Human flt3l

Immature dendritic cells (DCs) were obtained from the bone marrow of female Balb/c mice as previously described (Cohen STAT paper). Briefly, bone marrow was harvested from Balb/c mouse femur and tibia, cultured at 5 × 10⁵/ml for 6 days in culture RPMI (RPMI containing 10% FBS, 2 mM glutamine, .1 mM nonessential amino acids, 100 units/ml sodium pyruvate, and 100 mg/ml penicillin/streptomycin) supplemented with 30 ng/ml human Flt-3L and 25 ng/ml murine IL-6 (both from Peprotech, Rocky Hill, NJ). On day 6 cells were harvested, washed 2 times in PBS, resuspended at 2 × 10⁶/ml in culture RPMI supplemented with 50 ng/ml murine GM-CSF and 10 ng/ml of murine IL-4 (both from Peprotech), and incubated overnight. The next day the cells were harvested and frozen down at 2 × 10⁷ in FBS containing 10% DMSO. On day of vaccination cells were thawed, washed once to remove DMSO, and then resuspend at 2 × 10⁶ in culture RPMI containing only 1% FBS and supplemented with murine GM-CSF (50 ng/ml) and IL-4 (10 ng/ml). After 1 hour of incubation the cells were exposed to murine HER-2 peptides (2 class II and 1 class I), incubated for 2 additional hours, activated with 20 ng/ml LPS and 10 ng/ml CpG (Invivogen, San Diego, CA), incubated for 2 more hours, harvested, washed 3 times in PBS, and resuspended in PBS at a concentration of 1 × 10⁷/ml for vaccination.

Thymocyte subpopulations of interest were purified by MACS depletion followed by cell sorting and then plated onto OP9-DL1 monolayers (30 (link)). The OP9-DL1 cells were inactivated with Mitomycin C (Stem Cell) immediately prior to use. 10³ sorted thymocytes were seeded per well in 96-well plates in αMEM (Thermo Fisher) supplemented with 20% FCS (Bovogen Biologicals), penicillin/streptomycin/gentamycin (Sigma), 2 ng/mL murine IL-7 (Peprotech), and 5 ng/mL human FLT3L (Peprotech). The media were refreshed every 2 days and freshly inactivated OP9-DL1 cells were added every 4 days.

Murine bone marrow cells were isolated by flushing femur and tibia of the hind legs of two to three mice. Bone marrow cells from individual animals were pooled for further processing. Mouse bone marrow cells were cultured in DMEM with 10% h.i. FCS (Sigma-Aldrich), 2 mM L-Glutamine (Lonza), 100 U/ml Penicillin and 100 μg/ml Streptomycin (Life Technologies) supplemented with 10ng/ml murine recombinant IL3, 10 ng/ml human recombinant IL6, and 100ng/ml murine recombinant SCF (all Peprotech) (Argiropoulos et al., 2008 (link)).
CD34+ mononuclear cells were isolated from human bone marrow aspirates. Bone marrow aspiration from healthy donors was performed at the Institute of Transfusion Medicine and Immunohematology of Goethe University and German Red Cross Blood Donor Service in Frankfurt. Use of the bone marrow aspirates for research purposes was approved by the Ethics Committee of the University of Frankfurt (329-10) and donors gave written consent for use of the samples. Short term culture of CD34+ bone marrow cells from healthy donors was done in StemSpan™ Serum-Free Expansion Medium (SFEM, Stemcell Technologies) supplemented with 100 ng/ml murine SCF, 100 ng/ml murine TPO, 100 ng/ml human FLT3-L and 100 ng/ml human IL6 (all Peprotech).

A two-stage culture strategy was employed for production of dendritic cells from BM [12 (link)]. Briefly, BM cells were harvested from the femurs and tibia of Balb/c mice and cultured at 5 × 10⁵ cells/mL for 6 days in one of the seven culture media supplemented with 30 ng/mL human Flt-3L and 25 ng/mL murine IL-6 (both from Peprotech, Rocky Hill, NJ, USA) (Stage I). On day 6 cells were harvested, washed two times in phosphate buffered saline (PBS), resuspended in RPMI (without FCS) supplemented with 50 ng/mL murine GM-CSF and 10 ng/mL IL-4 (both from Peprotech), either plated in 12-well dishes for analytic studies or placed in culture flasks for vaccine production, and incubated overnight at 2 × 10⁶ cells/mL (Stage II). The next day, cells were activated by the addition of E. coli K12 lipopolysaccharide (LPS) (20 ng/mL) and CpG motif-containing oligonucleotide ODN1826 (10 ng/mL) (each from In VivoGen, San Diego, CA, USA).