Clone mp6 xt22

Splenocytes were prepared as indicated above. Stimulation assays were performed using 1 × 10⁶ live splenocytes per well in 96-well U-bottom plates. Pools of overlapping peptides spanning the entire coding sequences of brachyury, CEA and MUC1 were synthesized as 15-mers with 11-amino acid overlaps (JPT GmbH) and lyophilized peptide pools were dissolved in Dimethyl sulfoxide (DMSO). Similarly constructed peptide pools corresponding to SIV-Vif and SIV-Nef served as off-target controls. Splenocytes in R10 media (RPMI 1640, 10% fetal bovine serum, and antibiotics) were stimulated by the addition of peptide pools at 2 μg/mL/peptide for 6 h at 37°C and 5% CO₂, with protein transport inhibitor (GolgiStop, BD) added 2 hours into the incubation. Stimulated splenocytes were then stained for lymphocyte surface markers CD8α and CD4, fixed, permeabilized, and then stained for the intracellular accumulation of IFN-γ and TNF-α. Antibodies against mouse CD8α (clone 53–6.7), CD4 (clone RM4–5), IFN-γ (clone XMG1.2), and TNF-α (clone MP6-XT22) were purchased from BD and staining was performed in the presence of anti-CD16/CD32 (clone 2.4G2). Flow cytometry was performed using an Accuri C6 Flow Cytometer (BD) and analyzed in BD Accuri C6 Software.

ICS assays were performed using 10⁶ live splenocytes per well in 96-well U-bottom plates. Splenocytes in RPMI media supplemented with 10% FBS were stimulated by the addition of pools of overlapping peptide for S or N antigens at 2 μg/mL/peptide for 6 h at 37 °C in 5% CO₂, with protein transport inhibitor, GolgiStop (BD) added two hours after initiation of incubation. The S peptide pool (JPT: Cat #PM-WCPV-S-1) is a total of 315 spike peptides split into two pools comprised of 158 and 157 peptides each. The N peptide pool (JPT; Cat # PM-WCPV-NCAP-1) was also used to stimulate cells. A SIV-Nef peptide pool (BEI Resources) was used as an off-target negative control. Stimulated splenocytes were then stained for a fixable cell viability stain followed by the lymphocyte surface markers CD8β and CD4, fixed with CytoFix (BD), permeabilized, and stained for intracellular accumulation of IFN-γ, TNF-α and IL-2 (in studies 2 and 3). Fluorescent-conjugated antibodies against mouse CD8β antibody (clone H35-17.2, ThermoFisher), CD4 (clone RM4-5, BD), IFN-γ (clone XMG1.2, BD), TNF-α (clone MP6-XT22, BD) and IL-2 (clone JES6-5H4; BD), and staining was performed in the presence of unlabeled anti-CD16/CD32 antibody (clone 2.4G2; BD). Flow cytometry was performed using a Beckman-Coulter Cytoflex S flow cytometer and analyzed using Flowjo Software.

5x10⁶ splenocytes were incubated in 96-well round bottom plates in triplicate with Monensin (BioLegend), Brefeldin-A (BioLegend), CW3 peptide SWVPRLYQL (0.4 μg/mL), and the CD107a antibody (eBioscience, clone ABL-93) for four to five hours at 37°C. Cells were surface stained followed by fixation and permeabilization using the cytofix/cytoperm kit (BD biosciences). Permeabilized cells were stained with antibodies for TNFα (BioLegend, clone MP6-XT22), IFNγ (BD biosciences, clone XMG1.2) and analyzed by flow cytometry.