Anti tcrγ δ

Memory CD4+ T cells and non-CD4+ T cells were depleted from PBMCs by incubation with CD45RO, CD8, CD14, CD15, CD16, CD19, CD25, CD34, CD36, CD56, CD123, anti-TCRγ/δ, anti-HLA-DR, and CD235a (glycophorin A) antibodies (Miltenyi Biotech, Sunnyvale, CA). Naive CD4+ T cells were isolated using negative selection magnetic bead methods (Miltenyi Biotech, Sunnyvale, CA).

Primary human monocytes were isolated from buffy coats from healthy donors (Sanquin, Nijmegen, The Netherlands). Before sample collection, a written consent was obtained. Buffy coats were diluted 1:1 with phosphate-buffered saline (PBS) + 2% fetal bovine serum (FBS) (HyClone™ Fetal Bovine Serum, Fisher Scientific, Loughborough, UK) and loaded onto Greiner Bio-One™ LeucoSEP™ Polypropylene Tubes to obtain peripheral blood mononuclear cells (PBMCs). After being washed, the cells were diluted in MACS buffer, and a CD14 microbead kit was used to isolate the monocytes according to the manufacturer's protocol (Miltenyi Biotec, Leiden, The Netherlands). Naive CD4⁺ T-cells were isolated from the flow through using a negative selection. A naive CD4⁺ T Cell Isolation Kit, containing a cocktail of biotinylated CD45RO, CD8, CD14, CD15, CD16, CD19, CD25, CD34, CD36, CD56, CD123, anti-TCRγ/δ, anti-HLA-DR, and CD235a (glycophorin A) antibodies (Miltenyi Biotec, Leiden, The Netherlands), was used. All cells were frozen in 1:1 FBS and FBS with 20% dimethyl sulfoxide (DMSO) and stored at −80°C until further use.