Effect of anti–4-1BB costimulation on the differentiation of subset II cells.
Anti–4-1BB costimulation promotes the differentiation of subset II into III cells. Subset II cells were isolated by cell sorting from anti-CD28–stimulated CB CD8+ T cells after a 5-day culture in the presence of IL-15 and IL-12 as described in Figure 3A, and stimulated on plates precoated with isotype control IgG, anti-CD28, or anti–4-1BB plus anti-CD3 for 3 days. The resulting cells were analyzed for expression of CD28 and intracellular granzyme B as described in Figure 4. The cells were also analyzed for apoptosis by staining with annexin V (A). Percentages shown in the histograms represent the positive cells. Cells stimulated with the different antibodies were used as effectors in a standard 51Cr release assay at an effector cell–to–target cell ratio of 2:1 (B), as described above. The 51Cr release assays were performed in the presence or absence of anti-CD3 (1 μg/mL). The data are presented as the percentage of specific lysis. Subset II and III cells were purified and subjected to 51Cr release assays with the use of K562 cell line as target cells at an effector-to-target cell ratio of 2:1 in the presence or absence of anti-CD3. These data are representative of 3 experiments.
