Cd8 percp cy5.5 clone 53 67

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.

Cells were washed in PBS/2% TBS, adjusted to 10⁶/ml and 200 μl/well transferred into 96 well plates (Falcon) for staining with 50μl of antibodies: CD3-PE (clone 145-2C11, hamster IgG1, Beckton Dickinson (BD)), CD8-PerCP-Cy5-5 (clone 53-6.7, rat IgG2a, BD) and CD4-APC-Cy7 (clone GK1.5, rat IgG2b BioLegend) or their isotype controls (BioLegend). Cells were incubated for one hour at 4°C, washed in PBS and fixed in 2% paraformaldehyde for 15 minutes at 4°C, all in the dark. Fixed cells were washed in PBS/2% TBS and data acquired on a FACSCanto II (BD) using Diva software and analyzed using FlowJo v10 software (BD).

ATOR-1015 (anti-CTLA-4 x anti-OX40 bsAb), anti-OX40 mAb, anti-CTLA-4 (anti-GFP x anti-CTLA-4 bsAb) and IgG1 isotype control (anti-GFP) were developed using the proprietary ALLIGATOR-GOLD® library and FIND® optimization technology (Additional file 1: Supplementary Methods). For flow cytometry analysis of human cells, the following anti-human antibodies were used: CD3-PECy5 (clone SP34–2), CD4-APC-H7 (clone RPA-T4), CD25-BV421 (clone 2A3), CD127-FITC (clone HIL-7R-M21), CD134 (OX40)-PE (clone L106) and CD152 (CTLA-4)-APC (clone BNI3) (all from BD) and anti-human IgG-PE (Jackson Immuno-Research).
For flow cytometry analysis of murine cells, the following anti-mouse antibodies were used: CD25-PerCPCy5.5 (clone PC61.5), CD45-APCeFluor780 (clone 30-F11), NK1.1-FITC (clone PK136) and Foxp3-APC (clone FJK-16 s) from eBioscience, CD3-PE (clone 145-2C11), CD3-PECy7 (clone 145-2C11), CD4-BV510 (clone RM4–5), CD4- PECy7 (clone RM4–5), CD8-PE (clone 53–6.7), CD8-PerCPCy5.5 (clone 53–6.7), CD11b-FITC (clone M1/70), CD19-FITC (clone 1D3), CD107a-APC (clone 1D4B), MHC-II-FITC (clone 2G9), Granzyme B-PE (clone GB11) and FVS450 viability stain from BD.