Monesin

Spleens were harvested at 7 weeks post tumor inoculation and placed in cold PBS and then mashed by two baked frosted glass slides. Splenocytes were passed through 40 μm cell strainers to get rid of connective tissue and then lysed by RBC lysis buffer. 2 × 10⁶ splenocytes were placed per well in 24-well plates cultured with RPMI 1640 medium supplemented with L-glutamine and FBS. 2 μg/ml recombinant mouse mesothelin (BioLegend, #594006) was applied for 72-hour stimulation. Brefeldin A and Monesin (BioLegend, #420601 and #420701) were added into the culture medium during the last five hours. The supernatant was collected to detect IFNγ by ELISA. The splenocytes were then harvested and prepared for flow cytometry.

For in vitro assays, peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll (Lymphoprep) density-gradient centrifugation and incubated on plates coated with anti-CD3 (3 μg/ml) and anti-CD28 (1 μg/ml) mAbs (Biolegend) or with IL-2 (100 U/ml) and IL-15 (10 ng/ml)(PeproTech EC Ltd, London, UK) for activation of CD8⁺ T and NK cells, respectively. CD107a-FITC (clone H4A3) antibody was added to the wells followed by monesin (6 μg/ml, Biolegend) and cultured for 6 h before analysis on a Gallios Flow Cytometer using Kaluza 1.3 analysis software (Beckman Coulter, CA, USA). For IFN-γ production, activated CD8⁺ T lymphocytes were treated for 5 h with brefeldin A (10 μg/ml, Biolegend), and cell surfaces were stained with CD8-PE and CD3-PerCP mAbs, followed by intracellular staining with anti-INFγ-FITC for 30 min at 4°C. Similarly, PBMCs were incubated with CD8-PE, CD3-PerCP and CD16-APC mAbs, followed by intracellular staining with granzyme B-PE and perforin-FITC mAbs (Biolegend). In both cases, a fixation / permeabilization kit (Immunostep, Salamanca, Spain) was used and the 7-Aminoactinomycin D (7AAD) fluoresecent marker (Biolegend) was included to excluded dead cells from the analysis. Cells were further analyzed by flow cytometry.

Cell surface staining was performed for 15 min in the dark using PBS. The exclusion of dead cells was achieved using Zombie UV dye (BioLegend). Cells were stimulated with Ionomycin (500 ng/ml), PMA (25 ng/ml), Monesin (1×, BioLegend) and Brefeldin A (2 μg/ml) diluted in IMDM buffer and incubated for 3 h at 37 °C to detect cytokines and FasL expression. For intracellular stainings BD Cytofix/Cytoperm Fixation/Permeabilization kit was used. Surface and intracellular stainings were performed using following antibodies: CD3 (17A2, eBioscience), CD43 (1B11, BioLegend), CD49b (Dx5, eBioscience), CD69 (H1.2F3, eBioscience) CD86 (GL1, BioLegend), CD107a (ID4B, BioLegend), FasL (MFL3, BD Pharmingen), IFNγ (XMG1.2, eBioscience), IL-10 (JES5-16E3, eBioscience) IL-13 (eBio13A, eBioscience), NK1.1 (PK136, eBioscience), and TNFα (MP6-XT22, BioLegend).

For splenic cDC purification, splenocytes were labeled with CD11c-microbeads (Miltenyi Biotec, Sunnyvale, CA, USA) for positive selection, according to the manufacturer’s instructions (autoMACS^® Separator, Miltenyi Biotec, Bergisch Gladbach, Germany). Naïve CD4⁺ T cells were purified using a CD4⁺CD62L⁺ T Cell Isolation Kit (Miltenyi Biotec), according to the manufacturer’s instructions. Purified naïve CD4⁺ T cells were co-cultured with splenic cDCs for 5 d, in RPMI-1640 containing 10% FBS supplemented with anti-mouse CD3 (1 μg/ml) and recombinant mouse IL-2 (10 ng/ml, R&D Systems). On day 5, the cells were re-stimulated with a cell activation cocktail (BioLegend) and monesin (2 μM, BioLegend), for a further 4 h. The cells were then stained with PE-conjugated IL-4 (11B11, BD Biosciences) and APC-conjugated interferon gamma (IFN-γ; XMG1.2, BD Biosciences) using a Fixation/Permeabilization Kit (BD Biosciences), according to the manufacturer’s instructions.

Functional analysis of NK and T cells was performed as recently described by using isolated splenic mononuclear cells (MNC) (23 (link)). Cells were stimulated in the presence of 200 U/ml mIL-2 either with 50 ng phorbol 12-myristate 13-acetate (PMA), 1 μg ionomycin calcium salt (Sigma-Aldrich, St. Louis, MO, USA), alternatively with murine YAC-1 cells as target cells (with an effector:target ratio 2:1), 5 μg/ml brefeldin A (Sigma Aldrich), and 2 μM monesin (Biolegend) for 4 h at 37°C and 5% CO₂. After stimulation, cells were stained with antibodies listed in Supplemental Table 1. Degranulation capacity was assessed by CD107a lysosome-associated membrane protein-1 (LAMP-1) expression. Cell activation was assessed by first fixing and permeabilizing cells using Transcription Factor Staining Buffer Set (eBioscience) and then by staining intracellularly for IFNγ.