Fluorophore conjugated antibodies

VSTs were stained with fluorophore-conjugated antibodies against CD3, CD4, CD8, CD14, CD16, CD19, CD45RO, CCR7, IFN-γ and TNF-α (Miltenyi Biotec, Bergisch Gladbach, Germany; BioLegend, San Diego, CA). All samples were acquired on a CytoFLEX cytometer (Beckman Coulter, Brea, CA). Data was analyzed with FlowJo X (FlowJo LLC, Ashland, OR) (Supplemental Figure 2).

VSTs were stained with fluorophore-conjugated antibodies against CD4, CD8, TCRαβ, TCRγδ, CD16, CD19, and CD56 (Miltenyi Biotec, Bergisch Gladbach, Germany; BioLegend). All samples were acquired on a CytoFLEX cytometer (Beckman Coulter, Brea, CA). Intracellular cytokine staining was performed as follows: 1 × 10⁶ VSTs were plated in a 96-well plate and stimulated with pooled pepmixes or individual peptides (200 ng/peptide/well) or actin (control) in the presence of brefeldin A (Golgiplug; BD Biosciences, Cat#BD555029, San Jose, CA) and CD28/CD49d antibodies (BD Biosciences, Cat#347690) for 6 hours. T-cells were fixed, permeabilized with Cytofix/Cytoperm solution (BD Biosciences) and stained with IFN-γ and TNF-α antibodies (Miltenyi Biotec). Pentamer staining was performed using APC-conjugated pentamers (Proimmune, Oxford, UK) per manufacturer’s guidelines. Complete antibody panels and dilutions, as well as all utilized pentamers are listed in Supplementary Tables 6-12. All gating strategies are listed in Supplementary Fig. 7. Data was analyzed with FlowJo X (FlowJo LLC, Ashland, OR, USA).

VSTs were stained with fluorophore-conjugated antibodies against CD4, CD8, TCRαβ, TCRγδ, CD16, CD19, and CD56 (Miltenyi Biotec, Bergisch Gladbach, Germany; BioLegend). All samples were acquired on a CytoFLEX cytometer (Beckman Coulter, Brea, CA). Intracellular cytokine staining was performed as follows: 1 × 10⁶ VSTs were plated in a 96-well plate and stimulated with pooled pepmixes or individual peptides (200 ng/peptide/well) or actin (control) in the presence of brefeldin A (Golgiplug; BD Biosciences, Cat#BD555029, San Jose, CA) and CD28/CD49d (BD Biosciences, Cat#347690) for 6 h. T-cells were fixed, permeabilized with Cytofix/Cytoperm solution (BD Biosciences, Cat#554714) and stained with IFN-γ and TNF-α antibodies (Miltenyi Biotec). Concurrent sample replicates were performed when possible, based on availability of cells. Data was analyzed with FlowJo X (FlowJo LLC, Ashland, OR). Antibody panels and dilution details are listed in Supplementary Tables 5–9. Gating strategy is included in Supplementary Fig. 12A, B.