Abstract

Generation of large numbers of dendritic cells (DC) for research or immunotherapeutic purposes typically involves in vitro conversion of murine bone marrow precursors or humanblood monocytes to DC via cultivation with supraphysiologic concentrations of cytokines such as GM-CSF and IL-4 for up to 7days. Alternatively, our group has recently established a new approach, based on the underlying mechanism of action of a widely used cancer immunotherapy termed Extracorporeal Photochemotherapy (ECP). Our method of rapid and cytokine-free production of therapeutically relevant DC populations,leveragingthe innate physiologic programs likely responsible for DC differentiation from blood monocytes in vivo, potentially offers a novel, inexpensive, and easily accessiblesource of DC for clinical and research uses. This approach involves ex vivo physiologic reprogramming of blood monocytes to immunologically tunable dendritic antigen-presenting cells, which we term "phDC," for physiological DC. To facilitate access and utilization of these new DC populations by the research community, in this chapter, we describethe use of a scaled-down version of the clinical ECP leukocyte-treatment devicetermed the Transimmunization (TI) chamberor plate, suitable for processing both mouse and human samples. We highlight the methodological sequences necessary to isolate mouse or human peripheral blood mononuclear cell (PBMC) from whole blood, and to expose those PBMC to the TI chamber for facilitating monocyte activation and conversion to physiological DC (phDC) through interaction with blood proteins and activated platelets under controlled flow conditions. We thenprovide sample protocols for potential applications of the generated DC, including their use as vaccinating antigen-presenting cells (APC) in murine in vivo antitumor models, and in human ex vivo T-cell stimulation and antigen cross-presentation assays which mimic clinical vaccination. We additionally highlight the technical aspects of loading mouse or human phDC with tumor-associated antigens (TAA) in the form of peptides or apoptotic tumor cells. We provide a simple and clinically relevant means to reprogram blood monocytes into functional APC, potentially replacing the comparatively expensive and clinically disappointing cytokine-derived DC which have previously dominated the dendritic cell landscape.

Full Text
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