Op9 dll1

OP9 and OP9/DLL1 were purchased from RIKEN BRC. C1R-A∗24:02 was a gift from Dr. Masafumi Takiguchi (Kumamoto University). An autologous LCL was established from peripheral blood of a healthy donor from whom #3-3-WT1-T-iPSCs were established as described previously.⁹ (link) After obtaining the cell lines, frozen stocks were prepared within one to five passages and new stocks were thawed frequently to maintain the original condition. The cell lines were passaged for less than 3 months after receipt or resuscitation. They were also authenticated by morphology, growth rate, and surface phenotype, especially by the expression of HLA class I.

All iPSC lines were cultured with mTeSR1 (StemCell Technologies, Vancouver, BC, Canada, http://www.stemcell.com) on Matrigel (BD Biosciences, San Diego, CA, http://www.bdbiosciences.com) -coated 6-well plates. Cell lines OP9, T98G, U-87, MCF7, BT-474, MDA-MB-453, Daudi, Hep G2, SK-OV-3, SCC-25, Raji, FHs 74 Int, HCT 116, Malme-3, Malme-3M, RPMI 8226, K562, THP-1, SW480, MCF 10A, MRC-5, NCI-H460 [American Type Culture Collection (ATTC), Manassas, VA, http://www.atcc.org] were cultured as recommended by ATCC. Cell line FM-57 [European Collection of Authenticated Cell Cultures (ECACC)] was culture cultured in RPMI 1640 with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, Waltham, MA, http://www.thermofisher.com). Cell line OP9-DLL1 (Riken BRC Cell Bank, Ibaraki, Japan, http://cell.brc.riken.jp/en/) was cultured in MEMα (Thermo Fisher Scientific) with 20% FBS. Frozen human peripheral blood CD14+ monocytes (Lonza, http://www.lonza.com) were thawed and maintained in RPMI 1640 with 10% FBS.

Human peripheral blood mononuclear cell (PBMC)-derived iPSC lines were generated as reported previously [11 , 22 (link)]. iPSCs were cultured with mTeSR1 (StemCell Technologies, Vancouver, BC, Canada) on six-well plates coated with Matrigel (BD Biosciences, Franklin Lakes, NJ) and mechanically passaged every 7 days by treating with 1 mg/ml Dispase (StemCell Technologies) at 37 °C for 5 min. Murine bone marrow-derived stromal cell line OP9-DLL1 (Riken BRC Cell Bank, Ibaraki, Japan) was cultured in Minimum Essential Medium α (MEM α) (Gibco, Waltham, MA) supplemented with 20% foetal bovine serum (FBS, Gibco). Tumour cell lines including breast ductal carcinoma cell line BT474 (ATCC HTB-20), breast metastatic carcinoma cell line MDA-MB-453 (ATCC HTB-131), breast adenocarcinoma cell line MCF7 (ATCC HTB-22) and breast ductal metastatic carcinoma cell line MDA-MB-435S (ATCC HTB-129) were cultured as recommended by ATCC. Primary NK cells were expanded from fresh PBMCs by co-culturing with inactivated modified K562 feeder cells as described previously [23 (link)]. NK cells were harvested as population of over 90% CD3-CD56+ cells after the co-culture.