Anti cd3 cd28

Peripheral blood mononuclear cells from healthy donors were separated on a Ficoll-Hypaque gradient. Monocytes were eliminated by plastic adherence. Lymphocytes were resuspended in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (BioWhittaker) and stimulated either with anti-CD3/CD28, anti-CD3 alone (both from Thermo Fisher Scientific, USA), or human recombinant IL-2 at the final concentration of 20 U/ml (equal to 10 ng/ml; Roche, USA). The anti-CD3 antibody was pre-coated to plates (1 µg/ml). Anti-CD28 (1 µg/ml) was added in soluble form. The anti-CD3 and anti-CD-28 antibodies were purchased from Beckman-Coulter, USA.

Tumor tissues were first minced and passed through 70 μm filters to obtain single-cell suspension. Cells were then stained with Viability Ghost Dye 510 (Tonbo Biosciences, 13-0870) for 10 min at 4 °C and blocked by anti-CD16/32 (Tonbo Bioscience, 70-0161) (1 to 100 dilution) at 4 °C for 10 min. Cells were further stained with anti-CD45 (Invitrogen, 11-0451-82), anti-CD3 (Tonbo Biosciences, 65-0031-U100), anti-CD8 (BD Pharmingen, 557654), anti-CD44 (BioLegend, 103041), anti-CD62L (BioLegend, 104424), anti-PD1 (BD Biosciences, 563059), anti-TIM3 (BioLegend, 119704), and anti-LAG3 (Invitrogen, 25223182) in staining buffer (2% FBS in PBS). For nuclear transcription factor staining, cells were permeabilized using a FoxP3/transcription factor staining kit (eBioscience, 00-5523-00) and then stained with anti-Ki67 (Invitrogen, 48-5698-82). For cytokine staining, cells were stimulated by anti-CD3/CD28 (Thermo Fisher, 11452D) at 37 °C overnight and then treated with BD GolgiPlug (BD Biosciences, 550583) at 37 °C for 5 h. After surface staining, cells were permeabilized using a BD Cytofix/Cytoperm kit (BD Biosciences, 554714) and stained with anti-IFNγ (BioLegend, 505826) and anti-TNFα (BioLegend, 506314). All antibodies were used at 1:150 dilution. Cells were then fixed with 1% paraformaldehyde and analyzed by BD FACSCelesta. Data were analyzed using BD FACSDiva and FlowJo software.

After the peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors using Polymorphprep (1,114,683, Axis-Shield, Norway) with density gradient centrifugation, the CD4⁺ T cells and CD8⁺ T cells were further purified using anti-CD4 MicroBeads (130–045-101, MilteniyBiotec, Germany) and anti-CD8 MicroBeads (130–045-201, MilteniyBiotec, Germany) following the manufacturer’s instructions. All the cells were cultured in RPMI 1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA) and 1% penicillin/streptomycin (Gibco, Grand Island, NY, USA) at 37 °C. These isolated cells were stimulated with or without IL-8 (100 ng/ml, Peprotech, NJ, USA) and plate-bound anti-CD3/CD28 (5 μg/ml, Thermo Scientific, Waltham, MA, USA) in the presence of IL-2 (10 ng/ml) for the indicated time. All samples were collected with the informed consent of the donors, and all related procedures were approved by the Institutional Review Board of Nanjing University of Chinese Medicine. MFC, a mouse stomach adenocarcinoma cell line, was purchased from Shanghai Cafa Biological Technology Co. Ltd. (Shanghai, China).

PBMC will be isolated over lymphoprep and T cells will be isolated using magnetic selection (Miltenyi Biotec). Cells will be cultured with stimuli such as PMA/ionomycin and anti-CD3/CD28 (Invitrogen). Cells will be transfected in order to silence or overexpress miR-21 or mir-210 (see paragraph “Transfection experiments”).

Directly conjugated monoclonal antibodies against CD8 and CD45RA and their isotypes and unconjugated CD8 antibodies were obtained from BD Biosciences (San Diego, CA). Anti-CCR7 and isotypes were purchased from R & D systems, Minneapolis, MN, and anti-CD3/CD28 was Life Technology, Camarillo, CA. TNF-α was obtained from Laguna Scientific, Laguna Niguel, CA. Antibodies to FLIP and IAP were purchased from Transduction Laboratories, San Diego, CA, and antibodies to phospho IKKα/β, phospho IκB, phospho JNK, phosphor TAK1 TAK1, and TAB2 were purchased from Cell Signaling Technologies, Inc. Beverly, MA. Antibodies to A20, TRAF2 and RIPK1 were obtained from Santa Cruz Biotechnology, Dallas, TX. In Situ Cell Death Detection Kit was purchased from Boehringer-Manheim, Indianapolis, IN.