Inhibition And Mechanism Of T Cell Activation By Exosomal MiRNA-A And MiRNA-B Derived From Lung Cancer

Background:Exosomes are important mediators of intercellular communication.One of their components,exosomal mi RNAs,can affect functions of their recipient cells by inhibition of protein translation.In the tumor microenvironment,tumor-derived exosomal mi RNAs have been found to be able to modulate anti-tumor functions of immune cells,resulting in immune escape of tumor cells.In the previous study,our team identified several exosomal mi RNAs including 2 novel mi RNAs（for the convenience,tentatively named as mi RNA-A and mi RNA-B）in the plasma of lung cancer patients.In the present study,we focused on the inhibitory effect of mi RNA-A and mi RNA-B on the T cell activation and the associated mechanisms.Methods:1)Exosomes in the culture medium of H1975,a human lung adenocarcinoma cell line,were extracted by the PEG method and characterized.2)The isolated exosomes were then added to the PHA-L-activated Jurkat T cells in the presence or absence of mi RNA-A-or mi RNA-B-specific inhibitors to observe whether the exosomes have inhibitory effect on T cell activation and whether the inhibition is associated with exosomal mi RNA-A and mi RNA-B.3)Jurkat-T cells were transfected with mi RNA-A and mi RNA-B at different doses,and then collected at different time points;Total RNA was extracted to detect the expression of signature molecules of activated T cells（IL-2,IFN-g,CD40 L and TNF-a）,so as to determine the optimal action concentration and time.4)Under the optimal transfection conditions（200 pmol,72 hours）,the signature molecules of T cell activation were detected by q PCR and ELISA,and proportion of the activated T cells was detected by Flow cytometry and Immunofluorescence.5)The target genes of mi RNA-A and mi RNA-B were analyzed by mi Randa,the common target genes for both mi RNA-A and mi RNA-B were chosen,and based on q PCR and Western Blot analyses two common target genes（TRIOBP and NFATC1）were chosen for further study.6)The expression of TRIOBP and NFATC1 in the T cells was knocked down by specific si RNA and the expression of the signature molecules of activated T cell and the proportion of activated T cells were detected to observe whether transfection of mi RNA-A and mi RNA-B and knockdown of TRIOBP and NFATC1 have similar inhibitory effects on T cell activation.7)High-throughput transcriptome sequencing in the TRIOBP-and NFATC1-knocked down Jurkat T cells was performed to explore the underlying molecular mechanisms.8)Single-cell suspensions were prepared from freshly resected human lung adenocarcinoma specimens with gentle MACS? Dissociator and immune cells were then isolated using anti-CD45 beads.Expression of mi RNA-A and mi RNA-B in the cancer tissues,proportion of activated T cells and expression of TRIOBP-and NFATC1 in the T cells were detected by q PCR,flow cytometry and immunofluorescence,respectively.Result:1)Exosomes derived from H1975 cells significantly inhibited T cell activation and pretreatment with the mi RNA-A-or mi RNA-B-specific inhibitor alleviated the inhibitory effect;2)Transfection of T cells with mi RNA-A or mi RNA-B expression decreased expression of the signature molecules for T cell activation（IL-2,IFN-g,CD40 L and TNF-a）as detected by q PCR and ELISA,and also reduced the proportion of activated T cells by flow cytometry and immunofluorescence.3)Among the 32 common target genes for both mi RNA-A and mi RNA-B which were chosen by the mi Randa software,decreased expression of TRIOBP and NFATC1 was induced by transfection of mi RNA-A or mi RNA-B as detected by q PCR and Western Blot.4)Similar to the inhibitory effect of mi RNA-A and mi RNA-B,knockdown of TRIOBP and NFATC1 by the gene-specific si RNA led to reduced expression of the signature molecules for T cell activation and decreased proportion of activated T cells,indicating that mi RNA-A and mi RNA-B commonly act on TRIOBP and NFATC1 to inhibit T cell activation.5)For further analysis of the underlying mechanisms,Jurkat-T cells were treated with TRIOBP-or NFATC1-specific si RNA and then subjected to high throughput transcriptome sequencing.71 genes were up-regulated and 33 genes down-regulated in both TRIOBP-knocked down and NFATC1-knocked down T cells.Pathway enrichment analysis by KEGG indicated that several signaling pathways including Th1 and Th2 differentiation were altered.that expression of IL-4R was increased,and the expression of p-AKT in IL-4R-related AKT pathway was decreased.Conclusion:Lung cancer-derived exosomal mi RNA-A and mi RNA-B inhibit T-cell activation via acting on the target genes TRIOBP and NFATC1,and the inhibitory effect is partially associated with the AKT signaling pathway.