Study On The Effect And Mechanism Of IL-7 Promoting CD19-directed CAR-T Cells Proliferation

Objectives:Cancer is one of the main causes of human death worldwide.Cellular immunotherapy can regulate the function of specific immune cells and target cancer cells more specifically with fewer side effects.In recent years,CAR-T cell(Chimeric Antigen Receptor T cell)has been engineered and proven to be the most promising strategy for the treatment of cancer.In particular,CAR-T cells targeting CD 19 have achieved remarkable success in the treatment of hematological malignancies including leukemia and lymphoma.However,how to further optimize the proliferation efficiency of CAR-T cells in vitro and extend their persistence in vivo is still an urgent problem to be solved.Magnetic beads and aAPC are two commonly used methods to stimulate CAR-T cells.We intended to compare the proliferation efficiency and exhausted molecule expression of CAR-T cells with two methods to expand.Cytokine IL-7,as a key component of T cell activation,proliferation and cytotoxicity,has been shown to significantly enhance the anti-tumor activity of CAR-T cells.However,whether to promote the proliferation of CAR-T cells and its specific mechanism remain to be verified and elucidated.miRNAs play a major role in T cell selection and increase survival rate,and participate in T cell activation and immune response.However,whether miRNA is involved in the activation of CAR-T cells by IL-7 has not yet been reported.Based on our previous research and literature research,we propose the following scientific questions:1)Do magnetic beads and aAPC stimulated anti-CD 19 CAR-T cells have the same proliferation efficiency,cell phenotype,memory fUNction,and exhaustion?2)Can IL-7 promote the proliferation of anti-CD 19 CAR-T cells?3)Look for key miRNAs involved in regulation of IL-7-stiMulated anti-CD 19 CAR-T cells and their specific regulatory mechanisms.In order to verify and clarify the above scientific questions,this study intended to prepare third-generation CAR-T cells constructed by transposon system electrotransduction and stimulated with two expansion methods(magnetic beads and aAPC)from 5-6 patients’ peripheral blood leukocytes,and compare the efficiency,memory phenotype and expression of exhaustion molecules of two CAR-T cells.Based on IL-7 cytokines enhancing T cell survival and steady-state proliferation,whole transcriptome sequencing is used to find key miRNAs and gene that regulate IL-7-stimulated anti-CD 19 CAR-T cells activation.It further elaborated that IL-7 induced the decrease of key gene CDKN1A by increasing the expression of miRNA-98-5p,thereby affecting the expression levels of downstream proteins CDKs,/Cyclins,and ultimately promoting the survival and steady-state proliferation of CAR-T cells.The results would greatly optimize the culture of anti-CD 19 CAR-T cells constructed by piggyBac electrotransformation,and explain the function and activity regulation molecular mechanism of CAR-T cells,so as to provide them with better clinical application basis and technical support.Methods:The first section:using electroporation transfection technology to transfer the constructed CD 19 PiggyBac transposon and transposase plasmid into T cells for modification and transformation,and then CAR-T cells was cocultured with artificial antigen-presenting cells(aAPC,artifiGialantigen-presenting cells))CD19 CD137L CD86 CD64 mIL-15-K562 or anti-CD3/CD28 magnetic beads according to a certain ratio,and the corresponding cytokines were added.The growth and proliferation of CAR-T cells in the two groups were observed under the microscope.qPCR and agarose gel experiments verified the residue of aAPC in the cocultured CAR-T cells,and flow cytometry observed the GFP expression of aAPC.In the process of stimulation,flow cytometry was used to detect the expression of CD3,CD4,CD8 and the myc expression(CAR expression marker),memory marker,exhaustion marker of CAR-T cells.The luciferase tumor killing experiment was used to detect the killing toxicity of CAR-T cells,and the Elisa experiment was used to verify the cytokines secreted by CAR-T cells stimulated with the two methods.The electron microscope observed process of CAR-T cells killing Raji tumor cells,and Western blot(WB)was used to verify the mechanism of CAR-T cells killing tumor cells.The second section:using electroporation transfection technology to transfer the constructed CD 19 PiggyBac transposon and transposase plasmid into T cells for modification and transformation,CAR-T cells was stimulated with anti-CD3/CD28 magnetic beads according to a certain ratio,and divide into two groups and added different cytokines(IL-7+IL-2+IL-21 VS IL-2+IL-21).The growth and proliferation of CAR-T cells in the two groups were observed under the microscope.In the process of stimulation,flow cytometry was used to detect the expression of CD3,CD4,CD8 and the myc expression(CAR expression marker),memory marker,exhaustion marker of CAR-T cells.The luciferase tumor killing experiment was used to detect the tumor killing toxicity of CAR-T cells,and the Elisa experiment was used to verify the cytokines secreted by CAR-T cells stimulated with the two methods.NAMALWA xenograft mouse model was constructed through tail vein injection of NAMALWA tumor cells,and 2×107 CAR-T cells and control T cells were again injected into the tail vein.The third section:Using the same method in the second part to stimulate two groups of CAR-T cells.Extracting the RNA of two groups of CAR-T cells,using whole transcriptome sequencing technology to sequence and bioinformatics technology to find different mRNA and miRNA.Expanding the number of sample to verify the expression of mRNA and miRNA used qPCR experiment and screen out significant highly expressed target mRNA and miRNA.Using GO and KEGG enrichment analysis to screen out the proliferation-related gene CDKN1A and WB experiment to detect expression of downstream proliferation-related genes:CDK2,CDK4,CyclinDl,CyclinE1.EdU experiment and flow cytometry to detect cell proliferation and cell cycle.TargetScan and miRanda software were used to predict CDKN1A related miRNAs,and then transfected the mimics and inhibitors of related miRNAs into CAR-T cells.WB experiment to detect again expression of downstream proliferation-related genes:C-myc,CDK2,CDK4,CyclinD1,CyclinE1,and EdU and flow cytometry to detect cell proliferation and cell cycle.Results:The first section:Two group of CAR-T cells were successfully obtained by two methods of aAPC or anti-CD3/CD28 magnetic beads.The growth state and proliferation multiple of CAR-T cells stimulated by aAPC were better and higher than those amplified by magnetic beads.qPCR and agarose gel experiments verified that aAPC did not have residues in cocultured CAR-T cells,and flow cytometry observed no GFP expression in aAPC.Flow cytometry detected that CAR-T cells stimulated by aAPC could redistribute the ratio of CD4+CAR/CD8+CAR-T cells,and CD45RO+CAR-T memory cell subsets expressed more.qRT-PCR verified that CAR-T stimulated by aAPC the expression lower of CTLA-4.The tumor killing experiment in vitro detected that the CAR-T cells stimulated by magnetic beads were more tumoricidal activity.The Elisa experiment verified that the cytokines secreted by the two groups of CAR-T cells were not significantly different,WB verified that CAR-T cells significantly increased the expression of STAT1,cleavage-caspase 3 and Bax,while the expression of caspase 3 dropped to an undetectable level.The second section:CAR-T cells were stimulated and expanded by magnetic beads,two groups of CAR-T cells were successfully obtained by adding two sets of different cytokine combinations.The growth status and proliferation of CAR-T cells stimulated by IL-7 were better and higher than control group.Flow cytometry detected that CAR-T cells stimulated by IL-7 can change the ratio of CD4+CAR/CD8+CAR-T cells,expression of memory cell subsets and the exhausted molecules were not significantly different comparing with control group.In vitro tumor killing experiments showed that CAR-T cells without IL-7 stimulation were more tumoricidal activity.Elisa experiments verified that there was no significant difference in the cytokines secreted by CAR-T cells between the two groups.NAMALWA tumor xenograft tumor model proved that the CAR-T cells stimulated by IL-7 showed significant anti-tumor effects in the follow-up in vivo experiments.The third section:Through whole transcriptome sequencing and bioinformatics analysis,the results showed that 52 mRNAs and 8 miRNAs had significant differences.we found that CDKN1A,FAM110A,GPC1,KLHL29,and TSN had significant differences.KLHL29 was up-regulated in IL-7-CAR-T group,and CDKN1A,FAM110A,GPC1,and TSN were down-regulated in IL-7-CAR-T group by qPCR verification.CDKN1A may affected proliferation of CAR-T cells by WB detection.At the same time,bioinformatics analysis and qPCR verification found that has-miR-320a-5p and has-miR-1973 were significantly different,which were down-regulated in IL-7-CAR-T group,but has-miR-98-5p were up-regulated in IL-7-CAR-T group.miR-98-5p mimics and inhibiter could both change the expression level of CDKN1A and downstream related proliferation proteins CDK2,CDK4,CyclinD1,CyclinE1.Flow cytometry found that IL-7 affected the DNA content and proportion in the S phase of the cell cycle.It was also found that IL-7 also affected the proliferation of T cells.We speculated that IL-7 might down-regulate the expression of CDKN1A through miRNA-98-5p,thereby affecting the proliferation of CAR-T cells.Conclusions:1.Compared with beads-CAR-T cells,aAPCs could significantly enhance CAR-T cell proliferation,and had higher expression of memory phenotype and lower expression of exhausted molecules.CAR-T cells stimulated by aAPCs could exert tumor-killing activity in vitro by activating the cytokine and other apoptosis-related pathways.It showed significant anti-tumor effects in NAMALWA xenograft mouse model.2.IL-7 could promote the proliferation of CAR-T cells,and there was no significant difference compared with the expansion fold of aAPCs-CAR-T cells,and could change the ratio of CD4+CAR/CD8+CAR-T cells,and showed significant anti-tumor effects both in vivo and in vitro.3.Through whole transcriptome sequencing and verification,it was found that IL-7 affected the proliferation of CAR-T cells by miRNA-98-5p mediating down-regulate the expression of CDKN1A proliferation genes.