Human il 4

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)].

Human monocytes were magnetically isolated using anti-CD19 microbeads. Isolated monocytes were cultured in RPMI medium supplemented with 10% FBS, human IL-4 and human GM-CSF (both from Biolegend) (as a DC differentiation medium) for 5 days. To allow maturation and vaccination of DCs, cells were further treated with conditioned media from BxPC3 cells for 24 h, followed by culturing with cell lysate of BxPC-3 cells. The next day, DCs were harvested and co-cultured with purified T + NK cells at a ratio of 1:20 (DCs:T cells) for priming.

Monocytes were isolated from PBMCS by adherence to plastic at 37 °C, 5% CO₂ for 2 h. Adherent cells were cultured in in 24-well plates in AIM-V serum-free medium (Gibco) supplemented with human GM-CSF and IL-4 (both at 50 ng/ml from Peprotech). After 5 days, human TNF-α (500 UI/ml, from Peprotech) was added for Mo-DCs maturation. On day 7, Mo-DCs were treated with 30 µg/mL of DnaK for 24 h. After that, cells harvested and analyzed by FACS or total RNA was extracted.
Bone marrow cells from healthy volunteers were obtained via BWH Tissue Bank and red blood cells were hemolyzed using ACK lysing buffer (Lonza). Single cell suspension was prepared in serum-free AIM-V medium (Gibco) at 1 × 10⁶ cells/ml and cultured for 5 days at 37 °C 5% CO₂, in the presence of 20 ng/ml human IL-4 and 20 ng/ml human GM-CSF (Biolegend). IL-4 and GM-CSF were supplemented again on day 3 of culture. DnaK was added to the media at 30 µg/ml on the 6th day of culture and the cells were incubated for another 24 h before used for mixed lymphocyte reaction.