Profiling Antigen-Specific T-cell Responses

Splenocytes were prepared as described above, plated in 96-well round-bottom plates, and stimulated using peptide pools for EBOV GP, SUDV GP, or MARV GP (as described above) at a final concentration of 5 µg/mL or media only. Stimulation and staining was then performed as described previously [33 (link)] except that the following antibodies were used: anti-CD4-Qdot605, anti-CD127-APCef780 (Invitrogen), anti-CD62L-PeCy7, and anti-CD8-PerCP/Cy5.5 antibodies (eBioscience), as well as LIVE/DEAD^® Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific), anti-TNF-Alexa488, anti-IL-2-PE, and anti-IFN-γ-e450 antibodies (eBioscience). Antigen-specific cells were identified by gating based on doublet negative, size, live cells, and either CD4⁺ or CD8⁺ surface expression. Background responses in unstimulated control samples were subtracted from responses of peptide stimulated T cells.