Experimental Research On Antitumor Effect Of T Cell After PD-1 Knocked Out

The immune system plays a critical role in cancer development.Under normal circumstances,the immune system can recognize,control,and even eliminate tumors.Cytotoxic T lymphocytes(CTLs,also known as CD8+T cells or killer T cells)are the primary immune cells responsible for killing tumor cells.T cells express programmed cell death protein 1(PD-1)on their surface.PD-1,when bound to its ligand PD-L1,generates inhibitory signals to repress T cell activity.PD-L1 is expressed by most types of cells;the PD-1/PD-L1 pathway plays a crucial role in the immune system to keep the self-tolerance and physiological immune responses in balance.Many tumor cells also express PD-L1;tumor cells can utilize the PD-1/PD-L1 pathway to attenuate T cell activity and thus escape the recognizing and killing capacity of CTLs.In this situation,the PD-1/PD-L1 checkpoint functions to interrupt cancer immunity cycle.Regulating effector T cell activity in response to tumor cells is one of the main functions of PD-1.Increased levels of PD-1 have been reported to be associated with exhausted or chronically stimulated T cells and poor survival in patients with cancer.PD-1/PD-L1 pathway blockade is an effective therapy for cancers and has gained remarkable progress in treating patients with lung cancer and advanced hematologic malignancies.In this study,we disrupted PD-1/PD-L1 pathway thought editing out PD-1(PD-1 KO)in CTLs with Clustered regularly interspaced short palindromic repeats(CRISPR)and CRISPR-associated protein 9(Cas9)system.We then investigated the effect of PD-1 KO on the anti-tumor activity of T cells.There are three parts of this experimental research.Part 1 Construction of crispr/cas9 vector transfected T cells to delete PD-1Objective:Construction of effective vector transfection T cell to deleted PD-1 by gene editing technique crispr/cas9.Methods:The non-target guided RNA sequence and three PD-1 guided RNA sequences were cloned into the vector lenticrispr V2 respectively.The293T cells were co-cultured with the vector to produce lentivirus.The lentivirus was co-cultured with T cells to transfect T cells to delete PD-1 gene to get PD-1 KO CTLs.Flow cytometry was used to detect the transfection efficiency of T cells,and the survival rate of T cells was detected by Western blot.The expression of PD-1 protein in CTLs cells was detected by Western blot.Results:1.The non-targeted guided RNA sequence and three PD-1 guided RNA sequences were successfully designed and cloned into vector lenticrisprV2.2.Lentivirus containing PD-1 guided RNA sequence was successfully obtained,and CTLs cells after PD-1 knockout were obtained after T lymphocyte transfection.The transfection efficiency was about 28.5%detected by flow cytometry.3.Western blot was used to detect the expression of PD-1 in CTLs cells after PD-1 knockout.The results showed that the total expression of PD-1 was significantly decreased,indicating that PD-1KO was effective in transduced CTLs.Part 2 The experiment on antitumor effect of PD-1 KO CTLs cells in vitroObjective:To observe the antitumor effect of PD-1 KO CTLs cells in vitro.Methods:PD-1 KO CTLs and CTLs(control group)were co-cultured with multiple myeloma(MM)cells respectively,the viability of MM.1S cells was detected.The specific cytotoxicity of two groups CTLs to MM.1S cells was detected by WST-1 method.The apoptosis of tumor cells was analyzed by AnnexinV/7-AAD staining of MM.1S cells and flow cytometry.Then MM.1S cell lysate was incubated with different caspase substrates to detect its caspase activity.Elisa assay was used to detect the cytokine secretion of TNF-αand IFN-γ.Results:1.Compared with the control group CTLs,the anti tumor cytotoxic effect of PD-1 KO CTLs was enhanced.2.Compared with the control group,PD-1 KO CTLs enhanced the apoptosis of tumor cells and enhanced the activities of caspase,caspase-3,caspase-8 and caspase-9,which were 8.21±0.47 and 4.92±0.35 times of those of the control group,respectively.3.TNF-αand IFN-γsecreted by PD-1 KO CTLs were 2.43±0.18 and1.92±0.21 times of those of control group,respectively.Part 3 The experiment on antitumor effect of PD-1 KO CTLs cells in vivoObjective:To investigate the antitumor effect of PD-1 KO CTLs cells to the MM graft mouse in vivo.Methods:To build humanized mouse models with the NOD/SCID mouse by the means of tail vein injection of human peripheral blood mononuclear cells(PBMC).Flow cytometry was used to detect the expression of human CD3~+T cells and human CD19~+B lymphocytes in peripheral blood of mice.Make MM grafted mouse models when they were humanized,and divided into two groups randomly.Two groups of mice received either human CTLs or PD-1 KO CTLs through tail vein four times.Tumor size was measured using a caliper every other day.The mean volume and SD of each group were calculated.Mice were killed when tumor volume reached 2000mm3.The overall survival was analyzed by the Kaplan–Meier method and calculated from the day of tumor cell injection to the day a mouse was found dead or killed.Results:1.The expression of human CD3~+T lymphocytes and CD19~+B lymphocytes was detected in peripheral blood of NOD/SCID mice after being humanized,and the proportion of expression was at normal adult level,indicating the success of humanization.2.The tumor became detectable after 2 weeks and the tumor growth in the PD-1 KO CTLs treated mice was dramatically repressed compared to that in the control group.3.All the mice in the control group died from progressive tumors by 52days.In contrast,only 40%of PD-1 KO CTLs treated mice died at the same time.Conclusion:1.The CRISPR-Cas9 system could efficiently knock out PD-1 in CTLs.2.PD-1 knock out enhances the cytotoxicity of CTLs to tumor cells,induces apoptosis of tumor cells and enhances the activity of caspase,which may promote tumor apoptosis by affecting the caspase protein-related pathway.The secretion of cytokines TNF-αand IFN-γwas significantly increased.3.Humanized mouse models with the NOD/SCID mouse by the means of tail vein injection of human peripheral blood mononuclear cells were built successfully.MM grafted mouse models could be made using the humanized NOD/SCID mice.PD-1KO CTLs cells exhibit good antitumor activity in vivo.PD-1 KO in CTLs effectively inhibited human MM cell growth in vivo and significantly improved overall survival of the xenografted mice.