Anti cd107a antibody

Single cell suspensions of spleen and liver were cultured in 10% heat inactivated fetal calf serum and RPMI 1640 and stimulated with phorbol 12-myristate 13-acetate (PMA, 200 ng/ml) and Ionomycin (5 µg/ml) for 4 hr in the presence of Brefeldin A (BD Pharmingen, San Jose, CA) to analyze cytokine production. For the degranulation assay, a suspension of 10⁵ liver cells was plated with YAC-1 target cells at an effector:target (E:T) ratio of 1:1 in 96-well V-bottom plates. Anti-CD107a antibody, Brefeldin A, and monensin (eBioscience, San Diego, CA) were added to each well before incubation. Plates were incubated for 6 hr at 37°C, after which surface staining followed by intracellular staining for TNFα was performed for analysis by flow cytometry.

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).

Spleen cells were resuspended in RPMI-1640 supplemented with 8% FBS and 50 IU ml⁻¹ IL-2. They were then stimulated with OVA peptide (257–264) (1 μg ml⁻¹) for 4 h at 37 °C in the presence of GolgiStop (BD) and anti-CD107a antibody (eBioscience). Cells were then stained with anti-CD45.1, anti-CD45.2, anti-CD8 Abs and the expression of CD107a evaluated by flow cytometry.