Abstract:

Objective To discuss the relationship between CXC motif chemokine receptor 6 （CXCR6） expression in tumor tissue and the expression of immune checkpoint molecules as well as the infiltration abundance of CD4? T lymphocytes， and to clarify its impact in the function of regulatory T lymphocytes （Tregs） and underlying mechanism. Methods Bioinformatics analysis was performed using Tumor Immune Estimation Resource （TIMER） database. Transcriptomic and immune infiltration data of 10 solid tumors were collected. Pearson correlation analysis was used to analyze the correlations of CXCR6 with immune checkpoint molecules and the infiltration abundance of CD4? T lymphocytes. In the experiments， the human peripheral blood mononuclear cells （PBMCs） from healthy individuals were used. The CD4? T lymphocytes were isolated and expanded， and then induced into the Treg cells. Recombinant lentiviral vectors of CXCR6 shRNA1-4# were constructed. The experiment was divided into blank group （without Treg）， empty vector Treg group（control group）， and CXCR6 knockdown group. After lentiviral transfection， the Treg cells were co-cultured with CD8? cytotoxic T lymphocytes （CTLs） and T2 tumor target cells. Flow cytometry was used to detect the purity of Treg cells； fluorometric counting method was used to detect the lysis rate of target cells； enzyme-linked immunosorbent assay （ELISA） was used to detect the levels of cytokines in the cells； RNA sequencing was used to detect the expression levels of differential genes in the cells； real-time fluorescence quantitative PCR（RT-qPCR） method was used to detect the knockdown efficiency of CXCR6 and the expression level of peptidase inhibitor 16（PI16） mRNA. Results The bioinformatics analysis results showed that CXCR6 was highly positively correlated with immune checkpoint molecules and the infiltration abundance of CD4? T lymphocytes in 10 solid tumors （P<0.01）， and was negatively correlated with tumor purity （except for glioblastoma）（P<0.01）. The flow cytometry results showed that high-purity CD4? T lymphocytes （90.13%±3.7%） and CD25?Foxp3? Treg cells （69.6%±3.4%） were successfully obtained. The RT-qPCR results showed that CXCR6-KD3# had the highest knockdown efficiency （P<0.05）. The fluorometric counting method results showed that compared with control group， the lysis rate of target cells in CXCR6 knockdown group was increased （P<0.01）. The ELISA results showed that compared with empty control group， the levels of interferon-γ （IFN-γ） and tumor necrosis factor-α（TNF-α） in CXCR6 knockdown group were increased （P<0.01）. The RT-qPCR results showed that compared with control group， the expression level of PI16 mRNA in Treg cells in CXCR6 knockdown group was increased （P<0.01）. The RNA sequencing results showed that the differential genes were enriched in the extracellular matrix space， regulation of cell migration， and phosphatidylinositol 3-kinase（PI3K）/protein kinase B（Akt） pathway. Conclusion CXCR6 is highly correlated with the expression of immune checkpoint molecules and CD4? T lymphocyte infiltration in 10 solid tumors， and is mainly localized in the tumor immune microenvironment. Knockdown of CXCR6 expression in Treg cells can attenuate their immunosuppressive function and promote the secretion of related cytokines by CTLs， and the mechanism may be related to the upregulation of PI16 mRNA expression level in the Treg cells.

Key words: CXC Motif chemokine receptor 6, Immune checkpoint, Regulatory T lymphocyte, Cytotoxic T lymphocyte, Immune escape, Tumor microenvironment, Immunotherapy target