The Study Of Potentiating Antitumor Immune Response Through Targeting LAG-3 In Head And Neck Squamous Cell Carcinoma

Background: Head and neck squamous cell carcinoma(HNSCC)is one of the most common human malignancies and is frequently attributed to smoking,alcohol and human papillomavirus(HPV)infection.In recent years,although radiotherapy,chemotherapy,surgery and other treatments have made some progress,the 5-year survival rate of HNSCC is still limited to 50-60%.Head and neck squamous cell carcinoma is defined as an immunosuppressive disease,because it is often accompanied by the anergy of effector T cells,the accumulation of immunosuppressive cells and the abnormality of antigen presenting function.Tumor immunotherapy aims at restoring the killing effect of the patient's immune system to cancer cells.Compared with traditional treatment,tumor immunotherapy can produce sustained anti-tumor effects,especially for targeting immune checkpoint,which has brought about tremendous changes in the treatment of cancer.In 2016,FDA approved the use of PD-1 monoclonal antibody in the treatment of HNSCC,which opened a new era of immunotherapy for HNSCC.However,the response rate of PD-1 in head and neck squamous cell carcinomas is not high,which prompts us to find novel immune checkpoints and broaden the therapeutic strategies of targeting immune checkpoints.Lymphocyte activation gene-3(LAG-3),one the next generation of immune checkpoints,is expressed on various types of immune cells,including T cells,B cells,regulatory T cells(Tregs)and so on.LAG-3 are highly expressed on exhausted or dysfunctional T cells,inhibiting the proliferation and secretion of T cells,and subsequently impair the cytotoxicity of T cells.LAG-3,expressing on Tregs,potentiate the immunosuppressive function of Tregs.These effects are conducive to immune escape of cancer cells.Thus,we hypothesis that LAG-3 may be a novel therapeutic target of immunotherapy.Purpose: To detect the expression of LAG-3 in the samples of human HNSCC.To analyze the relationship between LAG-3 and clinic-pathological parameters in human primary HNSCC.To investigate the prognostic value of LAG-3 in human primary HNSCC.To explore the potential and related immune molecules with LAG-3 in human primary HNSCC.To evaluate the expression of LAG-3 on different types of immune cell in immunocompetent and spontaneous double gene knockout mice model.To validate the effect of anti-LAG-3 in the HNSCC mouse model and to explore the immune modulatory mechanism.Method: The expression of LAG-3 in human HNSCC tissue microarrays was determined by immunohistochemistry.The relationship between LAG-3 and tumor size,lymph node status,pathological grades in human primary HNSCC was analyzed.Then,the association between LAG-3 and gender,age,smoking habit,drinking,HPV(human papillomavirus)infection was investigated.Both Kaplan-Meier univariate and Cox multivariate analysis were used to assess the association between LAG-3 and the patient's survival.The relationship between LAG-3 and other immune molecules was analyzed using Pearson correlation analysis and Hierarchical clustering analysis.The immunocompetent and spontaneous double gene knockout mice model was generated.The expression of LAG-3 on CD4+ T cells,CD8+ T cells,CD4+Foxp3+ T cells,CD11b+Gr1+ MDSCs(myeloid derived suppressor cells),and CD11b+F4/80+ TAMs(tumor associated macrophages)was detected in tumor bearing HNSCC mouse model,compared with wild type mice,using flow cytometry.The tumor growth and the body weight were recorded after blockade of LAG-3 with monoclonal antibody.The population of CD4+T cells,CD8+ T cells,Tregs,MDSCs and TAMs was analyzed with flow cytometry.the secretion of IFN-? was detected using in vitro study.The cytokines in tumors and peripheral blood were determined with cytokines microarrays and western blot.Result: The expression of LAG-3 on tumor infiltrating lymphocytes was(TILs)is upregulated(p<0.001),and its overexpression is associated with pathological characteristic(grades,tumor size and lymph node status)in human primary HNSCC(n=122).The expression of LAG-3 is elevated in metastatic lymph nodes and HNSCC with pre-radiotherapy.K-M curve shows that LAG-3 confers poor outcome in the primary HNSCC patients without lymph node metastasis(negative lymph node status,p =0.0108).Highly expression of LAG-3 is identified as an independent prognosis factor in primary HNSCC patients without lymph node metastasis(p = 0.014)using Cox regression model.The correlation analysis shows the expression of LAG-3 is positively associated with CD8,Foxp3,CD11 b,CD33,CD68 and CD163.Study in transgenic HNSCC mouse model reveals that LAG-3 is significantly upregulated on T cells(CD4+,CD8+,regulatory T cells),compared with wild type mice,but not myeloid cells(MDSCs and TAMs)during tumor progression.Treatment with anti-LAG-3 antibody restrained the tumorigenesis in the head and neck area and tongue by strengthening immune reaction with the enhancement of CD8+ T cell-mediated anti-tumor response through increasing the population and IFN-? secretion of CD8+ T cells.However,the population of CD4+ T cells is almost unchanged.And blockade of LAG-3 could decrease the population of the immunosuppressive cells(Tregs,MDSCs and TAMs)in part of immune organs or tumors.The mechanism study shows that the decreased population of MDSCs and TAMs may be associated with the downregulation of CCL2 and CXCL1.Conclusion: We identify LAG-3 to be a potential prognostic indicator for HNSCC without lymph node metastasis and LAG-3 blockade significantly decreases tumorigenicity and reinvigorates immune response in HNSCC mouse model.These results allow for the speculation that LAG-3 may be a potential target for immunotherapy in HNSCC.