Small Extracellular Vesicle PD-L1 Inhibits Antigen Presenting Cells And Associates With The Immunotherapy Response In Oral Squamous Cell Carcinoma & Case Report

Part One.Research on the correlation between small extracellular vesicle PD-L1 and clinical outcome of immunotherapy in oral squamous cell carcinomaPurpose: Oral squamous cell carcinoma(OSCC)is the most frequent malignant tumor in the oral and maxillofacial region.Surgery-based traditional therapy failed to further improve the 5-year survival of patients with local advanced OSCC.As a new treatment,immunotherapy has been proven to be efficient in many types of malignant tumors.Previous studies including ours have indicated that small extracellular vesicles(sEVs)secreted by cancer cells carry bioactive PD-L1 on their surface and can systemically suppress the immune system.The level of PD-L1 on sEVs hold great potential to serve as a predictor for immunotherapy in malignant melanoma and lung cancer.However,the relationship between sEV PD-L1 and immunotherapy response in OSCC remains unknown.Moreover,the mechanisms underlying the immunosuppressive effect of sEV PD-L1 also need to be fully elucidated.The aim of this study is to investigate the relationship between sEV PD-L1 and immunotherapy response in OSCC patients and also to supplement the mechanisms by which sEV PD-L1 suppress immune cells.Methods: First,nanoparticle tracking analysis(NTA),nanoflow cytometry,BCA assay were used to characterize sEVs and quantify the level of PD-L1 on sEVs isolated from the peripheral blood of OSCC patients and healthy donors.Receiver operating characteristic(ROC)curve was used to screen the biomarkers on sEVs for the diagnosis of OSCC.Linear regression method was used to analyze the relationship between sEV PD-L1 and outcome of immunotherapy in OSCC patients.Second,immunohistochemistry was used to evaluate the expression pattern of Alix,a regulatory protein of sEV PD-L1,in OSCC tissue slides.Then,linear regression was used to analyze the relationship between the expression level of Alix and the response to immunotherapy of OSCC patients.Finally,sEVs were extracted from OSCC tissues and co-cultured with immunocytes.Flow cytometry was used to analyze the effects of OSCC-derived sEVs on immunocytes.Results: The level of PD-L1 on sEVs isolated from the peripheral blood of OSCC patients was significantly higher than that of healthy donors,which could serve as a potential biomarker to distinguish OSCC patients from healthy donors.Moreover,the level of sEV PD-L1 positively correlated with the tumor burden,and negatively correlated with the response to anti-PD-1 immunotherapy in OSCC patients.The sEV PD-L1 regulatory protein Alix was highly expressed in OSCC.The expression level of Alix positively correlated with the level of sEV PD-L1,and negatively correlated with the response to anti-PD-1 immunotherapy.It was also found that OSCC-derived sEVs inhibited the proliferation and maturation of B lymphocytes.Conclusion: The level of sEV PD-L1 correlates with the response to immunotherapy in OSCC patients.Alix may be involved in the response to immunotherapy in OSCC by regulating the secretion of sEV PD-L1.Also,sEV PD-L1 may suppress anti-tumor immune response by inhibiting the proliferation and maturation of B lymphocytes in OSCC.Part Two.Research on the inhibitory effects of oral squamous cell carcinoma-derived small extracellular vesicle PD-L1 on antigen presenting cellsPurpose: The mature antigen presenting cells(APCs)can activate T cells to initiate antitumor immunity.The former part of this study found that OSCC-derived sEVs inhibited APCs.Thus,this part aimed to further investigate the inhibitory effects of OSCC-derived sEVs on APCs.Methods: First,sEVs secreted by OSCC cell lines were isolated by ultracentrifugation,and the morphology,particle size and characteristic protein of sEVs were detected by transmission electron microscopy(TEM),NTA,nanoflow cytometry and western blotting.The co-culture of OSCC cell-derived sEVs and APCs was established,and flow cytometry was performed to evaluate the apoptosis and maturation of APCs.Second,OSCC-derived sEVs with low and high level of PD-L1 were obtained,then flow cytometry and western blotting were used to determine the effect of OSCC-derived sEVs carrying different amount of PD-L1 on APCs.Finally,sEVs carrying PD-L1-GFP fusion protein were co-cultured with APCs.The potential transfer of PD-L1-GFP from OSCC-derived sEVs to APCs was determined by flow cytometry and western blotting.Results: Human and mouse OSCC-derived sEVs were successfully isolated from cell culture supernatant,possessing typical biconcave disc like structures with different levels of PD-L1.The co-culture experiments showed that the uptake of OSCC-derived sEVs by APCs was timedependent,leading to inhibitory effects on the proliferation and maturation of APCs,and the upregulation of PD-L1 on the surface of APCs.The co-culture of APCs and OSCC-derived sEVs suggested that the suppression of APCs induced by OSCC-derived sEVs positively correlated with the level of PD-L1 on sEVs.Finally,by detecting the level of PD-L1-GFP fusion protein,it was found that the level of PD-L1 on APCs was increased by the horizontal transfer of PD-L1 from sEVs to APCs,rather than regulating the intracellular transcriptional level of PD-L1.Conclusion: The level of PD-L1 on APCs is increased by the horizontal transfer of PD-L1 from sEVs to APCs,inhibiting the proliferation and promoting the apoptosis of APCs.