Clone xmg1

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.

Draining lymph nodes from immunized mice were harvested 3 days post-boost (DOL 17). Nitrocellulose 96-microwell plates (Millipore) were coated with 75 µl/well of anti-mouse IFNγ (10 µg/ml in PBS, clone R4-6A2, BD Pharmingen) overnight at 4°C, washed twice with wash buffer (PBS + Tween-20 0.05%) and once with distilled water. Wells were blocked with 200 µl of complete culture medium for 2 h at RT. Single-cell suspensions of dLNs in complete culture medium supplemented with recombinant mouse IL-2 (5 ng/ml, PeproTech) were added to the wells in the presence or absence of 10 µg/ml of Flublok and 2 µg/ml anti-mouse CD28 (Biolegend) and cultured for 18 h. Wells were then washed and incubated with 100 ml of biotinylated anti-mouse IFNγ (5 µg/ml in PBS + FBS 10%, clone XMG1.2, BD Pharmingen) for 2 h at RT, washed again and incubated with 100μl of streptavidin-alkaline phosphatase (1:1000 dilution in PBS + FBS 10%, MabTech) for 1 h prior to color development using BCIP/NBT substrate (Biorad) as per manufacturer’s protocol. Spots on air-dried plates were counted on an ImmunoSpot Analyzer.

Naïve CD4 T cells stained with APC-labeled anti-CD4 (clone RM4-5) and FITC-labeled anti-CD62L (clone MEL-14) were isolated, with purity of over 98%, from IL-6 deficient OT-II mice using a FACSAria II. CD49b(DX5)⁺ c-kit⁻ basophils were isolated, with purity higher than 95%, from the BM culture supplemented with IL-3 (10 ng/ml, Peprotech) for 7–9 days using a FACSAria II. Conventional CD11c⁺ DCs were isolated, with purity higher than 90%, from BM cells cultured with GM-CSF (20 ng/ml, Peprotech) using CD11c microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany). 2.5 × 10⁴ CD4 T cells were cultured with 0.5 × 10⁴ basophils activated with IgE (10 μg/ml, BD biosciences) and anti-IgE (10 μg/ml, BioLegend) and mature CD11c⁺ DCs for 3 days. 10 μM of OVA_323–339 peptide (ISQAVHAAHAEINEAGR), anti-IL-4 (10 μg/ml, clone 11B11, BD biosciences), anti-IFN-γ (10 μg/ml, clone XMG1.2, BD biosciences), anti-IL-6 (5 μg/ml, clone MP5-20F3, BD biosciences), and TGF-β (5 ng/ml, Peprotech) were added as indicated. Cell culture was performed in 96-well round-bottomed plates in a total volume of 200 μl.

Cells from draining LN and mononuclear cells from the spinal cord and brain were further stimulated with PMA (5 nM) and ionomycin (500 ng ml⁻¹) for 4 h in the presence of Golgi Stop (554724, BD Biosciences), before being harvested. They were stained with Abs against cell surface Ag, fixed with Cytofix/Cytoperm solution (555028, BD Biosciences), and then stained with mAbs against intracellular IFN-γ (1:200, Clone XMG1.2, BD Bioscience) and IL-17 (1:200, Clone TC11-18H10, BD Bioscience). Stained cells were analysed by flow cytometry.

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.