Characterization of Antigen-Specific T Cell Responses

Flow cytometry and tetramer were performed as previously described^{55 (link)}. Briefly, single cell suspended splenocytes were incubated with gp33-tetramer and np396-tetramer for 15 minutes and gp-66 tetramer for 30 minutes at 37 °C. After the incubation, surface antibodies were added, including anti-CD8 or anti-CD4 along with anti-IL7R, anti-PD-1, anti-CD62L, anti-CD44, anti-KLRG-1, anti-TIM3, anti-Lag-3, and anti-2B4 antibodies (eBioscience) for 30 minutes at 4 °C. For intracellular cytokine stain single suspended splenocytes were incubated with the LCMV specific peptides gp33 and np396 or the MHC-II epitope gp61. After 1 h, Brefeldin A (eBioscience) was added, followed by an additional 5 h incubation at 37 °C. After surface stain with anti-CD8 or anti-CD4 antibodies (eBioscience) cells were fixed with 2% formalin and permeabilized with 0.1% Saponin and stained with anti-IFNγ and anti-TNF antibodies (eBioscience) for 30 min at 4 °C.