Kg1a cells

OCI-AML3 cells (German Collection of Microorganisms and Cell Cultures [DSMZ], Braunschweig, Germany) were cultured in RPMI 1640 medium (#01-100-1ACS; Biological Industries; Kibbutz Beit-Haemek, Israel) containing 20% fetal bovine serum (FBS) (#10270-106; Gibco, Waltham, MA, USA). OCI-AML2 cells (DSMZ) were cultured in MEM-alpha medium (#12561-056, Gibco) containing 20% FBS. U937 (American Type Culture Collection [ATCC], Manassas, VA, USA), HL-60 (ATCC), and THP-1 (ATCC) cells were cultured in RPMI 1640 medium containing 10% FBS. KG-1a cells (ATCC) were cultured in IMDM (#SH30228.01; Hyclone, Logan, UT, USA) supplemented with 20% FBS. Human mesenchymal stem cells (hMSCs, China National Collection of Authentic Cell Cultures [NCACC]) were cultured in a mesenchymal stem cell medium (#7501; ScienCell, Carlsbad, CA, USA) containing 5% FBS. The 293T cell line (ATCC) was cultured in DMEM (#SH30243.01, Hyclone) supplemented with 10% FBS. IMS-M2 cell line was a kind gift from Dr. Li He at Zhongnan Hospital of Wuhan University (Wuhan, China) and cultured in RPMI 1640 medium containing 20% FBS. All cell lines were cultured at 37 °C in a humidified chamber in the presence of 5% CO₂. Penicillin/streptomycin (#SV30010, Hyclone) was added to all cell cultures, except for the medium used for plasmid/siRNA transfection.

KG1a cells, a human acute myelogenous leukemia cell line, purchased from the American Type Culture Collection, were maintained in Iscove’s modified Dulbecco’s medium (IMDM; Gibco) supplemented with 20% fetal bovine serum, penicillin (100 U ml⁻¹), and streptomycin (100 μg ml⁻¹) at 37°C in a humidified atmosphere containing 5% CO₂. For the disruption of actin cytoskeletons, 10⁶ ml⁻¹ cells were treated with cytochalasin D (CytD; 3 μg ml⁻¹; Sigma) in a prewarmed Hanks’ balanced salt solution (HBSS; Gibco) for 30 min at 37°C. For the disruption of lipid rafts, 10⁶ ml⁻¹ cells were treated by a prewarmed 10 mM MβCD (Sigma) and 10 mM Hepes buffer (Sigma) in a serum/antibiotic-free IMDM at 37°C for 30 min. The viability of the cells after the treatments was confirmed by trypan blue staining.

EBV-transformed lymphoblastoid cell lines (LCLs) were made from PBMCs as previously described.17 (link) To generate muromonab-CD3 (OKT3)-expressing LCLs, LCLs were resuspended in nucleofection solution using the Amaxa Nucleofector kit T, OKT3-2A-Hygromycin_pEK plasmid was added to 5 µg/10⁷ cells, the cells were electroporated using the Amaxa Nucleofector I, and the resulting cells were grown in Roswell Park Memorial Institute (RPMI)-1640 with 10% fetal calf serum (FCS) containing 0.4 mg/mL hygromycin.14 (link) To generate enhanced green fluoresent protein (eGFP) +LCLs, LCL cells were transduced with lentiviral vector encoding eGFP and eGFP +cells were purified by FACS sorting and expanded for use in these experiments. KG1a cells were purchased from American Type Culture Collection (ATCC) and transduced with lentiviral vector encoding eGFP. Banks of all cell lines were authenticated for the desired antigen/marker expression by flow cytometry prior to cryopreservation and thawed cells were cultured for less than 6 weeks prior to use in assays. To generate cells capable of stimulating the CMV-CD19CAR T cells via the CMV-specific TCR, autologous PBMCs were loaded with human cytomegalovirus (HCMV) PepMix (pp65pepmix) (>90%) (JPT peptide Technologies GmbH) at 1 µg/mL for 2 hours at 37C°. Cells were washed in PBS twice before use.

Human acute promyelocyte leukemia HL-60 cells and acute myeloid leukemia KG1a cells were purchased from the American Type Culture Collection (ATCC)(Manassas, VA, USA) and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) (Gibco, USA), penicillin (100 U/mL, Gibco), and streptomycin (100 µg/mL, Gibco). These cells were maintained at 37 °C in an incubator with 5% CO₂. Art was purchased from Sigma-Aldrich and dissolved in phosphate-buffered saline (PBS).