The Study Of Plasmacytoid Dendritic Cell And CD317 In Regulating Immune Evasion Of Head And Neck Squamous Cell Carcinoma

Head and neck squamous cell carcinoma(HNSCC)mainly originate from the squamous epithelium of the oral,pharynx,and larynx.Smoking,excessive alcohol use,and human papilloma virus(HPV)infection are the main risk factors for developing HNSCC.According to the cancer statistics in 2020,about 900 thousand people were diagnosed with HNSCC and 450 thousand patients died from HNSCC each year,which is a huge burden for the health care system all over the world.For now,the main therapeutic method for HNSCC is surgery,radiotherapy,and chemotherapy.Recently,cetuximab targeting the epidermal growth factor receptor(EGFR;also known as HER1)was approved by the U.S.Food and Drug Administration(FDA)for the treatment of metastatic or recurrent HNSCC.Although much progress has been made for the current treatment methods,there is hardly any promotion for the overall 5-year survival rate of HNSCC.Therefore,it's of great significance to deeply understand the biological and molecular mechanism underlying HNSCC and develop a new method to cure it.Immune evasion is one of the main features of cancer.HNSCC could evade immune attack through multiple pathways including secreting a large amount of immune-suppressive cytokines,recruit suppressive immune cells,and expressing immune checkpoints.Recently,several PD-1 antibodies including pembrolizumab and nivolumab have been approved for HNSCC treatment.However,less than 20% of HNSCC patients could respond to PD-1 blockade therapy.The immune suppression mechanism in the tumor microenvironment is complex,which is responsible for the low response rate of immunotherapy.Thus,its urgent to further explore the specific mechanism for HNSCC to evade the immune attack and provide more targets to improve the response rate.Plasmacytoid dendritic cell(p DC)is a type of dendritic cell(DC),which could secret a large amount of type I interferon(IFN-I)and play a key role in initiating immune response for combating virus infection.However,p DC was recently found infiltrated in the tumor microenvironment and remains dysfunctional.CD317 is a typical IFN induced protein and overexpressed in multiple types of tumor.Through interacting with the immunoglobulin-like transcript 7(ILT-7)receptor on p DC,CD317 could inhibit the secretion of IFN-I by p DC and induce immune tolerance.Additionally,the phenotype of p DC was altered and tend to secret less IFN-I.In the microenvironment of HNSCC,the specific role of CD317 and p DC has not been clarified.In this study,we would investigate the existence and specific function of CD317 and p DC in HNSCC.Part ?.Study on the Role of Plasmacytoid Dendritic Cell(p DC)in HNSCCObjective: A large amount of immune suppressive cells could be recruited into the tumor microenvironment of HNSCC.Meanwhile,antigen-presenting cells(APC)and T cells tend to be dysfunctional and exhausted.As the professional IFN-I secreting cells,the phenotype and function of p DC could be altered in the microenvironment of HNSCC.The infiltration characteristic,phenotype,and specific role of p DC in HNSCC need to be further investigated.Methods: Flow cytometry analysis was used to detect the phenotype of p DC in HNSCC fresh samples.Immunohistochemical staining was applied in the HNSCC tissue microarray to analyze the infiltration of p DC in a cohort of HNSCC patients.The corresponding histological and clinical parameters were exploited to investigate the survival implications of p DC infiltration in HPV positive and HPV negative HNSCC.Cervical carcinoma microarray was used to confirm the survival implication of p DC in HPV positive cancer.Other immune-related markers including Foxp-3,PD-1,TIM-3,and LAG-3 were also detected in the serial sections of tissue microarray.Correlation analysis between p DC and immune-related markers was subsequently conducted.Results: p DC was highly infiltrated in the HNSCC microenvironment compared with normal mucosa and dysplasia.Meanwhile,p DC in HNSCC patients expressed low levels of CD80,CD83,and CD86.The survival implication of p DC is different in HPV positive or HPV negative HNSCC.In HPV-positive cervical carcinoma,patients with p DC high infiltration tend to have a better overall survival rate.p DC infiltration in HPV negative HNSCC patients indicates poor overall survival and positively correlated with the expression of Foxp-3,TIM-3,LAG-3,and PD-1.Conclusions: The role of p DC may be different in HPV positive and HPV negative HNSCC patients.In HPV-positive tumor,p DC high infiltration patients may have a good prognosis.In HPV negative HNSCC,p DC indicates poor overall survival and could be a potential negative regulator in antitumor immune response.Part ?.Study on the Immunological Role of CD317 in HNSCCObjective: CD317 could act as a ligand and bind with the receptor on p DC for inhibiting the secretion of IFN-I and other immune active cytokines by p DC,which may contribute to immune suppression in the tumor microenvironment.CD317 has been reported highly expressed in multiple types of tumor.However,there is no related reports concerning the immune-related role of CD317.Thus,we tend to investigate the expression,clinical implication,and immunological role of CD317 in HNSCC.Methods: TCGA and Oncomine database were analyzed to preliminarily profile the expression of CD317 in different cancer types.Opal multiplex staining of CD317,CD11 b,CD3,and DAPI was applied in HNSCC tissue to study the specific location of CD317.HNSCC tissue microarray and immunohistochemical staining were used for investigating the protein expression level of CD317 in a patient cohort.Through Kaplan-Meier analysis and COX regression model,patients' survival information and the expression data of CD317 were combined to reveal the prognostic role of CD317.The correlation of CD317 expression with immune-related pathway was preliminarily investigated by Gene Set Enrichment Analysis(GSEA)based on the TCGA data.In the serial sections of tissue microarray,other immune-related markers including PD-L1,B7-H3,B7-H4,CD68,and CD163 were also detected.Correlation analysis between CD317 and immune-related markers was subsequently conducted.Results: We found that the m RNA expression level of CD317 is upregulated across 19 types of cancer and specifically overexpressed in HNSCC.In HNSCC tissue,CD317 was mainly colocalized with the tumor nests,where CD317 was also found co-localized with CD11 b and CD3.Further,in an HNSCC patient cohort,CD317 high expression independently confers poor overall survival.GSEA results showed that CD317 was closely correlated with negative innate immune response and negative adaptive immune response.Meanwhile,CD317 was positively correlated with immune suppressive markers including PD-L1,B7-H3,B7-H4,CD68,and CD163 in the tumor microenvironment.Conclusions: Our study revealed that CD317 is highly expressed in human and mouse HNSCC tissue.In human HNSCC,CD317 high expression indicates a poor prognosis.CD317 is closely associated with immune suppressive status in human and mouse HNSCC.Based on our results and other publications,we hypothesis that CD317 may negatively regulate immune response through binding with p DC in HNSCC.Part ?.Blocking CD317 and p DC Enhance Anti-tumor Immune Response in HNSCCObjective: CD317 binding with p DC may be exploited by tumor cells of HNSCC to induced immune evasion.In this part,HPV negative Tgfbr1/Pten 2c KO HNSCC mouse model would be used to investigate the role of CD317-p DC function and its impact on the immune status of HNSCC.Methods: Tamoxifen was applied to induce Squamous cell carcinoma in the oral mucosa and the skin of the head and neck in Tgfbr1/Pten 2c KO mice.Then CD317 monoclonal antibodies were intravenously treated.The tumor growth and body weight were monitored.At the end of this study,tumor tissue,tumor-draining lymph nodes,and spleen were collected for flow cytometry analysis.Tumor tissue was also used for immunohistochemical staining.p DCs,T cells,immune checkpoints expression,Tres,and MDSCs were detected in the peripheral immune organs and tumor tissue.CD317 was knocked down in SCC7,an HNSCC cell line.Then,SCC7 cells with no CD317 expression and wild-type SCC7 cells were implanted in immune-deficient NOG mouse to evaluate the role of CD317 on tumor growth without the immune system.Results: In the Tgfbr1/Pten 2c KO mice,after CD317 monoclonal antibodies were applied,tumor growth was significantly inhibited.Meanwhile,flow cytometry results showed that the population of p DC was effectively depleted in the CD317 blockade group.Meanwhile,T cell populations in the tumor harboring mice were increased and the immune checkpoints including PD-1,TIM-3,and LAG-3 expressed on T cells were decreased after CD317 blockade.Besides,the proportion of Tregs and MDSCs was decreased after CD317 blockade.In the immune-deficient NOG mice,we observed that SCC7 tumors with CD317 knockdown have the same growth speed as wild-type SCC7 tumors.Conclusions: In HPV negative Tgfbr1/Pten 2c KO HNSCC mouse model,CD317 blockade could alleviate the immune-suppressive status of tumor harboring mice and delayed tumor growth.However,CD317 could not affect tumor growth without the immune system.Thus,we conclude that CD317 antibodies may alleviate immunesuppressive status through disrupting the function of CD317-p DC in HNSCC.