Dendritic Cells Generated in Clinical Grade Bags Strongly Differ in Immune Functionality When Compared With Classical DCs Generated in Plates Re ´douane Rouas, Haidar Akl, Hussein Fayyad-Kazan, Nabil El Zein, Bassam Badran, Be ´range `re Nowak, Hugues Duvillier, Philippe Martiat, and Philippe Lewalle Summary: Mature dendritic cells (DCs) represent, by far, the most potent antigen-presenting cells. The development of clinical grade techniques to produce them in large numbers has rendered possible their use in clinical trials. It is therefore crucial to assess the DCs characteristics according to the methodology used to generate them, to improve the comparison and standardization of these trials. We thus compared DCs generated and matured in culture plates (pla-DCs) or in clinical grade bags (bag-DCs) by analyzing, their secretion of bioactive interleukin (IL)-12 and their capacity to induce in-vitro primary responses. We also used several molecular techniques to better characterize the functional differences between the 2 type of DCs. Mature bag-DCs displayed a mature phenotype, but did not secrete significant amounts of IL-12 and failed to initiate primary immune responses. Molecular analyses performed on immature bag-DCs showed them already engaged in a particular maturation process (early activation of nuclear factor k B and b-catenin). Using microarrays, we found underexpres- sion of receptors for the maturation cocktail in bag-DCs. In mature bag-DCs, we found crucial genes (IL-12, chemokines, and costimulatory and adhesion molecules) down-regulated. Electro- phoertic mobility shift assay and Western blots showed a normal activation profile in mature pla-DCs, but not in bag-DCs where the Mek/Erk pathway was still activated. Our results strongly suggest that differentiation of monocytes into DCs in bags generates immature DCs already engaged in an inefficient type of activation, with down-regulation of genes involved in response to the maturation cocktail. This results in mature DCs unable to induce T H 1-type responses. Key Words: dendritic cells generation and maturation, bags or plates, functionality (J Immunother 2010;33:352–363) F or dendritic cells (DCs) to be used as a cancer vaccine, it is critical that they can be efficiently loaded with tumor- associated antigens and can elicit specific T H 1 and Tc1 lymphocytes response against these antigens. 1–7 Tumor- associated antigens-specific T cells can be detected in patients, and, albeit unable to properly function in the tumoral microenvironment, are capable to lyse tumor cells in vitro. 8–10 Adoptive cell therapy, by ex-vivo stimulated tumor infiltrating lymphocytes is already efficient in some patients, 11 and its combination with DCs vaccine could be a promising new approach to cancer treatment. Importantly, in the context of allogeneic stem cell transplantation, antitumor specific donor T cells can be ex-vivo expanded to enrich T lymphocytes infusion to increase the graft- versus-tumor effect 12 ; tumor specific T cells can be also detected in normal donors. 13 Nevertheless, most of the lymphocytes that we try to expand ex vivo for cellular immunotherapy, are naive and/or have a low avidity for their cognate antigen, and therefore are expected to be critically dependent on the high levels of costimulatory molecules and cytokines production associated with mature DCs for their expansion. 14–19 For lymphocytes priming, the obtention of mature DCs expressing appropriate matura- tion markers but also capable of secreting large amount of interleukin (IL)-12p70 upon restimulation by CD40L, mimicking the in vivo T cells DCs interaction, is critical. 20–29 Therefore, characterizing the optimal way to generate mature DCs ex vivo, for clinical application is of utmost importance. 30,31 We focused on the comparison of monocytes-derived DCs generated and matured in closed bags (bag-DCs) with DCs generated and matured in plates (pla-DCs). One characteristic of mature DCs is a propensity to adhere to plastic surfaces. Recovery of these cells requires a time-consuming procedure that may compromise cell viability and sterility. To facilitate efficient recovery of DCs and maintain a closed system less vulnerable to microbial contamination, several groups have developed methods for generating and maturing monocytes-derived DCs in gas permeable cell culture bags. The theoretical advantage is that, in contrast to cells cultured in con- ventional polystyrene flasks or plates (pla-DCs), those cultured in bags bind less firmly to container surfaces and therefore can be harvested more easily while being perfectly adequate for clinical grade applications. The ideal way to process these cells in a clinical grade environment is to differentiate monocytes into immature DCs in bags and to perform the maturation and the antigen-loading steps in bags. This results in a completely closed-system procedure, avoiding the risk of patho- gens contamination as much as possible. In phenotypic Copyright r 2010 by Lippincott Williams & Wilkins Received for publication June 13, 2009; accepted October 29, 2009. From the Laboratory of Experimental Hematology, Department of Hematology, Jules Bordet Institute, ULB, Brussels, Belgium. All authors have declared that there are no financial conflicts of interest in regards to this study. Re´douane Rouas and Haidar Akl equally contributed to this study. Philippe Martiat and Philippe Lewalle are the senior co-authors. This study has been rendered possible thanks to the support of the Medic Foundation, Les Amis de l’Institut Bordet, and of the FRSM and Te´le´vie. Reprints: Philippe Martiat, Laboratory of Experimental Hematology, Jules Bordet Institute, ULB, 121, Boulevard de Waterloo, 1000, Brussels, Belgium (e-mail: pmartiat@ulb.ac.be). Supplemental Digital Content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s website, www.immunotherapy.journal.com. BASIC STUDY 352 | www.immunotherapy-journal.com J Immunother  Volume 33, Number 4, May 2010