The human T-ALL cell lines (Jurkat, CCRF-CEM, MOLT-4) and NSCLC cell line NCI-H1299 were cultured in the RPMI 1640 medium supplemented with 10% FBS. The human cervix adenocarcinoma, HeLa cells were cultured in Eagle's minimum essential medium supplemented with 10% FBS and 1% NEAA (10 mM). The breast cancer cell line, MCF-7 cells were cultured in the DMEM supplemented with 10% FBS.
T-ALL reprogramming and preparation of the T-ALL-iPSC lysates
The peripheral blood mononuclear cells (PBMC) from the T-ALL patients and healthy donors were infected with the Cyto Tune-iPS 2.0 Sendai virus reprogramming Kit (Thermo Fisher). The obtained ESC -like clones were identified for pluripotency, using methods like alkaline phosphatase staining, flow cytometry (2.4), and immunofluorescence staining for the pluripotency markers, and teratoma formation. The T-ALL-iPSCs were cultured with mTeSR plus medium (STEMCELL Technologies).
To prepare cell lysates, the iPSCs derived from the healthy cells (H-iPSCs) (1×107/ml) and T-ALL-iPSCs (1×107/ml) were frozen in liquid nitrogen and disrupted by three freeze-thaw cycles. The cell lysates were then spun at 13000g for 20 min, and the supernatants were collected as iPSC antigens. The total protein concentration of the cell lysates was quantified using the BCA Protein Assay Kit (Beyotime Institute of Biotechnology, China).
Generation of the different types of DC-CTLs
The DCs were prepared from the monocytes of the healthy donors. The PBMC were isolated by Lymphoprep (Stemcell, USA) density gradient centrifugation at 800g for 15 min at 20 ℃. After 2 h of incubation, the adherent cells were cultured in an Alys-505 complete culture medium (Zhuhai Baso Cell Science & Technology Co. Ltd, China) supplemented with 100 ng/ml GM-CSF and 30 ng/ml IL-4 induction of DCs. The non-adherent cells were cultured in the Alys-505 complete culture medium containing 1000 U/ml IL-2 and 200 ng/ml CD3 monoclonal antibody for generating the CTLs.
On day 3, the immature DCs were treated with 30µg/ml of different antigens and 1000 U/ml TNF-α. On day 7, the CTLs were co-cultured with autologous DCs at a 5:1 ratio. There were four groups, T group (only CTLs), DC-T group (CTLs were co-cultured with DCs), DCH-iPSC-T group (CTLs were co-cultured with H-iPSC lysates-stimulated DCs), and DCT-ALL-iPSC-T group (CTLs were co-cultured with T-ALL-iPSC lysates-stimulated DCs). All the groups were cultured at 37˚C in a humidified 5%CO2 incubator and passaged every 3 days in an Alys-505 complete culture medium with 1000 U/ml IL-2.
The T-ALL-iPSC were stained with PE anti-human TRA-1-60, APC anti-human SSEA-4 (Biolegend, USA) for 20 min at 4˚C and analyzed by the flow cytometry software. T cells were stained with FITC anti-human CD3, PerCp/Cyanine5.5 anti-human CD4, APC anti-human CD8a, PE anti-human CD25, PE anti-human PD-1, and PE anti-human CD57 (Biolegend, USA) for 20 min at 4˚C and analyzed by the flow cytometry software.
In vitro cytotoxicity assay
The CTLs from each group were examined for the cytotoxic activity toward the target tumor cells by performing a lactate dehydrogenase (LDH) release assay using the LDH cytotoxicity assay kit (Beyotime Institute of Biotechnology). The CTLs and tumor cells were co-cultured at the effector‑to‑target (E/T) ratios of 5:1, 10:1, 20:1 for 24 h. The specific lysis was calculated as follows: Percentage of cytotoxicity= [ (experimental release‑effector spontaneous release-target spontaneous release)] / [(target maximum release-target spontaneous release)] ×100%.
Cytokine Release Assay
The T cells were co-cultured with or without DCs for 24 h. The supernatants from each group were collected for assessing IL-6, IL-13, IFN-γ, and TNF-α (Biolegend Human Th Cytokine Panel (12-plex) with V-bottom Plate V02, USA) using flow cytometry.
CTLs from each group were co-incubated with the target cells at an E:T ratio of 10:1. Following 24 h of incubation at 37˚C, the supernatant was harvested and the concentration of the released IFN-γ was measured using the human IFN-γ Precoated ELISA Kit (Dakewe Bio-engineering Co. Ltd, Shenzhen, China).
The CTLs from each group were co-cultured with the target cells at an E:T ratio of 10:1. The PE anti-CD107a antibody (Biolegend, USA) was included during the culture. After 1 h, 2μM monensin (Biolegend, USA) was added to the co-cultured system and incubated for an additional 4 h, and then the cells were stained with the APC anti-human CD8a and analyzed using flow cytometry. All the CD8+ CD107a+ cells were regarded as the degranulated T cells.
Analysis of RNA-seq data
The human RNA-seq data for the cancer cell lines were downloaded from the Cell Model Passports Database (https://cellmodelpassports.sanger.ac.uk/). The cancer-related genes list was downloaded from the Network of Cancer Genes (http://ncg.kcl.ac.uk/cancer_genes.php). The list of over-expressed genes in T-ALL was screened from the Oncomine's Haferlach Leukemia Dataset. The heatmap was generated using GENE-E.
The GraphPad Prism 5 software was used for data analysis. All data are presented as the mean ± the standard deviations. The one-way ANOVA with Bonferroni’s post hoc test was used to analyze the differences between the groups. P < 0.05 indicates a statistical difference.