Study On The Role And Regulation Mechanism Of Follicular Cytotoxic T Cells In Non-small Cell Lung Cancer

Early studies have found that Follicular cytotoxic T cells(Tfc)are a subset of T cells that play a key role in chronic viral infections.Tfc cells have cytotoxic activity.Which are like follicular helper T cells(Tfh),Tfc cells express CXCR5 and can respond to CXCL13 to locate B cell follicles.More and more evidences have shown that Tfc cells present proinflammatory functions and have therapeutic potential in a variety of inflammatory diseases.However,whether Tfc cells play a role in human cancer,and its possible regulatory mechanism,are not completely clear.In this research,the applicant studied the distribution of Tfc cells in the peripheral blood and tumor tissues of non-small cell lung cancer(NSCLC),and investigated their role in anti-tumor immune response.The applicant further explored the effects of regulatory B cells(Breg)of modulating Tfc.This study provides theoretical support for the future clinical application of Tfc cells in non-small cell lung cancer.Part I:Distribution of Tfc in patients with NSCLCObjective:The main purpose of this section was to define the phenotype of Tfc cells in patients with NSCLC,and to clarify the distribution of Tfc cells in peripheral blood and tumor tissues,which could lay the foundation for follow-up research.Methods:Flow cytometry was used to define the phenotype of Tfc cells in NSCLC patients,and to detect Tfc cells in peripheral blood and tumor tissues in order to determine their distribution in peripheral blood and tumor microenvironment.Results:The proportion of Tfc cells in total tumor infiltrating cells and tumor infiltrating CD8+T cells in NSCLC patients was higher than that in peripheral blood.Conclusion:Compared with peripheral blood,Tfc cells were enriched in tumor tissues,suggesting that Tfc cells may participate in the anti-tumor immune response in NSCLC.Part Ⅱ:Function of Tfc cells in patients with non-small cell lung cancerObjective:The main purpose of this section was to explore the role of Tfc cells in the tumor tissues of patients with non-small cell lung cancer,and to investigate the possible mechanism by which Tfc cells can participate in the anti-tumor immune response.Methods:Flow cytometry was used to detect the degranulation ability of Tfc cells in peripheral blood and tumor tissues.Meanwhile,multiple detection methods were used to examine the secretion ability of pro-inflammatory cytokines,granzyme B and perforin by Tfc in peripheral blood and tumor tissues.Results:In the tumor microenvironment and peripheral blood of patients with NSCLC,Tfc cells expressed higher level of CD 107a.At the same time,Tfc cells in the tumor microenvironment and peripheral blood secreted higher levels of pro-inflammatory cytokines.However,the secretion levels of granzyme B and perforin were significantly reduced.Conclusion:In patients with non-small cell lung cancer,Tfc cells in the tumor microenvironment and peripheral blood presented stronger degranulation ability and proinflammatory cytokine secretion,but the cytotoxic activity was significantly reduced.It also suggested that Tfc cells did not participate in the body’s anti-tumor immune response by directly killing cancer cells.Part Ⅲ:Effect of Breg cells on Tfc in patients with non-small cell lung cancerObjective:The main purpose of this section was to explore the regulation of Breg cells on Tfc cells in patients with non-small cell lung cancer,and to provide new ideas for the treatment of tumors.Methods:The phenotype of Breg cells in NSCLC patients was defined,and the correlation between Tfc and Breg cells in peripheral blood and tumor microenvironment was detected.At the same time,Tfc cells and Breg cells were isolated from patients and were co-cultured in vitro to detect the cytokine secretion level of Tfc cells.In addition,the applicant explored the role of interleukin 10(IL-10)in the regulation of Tfc by Breg cells.Results:In patients with non-small cell lung cancer,there was no correlation between the distribution of Breg cells and Tfc cells in peripheral blood mononuclear cells and tumor infiltrating lymphocytes.Co-cultivation with Breg cells in vitro did not affect the secretion of granzyme B and perforin by Tfc cells.But the secretion of pro-inflammatory cytokines was significantly inhibited.After the blocking by IL-10,the cytokine secretion level of Tfc cells was significantly reduced.Conclusion:Breg cells had no obvious effect on the infiltration of Tfc cells into tumor tissues and cytotoxic activity in NSCLC patients,but they could significantly inhibit the secretion of pro-inflammatory cytokines of Tfc cells.The inhibitory effect was IL-10 dependent.Part Ⅳ:Anti-tumor immune response of Tfc cells and its regulatory mechanismObjective:The main purpose of this section was to further confirm the role of Tfc in anti-tumor immunity and its regulatory mechanism in a mouse model.Methods:The LLC-OVA subcutaneously transplanted tumor model was established in mice,and then Tfc and CXCR5-CD8+T cells were transferred to observe the tumor growth.Flow cytometry was used to detect CD8+T cells infiltration in the tumor microenvironment.Then,Breg cells and CXCR5+Tfc cells were co-transfused into mice inoculated with LLC-OVA to observe the tumor growth.Finally,the Breg and Tfc cotransfused mice were blocked with IL-10 to observe the tumor growth.Results:Transfusion of Tfc cells could significantly inhibit the growth of mouse lung tumors,and could effectively promote the infiltration of CD8+T cells in tumor tissues.When Breg cells and Tfc cells were co-transfused,mouse lung tumor growth was not significantly inhibited,but,the blockage of IL-10 could partially inhibit the tumor growth.Conclusion:In the LLC mouse tumor model,Tfc cells could significantly inhibit tumor growth and had strong anti-tumor immunity.Breg cells could inhibit Tfc cells’macro-effects in anti-tumor immune responses,which was was IL-10 dependent.