In vitro functional validation of anti-CD19 chimeric antigen receptor T cells expressing lysine-specific demethylase 1 short hairpin RNA for the treatment of diffuse large B cell lymphoma
BackgroundChimeric antigen receptor T (CAR-T) cell therapy is more effective in relapsed or refractory diffuse large B cell lymphoma (DLBCL) than other therapies, but a high proportion of patients relapse after CAR-T cell therapy owing to antigen escape, limited persistence of CAR-T cells, and immun...
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Frontiers Media S.A.
2025-01-01
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fimmu.2024.1521778/full |
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| author | Zhi Guo Zhi Guo Mingxin He Ning Liu Yiqing Yang Rui Sun Jianxun Wang Qiang Wang |
| author_facet | Zhi Guo Zhi Guo Mingxin He Ning Liu Yiqing Yang Rui Sun Jianxun Wang Qiang Wang |
| author_sort | Zhi Guo |
| collection | DOAJ |
| description | BackgroundChimeric antigen receptor T (CAR-T) cell therapy is more effective in relapsed or refractory diffuse large B cell lymphoma (DLBCL) than other therapies, but a high proportion of patients relapse after CAR-T cell therapy owing to antigen escape, limited persistence of CAR-T cells, and immunosuppression in the tumor microenvironment. CAR-T cell exhaustion is a major cause of relapse. Epigenetic modifications can regulate T cell activation, maturation and depletion; they can be applied to reduce T cell depletion, improve infiltration, and promote memory phenotype formation to reduce relapse after CAR-T cell therapy.PurposeWe propose to develop and validate in vitro the function of novel CAR-T cells for the treatment of DLBCL, which simultaneously express an anti-CD19 CAR with lysine-specific demethylase 1 (LSD1) short hairpin (sh)RNA to prevent depletion and prolong the survival of CAR-T cells.MethodsWe designed an shRNA sequence targeting LSD1 mRNA, and created a vector with the following elements: the U6 promoter driving expression of the LSD1 shRNA sequence, the EF1a promoter driving a second-generation anti-CD19 CAR sequence encoding an anti-CD19 single-chain variable fragment (FMC63), the CD8 hinge and transmembrane structural domains, the CD28 co-stimulatory structural domain, and the CD3ζ-activating structural domain. The MFG-LSD1 shRNA anti-CD19 CAR plasmid was first constructed, then packaged in retroviral vectors and transduced into human primary peripheral blood mononuclear cell-derived T cells to generate the corresponding CAR-T cells. We examined by flow cytometry the efficiency of two CAR-T cells in killing U-2932 cells (a human DLBCL line) upon co-culture with RNAU6 anti-CD19 CAR-T cells or LSD1 shRNA anti-CD19 CAR-T cells. We analyzed Ki-67 staining of the CAR-T cells by flow cytometry on days 0, 5, and 10, and counted the cells to assess expansion. We also used flow cytometry to detect the central memory T cell (TCM) proportion.ResultsWe detected the expression of the CAR in the CAR-T cells by flow cytometry, and observed transduction rates of 31.5% for RNAU6 anti-CD19 CAR-T cells and 60.7% for LSD1 shRNA anti-CD19 CAR-T cells. The killing efficiency of LSD1 shRNA anti-CD19 CAR-T cells was significantly higher than that of RNAU6 anti-CD19 CAR-T cells at the low effector target ratio. We further found that LSD1 shRNA anti-CD19 CAR-T cells secreted more IFN-γ and granzyme B than RNAU6 anti-CD19 CAR-T cells. CAR-T cells proliferated after U-2932 cell stimulation and were able to sustain proliferation. After stimulation via U-2932 cell co-culture, both RNAU6 anti-CD19 CAR-T and LSD1 shRNA anti-CD19 CAR-T populations had increased proportions of cells with the TCM phenotype, with a higher percentage among LSD1 shRNA anti-CD19 CAR-T cells.ConclusionWe developed a novel, feasible CD19-LSD1 shRNA CAR-T cell strategy for the treatment of DLBCL. Our in vitro assay results showed that LSD1 shRNA anti-CD19 CAR-T cells more effectively killed target cells than RNAU6 anti-CD19 CAR-T cells, and developed a higher proportion of TCM phenotype cells. LSD1 shRNA anti-CD19 CAR-T cells may represent a potential treatment for DLBCL. |
| format | Article |
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| institution | Kabale University |
| issn | 1664-3224 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Frontiers Media S.A. |
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| series | Frontiers in Immunology |
| spelling | doaj-art-484959bf3c044ee6adf73dbd64a04c772025-01-13T06:11:32ZengFrontiers Media S.A.Frontiers in Immunology1664-32242025-01-011510.3389/fimmu.2024.15217781521778In vitro functional validation of anti-CD19 chimeric antigen receptor T cells expressing lysine-specific demethylase 1 short hairpin RNA for the treatment of diffuse large B cell lymphomaZhi Guo0Zhi Guo1Mingxin He2Ning Liu3Yiqing Yang4Rui Sun5Jianxun Wang6Qiang Wang7Institute of Infection, Immunology and Tumor Microenvironment, Hubei Province Key Laboratory of Occupational Hazard Identification and Control, School of Medicine, Wuhan University of Science and Technology, Wuhan, ChinaDepartment of Hematology, The 6th Affiliated Hospital of Shenzhen University Health Science Center, Shenzhen, ChinaDepartment of Hematology, The 6th Affiliated Hospital of Shenzhen University Health Science Center, Shenzhen, ChinaDepartment of Hematology, The 6th Affiliated Hospital of Shenzhen University Health Science Center, Shenzhen, ChinaShenzhen Cell Valley Biomedical Co., LTD, Shenzhen, ChinaShenzhen Cell Valley Biomedical Co., LTD, Shenzhen, ChinaShenzhen Cell Valley Biomedical Co., LTD, Shenzhen, ChinaInstitute of Infection, Immunology and Tumor Microenvironment, Hubei Province Key Laboratory of Occupational Hazard Identification and Control, School of Medicine, Wuhan University of Science and Technology, Wuhan, ChinaBackgroundChimeric antigen receptor T (CAR-T) cell therapy is more effective in relapsed or refractory diffuse large B cell lymphoma (DLBCL) than other therapies, but a high proportion of patients relapse after CAR-T cell therapy owing to antigen escape, limited persistence of CAR-T cells, and immunosuppression in the tumor microenvironment. CAR-T cell exhaustion is a major cause of relapse. Epigenetic modifications can regulate T cell activation, maturation and depletion; they can be applied to reduce T cell depletion, improve infiltration, and promote memory phenotype formation to reduce relapse after CAR-T cell therapy.PurposeWe propose to develop and validate in vitro the function of novel CAR-T cells for the treatment of DLBCL, which simultaneously express an anti-CD19 CAR with lysine-specific demethylase 1 (LSD1) short hairpin (sh)RNA to prevent depletion and prolong the survival of CAR-T cells.MethodsWe designed an shRNA sequence targeting LSD1 mRNA, and created a vector with the following elements: the U6 promoter driving expression of the LSD1 shRNA sequence, the EF1a promoter driving a second-generation anti-CD19 CAR sequence encoding an anti-CD19 single-chain variable fragment (FMC63), the CD8 hinge and transmembrane structural domains, the CD28 co-stimulatory structural domain, and the CD3ζ-activating structural domain. The MFG-LSD1 shRNA anti-CD19 CAR plasmid was first constructed, then packaged in retroviral vectors and transduced into human primary peripheral blood mononuclear cell-derived T cells to generate the corresponding CAR-T cells. We examined by flow cytometry the efficiency of two CAR-T cells in killing U-2932 cells (a human DLBCL line) upon co-culture with RNAU6 anti-CD19 CAR-T cells or LSD1 shRNA anti-CD19 CAR-T cells. We analyzed Ki-67 staining of the CAR-T cells by flow cytometry on days 0, 5, and 10, and counted the cells to assess expansion. We also used flow cytometry to detect the central memory T cell (TCM) proportion.ResultsWe detected the expression of the CAR in the CAR-T cells by flow cytometry, and observed transduction rates of 31.5% for RNAU6 anti-CD19 CAR-T cells and 60.7% for LSD1 shRNA anti-CD19 CAR-T cells. The killing efficiency of LSD1 shRNA anti-CD19 CAR-T cells was significantly higher than that of RNAU6 anti-CD19 CAR-T cells at the low effector target ratio. We further found that LSD1 shRNA anti-CD19 CAR-T cells secreted more IFN-γ and granzyme B than RNAU6 anti-CD19 CAR-T cells. CAR-T cells proliferated after U-2932 cell stimulation and were able to sustain proliferation. After stimulation via U-2932 cell co-culture, both RNAU6 anti-CD19 CAR-T and LSD1 shRNA anti-CD19 CAR-T populations had increased proportions of cells with the TCM phenotype, with a higher percentage among LSD1 shRNA anti-CD19 CAR-T cells.ConclusionWe developed a novel, feasible CD19-LSD1 shRNA CAR-T cell strategy for the treatment of DLBCL. Our in vitro assay results showed that LSD1 shRNA anti-CD19 CAR-T cells more effectively killed target cells than RNAU6 anti-CD19 CAR-T cells, and developed a higher proportion of TCM phenotype cells. LSD1 shRNA anti-CD19 CAR-T cells may represent a potential treatment for DLBCL.https://www.frontiersin.org/articles/10.3389/fimmu.2024.1521778/fullLSD1 shRNAdiffuse large B cell lymphoma (DLBCL)anti-CD19 CAR-T cellsin vitro functional validationcell therapy |
| spellingShingle | Zhi Guo Zhi Guo Mingxin He Ning Liu Yiqing Yang Rui Sun Jianxun Wang Qiang Wang In vitro functional validation of anti-CD19 chimeric antigen receptor T cells expressing lysine-specific demethylase 1 short hairpin RNA for the treatment of diffuse large B cell lymphoma Frontiers in Immunology LSD1 shRNA diffuse large B cell lymphoma (DLBCL) anti-CD19 CAR-T cells in vitro functional validation cell therapy |
| title | In vitro functional validation of anti-CD19 chimeric antigen receptor T cells expressing lysine-specific demethylase 1 short hairpin RNA for the treatment of diffuse large B cell lymphoma |
| title_full | In vitro functional validation of anti-CD19 chimeric antigen receptor T cells expressing lysine-specific demethylase 1 short hairpin RNA for the treatment of diffuse large B cell lymphoma |
| title_fullStr | In vitro functional validation of anti-CD19 chimeric antigen receptor T cells expressing lysine-specific demethylase 1 short hairpin RNA for the treatment of diffuse large B cell lymphoma |
| title_full_unstemmed | In vitro functional validation of anti-CD19 chimeric antigen receptor T cells expressing lysine-specific demethylase 1 short hairpin RNA for the treatment of diffuse large B cell lymphoma |
| title_short | In vitro functional validation of anti-CD19 chimeric antigen receptor T cells expressing lysine-specific demethylase 1 short hairpin RNA for the treatment of diffuse large B cell lymphoma |
| title_sort | in vitro functional validation of anti cd19 chimeric antigen receptor t cells expressing lysine specific demethylase 1 short hairpin rna for the treatment of diffuse large b cell lymphoma |
| topic | LSD1 shRNA diffuse large B cell lymphoma (DLBCL) anti-CD19 CAR-T cells in vitro functional validation cell therapy |
| url | https://www.frontiersin.org/articles/10.3389/fimmu.2024.1521778/full |
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