Inhibitory Effects of Cyclosporine on Human Regulatory T Cells In Vitro C. Miroux, O. Moralès, A. Carpentier, S. Dharancy, F. Conti, E. Boleslowski, P. Podevin, C. Auriault, V. Pancré, and N. Delhem ABSTRACT Background. Inevitable hepatitis C virus (HCV) recurrence after liver transplantation is a major barrier to the survival of a transplanted liver. It may be promoted by immuno- suppression and the emergence of CD4  CD25  regulatory T cells (Treg). Treg cells can mediate the induction and maintenance of immunological self-tolerance as well as transplant tolerance. We investigated the effects of cyclosporine (CsA), a widely used immunosuppressive agent, on human CD4  CD25  Treg cells. Methods. Human CD4  CD25  cells isolated from healthy donors were cultured in the presence of 40 or 400 ng/mL CsA. The suppressive activity of Treg was assessed in mixed leukocyte reactions (MLR) using CD25  and autologous activated peripheral blood mononuclear cells (PBMC). Phenotype analysis (flow cytometric, Q-PCR) and cytokine production (ELISA) of Treg cells were then performed on cultures. Results. CsA (40 or 400 ng/mL) inhibited the proliferative capacity of PBMC and CD4  CD25  Treg in a dose-dependent manner. Interestingly, addition of 40 ng/mL CsA in MLR impaired the suppressive activity of CD4  CD25  cells, whereas a higher dose of CsA had no effect on Treg function. It appears that a therapeutic dose of CsA (40 ng/mL) did not change the phenotype of CD4  CD25  T cells, but altered Treg activity by switching the regulatory to an inflammatory cytokine profile. Conclusion. CsA significantly impaired the function of CD4  CD25  Treg cells by inducing interleukin-2 (IL-2) and interferon- (IFN-) secretion. The present studies suggested that CsA may block the induction of immune tolerance and decrease the risk of hepatitis C recurrence. H EPATITIS C virus (HCV) is responsible for hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). The survival alternative for patients with end-stage liver disease associated with HCV infection is orthotopic liver transplan- tation (OLT). Because HCV persists not only in hepato- cytes but also in leukocytes and lymph nodes, 1 the virus is redistributed into the donor liver after OLT. The severity and clinical course of the resulting hepatic reinfection vary widely with progression to cirrhosis within 5 years. The main factors determining the severity of recurrent hepatitis C after transplantation may be immunosuppression 2 and the proportion of natural regulatory T cells CD4  CD25  (Treg). 3 Although Cyclosporine (CsA) is a frequently used immunosuppressive drug for OLT, this agent is associated with an increased risk of graft rejection 4 but less severe recurrence of hepatitis C. 4,5 The immunosuppressive effects of CsA are attributed to the inhibition of interleukin-2 (IL-2) transcription and T-cell activation. T-cell tolerance after OLT is achieved due to T-cell anergy and the effects of Treg, which constitutively express high-affinity IL-2 recep- tor -chain (CD25). 6 Moreover, it has been demonstrated From the CNRS (Unité Mixte de Recherche: 8161) - Institut de Biologie de Lille (C.M.,V.P., N.D.), Lille; Unité Propre de Recher- che de l’Enseignement Supérieur 1833 Hôpital Cochin (O.M., A.C., F.C.), Paris; Inserm U795 Hôpital Huriez (S.D., E.B., P.P.), Lille; and UMR 6097, Institut de Pharmacologie Moléculaire et Cellulaire (C.A.), Sophia Antipolis, France. V.P. and N.D. equally contributed to the work. Address reprint requests to Delhem Nadira, MD, 1 rue du Professeur Calmette 59021 Lille Cedex, France. E-mail: nadira. delhem@ibl.fr © 2009 Published by Elsevier Inc. 0041-1345/09/$–see front matter 360 Park Avenue South, New York, NY 10010-1710 doi:10.1016/j.transproceed.2009.08.043 Transplantation Proceedings, 41, 3371–3374 (2009) 3371