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

Vopenkova K, Mollova K, Buresova I, Michalek J - J. Cell. Mol. Med. (2012)

Bottom Line: 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 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.

View Article: PubMed Central - PubMed

Affiliation: Advanced Cell Immunotherapy Unit, Department of Pharmacology, Faculty of Medicine, Masaryk University, Brno, Czech Republic. katka.skalova@gmail.com

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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.
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fig01: 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.

Mentions: DC were pulsed with tumour lysate to initiate the maturation process and then further matured with different agents (DC1–DC5) or kept without further maturation stimulus (DC6) as shown in Table 1. Significant differences were observed in DC viability, CD80, CD83, CD86 and CD14 expression after 48 hrs of maturation (Fig. 1). DC3 and DC4 expressed higher levels of CD83 and also CD86 than did the four other DC alternatives (P < 0.05). Higher expression of CD80 was observed in DC3, DC4 and DC5 (P < 0.05). No significant differences were observed in HLA-DR expression. As expected, the monocyte marker CD14 was expressed in DC6 (85.3%), but partially also in DC2 (19.9%). Cells treated with other maturation cocktails contained less than 6.0% of CD14+ monocytes (P < 0.05). In summary, the fully mature phenotype (CD80+, CD83+, CD86+, HLA-DR+ and CD14-) was achieved only in DC3 and DC4. Viability over 93% was reached in all DC except of DC5 (87.9% living cells, P < 0.05).


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

Vopenkova K, Mollova K, Buresova I, Michalek J - J. Cell. Mol. Med. (2012)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4118250&req=5

fig01: 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.
Mentions: DC were pulsed with tumour lysate to initiate the maturation process and then further matured with different agents (DC1–DC5) or kept without further maturation stimulus (DC6) as shown in Table 1. Significant differences were observed in DC viability, CD80, CD83, CD86 and CD14 expression after 48 hrs of maturation (Fig. 1). DC3 and DC4 expressed higher levels of CD83 and also CD86 than did the four other DC alternatives (P < 0.05). Higher expression of CD80 was observed in DC3, DC4 and DC5 (P < 0.05). No significant differences were observed in HLA-DR expression. As expected, the monocyte marker CD14 was expressed in DC6 (85.3%), but partially also in DC2 (19.9%). Cells treated with other maturation cocktails contained less than 6.0% of CD14+ monocytes (P < 0.05). In summary, the fully mature phenotype (CD80+, CD83+, CD86+, HLA-DR+ and CD14-) was achieved only in DC3 and DC4. Viability over 93% was reached in all DC except of DC5 (87.9% living cells, P < 0.05).

Bottom Line: 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 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.

View Article: PubMed Central - PubMed

Affiliation: Advanced Cell Immunotherapy Unit, Department of Pharmacology, Faculty of Medicine, Masaryk University, Brno, Czech Republic. katka.skalova@gmail.com

Show MeSH
Related in: MedlinePlus