Effects of Rapamycin on proliferation and cytotoxicity of γδT cells in vitro
Objective To investigate the effects of Rapamycin and Torin2 on the proliferation and cytotoxicity of human γδT cells in vitro and the related mechanisms.Methods A total of 10 healthy volunteers were enrolled,which included 5 males and 5 females with mean age of 68.0 years old(standard deviation 4.7 years old).The human chronic lymphocytic leukemia(CLL)tumor cell line MEC-1 was selected,the peripheral blood mononuclear cells(PBMC)were obtained from peripheral ve-nous blood of volunteers,and γδT cells were obtained after treatment.The morphological characteristics of human γδT cells cultured in vitro were observed on the 10th day,and the percentage of human γδT cells was detected by flow cytometry.TheγδT cells were treated with 100 nmol/L Rapamycin(Rapamycin group)and Troin2(Troin2 group),respectively,and RPM1 1640 culture medium was used as blank control(control group).The cells were cultured for 24-hour and 48-hour,and live cell counts was calculated.The killing effect of human γδT cells on MEC-1 cells was detected by lactate dehydrogenase(LDH)re-lease method with different effector-target ratios(1∶1,3∶1,9∶1,18∶1,36∶1).The expression of interleukin(IL)-17 in each group was detected by flow cytometry,and Western blot was used to detect the expression of phosphatidylinositol 3-kinase(PI3K),serine threonine kinase(AKT),phosphorylated AKT(p-AKT),mammalian rapamycin target protein(mTOR),phosphorylated mTOR(p-mTOR)and initiation factor 4E binding protein 1(4EBP-1)protein.Results The percentage of γδT cells was 4.70％±1.17％,and the percentage of γδT cells was 94.20％±2.18％after 10-day amplification and purification.In Rapamycin group,the 24-hour amplification factor was 1.45±0.2,and 48-hour amplification factor was 2.71±0.06,and there was no significant difference between Rapamycin group and control group(P＞0.05).The amplification folds of Torin2 group at 24-hour and 48-hour were 1.14± 0.05 and 2.09±0.06,respectively,which were significantly lower than those of Rapamycin group and control group(P＜0.05).The killing activity of γδT cells in Rapamycin group were 0.05％±0.01％(1∶1),10.84％±0.88％(3∶1),23.02％±0.65％(9∶1),50.74％±2.96％(18∶1)and 77.75％±0.55％(36∶1),respectively,which were significantly higher than those in control group(P＜0.05).The killing activity of γδT cells in Torin2 group was 0.06％±0.01％(1∶1),4.06％±0.84％(3∶1),10.72％±2.97％(9∶1),18.20％±2.83％(18∶1)and 36.18％±2.19％(36∶1),respectively,which were lower than those in Rapamycin group and con-trol group(P＜0.05).The expression of IL-17 in Rapamycin group(32.7±1.7)％and Troin2 group(12.2±1.4)％was significant-ly lower than that in control group(73.1±2.7)％(P＜0.05).The inhibition of IL-17 secretion by γδT in Troin2 group was more ob-vious,and the difference was statistically significant.Western blot results showed that compared with control group,the expression of mTOR in Rapamycin group and Troin2 group decreased,and the difference was statistically significant(tRapamycin group=14.23,P＜0.05;tTroin2 group=11.67,P＜0.05).The expression level of p-mTOR in Rapamycin group and Troin2 group decreased,and the difference was statistically significant(tRapamyin group=16.78,P＜0.05;tTroin2 group=25.31,P＜0.05);the expression of PI3K in Ra-pamycin group was increased,and the difference was statistically significant(t=18.77,P＜0.05);the expression of AKT was in creased,and the difference was statistically significant(t=22.21,P＜0.05);the expression of p-AKT was increased,and the difference was statistically significant(t=19.33,P＜0.05);the expression of 4EBP-1 was increased,and the difference was statis-tically significant(t=14.37,P＜0.05).Conclusion It is demonstrated that Rapamycin could enhance the tumor killing activity of human γδT cells on MEC-1 cells and inhibit the level of IL-17 un-affecting the proliferation of human γδT cells in vitro,and mechanism may be related to the negative feedback mechanism between mTOR C1/C2 sites.
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维普
