Impact of Steroids on Natural Killer Cells Against Cytotoxicity and Hepatitis C Virus Replication

Abstract

Background: Natural killer (NK) cells play a pivotal role in tumor surveillance and viral infection, such as hepatitis C virus (HCV) infection after liver transplantation (LT). Immunosuppression is a major causative factor for the accelerated recurrence of HCV and hepatocellular carcinoma (HCC). We recently reported that adoptive immunotherapy with activated donor liver NK cells elicited anti-HCC and anti-HCV effects after LT. Despite the potential importance of NK cells in the context of LT, the impact of immunosuppressive strategies on NK cell functions is poorly understood. In the present study, we performed phenotypic and functional analyses of NK cells derived from peripheral blood mononuclear cells (PBMCs) of healthy volunteers and from donor liver graft perfusate (liver mononuclear cells [LMNCs]). Methods: To determine the effect of immunosuppressive agents on NK cell function, freshly isolated NK cells derived from PBMCs or LMNCs were cultured for 7 days in the presence of rhIL-2 (100 U/ml) with or without the following immunosuppressive drugs: tacrolimus (FK), cyclosporine A (CsA), prednisone (MP), mycophenolate mofetil (MMF), and rapamycin (RAPA), at concentrations ranging from 0 to 1 μg/ml, to determine the infratherapeutic, therapeutic, and supratherapeutic doses. The effect of drugs on NK cell activation was tested on the basis of the following parameters: (1) NK cell phenotype, (2) NK cell proliferation, (3) cytotoxicity against K562 cells, (4) cytokine production of NK cells, and (5) anti-HCV activity with HCV genomic replicon cells. Results: NK cells maintained robust proliferation in the presence of FK and CsA. In marked contrast, MP, MMF, and RAPA significantly inhibited the proliferation of both PBMC and LMNC NK cells (Fig. 1A, p< 0.05). To further examine whether immunosuppressive agents alter NK cell function, we used cultured NK cells as effectors in both killing assay against K562 tumor cells and anti-HCV replicon assay. FK, CsA, MMF, and RAPA did not affect NK cell-mediated killing against K562 targets nor HCV replication while cytotoxicity and anti-HCV effect were significantly inhibited in the presence of MP (Fig. 1B, 1C, p< 0.01). Furthermore, MP specifically inhibited the expression of CD69, TRAIL, NKG2D, NKp30, NKp44, NKG2A, and CD132, as well as the production of IFN- γ (p < 0.05). These results were observed in both peripheral and liver NK cells.[Figure 1]Conclusions: Corticosteroids, especially at high doses, strongly inhibit the surface expression of some activating NK receptors, as well as the anti-tumor activity and anti-HCV effect of both liver and peripheral NK cells. These results may be used to improve the designs of steroid-related immunosuppressive regimens after LT.