Figure 4

Isolation of PD-1⁺, PD-1⁺⁺, and PD-1⁺⁺⁺ T cells using a novel antigen density-dependent magnetic sorting (ADD-MS). (A) ADD-MS procedure utilizing controlled release of antibody/beads based on antigen expression density. The key idea behind this method is to release the bound beads with short term incubation (30 min) at 37°C to differentially sort high and medium antigen-expressing cells. In a typical experiment, approximately (~) 100 million SRBC-rosetted T cells were processed to prepare ~4 million PD-1⁺⁺⁺, ~0.7 million PD-1⁺⁺, and ~3 million PD-1⁺ T cells. Anti-FITC or anti-PE microbeads (Miltenyi Biotec) were used at 2× volume of the primary FITC or PE-conjugated antibodies to sort the T cell subsets. (B) PD-1 and CD45RO expression by the CD4⁺ T cells isolated by the ADD-MS method.