H 2kb clone af6 88.5

The following cell surface antigens were detected by flow cytometry using the following antibodies: mouse CD4 (clone GK1.5), CD8 (clone 53–6.7), CD3ε (clone 145-2C11), CD69 (clone H1.2F3), I-A/I-E (clone M5/114.15.2), and H-2K^b (clone AF6-88.5) (all from Biolegend). BV421- and APC-conjugated, TB10.4₄₋₁₁-loaded, H-2K^b tetramers were obtained from the National Institutes of Health Tetramer Core Facility (Emory University Vaccine Center, Atlanta, GA). Zombie Violet Fixable viability dye (Biolegend) or the Live/Dead Fixable Far Red Dead Cell stain (ThermoFisher) were used for distinguishing live from dead cells. To stain for the Nur77 transcription factor, the Nur77 monoclonal antibody (clone 12.14) was used in combination with the Foxp3 Transcription Factor Staining Buffer Set (both from ThermoFisher) by the manufacturer’s protocol. Live/dead viability staining and surface staining were done for 20 minutes at 4°C, and intracellular staining was done for 30 minutes at room temperature. Samples were then fixed with 1% paraformaldehyde/PBS for 1 hour before being analyzed by a MACSQuant flow cytometer (Miltenyi Biotec). FlowJo Software (Tree Star, Portland, OR) was used to analyze the collected data. Single lymphocytes were gated by forward scatter versus height and side scatter for size and granularity, and dead cells were excluded.

For samples not sorted, cells were washed with PBS and stained in FACS buffer (2% FBS, 2 mM EDTA, and 0.001% NaN₃). All gating strategies are represented in Supplementary Fig. 9. Cells were first stained for H-2K^b/SIY-pentamer (PE; ProImmune) for 10 min at room temperature at a 1:20 dilution, followed by staining with remaining antibodies for 20 min on ice. Antibodies against the following molecules were used: CD3ε (clone 17A2, BioLegend, 100216), Thy1.2 (clone 30-H12, BioLegend, 105320), CD45.2 (clone 104, BioLegend, 109806), CD8α (clone 53-6.7, BioLegend, 100747), CD4 (clone RM4-5, BioLegend, 100547), PD-L1 (clone 10 F.9G2, BioLegend, 124312), CD19 (clone 6D5, BioLegend, 115545), I-A/I-E (clone M5/114.15.2, BioLegend, 107630), and H-2K^b (clone AF6-88.5, BioLegend, 742862). All antibodies were used at a 1:100 dilution. Fixable Viability Dye eFluor 506 or 780 (eBioscience) was used for live/dead discrimination and was used at a 1:200 dilution. All flow cytometric analysis was conducted on either an LSRFortessa or X20 (BD) and analyzed using FlowJo software (Tree Star).