MUTZ-3, a human cell line model for the cytokine-induced differentiation of dendritic cells from CD34+precursors by Allan J. Masterson, Claudia C. Sombroek, Tanja D. de Gruijl, Yvo M. F. Graus, Hans J. J. van der Vliet, Sinéad M. Lougheed, Alfons J. M. van den Eertwegh, Herbert M. Pinedo, and Rik J. Scheper Blood Volume 100(2):701-703 July 15, 2002 ©2002 by American Society of Hematology

MUTZ-3 DCs acquire characteristics of immature and mature DCs in the presence of cytokines.(A) Phenotype of unstimulated MUTZ-3, MUTZ-3 iDCs, and TNFα hi, matured MUTZ-3 mDCs. MUTZ-3 DCs acquire characteristics of immature and mature DCs in the presence of cytokines.(A) Phenotype of unstimulated MUTZ-3, MUTZ-3 iDCs, and TNFα hi, matured MUTZ-3 mDCs. Numbers refer to the percentage of positive cells stained for each CD marker. All cells were stained with PE- or FITC-labeled antigen-specific mAbs. Data are from one experiment representative of 5. (B) FACS analysis reveals up-regulation of costimulatory molecules CD80, CD86, and CD40, the adhesion molecule CD54, and HLA class II molecule HLA-DR during MUTZ-3 differentiation, unstimulated MUTZ-3 (dotted line), MUTZ-3iDC (solid line) and MUTZ-3 mDC (bold line). Isotype controls are represented by markers. Data are from one experiment representative of 5. (C) TGFβ1 induces the expression of LC-associated surface molecule langerin on MUTZ-3 cells. CD34+ MUTZ-3 cells were cultured in GM-CSF/TNFα followed by further culture in the presence or absence of TGFβ1. Numbers refer to the percentage of gated cells expressing both CD1a and langerin or background staining from an isotype control. Data are from one experiment representative of 3. (D) Morphology of MUTZ-3 mDCs is consistent with dendritic cell appearance (× 400, light microscopy). Allan J. Masterson et al. Blood 2002;100:701-703 ©2002 by American Society of Hematology

MUTZ-3 possesses functional antigen processing and presentation pathways.(A) MHC class I presentation. MUTZ-3 possesses functional antigen processing and presentation pathways.(A) MHC class I presentation. MUTZ-3 iDCs stimulate flu-specific cytotoxic T lymphocyte (CTL) responses by presentation of HLA-A2.1–restricted flu peptides. (Ai) MUTZ-3 DCs were loaded with the HLA-A2.1 binding peptide derived from the haeminfluenza matrix protein (M158-66) and cocultured with CD8+ T cells. The production of IFN-γ by CTLs (effector cell concentrations ranging from 0.5 × 105 to 0.125 × 105 per well) cocultured with M1 flu peptide–loaded (■) or HPV 16 E7–derived peptide-loaded T2 cells (□) as target cells (0.1 × 105per well) is shown. (Aii) Alternatively, MUTZ-3 DCs were infected with a recombinant adenovirus containing the M1 matrix protein gene and cocultured as described. CTLs were then restimulated overnight with M1 flu peptide–loaded (●) or HPV 16 E7–derived peptide-loaded T2 cells (○). Data are from one experiment representative of 3. (B) MHC class II antigen presentation. MUTZ-3 mDCs process and present the peptides derived from the common recall antigen TT, and stimulate TT-specific CD4+ T cells. Data are from one experiment representative of 3. (C) Presentation of alpha-GalCer via CD1d. MUTZ-3 iDCs were loaded with alpha-GalCer or vehicle control (dimethyl sulfoxide [DMSO]) and cultured in the absence or presence of TNFα hi for 48 hours. mDCs were then cocultured for 9 days with NKT cells isolated from healthy donors in the presence of IL-7 (10 ng/ mL) and IL-15 (10 ng/mL) and with or without CD1d51 blocking antibody. Results show the relative yield of NKT cells in vehicle and alpha-GalCer–loaded MUTZ-3 iDCs and mDCs with or without blocking of alpha-GalCer presentation using an anti–CD1d blocking antibody. Data are from one experiment representative of 3. Allan J. Masterson et al. Blood 2002;100:701-703 ©2002 by American Society of Hematology