title: Comparative study of clinical grade human tolerogenic dendritic cells.
abstract: BACKGROUND
  The use of tolerogenic DCs is a promising therapeutic strategy for transplantation and autoimmune disorders. Immunomodulatory DCs are primarily generated from monocytes (MDDCs) for in vitro experiments following protocols that fail to fulfil the strict regulatory rules of clinically applicable products. Here, we compared the efficacy of three different tolerance-inducing agents, dexamethasone, rapamycin and vitamin D3, on DC biology using GMP (Good Manufacturing Practice) or clinical grade reagents with the aim of defining their use for human cell therapy.
  
  
  METHODS
  Tolerogenic MDDCs were generated by adding tolerogenic agents prior to the induction of maturation using TNF-α, IL-β and PGE2. We evaluated the effects of each agent on viability, efficiency of differentiation, phenotype, cytokine secretion and stability, the stimulatory capacity of tol-DCs and the T-cell profiles induced.
  
  
  RESULTS
  Differences relevant to therapeutic applicability were observed with the cellular products that were obtained. VitD3-induced tol-DCs exhibited a slightly reduced viability and yield compared to Dexa-and Rapa-tol-DCs. Phenotypically, while Dexa-and VitD3-tol-DCs were similar to immature DCs, Rapa-tol-DCs were not distinguishable from mature DCs. In addition, only Dexa-and moderately VitD3-tol-DCs exhibited IL-10 production. Interestingly, in all cases, the cytokine secretion profiles of tol-DCs were not modified by a subsequent TLR stimulation with LPS, indicating that all products had stable phenotypes. Functionally, clearly reduced alloantigen T cell proliferation was induced by tol-DCs obtained using any of these agent. Also, total interferon-gamma (IFN-γ) secretion by T cells stimulated with allogeneic tol-DCs was reduced in all three cases, but only T cells co-cultured with Rapa-tol-DCs showed impaired intracellular IFN-γ production. In addition, Rapa-DCs promoted CD4+ CD127 low/negative CD25high and Foxp3+ T cells.
  
  
  CONCLUSIONS
  Our results demonstrate contrasting influences of different clinical-grade pharmacological agents on human tol-DC generation. This should be taken into account for decisions on the use of a specific agent for the appropriate cellular therapy in the context of a particular disease.
authors.affiliation: Laboratory of Immunobiology for Research and Diagnosis, Blood and Tissue Bank, Dept. of Cell Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, Institut Investigació Germans Trias i Pujol, Spain.
authors.forename: M
authors.initials: M
authors.lastname: Naranjo-Gómez
authors.forename: D
authors.initials: D
authors.lastname: Raïch-Regué
authors.forename: C
authors.initials: C
authors.lastname: Oñate
authors.forename: L
authors.initials: L
authors.lastname: Grau-López
authors.forename: C
authors.initials: C
authors.lastname: Ramo-Tello
authors.forename: R
authors.initials: R
authors.lastname: Pujol-Borrell
authors.forename: E
authors.initials: E
authors.lastname: Martínez-Cáceres
authors.forename: Francesc E
authors.initials: FE
authors.lastname: Borràs
mesh_terms: D000893:Anti-Inflammatory Agents / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002762:Cholecalciferol / Q000494:pharmacology; D003713:Dendritic Cells / Q000166:cytology / Q000187:drug effects / Q000276:immunology*; D003907:Dexamethasone / Q000494:pharmacology; D051858:Forkhead Transcription Factors / Q000378:metabolism; D006801:Humans; D007108:Immune Tolerance / Q000187:drug effects / Q000276:immunology*; D007371:Interferon-gamma / Q000378:metabolism; D016753:Interleukin-10 / Q000096:biosynthesis; D008070:Lipopolysaccharides / Q000494:pharmacology; D008213:Lymphocyte Activation / Q000187:drug effects; D010641:Phenotype; D020123:Sirolimus / Q000494:pharmacology; D018417:Th1 Cells / Q000166:cytology / Q000187:drug effects / Q000276:immunology / Q000378:metabolism
chemical_list: D000893:Anti-Inflammatory Agents; D015415:Biomarkers; C418974:FOXP3 protein, human; D051858:Forkhead Transcription Factors; D008070:Lipopolysaccharides; D016753:Interleukin-10; D002762:Cholecalciferol; D003907:Dexamethasone; D007371:Interferon-gamma; D020123:Sirolimus