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Archive > Volume 31 Issue 5 > 2024,31(5):493-500. DOI:10.3872/j.issn.1007-385X.2024.05.009 Prev Next
Technigue and Method
Establishment of an in vitro cytotoxicity evaluation model for BCMA CAR-T cells based on BCMA mutants
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Abstract:
Objective:To engineer a BCMA mutant resistant to γ-secretase cleavage in order to stabilize wild-type BCMA expression after γ-secretase cleavage and to generate target cells for measuring BCMA CAR-T cell cytotoxicity. Methods: Our study aimed to engineer a mutant BCMA protein (BCMA-CD8α TM) that could resist γ-secretase cleavage by replacing the transmembrane domain of the wild-type BCMA protein with the human CD8α sequence. Four different types of cells in which this mutant gene was expressed excessively were engineered, including U266 (U266BCMA Mut), K562 (K562BCMA Mut), SKOV3 (SKOV3BCMA Mut), and CHO (CHOBCMA Mut)cells. BCMA CAR Jurkat cells, loaded with the NFAT-EGFP reporter gene (BCMA-CAR-Jurkat-Reporter), were engineered and co- cultured with U266BCMA Mut cells. The expression level of EGFP was detected by FCM in order to indicate the activation level of NFAT.The cytotoxicity of BCMA CAR-T cells against Luciferase-labeled K562BCMA Mut cells was detected by the luciferase assay. Additionally,real-time cell analysis (RTCA) technique was employed to detect the cytotoxicity of BCMA CAR-T cells against SKOV3BCMA Mut and CHOBCMA Mut cells. Results: Application of γ -secretase inhibitor LY411575 to inhibit γ -secretase activity significantly enhanced the expression level of BCMA on the surface of wild-type U266 cells, and the average fluorescence intensity was increased by more than 10 times. However, the expression level of BCMA gradually decreased after removal of inhibitors (P<0.01). BCMA-CD8α TM mutant could resist the cleavage of γ -secretase and expressed stably on the surface of U266 cells (P>0.05). U266 cells and U266 cells overexpressing BCMA-CD8α TM were co-incubated with BCMA-CAR-Jurkat-Reporter cells, both of which could activate the Reporter system and enhance the expression of EGFP, but the effect was more significant in U266 cells overexpressing BCMA-CD8α TM (P<0.01). BCMA-CD8α TM mutants were successfully overexpressed in 3 BCMA-negative target cells, namely K562, SKOV3 and CHO cells, and the expression level of the mutant was only slightly increased under LY411575 treatment. Luciferase assay results showed that under different target-effect ratios BCMA CAR-T cells could all be specific and efficient in killing K562 cells overexpressing BCMA-CD8α TM. RTCA results showed that under different target ratios BCMA CAR-T cells could all effectively recognize and kill SKOV3 and CHO cells overexpressing BCMA-CD8α TM, but Mock-T cells with the same target ratio had no such effect. Conclusion: The BCMA-CD8α TM mutant engineered in this study can resist γ-secretase cleavage and exhibits stable surface expression on various target cells, thus offering various methods for evaluating the efficacy and specificity of BCMA CAR-T cell cytotoxicity in vitro.
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