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Isolation of monocytes from leukapheretic products for large‐scale GMP‐grade generation of cytomegalovirus‐specific T‐cell lines by means of an automated elutriation device
Author(s) -
Perseghin Paolo,
D'Amico Giovanna,
Dander Erica,
Gaipa Giuseppe,
Dassi Maria,
Biagi Ettore,
Biondi Andrea
Publication year - 2008
Publication title -
transfusion
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.045
H-Index - 132
eISSN - 1537-2995
pISSN - 0041-1132
DOI - 10.1111/j.1537-2995.2008.01756.x
Subject(s) - monocyte , cd8 , peripheral blood mononuclear cell , immunology , cd14 , biology , microbiology and biotechnology , antigen , cytokine , granulocyte macrophage colony stimulating factor , tumor necrosis factor alpha , immune system , in vitro , biochemistry
BACKGROUND: Dendritic cells (DC) act as antigen‐presenting cells in immune response–mediated mechanisms against malignant cells and/or viral or fungal pathogens. CD14+ monocytes have been so far isolated by techniques of plastic adherence or by using immunomagnetic methods. Here the effectiveness of a commercially available cell separation system (Elutra, Gambro BCT) in the separation of monocytes and the large‐scale production of cytomegalovirus (CMV)‐specific T‐cell lines were investigated. STUDY DESIGN AND METHODS: Six mononuclear cell (MNC) collections were processed with the Elutra system. Monocyte‐enriched fraction was differentiated into DCs by addition of granulocyte‐macrophage–colony‐stimulating factor and interleukin (IL)‐4. After 6 days of culture, DCs were matured in the presence of interferon (IFN)‐γ, IFN‐α, IL‐1β, tumor necrosis factor‐α, and poly(I:C) and pulsed with a pool of 48 MHC Class I and II–binding CMV peptides. Lymphocytes were then stimulated with mature autologous CMV peptide–pulsed DCs. RESULTS: After elutriation, the mean monocyte yield was 0.89 × 10 9 ± 0.65 × 10 9 , with a 51.0 ± 31.6 percent recovery and a 51.1 ± 35.4 percent purity. A significant correlation was observed when basal monocyte content was related to the postelutriation recovery (p < 0.0116). More than 60 percent of plated monocytes were differentiated into DCs, which after pulsing with CMV peptides, were able to stimulate a robust enrichment in CMV antigen–specific T cells in all tested samples (mean percentage of pentamer‐positive CD8+ cells, 35% compared to the initial 2%). CONCLUSION: Our findings might be helpful for an appropriate MNC collection, to maximize the efficiency of the elutriation system and subsequently obtain an optimal monocyte‐enriched yield for further DC generation and T‐cell stimulation.