OBJECTIVE To 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.METHODS CD8~+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~(-1)IL-2 and activated by the addition of anti-CD3 and anti-CD28(1 g·L~(-1) each 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(Ca N)levels were assayed with ELISA in CD8~+T cells after 48 h incubation with 10μm 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.RESULTS Intracellular 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.CONCLUSION CAI 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. OBJECTIVE To evaluate whether the IDO1 inhibitor 1-methyl-L-tryptophan (1-MT) calcium coupling 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. METHODS CD8 ~ + T cells were isolated from normal mice spleen by negative selection using magnetic cell separation. CD8 ~ + T cells were cultured in RPMI 1640 medium containing 10% FBS and 100 U · mL ~ (-1) IL-2 and activated by the addition of anti-CD3 and anti-CD28 (1 g · L -1 each 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 (Ca N) 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. R. The ESULTS Intracellular fluorescent intensity was markedly debase due to CAI treatment (P <0.01). While, 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 reduced. Flow cytometry revealed that after the combination caused a significant decrease in PD-1 expression in CD8 + T cells. CONCLUSION CAI 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.