Cell stimulation cocktail

To measure the levels of intracellular cytokines of CD4⁺ and CD8⁺ T cells as well as NK cells, PBMCs (1×10⁶ cells/mL) were cultured for 4 hours with and without 2 μl Cell Stimulation Cocktail [containing PMA (40.5 μM), ionomycin (669.3 μM), and Brefeldin A (2.5 mg/ml)] (Biolegend, San Diego, USA). Following cell culture, PBMCs were washed with PBS and supernatants were discarded. Prior to the detection of intracellular cytokine levels, cell surface staining was performed using anti-CD3-BV785, -CD4-BV510, -CD8-PE/Cy5, -CD28-PE, -CD56-FITC and -CD57-BV711 mAbs (all from Biolegend, San Diego, USA). For intracellular staining, Fix&Cell Permeabilization Kit (Invitrogen, California, USA) was used according to the manufacturer’s protocol. Briefly, cells were fixed and then permeabilized together with addition of anti-IFN-γ-PE/Cy7, -TNF-α-APC/Cy7 and -IL-10-BV421 mAbs. Stained PBMCs were washed after the incubation and samples were analyzed on a NovoCyte flow cytometer running NovoExpress software (Agilent Technologies, USA).

Tumors collected from mice were divided into small pieces and homogenized in cold staining buffer to form single-cell suspensions in the presence of digestive enzyme. Cells were stained with fluorescence-labeled antibodies against CD45 (Biolegend, Cat. no. 103116, clone 30-F11), CD3 (Biolegend, Cat. no. 100217, clone 17A2), CD4 (Biolegend, Cat. no. 100429, clone GK1.5), CD8a (Biolegend, Cat. no. 100721, clone 53–6.7), Foxp3 (Biolegend, cat. no. 126404, clone MF-14), NK-1.1 (Biolegend, Cat. no. 108709, clone PK136), CD62 L (Biolegend, Cat. no. 104407, clone MEL-14), CD44 (Biolegend, Cat. no. 103005, clone IM7), CD11b (Biolegend, Cat. no. 101205, clone M1/70), CD206 (Biolegend, Cat. no. 141731,clone C068C2), CD80 (Biolegend, Cat. no. 104733, clone 16-10A1), F4/80 (Biolegend, Cat. no. 123131, clone BM8), Ly-6G (Biolegend, Cat. no. 127615, clone 1A8), Gr-1 (Biolegend, Cat. no. 108407, clone RB6-8C5), CD86 (Biolegend, Cat. no. 105011, clone GL-1), CD103 (Biolegend, Cat. no. 121415, clone 2E7), and CD11c (Biolegend, Cat. no. 117307, clone N418), and were treated with Fixable Viability Dye (Biolegend, cat. no. 423101), Cell Stimulation Cocktail (Biolegend, cat. no. 423303), Intracellular Staining Perm Wash Buffer (10X) (Biolegend, Cat. no. 421002), and Fixation Buffer (Biolegend, Cat. no. 420801).

Isolated PBMCs were cultured and stimulated with soluble Purified NA/LE anti-human CD3 (UCHT1, 3 μg/ml)/CD28 (CD28.2, 1 μg/ml) or PMA (20 ng/ml)/Ionomycin (1 g $\mu$ /ml) (Cell stimulation cocktail, Biolegend) in the presence of the protein transport inhibitor Brefeldin A (BD Biosciences, 1/1000) for 5 h. Intracellular cytokine staining was performed according to our protocols [54 (link)]. For cytokine-dependent cultures, PBMCs were treated with a cocktail of cytokines including recombinant human IL-12 (Cedarlane,100 ng/ml), IL-18 (Biolegend,100 ng/ml), and IL-15 (Biolegend, 100 ng/ml) for 18 h. Brefeldin A (1/1000) was added 5 h before the intracellular staining. In other experiments, the effects of different cytokines including TNF-α (50 ng/ml), IFN-γ (100 ng/ml), IL-10 (100 ng/ml), IL-16 (500 ng/ml), IFN-α (100 ng/ml), IL-2 (20 ng/ml), IL-6 (100 ng/ml), and TGF-β (20 ng/ml) on CD26 expression was analyzed.

Cytokine release assays were performed upon stimulation with Cell Stimulation Cocktail (BioLegend) containing phorbol 12-myristate 13-acetate (PMA), ionomycin, and brefeldin A for 4 hours at 37°C. Cells were labeled using surface antibodies followed by fixation, permeabilization, and intracellular cytokine staining using the Intracellular Cytokine Staining kit (BioLegend) according to the manufacturer’s protocol.

In order to determine the intracellular cytokine levels of T lymphocytes, PBMCs (1 × 10⁶ cells/mL) were pre-cultured with the presence or absence of C-Vx for 72 h and further stimulated with Cell Stimulation Cocktail [PMA (40.5 μM), Ionomycin (669.3 μM), and Brefeldin A (2.5 mg/mL)] (Biolegend, San Diego, USA) for 4 h at 37°C incubator. After the culture, cell surface staining was performed using anti-human-CD3-BV785, -CD4-PE, and -CD8-FITC monoclonal antibodies (mAbs) (all from Biolegend, San Diego, USA), prior to the detection of intracellular cytokine levels. Intracellular staining was performed using Fixation/Permeabilization Kit (BD Cytofix/Cytoperm, California, USA) according to the manufacturer’s protocol. Simply, cells were fixed and then permeabilized together with the addition of anti-IFN-γ-PE/Cy7, -TNF-α-APC/Cy7, -IL-4-APC, -IL-10-BV421, -IL-17-Alexa Fluor 700 mAbs. After washing, samples were measured by flow cytometry.

Fresh mouse tumors were digested with the Mouse Tumor Dissociation Kit (Miltenyi). Dissociated tumor samples were filtered through the 70 µm strainer and then disposed by 36% percoll reagent to remove cell debris. ACK Lysis Buffer was used to lyse the erythrocytes. Then cells were blocked with Fc block (anti‐mouse CD16/32, BioLegend) on ice for 30 min. The samples were stained for lymphoid and myeloid immune populations with the following antibodies listed in Table S9 (Supporting Information). For the assessment of intracellular markers, cells were incubated with a Cell Stimulation Cocktail (BioLegend, 423 303) and stained with anti‐mouse IFN‐γ after fixation/permeabilization.
Other experimental information is presented in Supporting Information.