Use of CD137 expression for enrichment of antigen-specific T cells. (A) Experimental outline: Naive CD45RO⁻CD8⁺ T cells were stimulated with peptide-pulsed DCs and cultured for 1 week with IL-7 and IL-15 added on day 4. On day 8, the cells were restimulated with irradiated, autologous, peptide-pulsed PBMCs. Twenty-four hours later, CD137⁺ cells were isolated with magnetic beads and cultured for an additional 8 days in medium containing IL-2, IL-7, and IL-15 to allow both expansion and re-expression of the TCR. The cells were then analyzed for specificity using pMHC multimers. (B) Enrichment of an HCV/NS3_(1406-1415)-specific T-cell line by selection of CD137⁺ cells. Eight days after primary stimulation of naive T cells, an aliquot was stained with an NS3_(1406-1415)/MHC multimer to determine the frequency of antigen-specific cells before restimulation and/or enrichment (i). These cells were then split and either cultured in IL-2–, IL-7–, and IL-15–containing medium without any restimulation (ii), restimulated and cultured but not enriched (iii), or restimulated and enriched for CD137⁺ cells 24 hours later and then cultured for an additional 8 days (iv), and then stained with anti-CD8 antibody and the pMHC multimer. (C) Parallel to the pMHC-multimer staining, an aliquot of the enriched T-cell population was also restimulated with peptide-pulsed T2 cells and cytokine production was assessed after 5 hours (left). T cells incubated with unloaded T2 cells served as control (right). (D) Nine different HCV/NS3-specific T-cell lines were generated and enriched 24 hours after the second stimulation as above, and the number of pMHC multimer⁺ T cells assessed 8 days later and compared with lines not enriched (2-tailed, paired t test: P = .002).