Human cd8 t cell enrichment kit

CD8⁺ T cells were purified from PBMCs by negative selection using magnetic beads, resulting in depletion of CD4⁺, CD14⁺, CD16⁺, CD19⁺, CD20⁺, CD36⁺, CD56⁺, CD66b⁺, CD123⁺, TCRγ/δ⁺ and glycophorin A⁺ cells (Human CD8⁺ T Cell Enrichment Kit, Stemcell Technologies, Vancouver, BC, Canada). Purified CD8⁺ T cells were labeled with CFSE (2 µM, Life Technologies, Darmstadt, Germany), the CD8^- fraction was irradiated at 30 Gy and added to the culture as feeder cells. Specific p:HLA-E presenting APCs were obtained from incubation of T2E cells with individual peptides (300 µM) for 18 h at 37 °C. Peptide loaded target cells were irradiated at 40 Gy. 2 × 10⁵ CFSE labeled CD8⁺ T cells were co-cultured with 1 × 10⁵ peptide loaded T2E cells in the presence of 5 × 10⁵ CD8⁻ feeder cells in 96 well roundbottom plates. Cells were cultured in RPMI supplemented with 10% human serum (C-C-Prom, Oberdorla, Germany) and 5 ng/mL IL-7 (Peprotech, NJ, USA). On day 7 of co-culture, IL-2 (Peprotech) was added to a final concentration of 10 U/mL. CD8⁺ T cells co-incubated with T2E cells without peptide or with medium only were carried along as controls. On day 10 of co-culture, cells were harvested and stained with CD3-PE-Cy7 and CD8-PerCP (both from BD Bioscience, Heidelberg, Germany). Cells were gated on CD3⁺CD8⁺ and CFSE dilution was analyzed (Supplementary Figure S4).

Lymphocyte subsets were purified from freshly prepared PBMC using EasySep (StemCell Technologies) negative magnetic selection kits: Human CD4⁺ T Cell Isolation Kit (StemCell Technologies, cat no. 17952), Human CD8⁺ T Cell Enrichment Kit (StemCell Technologies, cat no. 19053), Human NK Cell Enrichment Kit (StemCell Technologies, cat no. 19055) and Human Pan-B Cell Enrichment Kit (StemCell Technologies, cat no. 19554), per manufacturer’s instructions. After purification, 2 million (Pan B) or 3 million (CD4⁺ T, CD8⁺ T, and NK) isolated cells were lysed in Buffer RLT Plus (RNeasy Plus Mini Kit, Qiagen, cat no. 74134), per the manufacturer’s protocol, and lysates were kept at 4°C until RNA extraction. The remaining isolated cells were used for flow cytometric staining of markers to assess cell viability and purity as described below.