Monensin a

Monensin A (95%) was sourced from Sigma-Aldrich Chemical Co. The Jacobsen catalyst, 3-chloroperoxybenzoic acid (m-CPBA), and tert-butyl hydroperoxide (t-BOOH, 70% solution in water) were all acquired from Acros-Organics. Hydrogen peroxide (30% in water) was supplied by Fluka and stored at 5°C, and it was periodically titrated to confirm its purity. Iodosylbenzene (PhIO) was obtained through iodosyl benzenediacetate hydrolysis and its purity was measured by iodometric assay.

Lymphocytes were isolated from spleen and lungs as described before [83 (link)]. Cells were isolated from ovaries by mincing the tissue through a 70 μm cell strainer. Before lungs and ovaries were removed, mice were perfused with PBS. Blood samples were obtained from the tail vein. Red blood cells were lysed using ACK lysis buffer for 1 minute at room temperature. Surface staining of cells was performed for 20 min at room temperature in PBS supplemented with 2% FCS. For i. v. labelling of CD8 T cells, 5 μg of a fluorescently conjugated anti-CD8α antibody was injected i. v. 3 minutes prior to euthanasia. For peptide restimulations, cells were incubated with 1 μg/ml peptide in the presence of 20 μM Monensin A (Sigma Aldrich) for 6 hours at 37° C. Cell surface staining was performed as described above and cells were fixed with 1% PFA for 20 minutes. Cells were permeabilized using 2x BD lysis buffer (BD Biosciences) containing 0.05% Tween 20 (Sigma Aldrich) for 10 minutes. Intracellular staining was performed at room temperature for 20 minutes. Multiparametric flow cytometric analysis was performed using LSRII flow cytometer (BD Biosciences) and FACSDiva software. Data was analysed using FlowJo software (Tree Star).

Intracellular staining was performed after incubation with brefeldin A (1 μg/ml, Sigma-Aldrich, Schnelldorf, Germany) for 4 h. Subsequently, the cells were surface stained and fixed of the cells with PBS/ 4% paraformaldehyde. Then cells were permeabilized with 0.5% saponin (Carl Roth, Karlsruhe, Germany) and intracellularly stained with IFN-γ-Alexa647 (BD Bioscience, Pharmingen, Heidelberg, Germany). To determine the expression of the activation marker CD69, the cells were harvested and stained in PBS containing 2% FCS (Thermo Fisher Scientific, Oberhausen, Germany) with CD69-PE-Cy7, CD3-Horizon V450, CD56-PE (BD Pharmingen, Heidelberg, Germany) for 15 min at 4°C in the dark, washed and analyzed via flow cytometry. Degranulation of NK cells was determined by measuring CD107a-PE, CD3-FITC, CD56-APC (BD Pharmingen, Heidelberg, Germany) and Vγ9-Alexa488 (for γδT cell staining (78 (link)) as generous gift from Dietrich Kabelitz and Daniela Wesch). Human PBMCs were co-cultured with tumor cells and CD107a-PE was added for 1 h. Degranulation was blocked with 5 μg/ml Monensin A (Sigma-Aldrich, Schnelldorf, Germany) for an additional 3 h. Flow cytometric analysis was performed using the LSR II from BD Bioscience and data were analyzed with FlowJo® (Tree Star, Switzerland).

These assays were performed as previously described [18 (link)]. Briefly, 2 × 10⁵ SARS-CoV-2-specific T cells were stimulated for 5 h in a 96 well plate with peptides. Monensin A (Sigma-Aldrich) and FITC-conjugated Abs for CD107a or isotype matched antibodies (BD Pharmingen, San Diego, CA, USA) were added 1 h after stimulation and incubated for 5 h. Cells were then stained with antibodies against CD3, CD8, and CD4 and fixed, permeabilized with Cytofix/Cytoperm solution, and stained with antibodies against IFN-γ, TNF-α, and IL-2 (all from BD Pharmingen) at 4 °C for 20 min. An unrelated peptide group was included as a negative control for spontaneous CD107a release and/or cytokine production.