Ifn γ af700

PBMCs were cultured in RPMI-1640 medium (GIBCO, Grand Island, NY, USA) containing 10% FBS, and stimulated with anti-CD3/CD28 (2 μg/mL and 5 μg/mL, Ebioscience) plus Golgiplug (BD Biosciences) for 5 h. The cells were surface-stained with CD3-BV786, CD4-APC-Fire750, CD8-BV421, CD70-PE, and intracellularly stained with TNF-α-BV711 (BD Biosciences), IFN-γ-AF700 (Ebioscience), or IL-2-BV650 (BioLegend) antibodies. For Ki67, perforin or Granzyme B staining, PBMCs were surface-stained with CD3-BV786, CD4-APC-Fire750, CD8-BV421, CD70-PE, and intracellularly stained with Granzyme B-AF700 (BD Biosciences), Ki67-BV711, or perforin-APC (BioLegend) antibodies. A fixable viability dye eFluor® 506 (Ebioscience) was used to assess cell viability.

PBMCs were stimulated with anti-CD3/CD28 (5 µg/mL, Ebioscience) for 5 h in the presence of anti-CD107a BV421 (BioLegend) and Golgiplug (BD Biosciences). CD107a expression was measured as a marker of degranulation on CD8⁺ T cells after stimulation. The cells were surface-stained with CD3-BV786, CD4-APC-Fire750, CD8-BV421, CD244-PE-D594, CD160-AF488, and intracellularly stained with TNF-α-BV711, IL-2-BV650 (BioLegend), or IFN-γ-AF700 (Ebioscience) antibodies. For Ki67, perforin, Granzyme B, T-bet, or Eomes staining, PBMCs were surface-stained with CD3-BV786, CD4-APC-Fire750, CD8-BV421, CD244-PE-D594, CD160-AF488, and intracellularly stained with Granzyme B-AF700, T-bet-BV421 (BD Biosciences), Ki67-BV711, perforin-APC (BioLegend), or Eomes-PE-CY7 (Ebioscience) antibodies. A fixable viability dye eFluor^® 506 (Ebioscience) was used to label dead cells.