the PD-L1checkpoint[J].Immunity,2018,48(3):434-452.
[14]田坤,何雯婷,李玉民.PD-1/PD-L1通路在肿瘤免疫逃逸中的研究进展[J].现代肿瘤医学,2016,24(21):3513-3516.
[15]吴介恒,杨安钢,温伟红.PD-1/PD-L1参与肿瘤免疫逃逸的研究进展[J].细胞与分子免疫学杂志,2014,30(7):777-780.
[16]TAUBE J M,ANDERS R A,YOUNG G D,et al.Colocalization of inflammatory response with B7-H1expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape[J].Sci Transl Med,2012,4(127):127-137.
[17]马建辉,鲍镇美.干扰素治疗肾癌[J].中华泌尿外科杂志,2003,24(6):429-431.
[18]LAI Q,WANG H,LI A,et al.Decitibine improve the efficiency of anti-PD-1therapy via activating the response to IFN/PD-L1signal of lung cancer cells[J].Oncogene,2018,37(17):2302-2312.
[19]姜玉勃,刘昱辰,黄勇,等.PD-1/PD-L1免疫检查点抑制剂抗肿瘤治疗现状及其疗效预测标志物的研究进展[J].癌症进展,2018,16(11):1319-1327.
[20]HOGG S J,VERVOORT S J,DESWAL S,et al.BET-Bromodomain inhibitors engage the host immune system and regulate expression of the immune checkpoint ligand PD-L1[J].Cell Rep,2017,18(9):2162-2174.
[21]ABIKO K,MATSUMURA N,HAMANISHI J,et al.IFN-γfrom lymphocytes induces PD-L1expression and promotes progression of ovarian cancer[J].Br J Cancer,2015,112(9):1501-1509.
[22]DILLINGER B,AHMADI-ERBER S,LAU M,et al.IFN-gamma and tumor gangliosides:Implications for the tumor microenvironment[J].Cell Immunol,2018,325:33-40.
[23]PARRY R V,CHEMNITZ J M,FRAUWIRTH K A,et al.CTLA-4and PD-1receptors inhibit T-Cell activation by distinct mechanisms[J].Mol Cell Biol,2005,25(21):9543-9553.
[24]TAUBE J M,KLEIN A,BRAHMER J R,et al.Association of PD-1,PD-1ligands,and other features of the tumor immune microenvironment with response to anti-PD-1therapy[J].Clin Cancer Res,2014,20(19):5064-5074.
[25]SERRA P,PETAT A,MAURY J M,et al.Programmed cell death-ligand 1(PD-L1)expression is associated with RAS/TP53mutations in lung adenocarcinoma[J].Lung Cancer,2018,118(4):62-68.
[26]JI W M,KONG S K,KIM B S,et al.IFNγinduces PD-L1overexpression by JAK2/STAT1/IRF-1signaling in EBV-positive gastric carcinoma[J].Sci Rep,2017,7(1):17810-17819.
[27]LI L,YOKOYAMA S,NA H,et al.Lac water extract inhibits IFN-γsignaling through JAK2-STAT1-IRF1axis in human melanoma[J].RSC Adv,2018,8(38):21534-21540.
[28]NA L,FORMISANO L,GONZALEZERICSSON P I,et al.Melanoma response to anti-PD-L1immunotherapy requires JAK1signaling,but not JAK2[J].Oncoimmunology,2018,7(6):e1438106.
[29]GOWRISHANKAR K,GUNATILAKE D,GALLAGHER SJ,et al.Inducible but not constitutive expression of PD-L1in human melanoma cells is dependent on activation of NF-κB[J].PLoS One,2015,10(4):e0123410.
[30]FILIPPAKOPOULOS P,KNAPP S.Targetingbromodomains:Epigenetic readers of lysine acetylation[J].Nat Rev Drug Discov,2014,13(5):337-356.