Clone jes6 5h4

BM-DCs derived from BALB/c (H-2^d) female mice were used at day 6 of culture as antigen presenting cells. Cells were seeded in 96-well plates at 5 x 10⁴ cells/well and loaded with 1 μg/ml of homologous or negative control synthetic peptides, or infected with different mycobacterial strains with serial two-fold dilutions of M.O.I., starting at M.O.I. = 10, in RPMI 1640-GlutaMax medium (Invitrogen) containing 10% FBS. After 18h of infection at 37°C and 5% CO₂, cells were washed twice with RPMI medium to eliminate the IL-2 possibly produced by the infected DCs and then co-cultured with 1 x 10⁵ cells/well of T-cell hybridoma specific to EsxH/TB10.4₇₄₋₈₈ (1G1) or Ag85A_101-120 (2A1), respectively restricted by I-A^d or I-E^d. After over-night of co-culture at 37°C and 5% CO₂, the IL-2 secretion was quantified in the culture supernatants by ELISA (clone JES6-1A12 for coating and clone JES6-5H4 for detection, BD Pharmingen).

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.