For H2-Kb Ova257-264 (SIINFEKL)-specific CD8+ T-cell analysis, spleen cells were obtained 12 days after tumor inoculation and stained with AF647-coupled dextramer loaded with Ova257-264 peptide (DexOT–I; Immudex, Copenhagen, Denmark), FITC-anti-CD11b, PerCP-anti-CD43, BV421-anti-CD8 (all from BioLegend, San Diego, CA, USA), Fixable Viability Dye eFluor 780, PE-Cy7-anti-CD62L (eBioscience), and BV510-anti-CD44 (Becton-Dickinson).
The frequency of CD8+ T cells specific for the H2-Db-restricted Leader-Gag-derived epitope GagL85–93 [CCLCLTVFL (33 (link))] was analyzed 14 days after DNA-based immunization in peripheral blood cells after erythrocyte lysis or in spleen cells after tumor cell inoculation. Cells were stained with PE-coupled MHC I tetramer [TetIGagL; carrying the peptide AbuAbuLAbuLTVFL, in which cysteine residues of the original GagL85-93 amino acid sequence were replaced by aminobutyric acid (Abu) to prevent disulfide bonding; MBL, Woburn, MA, USA], PerCP-anti-CD43, BV421-anti-CD8, BV510-anti-CD44, PE-Cy7-anti-CD62L (all from BioLegend), and Fixable Viability Dye eFluor 780 (eBioscience).
Data were acquired on a BD FACSymphony A5 flow cytometer (Becton-Dickinson) and analyzed using FlowJo software (TreeStar). Exemplary plots showing the gating strategy are shown inSupplementary Figure 2
The frequency of CD8+ T cells specific for the H2-Db-restricted Leader-Gag-derived epitope GagL85–93 [CCLCLTVFL (33 (link))] was analyzed 14 days after DNA-based immunization in peripheral blood cells after erythrocyte lysis or in spleen cells after tumor cell inoculation. Cells were stained with PE-coupled MHC I tetramer [TetIGagL; carrying the peptide AbuAbuLAbuLTVFL, in which cysteine residues of the original GagL85-93 amino acid sequence were replaced by aminobutyric acid (Abu) to prevent disulfide bonding; MBL, Woburn, MA, USA], PerCP-anti-CD43, BV421-anti-CD8, BV510-anti-CD44, PE-Cy7-anti-CD62L (all from BioLegend), and Fixable Viability Dye eFluor 780 (eBioscience).
Data were acquired on a BD FACSymphony A5 flow cytometer (Becton-Dickinson) and analyzed using FlowJo software (TreeStar). Exemplary plots showing the gating strategy are shown in