| Objective CAR-T cell therapy is an adoptive cell therapy that genetically modifies patients’ T cells to endow them the ability to target and kill tumor cells.The research objectives of this project include: To optimize the culture system of primary T cells and lentivirus infection of primary T cells;To prepare novel Nb CAR-T cells that targeting CD19 and CD20 and sh RNA-mediated CAR-T cells with low expression of Blimp-1;To determine the anti-tumor activity of Nb CAR T cells and spermidine pretreated Nb CAR-T cells in vitro and in vivo;To determine the cytotoxicity of sh Blimp-1 CAR-T cells to B-lymphoma cell in vitro and in vivo and mechanism.Methods 1.Optimal the in vitro amplification system of primary T cells and infection conditions of primary T cells by lentivirus: Combine cytokines that affecting T cell proliferation and activation,then flow cytometry,enzyme-linked immunoassay,cell absolute count and cell morphology methods were used to explore the optimal system of T cell expansion culture in vitro;Combine Retronectin?,polybrene,anti-human CD3/CD28 monoclonal antibody,and IL-2,using fluorescence microscope and flow cytometry to explore the optimum condition for lentivirus infection of T cells;2.Prepare Nb CAR T cells and investigate their biological characteristics: CAR sequences targeting CD19,CD20 and targeting double were designed to construct Nb CAR overexpressed lentivirus vector.Lentivirus was packaged by 293 T cells,and then Nb CAR-T cells were prepared by infected T cells after concentration and purification of Nb CAR lentivirus;Flow cytometry were used to detect the positive rate of Nb CAR-T cells,CD8/CD4 ratio and activation level;The Nb CAR-T proliferation weas determined by Cell Trace fluorescent dye method and Cell absolute counting method;The secretion of IL-2 and IFN-γ under Daudi and K562 cell stimulation was analyzed by ELISA;3.Investigate the in vitro and in vivo antitumor activity of Nb CAR-T cells and spermidine pretreated CAR-T cells: Using the flow cytometry and enzyme-linked immunoassay to determine the working concentration of spermidine based on T cell proliferation,apoptosis resistance,and cytokine secretion level;Tumor cell lines with stable expression of luciferase were constructed,the lysis ability of Nb CAR-T cells on blood tumor cells was detected by luciferase assay or LDH assay,and the lysis ability of Nb CAR-T cells on primary tumor cells of ALL patients was detected by LDH assay;A mouse hematological tumor model was established,the cytotoxicity of Nb CAR-T cells and spermidine-pretreated Nb CAR-T cells in vivo was monitored by in vivo imaging;The effect of Nb CAR-T cells and spermidine pretreatment on B-lymphoma cells in vitro and in vivo was investigated;4.Analyze The correlation between Blimp-1 and T cell exhaustion: TIMER2.0 and TISIDB online software were used to analyze the expression of Blimp-1 in tumors,prognosis of patients and the correlation with TOX,NR4 A,NFAT,PD-1,CTLA-4,TIM-3,LAG-3,Bcl-2,Bcl-6,EOMES,T-bet that involved in T cell exhaustion,so as to clarify the effect of Blimp-1 on T cell effector function in tumor immunity;5.Prepare sh Blimp-1 CAR-T cells and detect it’s in vitro and in vivo cytotoxicity: sh RNA gene editing technology was used to down-regulate the expression of Blimp-1 in K562 cells and T cells,and the optimal sh RNA sequence targeting Blimp-1 was determined by RT-q PCR and Western Blot;Blimp-1 inhibitory lentivirus vector was constructed,lentivirus was packaged,and T cells were infected after concentration and purification of sh CAR lentivirus to prepare sh RNA-mediated Blimp-1 inhibitory CAR-T cells;CAR-T cells exhaustion level after Blimp-1 down-regulation was detected by flow cytometry,RT-q PCR,ELISA and LDH.Include proliferation potential,cytokine release level,ratio of memory phenotype and in vitro cytotoxicity of CAR-T cells.The in vivo circulation of sh Blimp-1 CAR-T cells was studied by detecting the proportion of CAR-T cells and tumor cells in peripheral blood of mice.The in vivo anti-tumor ability of sh Blimp-1 was determined by monitoring tumor load,survival time and body weight of mice;6.Elaborate the underlying biological mechanism of sh Blimp-1 CAR-T: RNA-seq data were used to analyze the transcriptional profiling of sh Blimp-1 CAR-T cells,and bioinformatics analysis methods such as GO,KEGG and GSEA were used to study the effect of Blimp-1 down-regulation on CAR-T cell signaling pathway,energy uptake.Therefor to explore the potential anti-tumor mechanism of sh Blimp-1 CAR-T cells.Results 1.T cells isolated and purified from human peripheral blood collected by heparin anticoagulant tube could be expanded 90-100 times within one week under anti-human CD3/CD28(1μg/m L)stimulation for 48 hours,supplement with autologous plasma or FBS(10%),IL-2(200U/m L)culture system.The optimal efficiency of lentivirus infection was obtained in the presence of Retronectin?(20μg/m L),polybrene(8μg/m L),anti-human CD3/CD28(5μg/m L)and IL-2(200U/m L)combined with physical centrifugation;2.The constructed CD19 Nb CAR-T cells,CD20 Nb CAR-T cells and Bispecific Nb CAR-T cells showed certain biological activity,killing 60% of primary tumor cells and cell line tumor cells.Meanwhile,the anti-tumor function of CD19 CAR-T and Nb CAR-T cells pretreated with spermidine(5μM)was improved in vivo and in vitro,and the cell lysis capacity was increased to more than 80%.The survival time of hematological tumor mice was significantly prolonged and Bispecific Nb CAR-T cells were superior to both CD19 Nb CAR-T cells and CD20 Nb CAR-T cells in terms of tumor killing ability.3.Blimp-1 is highly expressed in tumor-infiltrating T cells and is involved in regulating T cell depletion;Down-regulation of Blimp-1,TCM population of CAR-T cells increased from 40% to more than 60%,and the resistance to apoptosis was increased,promoted CAR-T cell antigen dependent proliferation and cytotoxicity,and increased IL-2 and IFN-γ secretion levels.In addition,the in vivo circulation time was increased,thus prolongating the survival time of hematological tumor mice.Mechanistically,Blimp-1 may regulate CAR-T cell exhaustion by inhibiting anti-apoptotic genes such as Bcl-2 and Bcl-6,cell memory genes such as 4-1BB and TCF7,and promoting immune checkpoints such as PD-1 and TIGIT,and meanwhile may by regulating lymphocyte proliferation,cytokine-cytokine receptor signaling pathways.Conclusion In this study,the preparation process of CAR-T cells was optimized to obtain the best system for in vitro culture and lentivirus infection,laying a foundation for further research on the biological function of CAR-T cells;The prepared CAR-T cells with novel nanobodies targeting CD19 and CD20 showed high lysis effect on primary tumor cells of ALL patients and B lymphoma cells,indicating the feasibility of using nanobody in CAR-T cell technology;Spermidine preconditioning can enhance the antitumor effect of CAR-T cells in vitro and in vivo,which can be a simple and convenient measure to enhance their activity;Blimp-1 inhibitory CAR-T cells can enhance the in vivo and in vitro clearance capacity that against B lymphoma cells by restoring the exhaustion state of CAR-T cells. |
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