IFN-? Regulates The Expression Of PD-L1 And NKG2D Ligands In Nasopharyngeal Carcinoma

ObjectiveAs a malignant tumor highly related to EB virus,nasopharyngeal carcinoma is characteristic by a large number of infiltrating lymphocytes and the high expression of PD-L1.It is generally believed that those solid tumors with a high level of PD-L1expression and a large number of infiltrating CD8~+T cells have a better response to PD-1/PD-L1 inhibitors.However,the so far clinical data show that the objective response rate of PD-1 inhibitors in advanced NPC is only 20%to 30%.Some studies found that IFN-?,the strongest inducer of PD-L1,can down-regulate the expression of NKG2D ligands(NKG2Ls)on the surface of tumor cells,whose specific receptor,NKG2D,is a kind of active protein expressing on the surface of NK cells and CD8~+T cells and can regulate both innate and specific immune responses.Therefore,the down-regulation of NKG2DLs on the surface of tumor cells may lead to immune escape and even immune resistance.The purpose of this study was to elucidate the regulatory effect and mechanism of IFN-?to the expression of PD-L1 and NKG2DLs in NPC,and what a role does this phenomenon play in the primary resistance to PD-1/PD-L1 inhibitors.MethodsThe RNA sequencing data of patients with non-NPC head and neck squamous cell carcinoma(HNSCC)and NPC was obtained from TCGA database and the GEO database.The correlation in the mRNA expression level between IFN-?and PD-L1and NKG2DLs was analyzed to screen out the most relevant NKG2DL molecule.Immunohistochemical staining was performed on NPC specimens obtained from the clinical sample bank of our center.The correlation between the expression of CD8,PD-L1 and ULBP3 protein and the clinical features was evaluated at the tissue level.Besides,the tumor microenvironment(TME)in baseline tumor specimens of NPC patients treated with PD-1 inhibitors were observed retrospectively to observe the expression of ULBP3,PD-L1 and CD8 protein.Exogenous IFN-?was administrated to three types of NPC cells,CNE-1,CNE-2 and 5-8F,and qPCR,flow cytometry and Western-blot were taken after 0,4,12,and 24 hours to verify the regulatory effect mediated by IFN-?to the expression of PD-L1 and NKG2DLs.Results1.In patients with non-NPC HNSCC and NPC,the expression of IFN-?was significantly correlated with the mRNA expression of PD-L1(HNSCC:R=0.65,P<0.001,NPC:R=0.35,P<0.001),and ULBP3(HNSCC:R=-0.176,P<0.001,NPC:R=-0.3,P=0.001).2.In NPC,PD-L1 was mainly distributed on the membrane of tumor cell,with annular or linear staining,and a small amount was expressed in the cytoplasm of interstitial immune cells,while ULBP3 was mostly distributed in cytoplasm,mainly in interstitial lymphocytes.The expression of PD-L1 on cell membrane was significantly positively correlated with the infiltration of CD8~+T cells and negatively correlated with the expression of ULBP3.3.In NPC cell lines,IFN-?could induce a significant up-regulation of PD-L1 at mRNA and protein level,and a significant down-regulation of ULBP3 at mRNA and protein level.4.Among patients with NPC at stage III~IVA who received standard treatments,the OS and DFS were significantly longer in patients with high expression of ULBP3than those with negative or low expression(OS:P=0.033;DFS:P=0.036).5.In the TME of a patient with advanced NPC resistant to PD-1 inhibitor,we found a large number of infiltrated CD8~+T cells,a strong expression of PD-L1 and a negative expression of ULBP3.In the TME of another patient who responded to PD-1 inhibitor,there were a similar large number of CD8~+T cell infiltration and a high expression of PD-L1,whearas a lack of ULBP3 expression at the same time.Conclusions1.At both mRNA and protein levels,IFN-?could up-regulate the expression of PD-L1 and down-regulate the expression of UBLP3 at the same time,with a significant time-effect relationship.These two molecular events may be mediated by the same signaling pathway,JAKs/STATs pathway,which still needs further experiments to verify.2.The level of ULBP3 expressed on tumor cell membrane is a prognostic factors for patients with NPC at stage III~IVA receiving standard treatment.3.The loss of UBLP3 protein expressed on cell membrane may be the unique mechanism of primary resistance to PD-1 inhibitors in some patients with NPC.Patients with highly-expressed PD-L1 and a ULBP3 deficiency may be a unique subgroup of immune escape.The new classification approach included in ULBP3 to the TME of patients with NPC may classify the sensitive population of PD-1inhibitors more accurately,and guide the clinical application and curative effect monitoring of PD-1 inhibitors better.