Imag anti human cd8 particles

Large-volume phlebotomies (180 mL) were performed on patients who were on suppressive ART for ≥16 months. PBMC were isolated by Ficoll-Paque density gradient centrifugation. CD8⁺ T cells were removed from PBMC by positive selection using BD iMag anti-human CD8 Particles (BD). Total CD4⁺ T cells were isolated from PBMC by negative selection using the EasySepCD4⁺ T cell Enrichment Kit (STEMCELL). Resting CD4⁺ T cells were isolated from PBMC by negative selection using the EasySep Human Custom Enrichment Kit (STEMCELL) that depletes cells expressing CD8, CD14, CD16, CD19, CD20, CD36, CD56, CD66b, CD123, TCRγ/δ, glycophorin A, CD69, CD25, and HLA-DR.

Tumor antigen-specific T cells were co-cultured with HLA-A2⁺ or HLA-A2^- melanoma cells (1–2 × 10⁶ lymphocytes/well in 200 microliter final volume) in U-shaped 96-well plates at a 1:1 effector-to-target ratio. Following incubation for 1 hour at 37°C, CD8⁺ T cells were purified using BD IMag anti-human CD8 particles (BD Biosciences). Sorted CD8⁺ T cells, designated CD8⁺T-APC, were labeled with the fluorescent lipophilic dye DiIC18 (Life Technologies), according to the manufacturer’s instructions. DiIC18-labeled T-APCs were used as targets for specified T cell clones. To enable separate gating of CD8⁺T-APC and effector CTL, the latter were surface-biotinylated with Biotin-7-NHS (Roche) according to the manufacturer’s instructions. CD8⁺-T-APCs were co-cultured with effector CTL either immediately or 6-, 24- or 48-hours post separation with anti-human CD8 particles, as specified in each experiment.