Pe cy7 anti slamf7

Tumor-specific CD4 T clones were stained systematically in PBS, 0.2% bovine serum albumin (BSA), 5 mM EDTA, and 0.2% NaN₃ with either (i) FITC anti-CD3 (BioLegend), BV605 anti-CD4 (BioLegend), ECD anti-CD8 (BD), PE-Cy7 anti-SLAMF7 (BioLegend), PerCP-Cy5.5 anti-CD57 (BioLegend), APC (allophycocyanin) anti-CD28 (BioLegend), BV711 anti–PD-1 (BioLegend), PE anti-TCRαβ (BioLegend), and LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific) or (ii) FITC anti-CD3 (BioLegend), BV605 anti-CD4 (BioLegend), APC anti-TRAIL (BioLegend), PE anti-FasL (BioLegend), and LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific) at 4°C for 30 min. Data were acquired on LSR II (BD Biosciences) and analyzed using FlowJo software (v.10.4.2).

Enriched CD4 T cells were purified by positive selection using MACS isolation microbeads (Miltenyi) and stained directly ex vivo using a combination of fluorescent pMHCII multimers, as described above and FITC anti-CD3 (BioLegend), BV605 anti-CD4 (BioLegend), PE-Cy7 anti-SLAMF7 (BioLegend), BV711 anti–PD-1 (BioLegend), and LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific). Irrelevant multimers were used as negative controls. Data were acquired on LSR II (BD Biosciences) and analyzed using FlowJo software (v.10.4.2).