Objective: To explore the stimulation conditions, optimal culture time and infection time of C57BL/6J mice CD3+ T cells in vitro, so as to improve the infection efficiency of CD19 chimeric antigen receptor T cells (mCD19 CAR-T).
Methods: Purified C57BL/6J mice CD3+ T cells were cultured in anti-CD3/CD28 coated, anti-CD3 coated+soluble anti-CD28 and anti-CD3 coated, respectively. The cells were stimulated in above three conditions for 12 h and 24 h, following with 24 h, 48 h and 72 h incubation and then the number of cell clones was recorded. C57BL/6J mice CD3+ T cells were stimulated for 12 h, 24 h, and 36 h under the above three conditions, then interleukin (IL)-2 (100 U/ml) was added. The number of cell clones was recorded under microscope at 24 h, 48 h, and 72 h of culture. After 24 h of stimulation, CD3+ T cells derived from C57BL/6J mice were infected with retrovirus for 48 h to establish mCD19 CAR-T cells, and the percentage of GFP+ CAR-T cells was detected by flow cytometry.
Results: The infection efficiency of mCD19 CAR-T cells derived from C57BL/6J mice was only 5.23% under the optimized conditions of mCD19 CAR-T cells derived from BALB/c mice. The number of clones of C57BL/6J mice CD3+ T cells was the highest in anti-CD3 coated+soluble anti-CD28 group after stimulated for 24 h and followed cultured for 48 h. After 24 hours of stimulation under the above conditions and 48 hours of culture with IL-2, the number of T cell proliferating clones in the anti-CD3 coated+soluble anti-CD28 group was significantly increased compared with the same group without IL-2, and the infection efficiency of CAR-T cells in this group reached 17.63%±4.17%.
Conclusion: The optimal conditions for constructing CAR-T cells from C57BL/6J mice CD3+ T cells are different from those of BABL/c mice. T cells stimulated by anti-CD3 coated+soluble anti-CD28+IL-2 can obtain mCD19 CAR-T cells with the highest efficiency after retrovirus infection.
题目: C57BL/6J小鼠背景的CD19嵌合抗原受体T细胞的制备及优化.
目的: 探索C57BL/6J小鼠CD3+ T细胞体外刺激条件、最佳培养和感染时间，以期提高CD19嵌合抗原受体T细胞（mCD19 CAR-T）的感染效率。.
方法: 将纯化的C57BL/6J小鼠CD3+ T细胞在包被CD3/CD28抗体（anti-CD3/CD28 coated）、包被CD3抗体+可溶性CD28抗体（anti-CD3 coated+soluble anti-CD28）和包被CD3抗体（anti-CD3 coated）3种不同条件下分别刺激12 h和24 h，后续培养24 h、48 h和72 h并记录细胞克隆数。在上述3种条件下分别刺激C57BL/6J小鼠CD3+ T细胞12、24和36 h，然后加入白介素（IL）-2（100 U/ml），镜下记录培养24 h、48 h和72 h的细胞克隆数；此条件下取刺激24 h的CD3+ T细胞，感染mCD19 CAR-T逆转录病毒，制备mCD19 CAR-T细胞，流式检测48 h时3组中GFP+CAR-T细胞的百分率。.
结果: 利用获得BALB/c小鼠mCD19 CAR-T细胞的最优化条件，制备C57BL/6J小鼠的mCD19 CAR-T细胞感染效率仅5.23%；anti-CD3+soluble anti-CD28 coated刺激C57BL/6J小鼠 CD3+ T细胞24 h，终止刺激继续培养至48 h时细胞克隆数最高；在上述条件刺激24 h后加入IL-2培养48 h，anti-CD3+soluble anti-CD28 coated组T细胞增殖克隆数量显著增加，且CAR-T细胞感染效率为17.63%±4.17%。.
结论: C57BL/6J小鼠来源T细胞制备CAR-T细胞的最佳条件与BABL/c小鼠不同；anti-CD3+soluble anti-CD28 coated+IL-2刺激条件下诱导的T细胞感染逆转录病毒后可获得最高效率的mCD19 CAR-T细胞。.
Keywords: C57BL/6J mice; CD19; chimeric antigen receptor T cell.