Ficoll hypaque density gradient centrifugation

The study participants comprised 23 HBsAg-positive and HBeAg-negative CHB patients treated by analogs who had undetectable HBV-DNA for at least one year and who were enrolled in a multicenter, randomized, phase 3 study of Peg-IFN-α (ANRS HB06-PEGAN: NCT01172392). Fourteen patients remained on NA alone (control group) and nine received an additional 180μg of Peg-IFN-α (Pegasys; F Hoffmann-La Roche) once a week for 48 weeks (Peg-IFN-α group) (Table 1). Seven out of 14 patients in the control group and 7 out of 9 patients in the Peg-IFN-α group were HLA-A*02:01+ (Table 1). The study protocol was conducted according to the Declaration of Helsinki and French law for biomedical research, and approved by the Ethics Committee “Comite de Protection des Personnnes” (CPP) Sud-Méditerranée-I and the French Regulatory Authority “Agence Nationale de Securite du Medicament et des produits de sante” (ANSM). All participants gave the written informed consent. Heparinized blood samples were obtained at baseline and after 4 (Peg-IFN-α only), 12, 24, 48, 96 and 144 weeks of treatment. Peripheral blood mononuclear cells (PBMCs) were purified by Ficoll-Hypaque density gradient centrifugation (Eurobio) and the total lymphocyte concentration was determined. All the experiments were performed with freshly purified PBMCs.

The study participants comprised 23 HBsAg-positive and HBeAg-negative patients with CHB treated by analogs who had undetectable HBV-DNA for at least one year and were enrolled in a multicenter, randomized, phase 3 study of Peg-IFN-α (ANRS HB06 PEGAN, registered as NCT01172392). Fourteen patients remained on nucleos(t)ide analogs alone (control group) whereas nine received an additional 180 μg Peg-IFN-α (Pegasys; F Hoffmann-La Roche, Basel, Switzerland) once a week for 48 weeks (IFN group). All participants signed informed consent forms. The study protocol was conducted according to the Declaration of Helsinki and French law for biomedical research. It was approved by the Ethics Committee CPP Sud Méditerranée I and the French Regulatory Authority (ANSM). The main features of the patients are shown in Table 1. Heparinized peripheral blood samples were obtained at baseline and after 4 (Peg-IFN-α only), 12, 24, 48, 96, and 144 weeks of treatment. Peripheral blood mononuclear cells (PBMCs) were purified by Ficoll-Hypaque density gradient centrifugation (Eurobio) and the total lymphocyte concentration was determined. Plasma samples were collected before and at each time point of treatment and stored frozen at -80°C.

This protocol was conformed to the French Blood Service’s (EFS-AuRA) Institutional Review Board and the ethics committee of Grenoble University Hospital (CHU-Grenoble) and declared under the reference #DC-2008-787. Written informed consent was obtained from all participants prior to their participation in this study. Blood samples were obtained from healthy donors (HD, n=20) and peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Hypaque density gradient centrifugation (Eurobio). Lymph node or cutaneous metastatic tumors were obtained from 24 melanoma patients (naïve of treatment by immunotherapies) and reduced to cell suspensions by enzymatic digestion with 2 mg.ml-1 collagenase-D (Roche) 20 U.ml-1 DNase (Sigma) and mechanical disruption. The resulting cell suspensions were filtered and washed. Blood and tissue samples were biobanked and stored at -150˚C. Clinical features of melanoma patients are stated in Supplementary Table 1.