Complex evaluation of human monocyte-derived dendritic cells for cancer immunotherapy

Abstract

Dendritic cell (DC) immunotherapy is capable of generating tumour-specific immune responses. Different maturation strategies were previously tested to obtain DC capable of anti-cancer responses in vitro, usually with limited clinical benefit. Mutual comparison of currently used maturation strategies and subsequent complex evaluation of DC functions and their stimulatory capacity on T cells was performed in this study to optimize the DC vaccination strategy for further clinical application. DC were generated from monocytes using granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4, pulsed with whole tumour cell lysate and then matured with one of five selected maturation strategies or cultured without additional maturation stimulus. DC were characterized with regard to their surface marker expression, cytokine profiles, migratory capacity, allogeneic and autologous T cell stimulatory capacity as well as their specific cytotoxicity against tumour antigens. We were able to demonstrate extensive variability among different maturation strategies currently used in DC immunotherapeutic protocols that may at least partially explain limited clinical benefit of some clinical trials with such DC. We identified DC matured with interferon-γ and lipopolysaccharide as the most attractive candidate for future clinical trials in cancer immunotherapy.

Fig. 1

DC surface markers and viability. Immature DC were differentiated from monocytes by 6-day culture in the presence of GM-CSF and IL-4. Maturation was induced by TNF-α (DC1); TNF-α, IL-1α, IL-6, PGE2 (DC2); TNF-α, IL-1β, IFN-γ, PGE2, R848 (DC3); IFN-γ, LPS (DC4) or IFN-γ, R848 (DC5). DC6 is a control using no maturation cocktail. Expression of CD80, CD83, CD86, HLA-DR, CD14 and cell viability were evaluated by flow cytometry after 48 hrs in the gate of cells with high FSC/SSC. (A) Data are presented as the median (▪), 25–75% quantiles (box), and non-outlier range (whiskers) of 17 donors. Marker ▲ indicates significant difference from all groups not indicated by this marker, P < 0.05, Wilcoxon matched pair test. (B) Representative gating strategy and representative histograms for particular tested parameters are shown.

Fig. 2

DC cytokine profiles. DC were matured with TNF-α (DC1); TNF-α, IL-1α, IL-6, PGE2 (DC2); TNF-α, IL-1β, IFN-γ, PGE2, R848 (DC3); IFN-γ, LPS (DC4); IFN-γ, R848 (DC5) or were cultured without maturation (DC6). Box plots represent (A) production of IL-12p70 and IL-10 after 24 hrs of maturation, and (B) the ratio of IL-12p70/IL-10 production. Data are presented as the median (▪), 25–75% quantiles (box), and non-outlier range (whiskers) of 17 donors. Marker ▲ indicates significant difference from all groups not indicated by this marker, P < 0.05, Wilcoxon matched pair test.

Fig. 3

DC adherence to plastic tested in a ‘scratch assay’. DC were matured with TNF-α (DC1); TNF-α, IL-1α, IL-6, PGE2 (DC2); TNF-α, IL-1β, IFN-γ, PGE2, R848 (DC3); IFN-γ, LPS (DC4); IFN-γ, R848 (DC5) or were cultured without maturation (DC6). Scratch assay was performed after 24 hrs of maturation. The contrast of all pictures has been increased artificially and equally to facilitate the observation of the differences among DC types. (A) Scratch assay, 0 hr: a scratch without cells created in a DC monolayer by a pipette tip. The scratch is marked with an arrow on its both ends. (B) Scratch assay, after 6 hrs: adherent cells do not fill in the scratch, whereas migrating cells do. Each scratch is marked with an arrow. Pictures represent results obtained from four different donors, magnification of 400.

Fig. 4

DC in vitro migratory capacity. DC were matured with TNF-α (DC1); TNF-α, IL-1α, IL-6, PGE2 (DC2); TNF-α, IL-1β, IFN-γ, PGE2, R848 (DC3); IFN-γ, LPS (DC4); IFN-γ, R848 (DC5) or were cultured without maturation (DC6). Box plots represent (A) spontaneous DC migration (no CCL21 added), (B) migration towards 100 ng/ml CCL21, (C) the ratio of CCL21-induced migration/spontaneous migration and (D) CCR7 expression by DC. Data are presented as the median (▪), 25–75% quantiles (box), and non-outlier range (whiskers) of three (A, B, C) or five (D) independent experiments, two donors per group. Marker ▲ indicates significant difference from all groups not indicated by this marker, P < 0.05, Wilcoxon matched pair test.

Fig. 5

Autologous and allogeneic T cell proliferation in a mixed leucocyte reaction. DC matured with TNF-α (DC1); TNF-α, IL-1α, IL-6, PGE2 (DC2); TNF-α, IL-1β, IFN-γ, PGE2, R848 (DC3); IFN-γ, LPS (DC4); IFN-γ, R848 (DC5) or without maturation (DC6) were mixed with CFSE-labelled (A) autologous or (B) allogeneic T cells. Proliferation of total (CD3⁺), helper (CD3⁺CD4⁺) and cytotoxic (CD3⁺CD8⁺) T cells was analysed by flow cytometry after 6 days of co-cultivation. CFSE-negative cells were considered as proliferating. The results are shown for DC:T cell ratio of 1:5. Data are presented as the median (▪), 25–75% quantiles (box) and non-outlier range (whiskers) of (A) 17 or (B) 6 donors. Marker ▲ indicates significant difference from all groups not indicated by this marker, P < 0.05, Wilcoxon matched pair test. (C) Representative gating strategies and representative histograms for particular tested parameters are shown.

Fig. 6

Cytokine production of autologous and allogeneic T cells activated by DC. DC matured with TNF-α (DC1); TNF-α, IL-1α, IL-6, PGE2 (DC2); TNF-α, IL-1β, IFN-γ, PGE2, R848 (DC3); IFN-γ, LPS (DC4); IFN-γ, R848 (DC5) or without maturation (DC6) were mixed with non-stained (A) autologous or (B) allogeneic T cells in a DC:T cell ratio of 1:5. Production of IFN-γ and TNF-α by activated T cells was measured with cytometric bead array. Data are presented as the median (▪), 25–75% quantiles (box), and non-outlier range (whiskers) of at least six donors. Marker ▲ indicates significant difference from all groups not indicated by this marker, P < 0.05, Wilcoxon matched pair test.

Fig. 7

Cytotoxic effect of autologous and allogeneic T cells induced by DC. DC were loaded with a tumour lysate of a T98G tumour cell line and matured with TNF-α (DC1); TNF-α, IL-1α, IL-6, PGE2 (DC2); TNF-α, IL-1β, IFN-γ, PGE2, R848 (DC3); IFN-γ, LPS (DC4); IFN-γ, R848 (DC5) or cultured without maturation (DC6). Autologous or allogeneic T cells, co-cultured with those DC for 6 days, were mixed in a ratio 10:1 with CFSE-labelled fresh tumour cells. Those were target cells T98G, or control cells HCT116 and RPMI-8226 (as indicated in the figure legend). After 24 hrs of co-incubation, dead tumour cells were quantified on a flow cytometer. Results for cytotoxicity induced by (A) autologous and (B) allogeneic T cells are shown. Data are presented as the median (▪), 25–75% quantiles (box), and non-outlier range (whiskers) of at least 6 donors for T98G and two donors for both HCT116 and RPMI-8226 cell lines. Marker ▲ indicates significant difference from all T98G groups not indicated by this marker, P < 0.05, Wilcoxon matched pair test.