Anti mouse cd3e antibody clone 500a2

For ex vivo CD8+ T cell analysis, spleens were removed from vaccinated mice and homogenized with a syringe plunger over metal grid with cell culture medium. Erythrocytes were lysed with 3 ml TAC buffer and washed. Cells were filtered by 70 μm cell strainer and counted. For a short T cell restimulation, 4 × 10⁶ splenocytes were further incubated with respective peptides (1 μg/ml) for 4 h for CD8+ T cells and for 15 h for CD4+ T cells in the presence of BFA. As a control, T cells were stimulated in a non-antigen-specific manner using anti-mouse CD3e antibody (clone 500A2, BD Pharmingen 553238) at 1.25 μg/ml for CD8+ T cells for 4 h and for CD4+ T cells for 15 h in presence of BFA.

Spleens of vaccinated animals were collected and processed into a single-cell suspension by mechanical disruption using a 70 μm cell strainer and a plunger. Erythrocytes were lysed by incubation in lysis buffer (BD Pharm Lyse^TM) for 1 min at room temperature. Cells were passed through a 70 μm cell strainer and counted using a Neubauer cell counting chamber. Thereafter, 4 × 10⁶ splenocytes were plated at 100 μl per well of a 96-well plate and further incubated with 2 μg/ml of MVA-specific or control peptides and 1 μg/ml brefeldin A (Merck) for 5 h. Peptides were A19₄₇₋₅₆ (VSLDYINTM), B8₂₀₋₂₇ (TSYKFESV), K3₆₋₁₅ (YSLPNAGDVI), A3_270−277 (KSYNYMLL), or D13_118−126 (NCINNTIAL) derived from MVA and OVA_257−264 (SIINFEKL) peptide derived from ovalbumin. K3 and D13-derived peptides are H2-D^b-restricted, all other peptides are H2-K^b- restricted. All peptides were purchased from Biosynthan (Germany). Beta-galactosidase (β-Gal) peptide was used as negative control as a non-cognate ligand. As an additional control, T cells were stimulated in a non-antigen-specific manner using anti-mouse CD3e antibody (clone 500A2, BD Pharmingen 553238) at 1,25 μg/ml. For the determination of CD107a expression, splenocytes were additionally incubated in the presence of anti-CD107a antibody (eBioscience).