Immunomagnetically selected tetramer-positive cells secrete IFN-γ in response to peptide only after in vitro culture.

Tetramer-stained cells from subject 606 were isolated from PBMC using α-PE Miltenyi beads and cultured for 5 days in the presence of IL-2 and IL-15. Fresh or cultured cells were stimulated with autologous BLCL alone (A, B) or with autologous BLCL pulsed with the relevant peptide (C, D) for 8 hours. Brefeldin A (10 μmol/L) was added during the last 4 hours of incubation. Cells were washed and stained with gag tetramer PE, CD8-Cy5, and IFN-γ FITC. Left panels depict tetramer-positive cells tested immediately after immunomagnetic selection. Right panels show IFN-γ–secreting cells after culture. Analyses are performed on the CD8-gated population.