Fixation and intracellular staining permeabilization wash buffer

PE‐labeled anti‐CCR7 (4B12; BioLegend), FITC‐labeled anti‐CD4 (GK1.5; TONBO Bioscience, San Diego, CA, USA), and PE‐Cy5‐labeled anti‐CD8a (53–6.7; TONBO Biosciences) antibodies were used to stain cell‐surface molecules after blocking the Fc receptors with 2.4G2 (BD Pharmingen, Franklin Lakes, NJ, USA). In the experiment described in Fig. 2C, CD4⁺ T cells were fixed and permeabilized with Fixation and Intracellular Staining Permeabilization Wash Buffer (BioLegend). Fluorescence intensity was acquired by MACSQuant flow cytometry (Miltenyi Biotec, Tubingen, Germany) and analyzed by FlowJo (TOMY Digital Biology, Tokyo, Japan).

Cells isolated from mouse spleen and thymus were stained with Ghost Violet™ 510 viability dye (TONBO Biosciences) for dead cell exclusion, blocked with Fc block (2.4G2, BD Biosciences) and stained with the following anti-mouse antibodies from Biolegend: CD4-BV785 (GK1.5), CD44-PE-Cy7 (IM7), TCRb-APC (H57-597), CD8-PerCP-Cy5.5 (53-6.7) and CD45-APC-Cy7 (30-F11). Cells were then fixed and permeabilized using fixation and intracellular staining permeabilization wash buffer from Biolegend following the manufacturer’s instructions. Cells were stained with mouse anti-IRAP-AF594 (F5, Santa Cruz Biotechnology, dilution 1/20) or with rabbit a-Stx6 (ProteinTech, dilution 1/100) or monoclonal rabbit IgG (Cell Signaling Technology, dilution 1/500) followed by staining with a-rabbit AF594 (Thermo Fisher Scientific, dilution 1/200).