Closing the system: production of viral antigen-presenting dendritic cells eliciting specific CD8+ T cell activation in fluorinated ethylene propylene cell culture bags

Jean-Philippe Bastien,Vibhuti Dave,David Juncker,Denis-Claude Roy,Ariane V Beland,Veronique Laforte,Corinne A Hoesli,Marie-Paule Lachambre,Natalie Fekete

doi:10.1186/s12967-020-02543-1

Abstract

BackgroundA major obstacle to anti-viral and -tumor cell vaccination and T cell immunotherapy is the ability to produce dendritic cells (DCs) in a suitable clinical setting. It is imperative to develop closed cell culture systems to accelerate the translation of promising DC-based cell therapy products to the clinic. The objective of this study was to investigate whether viral antigen-loaded monocyte-derived DCs (Mo-DCs) capable of eliciting specific T cell activation can be manufactured in fluorinated ethylene propylene (FEP) bags.MethodsMo-DCs were generated through a protocol applying cytokine cocktails combined with lipopolysaccharide or with a CMV viral peptide antigen in conventional tissue culture polystyrene (TCPS) or FEP culture vessels. Research-scale (< 10 mL) FEP bags were implemented to increase R&D throughput. DC surface marker profiles, cytokine production, and ability to activate antigen-specific cytotoxic T cells were characterized.ResultsMonocyte differentiation into Mo-DCs led to the loss of CD14 expression with concomitant upregulation of CD80, CD83 and CD86. Significantly increased levels of IL-10 and IL-12 were observed after maturation on day 9. Antigen-pulsed Mo-DCs activated antigen-responsive CD8+ cytotoxic T cells. No significant differences in surface marker expression or tetramer-specific T cell activating potency of Mo-DCs were observed between TCPS and FEP culture vessels.ConclusionsOur findings demonstrate that viral antigen-loaded Mo-DCs produced in downscaled FEP bags can elicit specific T cell responses. In view of the dire clinical need for closed system DC manufacturing, FEP bags represent an attractive option to accelerate the translation of promising emerging DC-based immunotherapies.

Highlights

A major obstacle to anti-viral and -tumor cell vaccination and T cell immunotherapy is the ability to produce dendritic cells (DCs) in a suitable clinical setting
No significant differences in monocyte-derived DCs (Mo-DCs) concentration or yield were observed at day 9 with the NLV differentiation protocol between the 7 mL fluorinated ethylene propylene (FEP) bags and tissue culture polystyrene (TCPS) plates (Additional file 1: Figure S1)
As observed after the first round of stimulation, CD25 expression was maintained on tetramer-specific CD8+ T cells cultured in presence of NLV-pulsed Mo-DCs whereas nonspecific T cells only displayed a modest increase in CD25 expression (Fig. 6c, d; Additional file 1: Figure S7). These results suggest that Mo-DCs differentiated in TCPS plates or FEP bags display similar capacity to activate and expand antigen-specific T cells

Summary

Introduction

A major obstacle to anti-viral and -tumor cell vaccination and T cell immunotherapy is the ability to produce dendritic cells (DCs) in a suitable clinical setting. DCs have been hypothesized to be the panacea for personalized immunotherapy owing to their unique capacity to activate T cells and initiate the adaptive immune response to eliminate target cells that are transformed or infected with pathogens [2,3,4]. Due to their low frequency of occurrence in peripheral blood, DCs are commonly differentiated ex vivo from monocytes in the presence of cytokines added to culture media, typically interleukin (IL)-4 and macrophage-colony stimulating factor (GM-CSF) [5]. The immature monocyte-derived DCs (Mo-DCs) generated are induced to mature when loaded with peptide antigen, usually in combination with toll-like receptor-activating inflammatory cytokine mixtures of varying composition [6]

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