Antigen-specific CD4+ T cell isolation

Fresh PBMCs (10–20 × 10⁶ cells, final concentration 10 × 10⁶ cells/ml) were stimulated for 3 h at 37°C with 0.6 nmol/ml PepTivator C. albicans MP65 (pool of 15-amino-acid-length peptides with 11 amino acid overlap, Miltenyi Biotec) in RPMI + 5% human serum in the presence of 1 μg/ml anti-CD40 (HB14, Miltenyi Biotec). After stimulation, the PBMCs were labeled with PE-conjugated anti-CD154 (5C8, Miltenyi Biotec) and enriched with anti-PE magnetic beads (Miltenyi Biotec) (41 (link)). After enrichment, the cells were stained with PerCP-Cy5.5 anti-CD4 (RPA-T4, Biolegend), AlexaFluor700 anti-CD3 (SK7, Biolegend), and APC-Cy7 anti-CD45RA (HI100, Biolegend), and the antigen-specific T cells were gated as CD3⁺CD4⁺CD45RA⁻CD154⁺ single live cells for single cell sorting.