MUTZ-3 cells
(ACC-295, DSMZ) were provided by Prof. T. de Gruijl (Amsterdam UMC,
The Netherlands). Cells were maintained at a cell density of 0.5–1
× 106 cells/mL in 12-well tissue culture plates (Corning)
in MEM-alpha (Gibco) with 20% FBS (Hyclone), 1% glutaMAX (Gibco),
10% spent medium from the renal carcinoma cell line 5637 (ACC-35,
DSMZ) and 100 U/mL penicillin–streptomycin (Gibco). Cells were
routinely cultured at 37 °C with 5% CO2. Differentiation
of MUTZ-3 cells into MUTZ-3-derived LCs (muLCs) was performed according
to described protocols.52 (link),53 (link) In short, MUTZ-3 cells
were differentiated in the presence of 100 ng/mL GM-CSF (Genway Biotech),
10 ng/mL TGF-β (R&D Systems), and 2.5 ng/mL TNF-α
(R&D Systems) for 11 days. Twice a week, half of the medium was
replaced with fresh medium and double concentration of cytokines.
To verify the differentiated muLC phenotype, cells were analyzed by
flow cytometry for expression of CD207 (clone DCGM4, Beckman Coulter)
and CD1a (clone Hl149, BD Biosciences) as well as the absence of CD34
(clone 581, BD Biosciences).
THP-1 cells, transfected with a
lentiviral human langerin construct or empty vector, were cultured
in RPMI-1640 (Lonza) supplemented with 10% heat-inactivated FBS and
100 U/mL penicillin-streptomycin (Gibco) as described in.21
(ACC-295, DSMZ) were provided by Prof. T. de Gruijl (Amsterdam UMC,
The Netherlands). Cells were maintained at a cell density of 0.5–1
× 106 cells/mL in 12-well tissue culture plates (Corning)
in MEM-alpha (Gibco) with 20% FBS (Hyclone), 1% glutaMAX (Gibco),
10% spent medium from the renal carcinoma cell line 5637 (ACC-35,
DSMZ) and 100 U/mL penicillin–streptomycin (Gibco). Cells were
routinely cultured at 37 °C with 5% CO2. Differentiation
of MUTZ-3 cells into MUTZ-3-derived LCs (muLCs) was performed according
to described protocols.52 (link),53 (link) In short, MUTZ-3 cells
were differentiated in the presence of 100 ng/mL GM-CSF (Genway Biotech),
10 ng/mL TGF-β (R&D Systems), and 2.5 ng/mL TNF-α
(R&D Systems) for 11 days. Twice a week, half of the medium was
replaced with fresh medium and double concentration of cytokines.
To verify the differentiated muLC phenotype, cells were analyzed by
flow cytometry for expression of CD207 (clone DCGM4, Beckman Coulter)
and CD1a (clone Hl149, BD Biosciences) as well as the absence of CD34
(clone 581, BD Biosciences).
THP-1 cells, transfected with a
lentiviral human langerin construct or empty vector, were cultured
in RPMI-1640 (Lonza) supplemented with 10% heat-inactivated FBS and
100 U/mL penicillin-streptomycin (Gibco) as described in.21