Cutting Edge Communication: In Vitro Generation of Dendritic Cells from Human Blood Monocytes in Experimental Conditions Compatible for In Vivo Cell Therapy

Abstract

DC are professional APC that are promising adjuvants for clinical immunotherapy. Methods to generate in vitro large numbers of functional human DC using either peripheral blood monocytes or CD34+ pluripotent HPC have been developed recently. However, the various steps of their in vitro production for further clinical use need to fit good manufacturing practice (GMP) conditions. Our study focused on setting up such a full procedure, including collection of mononuclear cells (MNC) by apheresis, separation of monocytes by elutriation, and culture of monocytes with GM-CSF + IL-13 + autologous serum (SAuto) in sterile Teflon bags. The procedure was first developed with apheresis products from 7 healthy donors. Its clinical feasibility was then tested on 7 patients with breast cancer. The characteristics of monocyte-derived DC grown with SAuto (or in some instances with a pooled AB serum) were compared with those obtained in the presence of FBS by evaluation of their phenotype, their morphology in confocal microscopy, and their capacity to phagocytize latex particles and to stimulate allogeneic (MLR) or autologous lymphocytes (antigen-presentation tests). The results obtained demonstrate that the experimental conditions we set up are easily applicable in clinical trials and lead to large numbers of well-defined SAuto-derived DC as efficient as those derived with FBS.

Directed Differentiation of Human Embryonic Stem Cells into Functional Dendritic Cells through the Myeloid Pathway

“…Immature DC precursors isolated from peripheral blood, or DCs generated from PBMC and CD34 ϩ hemopoietic progenitors have been used in clinical trials of dendritic cell-based vaccines (7,43,(45)(46)(47)(48). However, these techniques are laborious, require repeated generation of new DCs for each vaccination, and are difficult to standardize (43).…”

“…Human ES cell lines H1 (NIH code WA01; passages [32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51] and H9 (NIH code WA09; passages 40 -44) were maintained in an undifferentiated state by weekly passages on mouse embryonic fibroblasts as previously described (25). A mouse bone marrow stromal cell line OP9 was obtained from Dr. T. Nakano (Research Institute for Microbial Diseases, Osaka University, Osaka, Japan).…”

Massive expansion of regulatory T-cells following interleukin 2 treatment during a phase I-II dendritic cell-based immunotherapy of metastatic renal cancer

“…They elicit T-cell immune responses by presenting antigens on major histocompatibility complex (MHC) class I and class II molecules to both naive and memory CD8 and CD4 T-cells (10)(11)(12). The possibility to generate in vitro functional DCs from peripheral blood monocytes (13)(14)(15)(16) or from CD34 + progenitors obtained from cord blood (17)(18)(19), bone marrow (BM) or cytokine mobilized peripheral blood (20)(21)(22)(23) has opened a new field of vaccine-based immunotherapy for cancer. Thus, various DC-based clinical trials have been performed for treatment of metastatic RCCs using either tumor lysate-pulsed DCs (24)(25)(26), tumor cell-DC hybrids (27)(28)(29), tumor RNA-transfected DCs (30) or more recently DCs loaded with HLA-A2-restricted MUC1-derived peptides (31).…”

Myeloid blood CD11c+ dendritic cells and monocyte-derived dendritic cells differ in their ability to stimulate T lymphocytes

“…However, the use of different starting monocyte preparations, serum supplements, cytokines, and differentiating/activating agents has contributed to marked heterogeneity in opinion as to the optimal phenotype and functional characteristics of Mo-DCs for immunotherapy. 20 A recent report suggests that Mo-DCs generated with GM-CSF/ IL-4 may reduce rather than enhance immune responses. 21 Accumulating data suggest that the quality of immune responses may also be affected by the route of administration of DCs.…”