*Purpose: Natural killer (NK) cells are enriched in lymphocytes within the liver and are considered to be main regulators of liver transplantation (LT) rejection and tolerance. 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 in long term patients on NK cell activation and function.

*Methods: Peripheral blood mononuclear cells (PBMC) of 14 LT recipients and 14 healthy individuals (control) were used. Flow cytometry was done to analyse lymphocyte subsets and the phenotype of NK cells for in vivo and vitro. PBMC were exposed to single drugs [Everolimus(EVR), 5ng/ml; Sirolimus(SIR), 5ng/ml; Tacrolimus(TAC), 5ng/ml; Cyclosporine A (CSA), 125ng/ml; Mycophenolic acid (MPA), 15ug/ml; Steroid, 0.5ug/ml], and in typical combinations of [TAC, 0.5ng/ml + MMF, 15ug/ml + Steroid, 0.5ug/ml; TAC, 5ng/ml + SIR, 5ng/ml + Steroid, 0.5ug/ml; TAC, 5ng/ml + EVE, 5ng/ml + Steroid, 0.5 ug/ml] for 3 days. NK cells were sorted from PBMC under the same culture conditions, and RNA-seq was performed analysing genes for inhibition and activation.

*Results: 1. Immunosuppressants reduced in vivo the number of CD4T, CD8T and NK cells, but did not change the percentage. For NK cells, the number of CD56dimCD16+/- subgroups were mainly reduced. 2. It is worth noting that NK cells activity increased in LT 3. In vitro, Clinical drug treatment impaired the ability of NK cells to secrete interferon-r and perforin by flow cytometry and RNA- seq, and reduced expression of NKp44, CD69, NKG2D. In particular, no difference of NK cell activity was noted, when exposed to the three combination groups, despite their different impact on NK cells when exposed to the single drug.

*Conclusions: This is the first time to use drug plasma concentrations simulating the clinical microenvironment in stable liver transplant recipients under in vitro conditions. Clinical drug treatment severely impairs NK cell activation and function. Despite of distinct actions on NK cells if given as single drugs, no differences are observed between all three different combinations of TAC neither with an mTOR nor MMF.

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