Anti cd45ro

CD8⁺ T cells were isolated from monocyte-depleted PBMCs by magnetically-activated cell sorting (MACS) using positive selection with human anti-CD8 microbeads according to the manufacturer’s instructions (Miltenyi Biotec). When indicated CD8⁺ T_N cells were isolated from monocyte-depleted PBMCs by MACS using the CD8⁺ T-cell isolation kit, anti-CD45RO and anti-CD57 microbeads (Miltenyi Biotec). In that case, the monocyte-depleted PBMCs were depleted from CD45RO and CD57 positive cells, after which a positive selection was performed for CD8⁺ T cells. The T cells were co-cultured with moDCs that were electroporated with mRNA encoding GFP, tNGFR, TriMix, TetraMix or Melan-A/MART-1 following the protocol described by Bonehill et al (53 (link)). Evaluation of the CD8⁺ T-cell specificity for Melan-A/MART-1 was assessed at different time points using flow cytometry. When indicated the CD8⁺ T cells were phenotyped by flow cytometry or used for the in vitro killing assay.