Experimental Study Of SNX25 Regulating The Expression Of Exosomal PD-1 In CD8~+T Cells

Objective:To observe the expression level of inhibitory molecule PD-1(programmed death1,PD-1)on tumor-infiltrating CD8~+T cell,investigate the ability of SNX25(sorting nexin 25,SNX25)to sort PD-1 into exosomes in tumor-infiltrating CD8~+T cells and its role in anti-tumor immunity.Methods:(1)CD8~+T cells derived from wild type mouse spleen,tumor-bearing mouse spleen and tumor-bearing mouse tumor tissue were isolated by immune magnetic bead method and purified by flow cytometry.We use flow cytometry and western blot to detect the expression of PD-1 in CD8~+T cells derived from different sources.The tumor cell conditioned medium(TCCM)of mouse CT26 colon cancer cells was added to the culture wells of mouse spleen-derived CD8~+T cells to detect the change of PD-1 expression on the cell surface.(2)Inoculate the CD8~+T cells derived from wild-type mouse spleen(WT-sp-CD8),CD8~+T cells derived from tumor-bearing mouse spleen(TU-sp-CD8)and CD8~+T cells derived from tumor-bearing mouse tumor tissues(TIL-CD8)separated by immunomagnetic bead in the 24-well plates with 1?g/mL anti-mouse CD3 and CD28 antibodies.Incubate at 37?,5%CO2 for 24h,collect the culture supernatant,and extract exosomes from different CD8~+T cells.Transmission electron microscopy was used to observe the morphology of exosomes derived from CD8~+T cells,and nanoparticle tracking analyzer was used to detect the size distribution of exosomes.Western blot detected the expression of exosomes-related proteins and PD-1,and FCM detected the surface molecules of exosomes.Western blot and FCM detected the expression of PD-1 in exosomes derived from spleen CD8~+T cells of wild-type mice by TCCM treatment.(3)Western blot was used to detect the expression of SNX25 in CD8~+T cells from wild-type mouse spleen,tumor-bearing mouse spleen,and tumor-bearing mouse tumor tissue.Co-IP detects whether PD-1 and SNX25 bind to each other in CD8~+T cells,and further observes the changes in the binding of WT-sp-CD8,TU-sp-CD8 and TIL-CD8.Western blot was used to detect the changes in the expression of SNX25 in CD8~+T cells and the combination of PD-1 and SNX25 after TCCM treatment.(4)To investigate the role of SNX25 in exosomal PD-1 sorting,specific siRNA was used to knock down the expression of SNX25 in CD8~+T cells.Use flow cytometry o detect the expression of PD-1 on CD8~+T cells and Use western blot to detect the expression of PD-1 in CD8~+T cells derived exosomes.(5)To investigate the role of SNX25 in exosomal PD-1 sorting,lentivirus was used to overexpress SNX25.Use flow cytometry o detect the expression of PD-1 on CD8~+T cells and Use western blot to detect the expression of PD-1 in CD8~+T cells derived exosomes.(6)Use immunofluorescence technology to observe the localization of PD-1,SNX25 and early endosomes(EEA1),late endosomes(CD63),in CD8~+T cells to understand the sorting of SNX25 in exosomal PD-1 Possible role.Results:(1)The purity of CD8~+T cells derived from mouse spleen and tumor tissues sorted by immunomagnetic bead method is more than 90%.Compared with CD8~+T cells derived from spleens of wild-type mice and tumor-bearing mice,the expression of PD-1 on the surface of tumor-infiltrating CD8~+T cells was abnormally high(p<0.05).After TCCM treatment,the MFI value of PD-1 on the surface of CD8~+T cells derived from wild-type mouse spleen reached 23826±2153.6 on the second day,while the MFI value of PD-1 in the control group was 2731±226.27.(2)Under transmission electron microscopy,exosomes derived from CD8~+T cells have a round or elliptical saucer-like structure,and the diameter of exosomes is mainly distributed around 100 nm.Western blot analysis showed that it expressed CD63,Hsp70 and other characteristic molecules,but did not express Calnexin molecules.Flow cytometry showed that exosomes expressed CD8,CD25 and PD-1molecules.And we found that the content of PD-1 in exosomes derived from tumor-infiltrating CD8~+T cells was reduced compared with exosomes derived from CD8~+T cells in spleens of wild-type mice and tumor-bearing mice.After TCCM treatment,PD-1 in exosomes derived from CD8~+T cells of wild-type mouse spleen was significantly reduced.(3)Co-IP detection found that PD-1 and SNX25 in CD8~+T cells combined with each other.Compared with CD8~+T cells derived from the spleens of wild-type mice and tumor-bearing mice,the binding of PD-1 and SNX25 in CD8~+T cells derived from tumor tissue was reduced(p<0.05).After TCCM treatment,the binding of PD-1and SNX25 in CD8~+T cells of wild-type mouse spleen decreased(p<0.05).(4)In CD8~+T cells,PD-1 and SNX25 are colocalized with early endosomes(EEA1),late endosomes(CD63).(5)After knocking down SNX25 in CD8~+T cells of wild-type mouse spleens,the content of PD-1 in exosomes secreted by CD8~+T cells decreased,while the expression of PD-1 on the surface of CD8~+T cells increased.(6)After overexpressing SNX25 in CD8~+T cells of wild-type mouse spleens,the content of PD-1 in exosomes secreted by CD8~+T cells increased,while the expression of PD-1 on the surface of CD8~+T cells decreased.Conclusion:The reduction of PD-1 molecules sorted into exosomes by SNX25 in tumor infiltrating CD8~+T cells leads to the reduction the transmission of PD-1 molecules released by CD8~+T cells to the extracellular environment through the exosomes pathway,which increases the expression level of PD-1 on the surface of CD8~+T cells.