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

Abstract

Background: 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. 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.

Methods: Mo-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.

Results: Monocyte 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.

Conclusions: Our 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.

Keywords: Cellular therapy; Dendritic cell; Fluorinated polymers; Immunotherapy; Monocyte; Polystyrene; Scale-down.

Fig. 1

Overview of cell culture methods for Mo-DC production, phenotypic and functional assessment. a Mo-DC differentiation protocol with maturation in the presence of LPS. b Mo-DC differentiation protocol with NLV peptide (derived from CMV pp65 matrix protein) loading. c T cell and Mo-DC co-culture protocol to determine the ability to generate specific T cell using NLV-loaded DCs produced in two batches (DC-A and DC-B) for T cell re-stimulation

Fig. 2

Mo-DC viability in FEP culture bags and TCPS plates. a LPS treated Mo-DCs were generated in FEP culture bags (of 1 mL, 2 mL or 7 mL volumes) or 24-multiwell TCPS plates for 9 days (except 2 mL data available only for day 2). b Cell viability was assessed before (Start) and after 2, 7 and 9 days of culture in 7 mL FEP bags or TCPS plates. Shown are Tukey's Box-and-whisker plots of 4 to 17 independent experiments per condition. ns: no statistically significant differences for both paired (n ≥ 4) and unpaired comparisons. Using unpaired comparisons, a significantly reduced viability was observed in the 1 mL bags compared to the 2 mL bags (p = 0.02, Tukey HSD test)

Fig. 3

LPS treated Mo-DCs can be efficiently generated in FEP bags and TCPS plates. Flow cytometry was performed before culture (Start) and after 2, 7 and 9 days of differentiation and maturation culture on FEP or TCPS surfaces. Shown are Tukey's Box-and-whisker plots. ns: no statistically significant differences for both paired (n ≥ 4 donors for all surface markers at all time points except n = 3 for CD86 at day 7) and unpaired (n = 14 donors at day 0; n = 4 to 10 donors at other time points except n = 3 for CD86 day 7) comparisons

Fig. 4

Mo-DC cytokine production in vitro. LPS treated Mo-DCs were generated in FEP culture bags or TCPS plates. Levels of IL-10 and IL-12 in cell culture supernatants collected at day 7 and 9 of culture were compared to medium controls. Shown are min–max Box-and-whisker plots with individual data points from n = 6 donors for FEP and n = 4 donors for TCPS. Dashed lines between day 9 data points represent matched measurements from a given donor. ns: no statistically significant differences for both paired and unpaired comparisons

Fig. 5

Impact of pulsed Mo-DCs cultured in FEP bags or TCPS plates on T cell expansion. Following Mo-DC differentiation, 15 × 10⁶ T cells were cultured in the presence of CMVpp65 peptide (NLV)-pulsed Mo-DC at 1:10 T cell: Mo-DC ratio. T cells were enumerated at day 0, 7 and 14. Each data point represents the mean ± SEM of 7 independent experiments, each from an independent donor

Fig. 6

Pulsed Mo-DCs cultured in FEP bags or TCPS plates exhibit the same T cell activation capacity. Following Mo-DC differentiation, 15 × 10⁶ T cells were cultured in the presence of unpulsed or CMVpp65 peptide (NLV)-pulsed Mo-DC at 1:10 T cell: Mo-DC ratio. a, b At day 14, the proportion of NVL-specific T cells was assessed by flow cytometry. c, d The activation status of CD8⁺ T cells from the donor was assessed by the evaluation of CD25 expression. e, f The functionality of CD8⁺ T cells was evaluated through the expression of cytotoxic degranulation marker CD107a upon restimulation with CMVpp65 (NLV) antigen. g, h Granzyme B expression was evaluated upon re-stimulation with CMVpp65 (NLV) antigen. Panels show representative examples (a, c, e, g) or the mean ± SEM of 4 independent experiments (b, d, f, h). P-values shown are for paired comparisons