CD19嵌合抗原受体T细胞靶向B细胞肿瘤细胞株过程中糖皮质激素对其扩增影响的体外研究

Effects of glucocorticoids on the proliferation of CD19 CAR-T cells targeting B-cell tumor cell lines

目的评估糖皮质激素对CD19嵌合抗原受体T细胞(CAR-T细胞)靶向杀瘤过程中增殖的影响。方法健康志愿者的外周血单个核细胞(PBMC)作为T细胞来源,经CD3磁珠分选及CD19 CAR慢病毒转染制备CD19 CAR-T细胞;采用流式细胞术(FCM)测定CD19 CAR-T细胞转染率及其在培养体系中所占比例;采用CFDA SE细胞增殖示踪荧光探针试剂标记CD19 CAR-T细胞后测定其荧光强度;LDH细胞毒性检测方法检测不同浓度的糖皮质激素对B细胞肿瘤细胞株杀伤活性的影响。结果①CD19 CAR-T细胞CD19 CAR转染率为(51.34±5.28)%。②24 h后不同剂量甲泼尼龙对Nalm6、Pfeiffer及U2932细胞的杀伤活性高于地塞米松,48 h后低浓度(4 mg/ml)甲泼尼龙对Nalm6、Pfeiffer、U2932细胞杀伤活性高于低浓度(0.75 mg/ml)地塞米松,而高浓度(12 mg/ml)甲泼尼龙杀伤活性低于高浓度(2.25 mg/ml)地塞米松。但各个时间不同剂量甲泼尼龙组对EHEB细胞的杀伤活性均低于对应时间、剂量的地塞米松。③CD19 CAR-T细胞在不同浓度糖皮质激素作用下的比例及平均荧光强度比较,地塞米松对CD-19 CAR-T细胞的增殖抑制作用强于甲泼尼龙,两种糖皮质激素的高浓度组较低浓度组对CD19 CAR-T细胞的增殖抑制作用更明显。④CD19 CAR-T细胞与不同肿瘤细胞共培养体系中,其比例及平均荧光强度比较,地塞米松对CD19 CAR-T细胞的增殖抑制作用强于甲泼尼龙,两种糖皮质激素的高浓度组较低浓度组对CD19 CAR-T细胞的增殖抑制更明显。结论CD19 CAR-T细胞靶向不同肿瘤细胞株过程中地塞米松对CD19 CAR-T细胞的扩增和增殖抑制作用大于甲泼尼龙,高剂量组该抑制作用更加明显。

Objective To evaluate the effects of glucocorticoids(dexamethasone and methylprednisolone)on the proliferation of CD19 Chimeric antigen receptor(CAR)modified T cells in vitro.Methods Peripheral blood mononuclear cells from healthy volunteers were collected as T cells.CD19 CAR-T cells were prepared by CD3 magnetic beads sorting and CD19 CAR lentivirus transfection.The transfection rates and the proportion of CD19 CAR-T cells in the culture system were analyzed using a flow cytometer.The mean fluorescence intensity(MFI)of CD19 CAR-T cells was measured after staining with Carboxyfluorescein diacetate succinimidyl ester cell proliferation tracer fluorescent probe,Lactate dehydrogenase(LDH)cytotoxicity assay was used to detect the effects of different concentrations of glucocorticoid on the killing activity of B-cell tumor cell lines.Results In this study,the CD19 CAR transfection rate of CD19 CAR-T cells was(51.34±5.28)%.The killing activities of different doses of methylprednisolone on Nalm6,Pfeiffer,and U2932 tumor cells were higher than that of dexamethasone at 24 h.The killing activities of 4 mg/mL methylprednisolone on Nalm6,Pfeiffer,and U2932 were higher than that of 0.75 mg/ml group,while the killing activity of 12 mg/ml methylprednisolone was lower than that of 2.25 mg/ml dexamethasone at 48 h.However,the killing activities of different doses of methylprednisolone on EHEB tumor cells were lower than those of different doses of dexamethasone at 24 and 48 h.The average MFI and proportion of CD19 CAR-T cells under different concentrations of glucocorticoid the proliferation inhibition of CD19 CAR-T cells by dexamethasone was higher than that of methylprednisolone.The proliferation inhibition of CD19 CAR-T cells of the two glucocorticoids in high concentration groups were more obvious than that in low concentration groups.When CD19 CAR-T cells were co-cultured with different tumor cells,the proportion and average MFI of CD19 CAR-T cells showed that the proliferation inhibition of dexamethasone was higher than that of methylprednisolone.The proliferation inhibition of CD19 CAR-T cells of the two glucocorticoids in high concentration groups was more obvious than that in low concentration groups.Conclusion Dexamethasone inhibits the cell proliferation of CD19 CAR-T cells more than methylprednisolone during the targeting of different tumor cell lines.The inhibition effect of dexamethasone on the proliferation and amplification of CD19 CAR-T cells was greater than that of methylprednisolone during the targeting of CD19 CAR-T cells to different tumor cell lines.Moreover,the inhibition effect of the high dose group was more obvious.