Study In Immunosuppressive Mechanism Of Treg,MDSC And PD-1 Positive T Cell In Head And Neck Squamous Cell Carcinoma

Objective: 1.To investigate the interaction of suppressing cells in the HNSCC microenvironment and to evaluate their interactions and relationships.2.To study the relationship between the main components of Treg,MDSC and PD-1 positive T cells in HNSCC immunosuppressive network and the common inhibition mechanism.3.To evaluate the functional status of effector T cells in local and peripheral environment,to explain the possible causes and functional recovery mechanisms,and to explore the feasibility of multi-target combination therapy for immunotherapy.Method: 1.Peripheral blood PBMC were isolated by Ficoll density gradient centrifugation.Peripheral blood PBMC of HNSCC group and healthy adult group were isolated.Treg,MDSC and PD-1 positive T cells in PBMC were detected by multi-channel fluorescence flow cytometry and quantified.For analysis,the marker of Treg is CD4+CD25+Fox P3+GARP+CTLA-4+,the marker of T cell is CD4+PD-1+/CD8+PD-1+,and the marker of MDSC is CD14-HLA-DR-CD11b+CD33+.2.Isolation of tumor TILs,surgical removal of HNSCC specimens will be used to separate cancer cells and TILs.TILs and tumor cells were isolated using Ficoll density gradient centrifugation.Treg,MDSC and PD-1 positive T cells in TILs were detected by multi-channel fluorescence flow cytometry.3.The expressions of IL-10,TGF-β1,INF-γ and granzyme B in TILs in peripheral blood PBMC,serum and tumor tissues were detected by ELISA and flow cytometry.4.Screening of immunologically tolerant cases,ie,PD-L1 and TILs double positive cases,observe the expression of PD-L1 and TILs in tumor tissues,and detect PD-L1,CD3,CD8,Foxp3 in serial sections by immunohistochemistry.CD11 b,CD33,CD68 antibody staining,Treg,MDSC,PD-1 positive T cells in tumor tissues and surrounding stroma were examined,and the expression types were summarized and analyzed.Results:1.Tregs in HNSCC exhibit a highly immunosuppressive phenotype.The proportion of CD4+Foxp3+Tregs in TILs and peripheral blood of HNSCC patients increased significantly,and the CTLA4+Foxp3+Tregs and GARP+Foxp3+Tregs phenotypes increased to different extents.Compared with normal control peripheral blood,the difference was significant.2.The proportion of MDSCs in HNSCC patients is increased and positively correlated with the increase of CD4+Foxp3+Tregs.The proportion of MDSCs in TILs and peripheral blood of HNSCC patients was significantly increased,which was significantly higher than that in normal tonsil tissues and normal controls.The difference was significant.There was a correlation between elevated %CD4+Foxp3+Treg and elevated %MDSC,and there was a strong correlation between elevated %CD4+Foxp3+Treg and elevated %MDSC in HNSCC-TILs.3.The expression of PD-1 in CD4+ and CD8+ T cells increased in HNSCC patients.The proportion of PD-1+CD8+ T cells and PD-1+CD4+ T cells in TILs and peripheral blood of HNSCC patients increased,which was significantly higher than that in normal controls.The difference was significant.4.HNSCC patients have reduced anti-tumor cytokines and elevated levels of immunosuppressive cytokines.The content of IFN-γ in peripheral blood of patients with HNSCC decreased,which was significantly lower than that in normal controls.The difference was significant.The levels of IL-10 and TGF-β1 in peripheral blood of patients with HNSCC increased significantly compared with the levels of peripheral blood in normal controls,and the difference was significant.There was a correlation between elevated CD4+Foxp3+Treg ratio and high plasma levels of TGF-β1.5.The killing activity of CD8+CTL cells in HNSCC decreased.The GB of CD8+T cells in the TILs of patients with HNSCC was significantly inhibited,and the content was significantly reduced,which was significantly lower than that in the normal control,peripheral blood,HNSCC peripheral blood and normal tonsil tissue.The difference was significant.The GB of CD8+T cells in HNSCC peripheral blood PBMC was no significant decrease compared with it in peripheral blood PBMC of normal controls,and the difference was not significant.6.Expression of tumor PD-L1 in HNSCC tumor microenvironment and immunosuppression network of immune cells in TILsPD-L1 was mainly expressed in HNSCC tumor cells,and 23/32(71.7%)showed positive PD-L1 staining.Most of the specimens(22/23)showed staining confined to the edge of the tumor at the interface between the cancer nest and the inflammatory matrix,and only 1/23 of the cases showed diffuse PD-L1 expression in the cancer nest.Simultaneous detection of CD4+ T cells,CD8+ T cells,Fox P3+Tregs,MDSCs and CD68+ TAMs cells in TILs constitutes the main component of TILs.Tumor cells,CD68+TAMs and T cells form contact PD-L1:PD-1 co-suppression mechanism,Fox P3+Tregs,MDSCs may play a major role in non-PD-L1:PD-1 immunosuppressive mechanism.Conclusion:1.In HNSCC,the proportion of Fox P3+GARP+CTLA4+Treg,MDSC,PD-1+ T cell increased,IFN-γ decreased and the corresponding IL-10 and TGF-β1 increased.These factors are related to each other and interact with each other to form HNSCC local immunity.Suppress the main part of the network.2.HNSCC is accompanied by obvious TILs infiltration.The obvious inflammatory microenvironment is an important feature of head and neck squamous cell carcinoma.The inflammatory immune response of TILs may constitute the main cause of tumor microenvironment immunosuppression,and may also promote the occurrence and development of tumors.3.Tumor cells and CD68+TAM in most tumors induced the formation of PD-1:PD-L1 immunosuppressive pathway in local contact with T cells,and the increase of Tregs and the corresponding increase of MDSC also directly inhibited the tumoricidal effect of CD8+CTL.A non-PD-1:PD-L1 immunosuppressive mechanism is formed.4.GARP may be an ideal candidate target for elimination of Treg.5.HNSCC tumor immunotherapy focuses on the local immune microenvironment,combined with the elimination of Treg,MDSC,PD-1+ T cell inhibition in HNSCC tumors,can effectively relieve the immunosuppression of local tumor microenvironment,further strengthen endogenous Anti-tumor response It is necessary to adopt a “scientific combination” or “combination therapy” strategy in the future.