自噬促进B16细胞由IFN-γ诱导的MHC-Ⅰ类分子的表达
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摘要
人类和动物肿瘤细胞表面MHC-I类分子普遍存在下降和缺失,可能与参与MHC-I类分子抗原提成过程中各种成分的功能失调有关,越来越多的证据表明,自噬,在维持细胞稳态的过程中发挥着重要的作用,而且在细胞表面MHC-II类分子的表达中也起着重要的作用,但是有关自噬是否在调节MHC-I类分子表达中起作用还鲜有文献报道,本实验旨在研究自噬对肿瘤细胞B16细胞表面MHC-I分子的调控的作用,及这种作用是否对CTL杀伤B16细胞有一定的影响。
本实验首先用流式细胞术检测了自噬增强剂及抑制剂对小鼠巨噬细胞MHC-I类分子,及CD80,CD86表达量的影响,发现自噬对小鼠巨噬细胞MHC-I类分子阳性率没有明显的影响,但自噬增强剂可以降低MHC-I类分子平均荧光强度,自噬抑制剂则作用相反。而且自噬增强剂能够降低巨噬细胞共刺激分子CD86的阳性率和平均荧光强度,自噬抑制剂则作用相反,对CD80的表达没有影响。巨噬细胞转染Mouse Atg5, Atg7和Beclin 1 siRNAs后检测MHC-I类平均荧光强度升高。在IFN-γ存在的情况下,自噬增强剂增加B16细胞MHC-I类分子表达量,抑制剂则相反,B16细胞转染Mouse Atg5, Atg7和Beclin 1 siRNAs, MHC-Ⅰ类分子表达量与对照质粒组相比呈下降趋势,免疫胶体金电镜观察自噬增强剂RM处理的B16细胞,其内自噬小体或自噬溶酶体内有MHC-I分子的表达,然而IFN-y和RM同时刺激则抑制这种聚集,而且,PBS处理组并没有观察到MHC-I分子。自噬增强剂和抑制剂对IFN-γ诱导的MHC-I类分子抗原提成过程中LMP2, LMP7, TAP1,TAP2, Tapasin的mRNA水平没有影响。清除B16细胞膜表面MHC-I类分子表达及蛋白酶体的活性后,IFN-γ和自噬增强剂可以协同增加MHC-I类分子的表达,自噬抑制剂则降低表达。
在存在IFN-Y时,自噬增强剂可以增加CTL对B16细胞的杀伤能力,抑制剂则相反,稳转Beclin 1-siRNAB 16细胞,CTL对其杀伤能力则下降。采用RealtimePCR和ELISA检测肿瘤组织IFN-y水平随着接种日期增加呈先增高后下降趋势,在接种14d时最高,肿瘤细胞MHC-I抗原表达情况有相同趋势。取荷瘤12d小鼠脾细胞制备CTL,经自噬增强剂来预处理的荷瘤14d鼠的肿瘤组织24h,流式检测CTL对肿瘤细胞的杀伤能力,与未处理组相比增加,而自噬抑制剂处理组则下降；荷瘤25d组则有相反的结果。荷瘤25d小鼠脾细胞制备的CTL,对经自噬增强剂或抑制剂来预处理荷瘤25d鼠的肿瘤组织的杀伤能力与对照组相比均没有统计意义。
自噬不仅参与MHC-I类分子的降解,而且在MHC-Ⅰ类分子抗原肽的产生过程中起到重要作用,自噬在肿瘤的早期阶段,能够增加CTL对肿瘤细胞的杀伤能力,但是在肿瘤的晚期,则会阻碍这种杀伤作用,这种不同的效应可能与肿瘤的不同阶段IFN-γ的表达量不同。综上所述,我们发现自噬参与MHC-I类分子抗原肽的表达,因此自噬在肿瘤免疫过程中起到了不同的作用。
The reduction or loss of IFN-γsurface.expression in human and murine tumor cells is partly attributable to the dysregulation of various components of the MHC-I antigen-processsing machinery. Accumulating evidence suggests that autophagy, besides its vital role in maintaining the cellular homeostasis, plays an important role in MHC-II surface expression. In our study, in the present study, we ask whether autophagy is involved in the reduction or loss of MHC-I antigen in tumor cells and make clear that whether the cytolysis of CTL to melanoma cell is changed.
Here, we use autophagy inducer and inhibitor stimulate macrophage, detect the MHC-I antigen and CD80,CD86 expression by flow cytometry, The flow cytometric analysis showed that these agents did not alter the number of MHC-I antigen positive cells, but affected the mean fluorescence intensity (MFI) significantly Rapamycin decreased the MFI,others increased the MFI at different level, We found that CD80 was not changed by autophagy, but CD86 was significantly altered, knockdown of autophagy-related genes Atg5, Atg7 or Beclin 1 also increased MHC-I antigen expression by macrophages.In B16cell, rapamycin did not show inhibitory effect, but synergized with IFN-γto further upregulate MHC-I antigen expression On the other hand, the suppression of autophagy by 3-MA, BM, wortmannin and chloroquine, downregulated MHC-I antigen of B16 cells, The similar result was also obtained by electron microscopy, MHC-I antigen molecules were present in the autophagolysosomes of B16 cells treated with rapamycin but the addition of IFN-y inhibited such colocalization, MHC-I antigen was not observed in PBS-treated cells, Our results indicated that neither autophagy inducer nor inhibitors affected the expression of genes LMP2, LMP7,TAP1, TAP2, Tapasin induced by IFN-y, we using acid to remove surface class I peptide complexes induced by IFN-y, decreased expression of MHC-I antigen by proteasome inhibitors could be partially restored in the presence of rapamycin, while the autophagy inhibitors further suppressed MHC-I antigen surface expression.
The effcient killing to IFN-y-treated B16 cells was observed, which could be enhanced or impaired by addition of rapamycin or autophagy inhibitors the effect of IFN-y treatment on cytolysis was impeded in B16 cell line stably expressing Beclin 1 siRNA.The high level of tumor IFN-y could be detected on d5 after tumor cell inoculation and probably reached to the highest level on d15, then gradually decreased, evaluated by both real time RT-PCR and ELISA,In parallel, the similar expression pattern of MHC-I antigen in melanoma tumor cells was observed. We then treated melanoma (dl2)with rapamycin or autophagy inhibitors for 48 h, and performed the cytolysis assay, The spleen T cells from untreated d12 tumor-bearing mice were isolated and stimulated in vitro and used as the effector cells, The rapamycin treatment effectively strengthened the sensitivity of tumor cells to CTLs, and the treatment with autophagy inhibitors nevertheless decreased the sensitivity, leading to low cytolysis. We also tested the large melanoma 25d, rapamycin treatment decreased the cytolysis, and autophagy inhibitors increase the cytolysis.We then isolated the spleen T cells from d25 tumor-bearing mice and stimulated them in vitro for the use as effector cells. The impaired cytolysis to the tumor cells of large melanoma was observed, and neither rapamycin nor autophagic inhibitors had the effect on the cytolysis.
Autophagy not only participates in the degradation of MHC-I antigen but also plays a role in the generation of MHC-I binding peptides. For these two processes, IFN-y interferes with MHC-I antigen degradation, rather than affecting peptide generation. Using B16 melanoma mouse model, we further show that autophagy may enhance the cytolysis of CTL to melanoma cells at the early stage of melanoma, but impairs the cytolysis at the late stage. Such different consequences may be explained by the different levels of IFN-yduring tumor progression. Taken together, our fndings demonstrate that autophagy is involved in the regulation of MHC-I antigen expression, through which autophagy can play different roles in tumor immunity.
本实验首先用流式细胞术检测了自噬增强剂及抑制剂对小鼠巨噬细胞MHC-I类分子,及CD80,CD86表达量的影响,发现自噬对小鼠巨噬细胞MHC-I类分子阳性率没有明显的影响,但自噬增强剂可以降低MHC-I类分子平均荧光强度,自噬抑制剂则作用相反。而且自噬增强剂能够降低巨噬细胞共刺激分子CD86的阳性率和平均荧光强度,自噬抑制剂则作用相反,对CD80的表达没有影响。巨噬细胞转染Mouse Atg5, Atg7和Beclin 1 siRNAs后检测MHC-I类平均荧光强度升高。在IFN-γ存在的情况下,自噬增强剂增加B16细胞MHC-I类分子表达量,抑制剂则相反,B16细胞转染Mouse Atg5, Atg7和Beclin 1 siRNAs, MHC-Ⅰ类分子表达量与对照质粒组相比呈下降趋势,免疫胶体金电镜观察自噬增强剂RM处理的B16细胞,其内自噬小体或自噬溶酶体内有MHC-I分子的表达,然而IFN-y和RM同时刺激则抑制这种聚集,而且,PBS处理组并没有观察到MHC-I分子。自噬增强剂和抑制剂对IFN-γ诱导的MHC-I类分子抗原提成过程中LMP2, LMP7, TAP1,TAP2, Tapasin的mRNA水平没有影响。清除B16细胞膜表面MHC-I类分子表达及蛋白酶体的活性后,IFN-γ和自噬增强剂可以协同增加MHC-I类分子的表达,自噬抑制剂则降低表达。
在存在IFN-Y时,自噬增强剂可以增加CTL对B16细胞的杀伤能力,抑制剂则相反,稳转Beclin 1-siRNAB 16细胞,CTL对其杀伤能力则下降。采用RealtimePCR和ELISA检测肿瘤组织IFN-y水平随着接种日期增加呈先增高后下降趋势,在接种14d时最高,肿瘤细胞MHC-I抗原表达情况有相同趋势。取荷瘤12d小鼠脾细胞制备CTL,经自噬增强剂来预处理的荷瘤14d鼠的肿瘤组织24h,流式检测CTL对肿瘤细胞的杀伤能力,与未处理组相比增加,而自噬抑制剂处理组则下降；荷瘤25d组则有相反的结果。荷瘤25d小鼠脾细胞制备的CTL,对经自噬增强剂或抑制剂来预处理荷瘤25d鼠的肿瘤组织的杀伤能力与对照组相比均没有统计意义。
自噬不仅参与MHC-I类分子的降解,而且在MHC-Ⅰ类分子抗原肽的产生过程中起到重要作用,自噬在肿瘤的早期阶段,能够增加CTL对肿瘤细胞的杀伤能力,但是在肿瘤的晚期,则会阻碍这种杀伤作用,这种不同的效应可能与肿瘤的不同阶段IFN-γ的表达量不同。综上所述,我们发现自噬参与MHC-I类分子抗原肽的表达,因此自噬在肿瘤免疫过程中起到了不同的作用。
The reduction or loss of IFN-γsurface.expression in human and murine tumor cells is partly attributable to the dysregulation of various components of the MHC-I antigen-processsing machinery. Accumulating evidence suggests that autophagy, besides its vital role in maintaining the cellular homeostasis, plays an important role in MHC-II surface expression. In our study, in the present study, we ask whether autophagy is involved in the reduction or loss of MHC-I antigen in tumor cells and make clear that whether the cytolysis of CTL to melanoma cell is changed.
Here, we use autophagy inducer and inhibitor stimulate macrophage, detect the MHC-I antigen and CD80,CD86 expression by flow cytometry, The flow cytometric analysis showed that these agents did not alter the number of MHC-I antigen positive cells, but affected the mean fluorescence intensity (MFI) significantly Rapamycin decreased the MFI,others increased the MFI at different level, We found that CD80 was not changed by autophagy, but CD86 was significantly altered, knockdown of autophagy-related genes Atg5, Atg7 or Beclin 1 also increased MHC-I antigen expression by macrophages.In B16cell, rapamycin did not show inhibitory effect, but synergized with IFN-γto further upregulate MHC-I antigen expression On the other hand, the suppression of autophagy by 3-MA, BM, wortmannin and chloroquine, downregulated MHC-I antigen of B16 cells, The similar result was also obtained by electron microscopy, MHC-I antigen molecules were present in the autophagolysosomes of B16 cells treated with rapamycin but the addition of IFN-y inhibited such colocalization, MHC-I antigen was not observed in PBS-treated cells, Our results indicated that neither autophagy inducer nor inhibitors affected the expression of genes LMP2, LMP7,TAP1, TAP2, Tapasin induced by IFN-y, we using acid to remove surface class I peptide complexes induced by IFN-y, decreased expression of MHC-I antigen by proteasome inhibitors could be partially restored in the presence of rapamycin, while the autophagy inhibitors further suppressed MHC-I antigen surface expression.
The effcient killing to IFN-y-treated B16 cells was observed, which could be enhanced or impaired by addition of rapamycin or autophagy inhibitors the effect of IFN-y treatment on cytolysis was impeded in B16 cell line stably expressing Beclin 1 siRNA.The high level of tumor IFN-y could be detected on d5 after tumor cell inoculation and probably reached to the highest level on d15, then gradually decreased, evaluated by both real time RT-PCR and ELISA,In parallel, the similar expression pattern of MHC-I antigen in melanoma tumor cells was observed. We then treated melanoma (dl2)with rapamycin or autophagy inhibitors for 48 h, and performed the cytolysis assay, The spleen T cells from untreated d12 tumor-bearing mice were isolated and stimulated in vitro and used as the effector cells, The rapamycin treatment effectively strengthened the sensitivity of tumor cells to CTLs, and the treatment with autophagy inhibitors nevertheless decreased the sensitivity, leading to low cytolysis. We also tested the large melanoma 25d, rapamycin treatment decreased the cytolysis, and autophagy inhibitors increase the cytolysis.We then isolated the spleen T cells from d25 tumor-bearing mice and stimulated them in vitro for the use as effector cells. The impaired cytolysis to the tumor cells of large melanoma was observed, and neither rapamycin nor autophagic inhibitors had the effect on the cytolysis.
Autophagy not only participates in the degradation of MHC-I antigen but also plays a role in the generation of MHC-I binding peptides. For these two processes, IFN-y interferes with MHC-I antigen degradation, rather than affecting peptide generation. Using B16 melanoma mouse model, we further show that autophagy may enhance the cytolysis of CTL to melanoma cells at the early stage of melanoma, but impairs the cytolysis at the late stage. Such different consequences may be explained by the different levels of IFN-yduring tumor progression. Taken together, our fndings demonstrate that autophagy is involved in the regulation of MHC-I antigen expression, through which autophagy can play different roles in tumor immunity.
引文
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