Anti murine tcr β chain apc fire750

pMHC monomers were generated as previously described⁶⁶ (link). All pMHC-multimer reagents were generated by 45 min incubation of 4 μg biotinylated pMHC monomer with 1 μg streptavidin-BV421 (Biolegend, reference #405225) or Streptavidin-APC (eBioscience, reference #17-4317-82) in a total volume of 100 μl FACS buffer for staining of up to 1x10⁷ cells. pMHC-multimer staining was performed separately from surface antibody staining for 45 min on ice. Surface antibody staining was performed for 25 min on ice subsequent to pMHC-multimer staining. Depending on the respective experiments, a subset of the following antibodies were used: anti-human TCR α/β FITC (BioLegend, reference #306706), CD3 PC7 (Beckman Coulter, reference #737657), CD8 PE (Invitrogen, reference #MHCD0804), anti-murine TCR β-chain APC (BioLegend, reference #109212), anti-murine TCR β-chain APC/Fire750 (BioLegend, reference #109246), CD62L FITC (Biolegend, reference #304804), CD45-RO PC7 (BioLegend, reference #304230), Lag3 FITC (Biolegend, reference #369308) and PD-1 APC (Invitrogen, reference #17-2799-42). Live/dead discrimination was performed with propidium iodide (LifeTechnologies, reference #P1304MP).

One day before co-culture with T cells, K562 cells (retrovirally transduced to express the human MHC class-I molecule of interest) were irradiated (80 Gy) and loaded with peptide (10⁻¹² M, 10⁻¹⁰ M, 10⁻⁹ M, 10⁻⁸ M, 10⁻⁷ M, 10⁻⁶ M, 10⁻⁵ M, 10⁻⁴ M) overnight at 37°C, 5% CO₂. T cells were co-cultured in a 1:1 ratio with peptide-loaded K562 cells and 1x Golgi plug (BD PharMingen, reference #555029) for 4 h at 37°C. 25 ng ml^-1 Phorbol-12-myristat-13-acetat (PMA) (Sigma, reference #P1585-1mg) and 1μg ml^-1 ionomycin (Sigma, reference #I9657-1mg) were used for positive control. After co-culture, staining for live/dead discrimination was performed with ethidium-monoazide-bromide (LifeTechnologies, reference #E1374). Subsequently, surface marker antibody staining for CD8 (PE, Invitrogen) and anti-murine TCR β-chain APC/Fire750 (BioLegend, reference #109246) was performed and followed by permeabilization using Cytofix/Cytoperm (BD Biosciences, reference #554714), and intracellular staining of IFNγ FITC (BD PharMingen, reference #340449), TNFα PC7 (eBioscience, reference #25-7349-82) and IL-2 APC (BD PharMingen, reference #341116).