Abstract
BackgroundDendritic cells (DCs) are the most potent antigen-presenting cell population for activating tumor-specific T cells. Due to the wide range of methods for generating DCs, there is no common protocol or defined set of criteria to validate the immunogenicity and function of DC vaccines.MethodsMonocyte-derived DCs were generated during 4 days of culture with recombinant granulocyte-macrophage colony stimulating factor and interleukin-4, and pulsed with tumor lysate produced by hypochlorous acid oxidation of tumor cells. Different culture parameters for clinical-scale DC preparation were investigated, including: 1) culture media; 2) culture surface; 3) duration of activating DCs with lipopolysaccharide (LPS) and interferon (IFN)-gamma; 4) method of DC harvest; and 5) cryomedia and final DC product formulation.ResultsDCs cultured in CellGenix DC media containing 2% human AB serum expressed higher levels of maturation markers following lysate-loading and maturation compared to culturing with serum-free CellGenix DC media or AIM-V media, or 2% AB serum supplemented AIM-V media. Nunclon™Δ surface, but not Corning® tissue-culture treated surface and Corning® ultra-low attachment surface, were suitable for generating an optimal DC phenotype. Recombinant trypsin resulted in reduced major histocompatibility complex (MHC) Class I and II expression on mature lysate-loaded DCs, however presentation of MHC Class I peptides by DCs was not impaired and cell viability was higher compared to cell scraping. Preservation of DCs with an infusible cryomedia containing Plasma-Lyte A, dextrose, sodium chloride injection, human serum albumin, and DMSO yielded higher cell viability compared to using human AB serum containing 10% DMSO. Finally, activating DCs for 16 hours with LPS and IFN-γ stimulated robust mixed leukocyte reactions (MLRs), and high IL-12p70 production in vitro that continued for 24 hours after the cryopreserved DCs were thawed and replated in fresh media.ConclusionsThis study examined criteria including DC phenotype, viability, IL-12p70 production and the ability to stimulate MLR as metrics of whole oxidized tumor lysate-pulsed DC immunogenicity and functionality. Development and optimization of this unique method is now being tested in a clinical trial of autologous oxidized tumor lysate-pulsed DC in clinical-scale in recurrent ovarian, primary peritoneal or fallopian tube cancer (NCT01132014).
Highlights
Dendritic cells (DCs) are the most potent antigen-presenting cell population for activating tumorspecific T cells
We have recently demonstrated that Monocytederived DCs (MoDCs) cultured from an elutriated leukapheresis product for 4 days (Day-4 DCs) and pulsed with UVB-irradiated or freeze-thawed tumor cell lysate, produced higher levels of IL-12p70 and IP-10 upon activation with bacterial lipopolysaccharide (LPS) and interferon (IFN)-g compared to MoDCs cultured for 2 or 7 days [Cheryl L-L, Chiang, Andrea R
Research-scale DC preparations were set up by seeding each T25 cm2 NUNC Surface flasks with 1 × 106 healthy donor monocytes/ml media
Summary
Dendritic cells (DCs) are the most potent antigen-presenting cell population for activating tumorspecific T cells. Labor and expense for DC generation is necessary to move DC vaccines to later phase clinical trials [16,17,18] To this end, we have recently demonstrated that MoDCs cultured from an elutriated leukapheresis product for 4 days (Day-4 DCs) and pulsed with UVB-irradiated or freeze-thawed tumor cell lysate, produced higher levels of IL-12p70 and IP-10 upon activation with bacterial lipopolysaccharide (LPS) and interferon (IFN)-g compared to MoDCs cultured for 2 or 7 days [Cheryl L-L, Chiang, Andrea R. These mature lysate-loaded Day-4 DCs were highly immunogenic and stimulated the strongest allogeneic T cell proliferation, while inducing potent specific anti-ovarian tumor responses as Day-7 ‘classical DCs’ in T cells obtained from healthy volunteers and ovarian cancer patients
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