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The Culture Dish Surface Influences the Phenotype and Cytokine Production of Human Monocyte-Derived Dendritic Cells

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The Culture Dish Surface Influences the Phenotype and Cytokine Production of Human Monocyte-Derived Dendritic Cells

Alexander Sauter et al. Front Immunol.

Abstract

Monocyte-derived dendritic cells (moDC) are an important scientific and clinical source of functional dendritic cells (DC). However, the optimization of the generation process has to date mainly been limited to the variation of soluble factors. In this study, we investigated the impact of the cell culture dish surface on phenotype and cytokine profile. We compared a standard cell culture dish to a non-adherent culture dish for two immunogenic maturation conditions, two tolerogenic conditions, and an unstimulated control. Phenotype, cytokine profile and T cell stimulatory capacity were determined after a 3-day culture. Light microscopy revealed an increase in homotypic cluster formation correlated with the use of non-adherent surfaces, which could be reduced by using blocking antibodies against CD18. All surface markers analyzed showed moderate to strong differences depending on the culture dish surface, including significantly decreased expression of key maturation markers such as CD80, CD86, and CCR7 as well as PD-L1 on cells stimulated with the Jonuleit cytokine cocktail cultured on a non-adherent surface. Significant differences in the secretion of many cytokines were observed, especially for cells stimulated with LPS, with over 10-fold decreased secretion of IL-10, IL12-p40, and TNF-α from the cells cultured on the non-adherent surface. All immunogenic moDC populations showed similar capacity to induce antigen-specific T cells. These results provide evidence that the DC phenotype depends on the surface used during moDC generation. This has important implications for the optimization of DC-based immunotherapy development and underlines that the local surrounding can interfere with the final DC population beyond the soluble factors.

Keywords: adhesion; cytokines; homotypic clusters; immunogen; monocyte-derived dendritic cells; monocytes; non-adherent culture plate; tolerogen.

Figures

Figure 1
Figure 1
Homotypic cell clusters form on the non-adherent surface but less on the standard culture dish. Representative microscopy pictures of iDC (DMSO) at the end of the 3-day culture on (A) a standard cell culture dish and (B) a non-adherent culture dish. The increase in cell cluster formation on non-adherent dishes relative to standard dishes could be observed for all treatments (n = 8).
Figure 2
Figure 2
Influence of the culture dish on the phenotype of differentially stimulated moDC. The phenotype was analyzed by flow cytometry using the indicated surface molecules. % positive cells or median fluorescence intensity (MFI) are shown. Color code: Control (DMSO)—green, immunogenic DC populations (LPS & Jonuleit cytokine cocktail)—red, tolerogenic DC populations (Dexamethasone with vitamin D3 & IL-10)—blue. Squares (left): standard culture dish; circles (right): non-adherent culture dish. The median is marked with a line. Significance values are given in grades *P < 0.05, **P < 0.01, and ***P < 0.001. Black significance grades are the result of a collective 2 way ANOVA testing for all treatments, red grades are from separate tests for the iDC/tolDC and immunogen subgroups (n = 8).
Figure 3
Figure 3
Influence of the culture dish on the cytokine production of differentially stimulated moDC. Cytokines were measured in cell free culture supernatants of the generated moDC populations. Color code: Control (DMSO)—green, immunogenic DC populations (LPS and Jonuleit cytokine cocktail, respectively)—red, tolerogenic DC populations (Dexamethasone with vitamin D3 and IL-10, respectively)—blue. Circles (left): standard culture dish; squares (right): non-adherent culture dish. The median is marked with a line. Significance values are given in grades *P < 0.05, **P < 0.01, and ***P < 0.001. Black significance grades are the result of a collective 2 way ANOVA testing for all treatments, red grades are from separate tests for the iDC/tolDC and immunogen subgroups (n = 6).
Figure 4
Figure 4
Antigen-specific T cell induction is not influenced by the culture dish conditions of the moDC. Autologous PBMC depleted of monocytes were co-cultured with PPD-loaded DC generated on the indicated surface and matured with indicated stimuli for 7 days, and antigen-specific IFN-γ secretion by CD4+ and CD8+ cells was analyzed by flow cytometry with LPS-DC as stimulators (± PPD). Delta LPS: moDC cultured on a conventional culture dish, stimulated with LPS; Hydrocell LPS: moDC cultured on a non-adherent culture dish, stimulated with LPS; Delta Jonuleit: moDC cultured on a conventional culture dish, stimulated with Jonuleit cytokine cocktail (TNF, IL-6, IL-1β, and PGE2); Hydrocell Jonuleit: moDC cultured on a non-adherent culture dish, stimulated with Jonuleit cytokine cocktail. Each color-coded symbol represents results from one individual (n = 9).

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