慢性淋巴细胞白血病细胞损害CD8^(+)T细胞的线粒体代谢功能影响CAR-T细胞效能的体外试验研究

Chronic lymphocytic leukemia cells damage the mitochondrial metabolism of CD8^(+) T cells and influence the efficacy of CAR-T cells in vitro

目的通过体外试验探究慢性淋巴细胞白血病细胞损害CD8^(+)T细胞的线粒体代谢功能影响嵌合抗原受体的T淋巴细胞(chimeric antigen receptors modified T cells,CAR-T)的效能。方法将培养得到的CAR-T细胞和CD8^(+)T细胞作为正常组,感染组为加入慢性淋巴细胞白血病细胞进行侵染的CAR-T细胞和CD8^(+)T细胞。采用FACScan流式细胞仪检测CAR-T细胞的凋亡情况;Western blot检测细胞凋亡相关的蛋白Bcl-2、cleaved-caspase-3和Bax的相对表达量;酶联免疫吸附实验(enzyme linked immunosorbent assay,ELISA)检测炎症因子肿瘤坏死因子(tumour necrosis factor-alpha,TNF-α)、白细胞介素(interleukin,IL)-6和IL-10的变化;使用Pierce BCA蛋白检测试剂盒测定蛋白质浓度;化学发光发检测细胞内三磷酸腺苷(adenosine triphosphate,ATP)水平,分光光度计检测线粒体丙二醛(malondialdehyde,MDA)、还原型谷胱甘肽(glutathione,GSH)的含量;荧光探针法检测胞内钙离子浓度的变化,实时荧光定量PCR检测线粒体DNA表达水平的变化。结果正常组CAR-T细胞凋亡率显著低于感染组CAR-T细胞凋亡率,差异有统计学意义[(4.58±0.16)%比(7.06±0.74)%,χ^(2)=5.674,P<0.05]。感染组的Bax和cleaved-PARP表达量明显高于正常组,Bcl-2表达量明显低于正常组,差异具有统计学意义[(3.51±0.01)%比(1.07±0.01)%,(2.34±0.05)%比(1.36±0.04)%,(0.66±0.02)%比(1.17±0.02)%,χ^(2)值分别为298.838,26.509和31.231,P值均<0.05]。感染组的IL-6、TNF-α表达量较正常组明显升高,IL-10表达量较正常组明显降低,组间差异具有统计学意义[(87.69±21.07)ng/L比(43.25±5.36)ng/L,(65.12±15.75)ng/L比(33.14±5.23)ng/L,(16.02±1.69)ng/L比(24.33±2.33)ng/L,t值分别为3.54,3.338和4.988,P值均<0.05]。感染组的ATP和总钙离子浓度低于正常组,差异具有统计学意义[(0.15±0.03)μmol/g比(0.32±0.06)μmol/g,(12.46±2.74)μmol/L比(25.97±3.49)μmol/L,t值分别为4.389和5.274,P值均<0.05]。感染组的MDA含量高于正常组,GSH含量低于正常组,差异具有统计学意义[(1.84±0.23)μmol/L比(1.34±0.21)μmol/L,(1.07±0.17)pg/mg比(5.23±0.36)pg/mg,t值分别为2.781和18.098,P值均<0.05]。感染组CD8^(+)T细胞线粒体基因蛋白的表达量明显低于正常组,差异具有统计学意义[(0.62±0.03)%比(1.01±0.06)%,χ^(2)=10.070,P<0.05]。结论离体状态下慢性淋巴细胞白血病细胞通过损害CD8^(+)T细胞的线粒体代谢功能促进CAR-T细胞的凋亡。

Objective To explore the effects of chronic lymphocytic leukemia cells damage the mitochondrial metabolism of CD8^(+)T cells and the efficiency of chimeric antigen receptors modified T cells(CAR-T)in vitro.Methods The cultured CAR-T cells and CD8^(+)T cells were taken as the normal group,and the infection group was the CAR-T cells and CD8^(+)T cells infected with chronic lymphoblastic leukemia cells.FACScan flow cytometer was used to detect the apoptosis of CAR-T cells.Western blot was used to detect the relative expression of apoptosis-related proteins Bcl-2,cleaved-caspase-3 and Bax.Enzyme-linked immunosorbent assay(ELISA)was used to detect the changes of inflammatory factors tumor necrosis factor-α(TNF-α),interleukin(IL)-6 and IL-10.Pierce BCA protein assay reagent kit was used to determine protein concentration.Chemiluminescence was used to detect intracellular adenosine triphosphate(ATP)levels,and a spectrophotometer to detect mitochondrial malondialdehyde(MDA)and glutathione(GSH)content.Fluorescent probe method was used to detect changes in intracellular calcium ion concentration,and real-time fluorescent quantitative PCR was used to detect changes in mitochondrial DNA expression levels.Results The apoptosis rate of CAR-T cells in the normal group was significantly lower than that in the infection group[(4.58±0.16)%vs(7.06±0.74)%,χ^(2)=5.674,P<0.05].The expression levels of Bax and cleaved-PARP in the infection group were significantly increased than that of the normal group,and the Bcl-2 was significantly lower than that of the normal group[(3.51±0.01)%vs(1.07±0.01)%,(2.34±0.05)%vs(1.36±0.04)%,(0.66±0.02)%vs(1.17±0.02)%,χ^(2)values were 298.838,26.509 and 31.231 respectively,all P values<0.05].The expression levels of IL-6 and TNF-αin the infection group were significantly higher than that of the normal group,and the IL-10 was significantly lower than that of the normal group[(87.69±21.07)ng/L vs(43.25±5.36)ng/L,(65.12±15.75)ng/L vs(33.14±5.23)ng/L,(16.02±1.69)ng/L vs(24.33±2.33)ng/L,t values were 3.54,3.338 and 4.988 respectively,all P values<0.05].The concentration of adenosine triphosphate(ATP)and total calcium ion in the infection group were significantly lower than that of the normal group[(0.15±0.03)μmol/L vs(0.32±0.06)μmol/L,(12.46±2.74)μmol/L vs(25.97±3.49)μmol/L,t values were 4.389 and 5.274 respectively,both P values<0.05].The MDA content of the infection group was higher than that of the normal group,and the GSH content was significantly decreased[(1.84±0.23)μmol/L vs(1.34±0.21)μmol/L,(1.07±0.17)pg/mg vs(5.23±0.36)pg/mg,t values were 2.781 and 18.098 respectively,both P values<0.05].The expression level of CD8^(+)T cell mitochondrial gene protein in the infection group were significantly lower than that of the normal group[(0.62±0.03)%vs(1.01±0.06)%,χ^(2)=10.070,P<0.05].Conclusion Chronic lymphocytic leukemia cells in vitro promote the apoptosis of CAR-T cells by impairing the mitochondrial metabolism of CD8^(+)T cells.