*Purpose:

Natural killer (NK) cells as part of the innate immune system are residing in the liver and are considered to be main regulators in alloimmune reactivity as well as viral and tumor surveillance. However, the effects of immunosuppressants on NK cells are not clearly understood. The purpose of this study was to evaluate the impact of trough level conditions of immunosuppressants on human NK cell function.

*Methods: Cytolytic assayCytolytic activity of primary NK cells to lyse K562 or HL60 cells was detected using the carboxyfluorescein diacetate succinimidyl ester (CFSE, Molecular Probes, Eugene) staining method. NK cells sorted by MACS were exposed to single drugs [Everolimus(EVE), 5ng/ml; Sirolimus(SIR), 5ng/ml; Tacrolimus(TAC), 5ng/ml; Cyclosporine A (CSA), 125ng/ml; Mycophenolate Mofetil (MMF), 15ug/ml; Steroid, 0.5ug/ml], and typical combination drugs [Group 1:TAC, 5ng/ml + MMF, 15ug/ml + Steroid, 0.5ug/ml; Group 2: TAC, 5ng/ml+ SIR, 5ng/ml + Steroid, 0.5ug/ml; Group 3: TAC, 5ng/ml + EVE, 5ng/ml + Steroid, 0.5 ug/ml] for 3 days as effector cells. K562 cells and HL60 cells were selected as target cells. NK cells and K562 cells or HL60 cells were co incubated for 4.5 hours according to the effect target ratio of 2.5:1, 5:1 and 10:1. The ratio of CFSE and DAPI double positive was detected by flow cytometry.

*Results: 1. The killing function of NK cells was detected by flow cytometry. It was found that the killing efficiency increased with the increase of effect target ratio. When the effect target ratio was 10:1, the killing efficiency was the highest. 2. Combined immunosuppressive therapy (Group1: TAC, 5ng / ml + MMF, 15 µ g / ml + Steroid, 0.5 µ g / ml; Group2: TAC, 5ng / ml + SIR, 5ng / ml + Steroid, 0.5 µ g / ml; Group3: TAC, 5ng / ml + EVE, 5ng / ml + Steroid, 0.5 µ g / ml) had the greatest inhibitory effect on NK cell killing function. 3. In comparison among single drugs, Steroid (0.5 µ g / ml), MMF (15 µ g / ml) and mTOR inhibitors (EVE, 5ng / ml; SIR, 5ng / ml) had the strongest inhibition on the killing function. Interestingly, calcineurin inhibitors (TAC, 5ng / ml; CSA, 125ng / ml) had no significant effect on the killing function of NK cells. Compared with mTOR inhibitors, the IMPDH inhibitor MMF has stronger inhibitory effect.

*Conclusions: This is the first time to use drug plasma concentrations of immunosuppressive drugs to simulate the clinical microenvironment in stable liver transplant recipients under in vitro conditions. Exposure to immunosuppressants impaired the killing ability of NK cells in varying degrees. The inhibition effect of combination therapies is the strongest, followed by steroid, MMF and mTOR inhibitors, and steroid accounts for the main inhibitory component in the combination therapy. Interestingly, calcineurin inhibitors have little effect on the killing ability of NK cells.

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