4.1 Reagents and mice

The reagents used in T lymphocytes isolation were: RosetteSep™ Human T Cell Enrichment Cocktail (CAS#15021). The reagents for cytokines assay were used human ELISA kit: IL-1β (CAS: BMS224-2), IL-6 (CAS: EH2IL6), TNF-σ (CAS: KAC1751), IFN-γ (CAS: KHC4021), IL-12 (CAS: KHC0121), Perforin (PFP) (CAS: ab46068), Granzyme B (GZMB) (CAS: BMS2027). The CD28 functional monoclonal antibody (Invitrogen, CAS:16–0289-85). The mice used in investigation were the immune-deficient mouse NOD.Cg-PrkdcscidIL2rgtm1Sug/JicCrl (NOG) and purchased from the Beijing Vitalstar Biotechnology Co.,Ltd.

4.2 Cell lines and cell culture

The lung cancer cell (A549), human umbilical vein vascular endothelial cells (HUVEC) and human fetal lung fibroblast 1 (HEL1) were harvested from ATCC, which had been authenticated and free of mycoplasma, and together with these single clones were cultured in DMEM completed medium (DMEM + 10% FBS + 1% P/S) at 37℃ 5%CO2 cell incubators.

4.3 CD3+ total T lymphocytes isolation and cell culture

The CD3+ total naïve-T lymphocytes were isolated and purified directly from human peripheral whole blood by negative selection using the RosetteSep™ Human T Cell Enrichment Cocktail (CAS#15,021). The peripheral whole blood drawn into sodium heparin blood collection tubes and transferred to the 50 mL tube, then added the RosetteSep™ Human T Cell Enrichment Cocktail to the sample by 50μL/mL of sample. Mixed and incubated for 10 min at room temperature, then added equal volume recommended medium (PBS with 2%FBS) to dilute sample. Added 15 ml density gradient medium (CAS#07801) to the SepMate™ 50 tube (REF86450) and centrifuged it. Transferred the sample up to the density gradient medium gently and centrifuged it by 1200g for 10 min at room temperature. Poured supernatant into a new tube, then washed the enriched cells by recommended medium and centrifuged it by 300g for 10 min for twice. The enriched cells were the CD3+ T lymphocytes and cultured in DMEM 1640 + 10% FBS + 1% P/S + IL-2 (200U/ml) at 37℃ 5% CO2 cell incubator.

4.4 Generation of tumor sphering cell by clonal formation

The tumor sphering cells of lung cancer cell A549 were harvested by the clonal formation assay. The A549 cells (2*104cell/6-well-plate) were mixed in the 0.3% soft agar (1 ml), then put on the 0.8% soft agar (1 ml) and supplied with DMEM completed medium (2 ml), after 10 day, the sphering cells were harvested by the clonal picking.

4.5 The generation of the green fluorescent protein (GFP) cell

The tumor sphering cells, together with HUVEC and HEL1 were infected with lentivirus that stable expressed green fluorescent protein (GFP) for twice. Then, most of these cells could be conveniently observed and distinguished in the co-culture system by the fluorescence microscope.

4.6 Co-culture of tumor sphering cells and naïve-T cells

The tumor sphering cells of A549 (1*104cells/24-well-plate) and naïve-T cells (2*105cells) were mixed and put into 24-well-plate. Then, the CD28 functional monoclonal antibody was added at concentration of 100 ng/ml, and the same volume of PBS (PH7.2) acted as the control groups. The phenotypic observation was performed by the fluorescence microscope for 3-5 day. The cell viability assay of tumor cells was carried out by the MTS, and the supernatant medium was centrifuged and measured the effector cytokines.

4.7 Measurement of the cytokines

The cytokines: IL-1β (CAS: BMS224-2), IL-6 (CAS: EH2IL6), TNF-α (CAS: KAC1751), IFN-γ (CAS: KHC4021), IL-12 (CAS: KHC0121), Perforin (PFP) (CAS: ab46068), Granzyme B (GZMB) (CAS: BMS2027) was measured according to the ELISA kit described method from the supernatant.

4.8 Establishment of the single cell clone from tumor sphering cells

The single cell clones of A549 sphering cells were picked by the limited dilution method. The A549 sphering cells were limited diluted in medium (ratio: 1cell/well) and put into 96-well-plate, then, picked the single cell wells for further culture. At 15 day, these single cell clones were again limited diluted in medium (ratio: 1cell/well) and put into 96-well-plate, and choose the single cell wells for expansion.

4.9 Observation of scanning electron microscopic

The clone (F2-H3) cells (1*104cells/24-well-plate) together with naïve-T cells (2*105cells) were mixed and put on conductive silicon wafer, in which the CD28 functional monoclonal antibody (100 ng/ml) and the PBS (PH7.2) was added, respectively, and cultured in 24-well-plate for 2 day. Then, the sections were fixed, dehydrated and dried as the demands of scanning electron microscopic for further observation.

4.10 The flow cytometry sorting

The clone (F2-H3) cells (1*104cells/24-well-plate) together with naïve-T cells (2*105cells) were mixed and put into 24-well-plate, in which the CD28 functional monoclonal antibody (100 ng/ml) and the PBS (PH7.2) was added, respectively, and cultured for 2 day. Then, harvested all the cells for flow cytometry sorting. According to the GFP, the mix could be divided into GFP+ cells (survival cancer cell) and GFP− cells (T-cells), then, the GFP− cells further separated into activated T-cells and inactivated T-cells by cell size and cell volume.

4.11 The secondary killing test

The single cell clone (F2-H3) (1*104cells/24-well-plate) together with naïve-T cells (2*105cells) were mixed and put into 24-well-plate, in which the CD28 functional monoclonal antibody (100 ng/ml) and the PBS (PH7.2) was added, respectively, and cultured for 2 day. Then, the activated T-cells were harvested by the flow cytometry sorting, and subsequently used to evaluate the secondary killing of 7 single clones (F2-H3, A2-B2, A2-G5, A2-H11, A6-D11, B8-D1, B1-D9) and HUVEC, HEL1 (1*104cells/24-well-plate). The phenotypic observation was performed by the fluorescence microscope for 3 day.

4.12 Anti-tumor evaluation of tumor-specific T-cells in vivo

The immune-deficient mouse NOD.Cg-PrkdcscidIL2rgtm1Sug/JicCrl (NOG) used to test the anti-cancer efficacy in vivo and purchased from the Beijing Vitalstar Biotechnology Co.,Ltd. At the day 0, the clone cells F2-H3 (5*105 cells/mouse) was injected intravenously into the mice (8 weeks old, female). Then, the human peripheral blood T lymphocytes (1*107 cells) were co-cultured with cancer cells (F2-H3) (5*104cells/6-well-plate) supplying the CD28 signal for 48 h, and subsequently the T-cell repertoire was separated into activated group (T-F2-H3) and inactivated group (T- control) by flow cytometry sorting. Then, both group T-cells were further expanded proliferation in vitro supplied with Human T-Activator CD3/CD28 Dynabeads (CAS# 111.61D). At 5 day after engraftment, the T-cells (1*107 cells/mouse, 0.2 ml physiological saline) of both groups were injected intravenously into transplanted mice, and infused again at 10 day. The mice survival, body weight and fur state were recorded and analyzed after treatment.

These peripheral blood samples (10-20 ml) were donated from one healthy donor, and the informed consent was obtained after the nature and possible consequences of the studies were explained. The used mice were in accordance with institutional guidelines of animals' care. We complied with all ethical standards of the Research Ethics Committee of Yunnan University. The sample was isolated and purified using RosetteSep™ Human T Cell Enrichment Cocktail, (CAS#15021), as previous described method.

4.13 Statistics analysis

Differences between two groups were compared using Student’s or Welch’s t-test.