Influence of immunosuppressive drugs on NK cells in therapeutic drug exposure in liver transplantation

Natural killer (NK) cells are enriched in the liver and are main regulators in liver transplantation (LT) regarding rejection or tolerance, viral infection or tumor recurrence. Immunosuppression (IS) consists of a triple drug standard regimen, including Tacrolimus (TAC) and Corticosteroids (CS) with either Mycophenolate Mofetil (MMF) or Sirolimus (SIR)/Everolimus (EVE). In this study we simulated the immunosuppressive microenvironment in vitro. Peripheral blood mononuclear cells (PBMC) were cultured at the clinically effective plasma concentration of each drug for 3 days. CS seriously impaired the killing function of NK cells, followed by MMF and SIR/EVE. CS and TAC/CSA significantly decreased the secretion of IFN-γ. NK cell function in liver transplant (LT) recipients was most pronouncedly inhibited by a triple immunosuppressive regimen, CS playing the most prominent role compared to the other drugs. The MMF containing regimen demonstrated a significant increase in the expression of suppressive genes, especially of the Siglec7/9 family. The SIR group had stronger NK cell activity compared with the MMF group, although liver transplantation patients have lower NK cell activity and function. In conclusion, despite an overall comparable immunosuppressive efficiency in terms of prevention of acute rejection, a mTORI including regimen might be considered as having less impact on NK cell function and thus retaining their function. The enriched NK cells from PBMC of healthy volunteers were cultured in the presence of rhIL-2 (100 U/mL) in culture medium (RPMI 1640 medium supplemented with penicillin -streptomycin (100 µL/mL) and 10% human fetal bovine serum (FBS)), in a 37◦C, 95% humidity, 5%CO2 incubator. In addition, NK cells were exposed to single immunosuppressants EVE, 5ng/ml (n=2); SIR, 5ng/ml (n=2); TAC, 5ng/ml (n=2); CSA, 125ng/ml (n=2); MMF, 15 µg/ml (n=2); CS, 0.5 µg/ml (n=2)) and combined immunosuppressants (Group 1: TAC, 5ng/ml + MMF, 15 µg/ml + CS, 0.5µg/ml (n=3); Group 2: TAC, 5ng/ml +SIR, 5ng/ml + CS, 0.5 µg/ml (n=3); Group 3: TAC, 5ng/ml + EVE, 5ng/ml + CS, 0.5 µg/ml (n=3)) for 3 days, the CTL (n=5) did not contain drugs, and RNA was extracted for transcriptome sequencing.