Authors: Jones, K. L.; Brown, L. E.¹; Eriksson, E. M. Y.¹; Ffrench, R. A.; Latour, P. A.; Loveland, B. E.; Wall, D. M.²; Roberts, S. K.³; Jackson, D. C.¹; Gowans, E. J.

Source: Journal of Viral Hepatitis, Volume 15, Number 10, October 2008 , pp. 761-772(12)

Publisher: Wiley-Blackwell

Abstract:

Summary. 

Serum-free culture conditions to generate immature human monocyte-derived DC (Mo-DC) were optimized, and the parameters that influence their maturation after exposure to lipopeptides containing CD4⁺ and CD8⁺ T-cell epitopes were examined. The lipopeptides contained a single CD4⁺ helper T-cell epitopes, one of a number of human leucocyte antigen (HLA)-A2-restricted cytotoxic T-cell epitope and the lipid Pam2Cys. To ensure complete maturation of the Mo-DC, we examined (i) the optimal lipopeptide concentration, (ii) the optimal Mo-DC density and (iii) the appropriate period of exposure of the Mo-DC to the lipopeptides. The results showed that a high dose of lipopeptide (30 μm) was no more efficient at upregulating maturation markers on Mo-DC than a low dose (6 μm). There was an inverse relationship between Mo-DC concentration and the mean fluorescence intensity of maturation markers. In addition, at the higher cell concentrations, the chemotactic capacity of the Mo-DC towards a cognate ligand, CCL21, was reduced. Thus, high cell concentrations during lipopeptide exposure were detrimental to Mo-DC maturation and function. The duration of exposure of Mo-DC to the lipopeptides had little effect on phenotype, although Mo-DC exposed to lipopeptides for 48 rather than 4 h showed an increased ability to stimulate autologous peripheral blood mononuclear cells to release interferon-γ in the absence of exogenous maturation factors. These findings reveal conditions for generating mature antigen-loaded DC suitable for targeted immunotherapy.

Keywords: closed system; dendritic cells; free medium; hapatitis C virus; lipopeptides; serum

Document Type: Research article

DOI: http://dx.doi.org/10.1111/j.1365-2893.2008.01003.x

Affiliations: 1: Department of Microbiology and Immunology, The University of Melbourne, Parkville 2: Centre for Blood Cell Therapies, Peter MacCallum Cancer Institute 3: Department of Gastroenterology, The Alfred Hospital, Melbourne, Australia

Publication date: 2008-10-01

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