2.11. pMHC-I multimer cell staining and cell sorting

Cultured TCR⁺ J76^CD8 cells were washed once with pMHC staining buffer (DPBS supplemented with 2% FCS, 2 mM EDTA, 100 nM dasatinib) and were then stained with PE-labeled pMHC-I multimers diluted to approximately 50 nM in pMHC staining buffer at RT for 25 min. After pMHC-I multimer staining, TCR⁺ J76^CD8 cells were washed once with pMHC staining buffer followed by labeling with murine TCR-Cβ-APC (clone H57-597, BioLegend) and CD8-PacificBlue (Clone SK1, BioLegend).

Cultured ex vivo CD8⁺ T cells were washed once with DPBS/100 nM dasatinib and were then labeled with the Zombie Aqua™ Fixable Viability Kit (BioLegend 423102) 1:300 for 10 min at RT in DPBS + 100 nM dasatinib. Next, one volume of pMHC staining buffer + Human TruStain FcX (Fc receptor blocking solution, BioLegend 422302) was added 1:50 (v/v) following an incubation for 5 min at RT. Cells were then stained with prepared pMHC-I multimer libraries at RT for 25 min. After one wash, cells were stained using a cocktail containing optimally titrated antibodies (all from BioLegend) against human CD14 (M5E2, Cat. No. 301842), CD16 (3G8, 302048), CD19 (H1B19, 302242), and CD335 (9E2, 331924) (all Brilliant Violet 510-conjugated, defined as dump channel); CD8 (SK1, BioLegend 344714) APC-Cy7, and CD3 (UCHT-1) Alexa Fluor 700 (BioLegend 300424).

For the labeling of CD8⁺ T cells in freshly isolated PBMC with a DNA-barcoded pMHC-I multimer library, 0.1 µg/ml herring sperm DNA (Invitrogen™) was additionally added to the pMHC-I staining buffer and the above-mentioned antibody panel was appended by an antibody mix containing 30 DNA-barcoded TotalSeq™-C antibodies (BioLegend) as listed in Supplementary Table 4 .

Finally, the stained TCR⁺ J76^CD8 cells or PBMC-derived CD8⁺ T cells were stored in DPBS supplemented with 2.5% (v/v) paraformaldehyde and 1% FCS before flow cytometry measurement on a LSRFortessa flow cytometer and analyzed according to the gating strategy shown in Supplementary Figure 2 using FlowJo (BD Biosciences) v.10.9.0. In the dual color-encoded pMHC-I multimer-binding data shown, pMHC-I multimer binding CD8⁺ T cells were identified by a Boolean gating strategy as live CD8⁺ T cells stained positive in two pMHC multimer channels and negative in all other pMHC multimer color channels, as previously described (50, 51).

CD8⁺ T cells labeled with the DNA-barcoded pMHC-I multimer library were kept in pMHC staining buffer and pMHC-I multimer positive cells were sorted with a FACSAria™ Fusion cell sorter (BD Biosciences) according to the gating strategy shown in Supplementary Figure 2 into tubes containing 200 μl pMHC staining buffer.