The Study Of Dexs Overexpressing Tapasin Inducing CD8+TLC Autophagy To Promote T Cell Immune And Inhibit HBV Replication

Objective: To determine whether Dexs transfected with Tapasin lentivirus(TPN-Dexs)successfully expressed Tapasin;To explore the ability of TPN-Dexs to promote T lymphocytes activation and induce CTL immune response,and further verify its ability of antiviral effects in M-Tg HBV transgenic mice;To clarify whether TPN-Dexs promotes autophagy of CD8 + T lymphocytes,and detect changes in protein levels of signal pathway-related proteins in inducing CD8 + T lymphocytes autophagy,and clarify PI3 K / AKT / The mTOR pathway is involved in CD8 + T lymphocytes autophagy.Methods: 1.Replace the MCS gene fragment in H149:pLenti-EF1a-EGFP-P2A-Puro-CMV-MCS-3Flag lentiviral plasmid with Tapasin gene,and construct H8902 : pLenti-EF1a-EGFP-P2A-Puro-CMV-Tapasin-3Flag lentiviral plasmid was packaged as a lentiviral vector;Lentiviruses H149 and H8902 were transfected into DCs,respectively,and the expression of GFP was observed,and the secreted exosomes were collected by ultra high-speed differential centrifugation;Scanning electron microscopy,NTA,Western blotting,and exosome uptake experiments identified successful exosome extraction and high expression of Tapasin,that is,TPN-Dexs.2.In vitro verification of TPN-Dexs function: PBS,HBcAg,Dexs,TPN-Dexs,TPN-Dexs + Rap,TPN-Dexs + 3-MA were co-cultured with mouse spleen primary T lymphocytes,and CCK8 method was used to detect T lymphocytes proliferation ability,ELISA method was used to detect the secreted cytokines(IL-2,IFN-γ,IL-4,TNF-α and IL-10)levels,and LDH method was used to detect its ability to induce CTL immune response;The flow cytometry was used to sort mouse primary CD8 + T lymphocytes,cultured with PBS,Dexs,TPN-Dexs,TPN-Dexs + Rap.And autophagosomes were observed by transmission electron microscopy.The expression of autophagy related proteins LC3,p62 and Beclin-1 were detected by Western blotting.3.To verify TPN-Dexs’ function in vitro: To immunize M-Tg HBV transgenic mice with PBS,HBcAg,IFN-α,Dexs,TPN-Dexs,TPN-Dexs + Rap and TPN-Dexs + 3-MA through tail vein injection once a week.M-Tg HBV transgenic mice were sacrificed after immunization 4 times,and serum,liver and spleen were collected;Serum HBV DNA,ALT,AST levels were measured;HBcAg and HBsAg expression was detected by immunohistochemical staining;To extract spleen T lymphocytes,culture and test the ability of T lymphocytes to proliferate,secrete cytokines,and induce CTL immune response(the method is same as above);To sort CD8 + T lymphocytes to culture and detect autophagosomes and the expression of autophagy related proteins LC3,p62 and Beclin-1(the method is the same as above),and Western blotting and Real-Time PCR were used to detect the expression of AKT and mTOR protein under different autophagy conditions to verify the mechanism of autophagy.Results: 1.GFP fluorescent expression indicates successful lentivirus transfection;Exosomes were observed to be vesicle-like with electron microscopy,and the particle size was about 100 to 150 nm by NTA analysis.WB showed that H8902 successfully expressed Tapasin,while the control group did not express that.Exosomes all express Alix,HSP90 and CD81 to varying degrees.Confocal microscopy was used to observe the phenomenon of DCs engulfing PKH26-labeled exosomes,and gradually reached the nucleus over time,indicating that the extraction of TPN-Dexs was successful.2.The results of in vitro experiments found that T lymphocytes proliferation,secretion of cytokines IL-2,IFN-γ,IL-4,TNF-α and IL-10,and cytotoxic CTL responsess were enhanced in the TPN-Dexs group,and autophagy was induced.Increased autophagosome and increased expression of autophagy-related proteins LC3,p62,and Beclin-1;Compared with the TPN-Dexs group,in TPN-Dexs + Rap(autophagy inducer)group,T lymphocytes detection increased more,while in TPN-Dexs +3-MA(autophagy inhibitor)group,T lymphocytes detection decreased.These results show that in vitro TPN-Dexs can promote T lymphocytes proliferation,promote cytokines IL-2,IFN-γ,IL-4,TNF-α and IL-10 secretion,enhance the CTL responses,and induce CD8 + T lymphocytes to undergo autophagy.3.Results in M-Tg HBV transgenic mice,which is consistent with the results in vivo,indicating that TPN-Dexs can promote the proliferation of T lymphocytes and promote the cytokines IL-2,IFN-γ,IL-4,TNF-αand IL-10 secretion,enhance CTL responses,and induce CD8 + T lymphocytes to undergo autophagy;Liver immunohistochemical staining results show that TPN-Dexs can increase mouse serum ALT,AST levels and reduce liver HBsAg,HBcAg expression,while inhibiting HBV virus replication;From the AKT expression of the TPN-Dexs group and the TPN-Dexs + 3-MA group,it can be seen that TPN-Dexs can promote the upregulation of AKT expression,when the PI3 K inhibitor3-MA is added,AKT expression down-regulation,indicating that 3-MA inhibits the PI3 K / AKT pathway activated by TPN-Dexs;From the mTOR expression of the TPN-Dexs group and the TPN-Dexs + Rap group,it can be seen that TPN-Dexs can down-regulate mTOR expression,thereby promoting autophagy.It happened that mTOR expression was significantly down-regulated after adding the mTOR inhibitor Rap,indicating that TPN-Dexs and Rap synergistically down-regulate mTOR expression.These results indicate that the PI3 K / AKT / mTOR signaling pathway is involved in inducing autophagy of CD8 + T lymphocytes.Conclusions: TPN-Dexs can promote T lymphocytes proliferation in vivo and in vitro,promote the secretion of cytokines IL-2,IFN-γ,IL-4,TNF-α and IL-10,enhance CTL responses,and induce CD8 + T lymphocytes autophagy;PI3K / AKT / mTOR signaling pathway is involved in inducing CD8 + T lymphocytes autophagy in vivo;TPN-Dexs in M-Tg HBV transgenic mice can inhibit HBV virus replication,while increasing serum ALT and AST levels,and reduce liver HBsAg,HBcAg expression.