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.
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.


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)-gamma, IFN-alpha, IL-1beta, tumor necrosis factor-alpha, 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 x 10(9) +/- 0.65 x 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%).


CONCLUSIONS
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.
Dipartimento di Patologia Clinica, Servizio di Immunoematologia e Trasfusionale, Unità di Aferesi e Nuove Tecnologie Trasfusionali, Ospedale San Gerardo de' Tintori, Monza (MI), Italy. p.perseghin@hsgerardo.org
Paolo
P
Perseghin
Giovanna
G
D'Amico
Erica
E
Dander
Giuseppe
G
Gaipa
Maria
M
Dassi
Ettore
E
Biagi
Andrea
A
Biondi
D017951:Antigen Presentation; D000956:Antigens, Viral / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology* / Q000276:immunology / Q000821:virology; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology* / Q000276:immunology / Q000821:virology; D018929:Cell Culture Techniques; D002460:Cell Line; D003586:Cytomegalovirus Infections / Q000276:immunology*; D003713:Dendritic Cells / Q000166:cytology; D000939:Epitopes; D006801:Humans; D018189:Immunomagnetic Separation / Q000295:instrumentation* / Q000379:methods; D007937:Leukapheresis; D009000:Monocytes / Q000166:cytology*
D000956:Antigens, Viral; D000939:Epitopes