The Mechanism For APKC-ι Regulating The Changes Of CD4~+CD25~-FOXP3~+ T Cells And Immunosuppression In Cholangiocarcinoma

Objective:The immunosuppressive microenvironment provides a favorable condition for CCA metastasis,which is closely associated with a poor prognosis.Based on the previous foundation,this study was conducted to explore the mechanisms involved in the induction of immunosuppressive CD4~+CD25~-FOXP3~+T cells infiltration by a PKC-ιin CCA.Methods:Single-cell sequencing data was analyzed to characterize the CD4~+CD25~-FOXP3~+T cell population in CCA tissues,the results of which were validated by multiplex immunohistochemistry and flow cytometry.The immunosuppressive function of CD4~+CD25~-FOXP3~+T cells induced by overexpressing-a PKC-ιin CCA cells was measured by CFSE dilution assay.The in vitro assays were performed to determine the effects of a PKC-ιon regulating the HIF-1α/TGF-β1 signaling pathway in CCA cells.The role of HIF-1α/TGF-β1 in a PKC-ι-induced changes in the number of CD4~+CD25~-FOXP3~+T cells in CCA were examined by using flow cytometry assay.Clinical specimens were collected to verify the association between the expression of a PKC-ι,HIF-1αand TGF-β1and the correlation with CCA patients’prognosis.IP assay,protein degradation assay,and the ubiquitination level assay were performed to investigate the specific regulatory mechanism of a PKC-ιaffecting HIF-1αexpression.Furthermore,the role of this mechanism on the a PKC-ι/HIF-1α/TGF-β1 signaling axis and the changes in CD4~+CD25~-FOXP3~+T cell numbers was verified with in vitro and in vivo assays.Results:The percentage of CD4~+CD25~-FOXP3~+T cells was increased in CCA tissues.It showed high expression of CTLA4,BATF,and TNFRSF4/9/18 genes,which was similar to the regulatory T cells in fields of the classification and functional characterization.The CD4~+CD25~-FOXP3~+T cells induced by a PKC-ι-overexpressing CCA cells suppressed the proliferation of naive CD4~+T cells.a PKC-ιpromoted HIF-1αprotein accumulation via the deubiquitination pathway and activated the HIF-1 signaling pathway,which resulted in TGF-β1 upregulation and the increase in CD4~+CD25~-FOXP3~+T cells,and ultimately contributed to tumor immunosuppression.Conclusions:(1)Compared to the para-tumor,the percentage of CD4~+CD25~-FOXP3~+T cells showed a significant increase in CCA tissues,and the expression of immunosuppression-related genes was upregulated.(2)CD4~+CD25~-FOXP3~+T cells induced by a PKC-ι-overexpressing CCA cells exhibited immunosuppressive functions.(3)a PKC-ιregulated the HIF-1α/TGF-β1 signaling pathway via the de-ubiquitination pathway in CCA cells,which further promoted the number of CD4~+CD25~-FOXP3~+T cells,and finally contributed to tumor immunosuppression.(4)The expression of a PKC-ι,HIF-1αand TGF-β1 was upregulated and positively correlated with poor prognosis in CCA tissues.