Rpmi medium

Blood samples were collected once a day by jugular venepuncture into EDTA (10 mL) and dry tubes (10 mL) at D − 5 and from D + 1 to D + 20 just before treatment administration. Collected sera in dry tubes were frozen at −20 °C until use. Following this, 2 mL of EDTA-treated blood sample was used for viral DNA extraction, as described in Section 4.8. PBMCs were isolated from the remains of the EDTA tubes by density centrifugation using lymphocyte separation medium (Eurobio, Courtaboeuf, France). Cells were rinsed three times with phosphonate-buffered saline (Eurobio) and suspended in RPMI medium (Eurobio) supplemented with 10% foetal bovine serum (Eurobio), 100 IU/mL penicillin, 0.1 mg/mL streptomycin and 0.25 μg/mL amphotericin B (Eurobio). PBMCs were counted using an inverted microscope. In all, 2 × 10⁶ PBMCs were collected and frozen at −20 °C for viral quantitation, and 1 × 10⁶ PBMCs were collected and used for co-culture on RK13 monolayer cells.

Buffy coats from healthy donors were obtained from Zhongshan Hospital, Fudan University. This study has been approved by the Medical Ethics committee of Zhongshan Hospital, Fudan University. Peripheral blood mononuclear cells were separated by density-gradient centrifugation using Ficoll-Paque Plus (GE healthcare Bio-Science, Uppsala, Sweden) and were further processed for separation of T cells using CD3 MicroBeads (MiltenyiBiotec, Auburn, USA). T cells were cultured in RPMI medium (Eurobio, Les Ulis, France) supplemented with 20 IU/mL penicillin, 20 μg/mL streptomycin, and 10% decomplemented FBS (Life Technologies), and stimulated with Dynabeads® T-Expander beads coated with anti-CD3 and anti-CD28 Abs (Life Technologies) at a 1:1 cell/bead ratio in the absence of IL-2 (30 U/ml). Then IS stimulation experiments were conducted by treating T cells with different IS concentration for 96 h.

Peripheral blood mononuclear cells were isolated from blood donor buffy coats (written consent for the use of blood samples for the research protocol obtained according to the regulation for blood transfusion of the French blood organization Etablissement Français du Sang, Rennes (France)) by Ficoll (Thermofischer Scientific, Braunschweig, Germany) gradient centrifugation. After separation of monocytes by a 1-h adhesion step, T lymphocytes were purified from nonadherent cells by negative selection using Dynabeads^® Untouched™ Human T Cells Kit (Thermofischer Scientific). T lymphocytes were cultured in RPMI medium (Eurobio, Les Ulis, France) supplemented with 20 IU/mL penicillin, 20 μg/mL streptomycin, and 10% decomplemented fetal calf serum (Thermofischer Scientific), and stimulated with Dynabeads^® T-Expander beads coated with anti-CD3 and anti-CD28 antibodies (Thermofischer Scientific) before a 48-h treatment with 2 µM B[α]P (Sigma-Aldrich, St. Louis, MO, USA) as previously reported [16 (link)]. B[α]P was used as stock solutions in dimethylsulfoxide (DMSO). The final concentration of DMSO in culture medium was always <0.2% v/v and control cultures received a vehicle containing the same concentration of DMSO (CTR) as treated cultures.