Generation and maturation of dendritic cells for clinical application under serum-free conditions.
Monocyte-derived dendritic cells (MoDCs) in clinical use for cancer immunotherapy are ideally generated in serum-free medium (SFM) with inclusion of a suitable maturation factor toward the end of the incubation period. Three good manfacturing practice (GMP) grade SFMs (AIM-V, X-VIVO 15, and X-VIVO 20) were compared with RPMI-1640, supplemented with 10% fetal bovine serum or 10% human serum. DCs generated for 7 days in SFM were less mature and secreted less interleukin (IL) 12p70 and IL-10 than DCs generated in 10% serum. DC yield was comparable in SFMs, and a greater proportion of cells was viable after maturation. Toll-like receptor (TLR) ligands were compared for their ability to induce cytokine secretion under serum-free conditions in the presence of interferon (IFN) gamma. With the exception of Poly I:C, TLR ligands stimulated high levels of IL-10 secretion. High levels of IL-12p70 were induced by two TLR4-mediated stimuli, lipopolysaccharide and Ribomunyl, a clinical-grade bacterial extract. When T-cell responses were compared in allogeneic mixed leukocyte reaction, DCs stimulated with Ribomunyl induced higher levels of IFNgamma than DCs stimulated with the cytokine cocktail: tumor necrosis factor-alpha, IL-1beta, IL-6, and prostaglandin E2. In the presence of IL-10 neutralizing antibodies, DC IL-12p70 production and T-cell IFNgamma were increased in vitro. Similarly, DCs stimulated with Ribomunyl, IFNgamma, and anti-IL-10 induced high levels of tetanus toxoid-specific T-cell proliferation and IFNgamma secretion. Thus, MoDCs generated in SFM efficiently stimulate T-cell IFNgamma production after maturation in the presence of a clinical-grade TLR4 agonist and IL-10 neutralization.
Peng
Judy C
JC
Division of Chemical Engineering, University of Queensland, Brisbane, Queensland, Australia.
Thomas
Ranjeny
R
Nielsen
Lars K
LK
D000818:Animals; D019496:Cancer Vaccines; D002417:Cattle; D002454:Cell Differentiation; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D016207:Cytokines / Q000096:biosynthesis*; D003713:Dendritic Cells / Q000166:cytology* / Q000276:immunology; D006801:Humans; D009000:Monocytes / Q000166:cytology; D044967:Serum; D013601:T-Lymphocytes / Q000276:immunology; D051197:Toll-Like Receptor 4 / Q000378:metabolism
D019496:Cancer Vaccines; D016895:Culture Media, Serum-Free; D016207:Cytokines; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4