Interleukin 2 (il 2)

Human peripheral blood monocyte cells (PBMCs) from a healthy donor were isolated on Histopaque (1.077 g/ml, Sigma Aldrich, USA) density gradient centrifugation and resuspended in DMEM supplemented with 10% FBS, penicillin (100 IU/mL) and streptomycin (100μg/ml). For mixed lymphocyte reactions (MLR), 0.4μM pore size transwells (Greiner Bio-One, Germany) were used. To induce T cell proliferation, transwell plates were coated with anti-CD3 antibody (5μg/ml, AbD Serotec, UK). Then, PBMCs (2.5x10⁶) were resuspended in 500μl culture medium containing anti-CD28 antibody (1μg/ml, AbD Serotec, UK) and IL-2 (5U, Sino Biological, China) and added in the lower chamber. Control experiments were performed in the absence of stimulants. MSCs (0.5x10⁶) were resuspended in 200μl culture medium and placed onto the upper chamber of transwells. Cells were cultured for three days. Then, cell viability and total cell number or PBMCs in the lower chamber was measured using the Cyto Tox-Glo cytotoxicity kit (Promega, USA), according to manufacturer’s instructions.

The TLR7/8 agonists, D018, C008, 199–1, WA86, C142, B-130a, B-130b, WA175, C163 were synthesized by Zhisong Wang in Xuebin Liao’s Lab. R848, GS9620, and VTX2337 were purchased from MedChemExpress (MCE). IL-2, IL-4, GM-CSF, TNF-α and IFN-α were purchased from Sino Biological. BAY 11–7,082 was purchased from Sigma-Aldrich.

Peripheral blood mononuclear cells (PBMCs) were isolated from fresh peripheral blood of healthy donors by Ficoll-Paque density gradient medium (TBDSCIENCE, Tianjin, China), and then T cells were separated by CD3 positive selection kit (STEMCELL, Canada) in accordance with the manufacturer instructions. T cells were seeded at 1 × 10⁶/mL in X-vivo serum free medium (LONZA, Basel, Switzerland) supplemented with IL-2 (500 IU/mL, Sino Biological, Beijing, China) and activated with Human CD3/CD28 T Cell Activator (STEMCELL) for 24 h. After that, CD19-CAR T and CD19-s47-CAR T cells were obtained by transfecting with the aforementioned viruses at an MOI of 10 in the presence of polybrene (10 µg/mL), and the transfection rate of CAR T cells was detected 3 days after transfection by anti-CAR antibody CARGREEN (Yake, Shanghai, China). About 7 to 14 days after transfection, CAR T cells were collected for follow-up experiments.

The CD8⁺ T cell fractionation samples were derived from the aforementioned peripheral blood mononuclear cells (PBMC) extraction. Following the operational steps of the sorting reagent kit (STEMCELL, Canada), positive selection was conducted using a magnetic bead-based approach in conjunction with CD8 antibodies. The sorted CD8⁺ T cells were activated for 48 h using CD3/CD28 antibodies (STEMCELL, Canada), and subsequently, cultivation was continued in a serum-free medium (Lonza, Switzerland) supplemented with 20 ng/ml IL-2 (SinoBiological, China). We employed a CD8⁺ T cell to tumor cell ratio of 5:1, and after co-incubation for 48 h, cell collection was performed to analyze the functionality of CD8⁺ T cells and apoptosis of tumor cells. In the analysis of tumor cell apoptosis, a DIR (Beyotime, China) membrane stain was applied to the tumor cells.

The HCT116 cell line was purchased from ATCC and cultured with RPMI 1640 medium (Gibco) containing 10% fetal bovine serum (FBS, BI Industry). CD8⁺T-cells were isolated from peripheral blood mononuclear cells (PBMCs) and stimulated by IL2 (SinoBiological, Catalog Number: GMP-11848-HNAE-B) and OKT3 (BD Pharmingen, Catalog Number: 566685) as described in our previous work [14 (link)]. Effector T-cells were cocultured with HCT116 cells for 48 hours in a 96-well plate. The cells were incubated at 37°C with 5% CO₂ or hypoxic conditions (1% O₂, BioSpherix). Specific lactic dehydrogenase (LDH) released from tumor cells in cell-free supernatant was detected using a cytotoxicity LDH detection kit (Genmed), following the manufacturer's instructions. The amount of LDH released was used to assess the lysis of target cells, which can be translated into the lethal effect of effector cells. Percent cytotoxicity was calculated according to OD values using the following formula:
Cytotoxicity % = (Experimental-Effectorspontaneous-Target spontaneous)/(Target maximum-Target spontaneous)×100%.

The CD19-CAR retrovirus was kindly provided from Professor Jianxun Wang. CAR consisted of an extracellular single chain variable fragment (scFv) specific for CD19, followed by a CD8 hinge-transmembrane domain, CD28 costimulatory domain and CD3z intracellular signaling domain. The CAR-T cells were generated by retrovirus infection. In detail, after informed consent was obtained, whole blood samples derived from healthy donors were collected for isolating peripheral blood mononuclear cells (PBMCs) by Ficoll density gradient centrifugation (Lymphoprep, Stemcell, Canada). Then, OKT3 (soluble anti-human CD3 antibody) and IL-2 obtained from Sino Biological were added into the AIM-V medium (Gibco, USA) for the activation of PBMCs at the final concentrations of 100 ng/mL and 100 U/mL, respectively. 48 h later, activated T cells were infected with retroviral vectors carrying CD19 CAR by use of the spin inoculation method [36 (link)]. Finally, The CAR-T cells were expanded and the transduction efficiency was evaluated by flow cytometric analysis (CytoFLEX, Beckman Coulter, USA). Briefly, CAR-T cells were stained with PE-conjugated anti-myc (Invitrogen, USA) antibody, and FACS data was analyzed by CytoFLEX Software (Beckman Coulter, USA) and FlowJo (Version 10.0.7).