JAK-STAT Signaling Pathway Regulates IL-10 Expression And Function In CD8～+T Cells

ObjectiveIL-10 is a pleiotropic cytokine that plays an important role in antiviral and antitumor immunity.Efficacious CD8~+ T cells express IL-10 after stimulation with strong TCR signaling.When IL-10 acts directly on CD8~+ T cells,it promotes the killing effect of CD8~+ T cells.Up to now,the regulation of IL-10 expression in CD8~+ T cells and its signaling pathways that enhance CD8~+ T cell function are not clear.On the one hand,cytokine expression in T cells is regulated by the JAK-STAT signaling pathway;on the other hand,cytokines acting on T cells through their receptors also regulate downstream gene expression through the JAK-STAT signaling pathway.In this study,we used small molecule inhibitors and CRISPR-Cas9 gene editing technology combined with flow cytometry to explore the JAK-STAT signaling molecules that regulate IL-10 expression in CD8~+ T cells and the JAK-STAT signaling pathway through which IL-10 enhances CD8~+ T cell function via receptors.It provides new ideas and theoretical basis for tumor immunotherapy.Methods1.CD8~+ T cells were activated by adding respectively different JAK and STAT inhibitors,and IL-10 expression was analyzed by using flow cytometry.2.IL-10 reporter gene CD4Cre~+ CRISPR-Cas9 conditional knock-in mice breeding: T cell conditional expression Cas9 mice were obtained by crossing CD4 Cre mice with CRISPR-Cas9 conditional knock-in mice and then by using Tiger mice crossed with CD4Cre~+ CRISPR-Cas9 mice.3.sg RNA vector construction: design the corresponding sg RNA for the target gene exon,synthesize it and ligate it with the digested MSCV-sg RNAU6-PGK-BFP vector,transform,small extraction plasmid,sequencing,and select the correct clone for plasmid mid-extraction.4.Viral packaging and T cell transduction: The expression vector and packaging vector were transfected into 293 T cell-derived BOSC virus packaging cells using liposome lipo2000,and activated T cells derived from Cre~+Cas9+/+Tiger mice were infected with viral supernatant,and the effect of target gene knockout on IL-10 expression was detected by flow cytometry after48 h of culture.5.Addition of IL-10 to in vitro activated T cells,followed by expression analysis of effector molecules IFN-γ and Granzyme B by flow cytometry on in vitro activated T cells.Results1.We added different JAK and STAT inhibitors to activated CD8~+ T cells,and then examined the change of IL-10 expression,we found that JAK1 and STAT6 inhibitors could significantly reduce the expression of IL-10 in CD8~+ T cells.2.Gene and knockout of different JAKs using CRISPR-Cas9 technology revealed that JAK1 knockdown significantly reduced IL-10 expression in CD8~+T cells,which was also used to the knockout of potential STAT binding sites on the IL-10 promoter confirmed the STAT binding sites on the IL-10 promoter.3.Antibody blocking and CRISPR-Cas9 knockout of Tim3 both did not affect IL-10 expression in CD8~+ T cells.4.In vitro activated CD8~+ T cells were added with IL-10 in cell culture,and then different small molecule inhibitors of JAK and STAT were used to observe the changes in the expression of effector molecules IFN-γ and Granzyme B in CD8~+ T and found that exogenous IL-10 in CD8~+ T cells enhanced its effectivity mainly through JAK3 and SATA6.ConclusionsJAK1-STAT6 regulates IL-10 expression in CD8~+T cells,and STAT regulates IL-10 transcription by binding to multiple sites on the IL-10 promoter.Exogenous IL-10 enhances the effectivity of CD8~+T mainly through JAK3-STAT6.