LCMV-specific CD8+ T cell Characterization

Mice were infected with 2 x 10⁵ PFU of the LCMV Armstrong strain intraperitoneally (i.p.) (27 (link)). Eight days after infection mice were euthanized, and the single cell suspensions of splenocytes were prepared as previously described (11 (link)). Virus-specific CD8⁺ T cells were identified with R-phycoerythrin (PE) or allophycocyanin (APC)-labeled H2-D^b/GP33-41 and H2-D^b/NP396-404 dextramers (Immudex) and brilliant violet (BV) 421-labled H2-D^b/GP33-41 tetramers (kindly provided by the NIH Tetramer Core Facility). For the detection of cytokine production, splenocytes were incubated with 1 μM GP33 (KAVYNFATC) peptides (Anaspec) in the presence of GolgiStop (BD Biosciences) and GolgiPlug (BD Biosciences) for 4.5 hours at 37°C. In order to detect CD107a and CD107b surface externalization, anti-CD107a (1:1000; Biolegend) and anti-CD107b (1:1000; Biolegend) antibodies were added during peptide stimulation.