Anti cd27 bv510

Peripheral blood mononuclear cells (PBMCs) from healthy controls were isolated by the Ficoll gradient (GE Healthcare, Zwijndrecht, The Netherlands). Cells were further isolated using the CD4+ T Cell Isolation Kit on an autoMACS Pro Separator, according to the manufacturer’s instructions (Miltenyi Biotec, Leiden, The Netherlands). To isolate the naïve subpopulation, total CD4⁺ T cells were stained with anti-CD4 APC-eF780 (eBioscience, Nieuwegein, The Netherlands), anti-CD27 BV510 (Biolegend, Amsterdam, The Netherlands), anti-CD25 PE, anti-CD127 Alexa Fluor-647 and anti-CD45RO PE/Cy7 (BD Biosciences, Vianen, The Netherlands) antibodies and purified on a BD FACSAria™ III cell sorter (BD Biosciences, Vianen, The Netherlands). Naïve CD4 ⁺ CD25⁻CD27 ⁺ CD45RO⁻ T cells were defined according to the gating strategy shown in Figure S5. Purity was consistently > 99%.

Supernatants were removed and cell pellets were resuspended in 200 μL of staining buffer containing 1 μL each of anti-CD38-APC (BioLegend, clone HIT2), anti-CD13-PE-Cy7 (BioLegend, clone WM15), anti-CD21-PE-Dazzle (BioLegend, clone Bu32), anti-CD19-APC-efluor-780 (Invitrogen, clone HIB19), anti-IgD-PerCP-Cy5.5 (Biolegend, clone IA6–2), anti-IgM-FITC (Biolegend, clone MHM-88), anti-CD27-BV510 (Biolegend, clone M-T271), anti-CD11c-Alexa700 (BioLegend, clone Bu15). Staining buffer also contained Delta-S1-PE and S1-BV421 tetramers.