The Effect Of PD-L2 Glycosylation On Immune Evasion In Head And Neck Squamous Cell Carcinoma

Therapeutic antibodies based on PD-1/PD-L1 have achieved great success,but the function of another PD-1 ligand,programmed cell death 1 ligand 2(PD-L2),is poorly understood.Given that head and neck squamous cell carcinoma(HNSCC)are the sixth common cancer in the world and highly express PD-L2.Our research found that PD-L2 correlated with the patient’s clinical stage in HNSCC specimens,and interrelated with the poor prognosis.The abmormal glycosylation of preoteins have been known as the targets of many cancers.To elucidate the role of PD-L2 and its glycosylation is not only of great significance for the treatment of head and neck squamous cell carcinoma,but also can reveal the law of PD-L2 participating in the fine regulation of organisms.Methods1.Specimens of HNSCC patients were collected and analyzed the correlation between PD-L2 expression and AJCC staging by Western Blot.MRNA sequence detected HNSCC patients of PD-L2 expression.The expression levels of PD-L2 in different TNM stages were analyzed by immunohistochemistry(IHC).2.Animal vivo imaging technology tested the tumor growth after PD-L2 knockout by SCC7 cells in vivo,as well as the survival period and body weight change of tumorbearing mice,and flow cytometry tested the expression of active cytotoxic T cells.3.Western Blot analyzed PD-L2 expression in HNSCC tumor tissues and cancer cells.Glycoprotein staining and Coomassie blue staining were used to detect the positive staining of glycoprotein structure.Half-life analysis detected different processing forms of PD-L2 by using the protein synthesis inhibitor cycloheximide(CHX).4.The effect of PD-L2 glycosylation on the cell surface after treatment with glycosylation inhibitors was investigated.Co-immunoprecipitation(Co-IP)was used to analyze the binding of PD-1 in glycosylated PD-L2(PD-L2 WT)and nonglycosylated PD-L2(PD-L2 4NQ).In vivo experiments were performed to examine the effects of PD-L2 glycosylation effected tumor growth.5.A variety of cytokines associated with poor prognosis was detected and was identified cytokines which enhanced PD-L2 protein levels.PD-L2 glycosylation expression,PD-1 binding and T cell-mediated apoptosis were detected after adding inhibitor.6.Key glycosyltransferases that are positively correlated with tumor staging and PD-L2 are identified in the TCGA database.Aleuria Aurantia lectin(AAL)lectin blot analysis verified the staining of PD-L2 protein in defective cells.Western blot combined with chromatin immunoprecipitation(CHIP)experiment to investigate whether the upstream signaling pathway of PD-L2 can transcribe the key glycosyltransferase.MG132 combined with CHX half-life experiment to analyzed the effect of glycosyltransferase on PD-L2 ubiquitination.In the C3 H mouse in vivo model,the lack of glycosyltransferase was examined for PD-L2-mediated tumor growth.7.Flow cytometry,cell surface IP,and immunofluorescence analysis of glycosyltransferase-mediated PD-L2 cell membrane expression.Ubiquitination experiments investigated the degradation of PD-L2 in cells lacking glycosyltransferase.The Co-IP experiment tested the ability of PD-L2 to bind to the endosome sorting complex-0(ESCRT-0)required for transport in cells lacking glycosyltransferase.Immunofluorescence experiments examined the effect of knocking down ESCRT-0 on the colocalization signal of PD-L2 and lysosomal-associated membrane protein 2(LAMP2)and membrane abundance in cells lacking glycosyltransferase.Results1.PD-L2 level in the tumor tissue was significantly increased compared with the adjacent normal tissue.MRNA sequencing showed that the expression of PD-L2 in tumors was increased compared with normal tissues.2.Knockout of PD-L2 in mice in vivo experiments decreased tumor growth,prolonged the survival of mice,increased killer T cells.3.Two bands were observed in HNSCC tumor tissue and cancer cells.The results of glycoprotein staining and Coomassie blue staining showed that both bands represent PD-L2 and high molecular band was glycosylation.Glycosylation mass spectrometry confirmed that the 4 NXT motif sites(-Asn-X-Ser / Thr-)are all N-linked glycosylation sites of PD-L2.4.Half-life analysis showed the half-life of non-glycosylated PD-L2 was reduced.Glycosylation effectively reduces its ubiquitination level and improves the stability of PD-L2.Immunofluorescence combined with flow cytometry showed that in nonglycosylated mutant(PD-L2 4NQ)cells,the interaction of PD-L2 and PD-1 on the cell membrane was significantly reduced.In C3 H mouse model,the tumors expressing nonglycosylated PD-L2 4NQ cells grow slowly and the survival rate is significantly prolonged.In immunodeficient BALB/c Nude nude mice,no significant changes were observed in PD-L2-mediated tumor growth.The results show that the glycosylation modification of PD-L2 can enhance the binding to PD-1,inhibit tumor-specific killer T cell responses,and achieve immune escape.5.Western blot showed that EGF induces glycosylation of PD-L2.Examination of multiple downstream signal transduction pathways showed that EGF/STAT3-mediated PD-L2 up-regulation.When the STAT3 inhibitor Stattic is added,the glycosylation level of PD-L2 decreased,and PD-1 binding decreased and T cellmediated apoptosis of cancer cells increased.6.Western blot results showed that FUT8 knockout can eliminate the increase in PD-L2 glycosylation caused by STAT3.The addition of transcription inhibitor Actinomycin D(Act D)indicated that the glycosylation of PD-L2 may require EGF/STAT3-mediated transcriptional activation.CHIP data showed that EGF stimulates the direct binding of STAT3 to FUT8 promoter region.These results indicate that STAT3 transcriptionally up-regulates FUT8 expression and mediates PD-L2 glycosylation.7.AAL lectin blot analysis showed that PD-L2 is a downstream substrate of FUT8.After CHX treatment,the half-life of FUT8 binding motif mutation(PD-L2 REY)was significantly reduced,and FUT8 can promote PD-L2 fucosylation and mediate its stability.In vivo experiments have shown that tumors with PD-L2 REY mutants reduced tumor growth and increased activated cytotoxic T cells.It shows that FUT8 can catalyze the fucosylation of PD-L2,and mediate the immune evasion of PD-L2,and promote tumor development.8.Flow cytometry and cell surface IP analysis indicated FUT8 knockdown deducted EGF-mediated PD-L2 cell membrane expression.Immunofluorescence staining demonstrated that most of PD-L2 is located on the cell membrane in PD-L2 WT cells.In contrast,PD-L2 expression in the cytoplasm was observed in PD-L2 REY.Ubiquitination experiments combined with Co-IP results showed that REY can increase PD-L2 ubiquitination and enhance its ability to bind to the HRS which is component of the transport essential endosomal sorting complex-0(ESCRT-0),and FUT8 knock down or Stattic-treated had similar affect.Immunofluorescence showed that,the colocalization of lysosomal-associated membrane protein 2(LAMP2)and PD-L2 increased significantly in PD-L2 REY cells,and knocking down HRS weakened the co-localization signal of PD-L2 and LAMP2,and restored a amount of membrane abundance.ConclusionOur research suggests that PD-L2 is highly expressed in HNSCC and plays a key role in the immune evasion of tumor cells.At the same time,it is confirmed that PDL2 has glycosylation modification and the specific glycosylation site is clarified.In addition,the study also revealed the upstream signaling pathway that drives PD-L2 glycosylation and identified the key glycosyltransferase which regulates PD-L2 glycosylation.Molecular mechanism was elucidated that glycosylation increase the stability of PD-L2,which can provide a new target for tumor therapy.