CD4+ responder T (Tresp) cells were isolated with the CD4+ untouched T cell Isolation Kit (Miltenyi Biotec) and labeled with the CellTrace Violet Cell Proliferation Kit (Invitrogen, Molecular Probes) at a final concentration of 2.5 mM.
Tresp cells (5 × 104) were cocultured at different ratios with ex vivo–isolated polyclonal or A. fumigatus–specific CD137+ Tregs or CD154+ cells as a control in 384-well plates in RPMI-1640 supplemented with 5% AB serum. Cells were stimulated polyclonally with anti-CD3/-CD28 beads (Treg Suppression Inspector; Miltenyi Biotec). On day 6, dilution of proliferation dye and expression of CD25 (Miltenyi Biotec) were analyzed by flow cytometry. Allogenic Tresp cells were discriminated from Tregs via discordant HLA-A2 expression.
Tresp cells (5 × 104) were cocultured at different ratios with ex vivo–isolated polyclonal or A. fumigatus–specific CD137+ Tregs or CD154+ cells as a control in 384-well plates in RPMI-1640 supplemented with 5% AB serum. Cells were stimulated polyclonally with anti-CD3/-CD28 beads (Treg Suppression Inspector; Miltenyi Biotec). On day 6, dilution of proliferation dye and expression of CD25 (Miltenyi Biotec) were analyzed by flow cytometry. Allogenic Tresp cells were discriminated from Tregs via discordant HLA-A2 expression.
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