Combination of carboxyamidotriazole and 1-Methyl-L-tryptophan has synergistic inhibtory effects on programmed death 1 expression

Jing SHI,Lei GUO,De-chang ZHANG,Cai-ying YE

(Department of Pharmacology,Institute of Basic Medical Sciences,Chinese Academy of Medical Sciences and School of BasicMedicine,Peking Union Medical College,Beijing 10005,China）

T1-25

Combination of carboxyamidotriazole and 1-Methyl-L-tryptophan has synergistic inhibtory effects on programmed death 1 expression

Jing SHI,Lei GUO,De-chang ZHANG,Cai-ying YE

(Department of Pharmacology,Institute of Basic Medical Sciences,Chinese Academy of Medical Sciences and School of BasicMedicine,Peking Union Medical College,Beijing 10005,China）

OBJECTIVETo evaluate whether the IDO1 inhibitor 1-methyl-L-tryptophan(1-MT)combine calcium influx inhibitor carboxyamidotriazole(CAI)could further enhance the suppression of programmed death 1(PD-1)in CD8+T cells and investigate the curative effect of the combined use.METHODSCD8+T cells were isolated from normal mice spleen by negative selection using magnetic cell separation.The isolated CD8+T cells were cultured in RPMI 1640 medium containing 10%FBS and 100 U·mL-1IL-2 and activated by the addition of anti-CD3 and anti-CD28(1 g·L-1each mabs).CD8+T cells were pretreated for 48 h with drug and the fluo-3 as a marker of intracellular calcium concentration was detected by flow cytometry.The calcineurin(CaN)levels were assayed with ELISA in CD8+T cells after 48 h incubation with 10 μm CAI.The nuclear translocations of NFAT and AHR were detected by immunofluorescent staining after 48 h of drug treatment.The expression of PD-1 in CD8+T cells was analyzed by flow cytometry.RESULTSIntracellular fluorescent intensity was markedly debase due to CAI treatment(P＜0.01).Meanwhile,the changes of CaN content had a resembled correlation(P＜0.01).Immunofluorescence experiment showed that after combination therapy the transfer of NFAT and AHR in nuclear substantially reduced.Flow cytometry revealed that after the combination caused a significant decrease in PD-1 expression in CD8+T cells.CONCLUSIONCAI and 1-MT could inhibit markedly the expression of PD-1 in CD8+T cells by inhibiting the nuclear translocation of NFAT and AHR,respectively and the combination of them has synergetic effect.

carboxyamidotriazole;indoleamine 2,3-dioxygenase 1;nuclear factor of activated T cells;aryl hydrocarbon receptor;programmed death 1

The project supported by National Natural Science Foundation of China(81402943);CAMS Major Collaborative Innovation Project(2016-I2M-1-011);and PUMC Youth Fund(3332015168）

Lei GUO,Tel:69156405,E-mail：leiguo@ibms.cams.cn