MHC-I Epitope Prediction and T-cell Activation

A7(74) SFV genome was analysed in all three reading frames using peptide prediction websites (http://tools.iedb.org/mhci/, http://www.cbs.dtu.dk/services/HLArestrictor/)^{67 (link)} and peptide sequences were generated based on binding affinity prediction to mouse MHC-I (H2-K^b and H2-D^b). Peptides (GenScript USA Inc) were resuspended up to 1 mM with DMSO, aliquoted and stored at −20 °C. Enriched T cell populations from brain and spleen were stimulated with 1 μM of the selected peptide for 5 hrs at 37 °C, 5% CO₂ in the presence of 1000 U/mL recombinant IL-2 (Roche, Basel, Switzerland) and 1 μL/mL Golgi-Plug (BD Biosciences, San Jose, CA, USA) as described^{44 (link)}. Cells were washed and stained with CD8-PerCP Cy5.5 (clone 53-67, BD Biosciences 551162) for 30 mins on ice, fixed, permeabilised and stained for cytokines (IFN-γ-FITC, TNF-α-APC and IL-2-PE (Biolegend, San Diego, CA, USA)). Samples were acquired using BD Canto II, and the total cytokine production was calculated by subtracting background fluorescence using no peptide controls. PMA/ionomycin stimulated cells were used as positive controls. The known sequence of the peptides giving positive results were blasted against the NCBI database (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to determine the position of the peptide in SFV proteome.