Studies On The Effects Of Anti-CD 19 Chimeric Antigen Receptor T Cells In Vitro And The Molecular Mechanisms

ObjectiveWith the development of gene recombination technologies,adoptive cellular immunotherapy is considered to be the most promising treatment in the new century,in which chimeric antigen receptor(CAR)is expected to be more because of its advantages of eliminating the limitation of MHC.Thus the establishment of a safe,effective and stable anti-CD 19 CAR-T cell culture system for CD 19 positive hematologic tumor is of great significance.In order to provide experimental basis for the clinical research and the application of CAR-T technology in hematological malignancies,the targeting activities and quality control of CAR-T cells were evaluated in in vitro study.MethodsPiggyBac,a transposon/transposase system was used to transfer the CAR genes into peripheral blood mononuclear cells.Then,CAR-T cells were selectively propagated by artificial antigen presenting cells.After 18 days,the positive rate of CAR and the memory phenotype of CD44+CD62L+ were analyzed by flow cytometry.Meanwhile,the cytotoxicities of CAR-T on CD 19 expressing cell lines(NAMALWA,Raji,K562CD19+)were evaluated.The molecular mechanisms involved in above effects were explored by ELISA and Western Blot.The quality control of CAR-T cells were evaluated in in vitro study by detecting the residual of aAPC,bacteria,mycoplasma and endotoxinResults1.The anti-CD 19 CAR-T cells were stimulated by antigen presenting cells ex vivo and successfully expressed CAR and central memory phenotype.CD86/CD64/CD137L/mIL-15/CD19 aAPC could stably amplify anti-CD19 CAR-T cells.The average positive rate of CD3+CAR was 62.07±10.72%.The average positive rate of CD44+CD62L+memory phenotype was 48.32±10.01%.The ratio of CD4+CAR-T cells to CD8+CAR-T cells is about 1:1.2.Anti-CD19 CAR-T cells possessed selectively targeted anti-tumor effects on lymphoma cells.Anti-CD 19 CAR-T cells could target and kill CD 19 positive tumor cells,and the tumoricidal effects increased with the increase of the effective target ratio(2:1,4:1,8:1,16:1).It was found that CAR-T cells could kill tumor cells by releasing IFN-?,IL-2,IL-10,TNF-?,perforin and granzyme B.Under electron microscope,CAR-T cells enclosed Raji cells and perforated on the cell surface of tumor cell,and finally made tumor cells gradually cracked.Moreover,the CAR-T treatment significantly increased the expressions of cleaved-caspase-3,Bax and STAT1,meanwhile decreased the expression of caspase-3.Therefore,anti CD 19 CAR-T cells could kill tumor cells by activating IFN-?/STAT1 signaling pathway and caspase-3 apoptosis signaling pathway mediated by granzyme B.3.Quality control of anti-CD 19 CAR-T cells.DNA gel electrophoresis demonstrated that the PCR product from CAR-T cells with aAPC had no LTR sequence band,which was specific to lentivirus,indicating no residue of aAPC.In addition,the bacteria,mycoplasma and endotoxin in CAR-T cell suspension were negative.ConclusionThe aAPC culture system established by our research group could sucessfully amplify anti-CD 19 CAR-T cells which were modified genetically by piggyBac transposon/transposase system.The anti-CD 19 CAR-T cells could selevtively killed CD 19-positive hematologic malignant tumor cells inculding NAMALWA,Raji and K562CD19+cells in vitro by activating the IFN-?/STAT1 signal pathway and caspase-3 apoptosis signal pathway mediated by granzyme B.This study provided a preclinical experimental basis for anti-CD 19 CAR-T cell immunotherapy.