Isolation and Differentiation of DCs and CD8+ T Cells

Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors by Ficoll-Hypaque density gradient centrifugation and cultured in RPMI 1640 medium containing 10% FCS for 2 hours. DCs, which were generated from the adherent fraction of PBMCs [38 (link)], were cultured for 5 days in RPMI 1640 medium containing 10% FCS, 20 ng/mL human GM-CSF, and 10 ng/mL human IL-4 (Biolegend). Culture medium and cytokines were refreshed in every other day. On the day 5, the medium was replaced by rituximab-treated conditioned medium or control medium and cell culture was continued for 24 hours. Autologous CD8+ T lymphocytes were magnetically isolated using CD8 MicroBeads (Miltenyi Biotech, Germany) to obtain purity ≥95% CD8+ T cells. The isolated T lymphocytes were cultured in RPMI 1640 medium containing 10% FCS and 10 ng/mL human IL-2 (Biolegend) and the medium was replaced every other day [37 (link)].