Autophagy Facilitates Major Histocompatibility Complex Class Ⅰ Expression Induced By IFN-γ In B16 Melanoma Cells

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.