Ifn γ fitc

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.

The cells were extracted from lymph nodes, spleen, or colon tissue digested by collagenase. Isolated cells were stained with following antibodies: CD8-PE-CY7 (Thermo Scientific), CD4-APC-Fire™ 750 (BioLegend), IFN-γ-FITC (BioLegend), IL-17-APC (BioLegend), FOXP3-PE (BioLegend), CD19-PE (BioLegend), NK1.1-FITC (BioLegend), CD11B-APC (BioLegend), Gr-1-FITC (BioLegend), F4/80-PE (BioLegend), CD11C-APC-CY7 (BioLegend), MHC-II-PE/Dazzle™ 594 (BioLegend), CD8-APC (BioLegend), Ki67-PerCP-CY5.5 (BioLegend), Annexin V-FITC (BioLegend), CD103-PE (BioLegend), CD69-PE-CY7 (BioLegend), CD44-PE-CY7 (BioLegend), CD62L-FITC (BioLegend), CD107-FITC (BioLegend), IFN-γ-APC (BioLegend), CD107-PE (BioLegend), IFN-γ-PE (BioLegend), CD3-FITC (BioLegend), CD3-APC/Fire™ 750 (BioLegend), CD8-APC (BioLegend), IFN-γ-PE-CY7 (BioLegend), CD69-APC/Fire™ 750 (BioLegend), Tetramer-SIINFEKL-PE (MBL), p-JNK-PE (Cell Signaling Technology), p-IRAK4-Alexa Fluor 488 (Cell Signaling Technology). Intracellular staining was carried out using the Cytofix/Cytoperm kit (BD Pharmingen) following a 4 h restimulation by PMA/ionomycin (Sigma) in the presence of GolgiPlug (BD Pharmingen). Flow cytometric data acquisition was performed on a NovoExpress flow cytometer and analyzed with NovoExpress software (ACEA Biosciences).

For functional assays on cytokine production by T cells, thawed isolated PBMCs were stimulated for 16 h at 37 °C in a 5% CO₂ atmosphere with anti-CD3/CD28 (1 μg/mL) in complete culture medium (RPMI 1640 supplemented with 10% fetal bovine serum and 1% each of l-glutamine, sodium pyruvate, nonessential amino acids, antibiotics, 0.1 M HEPES, 55 μM β-mercaptoethanol). For each sample, at least 2 million cells were left unstimulated as negative control, and 2 million cells were stimulated. All samples were incubated with a protein transport inhibitor containing brefeldin A (Golgi Plug, Becton Dickinson) and previously titrated concentration of CD107a-PE. After stimulation, cells were stained with LIVE-DEAD Aqua (ThermoFisher Scientific) and surface mAbs recognizing CD3 PE- Cy5, CD4 AF700, and CD8 APC-Cy7 (Biolegend, San Diego, CA, USA). Cells were washed with stain buffer, and fixed and permeabilized with the cytofix/cytoperm buffer set (Becton Dickinson) for cytokine detection. Cells were next stained with previously titrated mAbs recognizing IL-17 BV421, TNF BV605, IFN-γ FITC, IL-2 APC, or granzyme-B BV421 (all mAbs from Biolegend). Then, a minimum of 100,000 cells per sample were acquired on a Attune NxT acoustic cytometer (ThermoFisher).