Proliferative response of spleen and BM antigen-specific memory CD8 cells to IL-15, IL-7, and IL-21. OT-I-B6 mice were immunized against OVA as described in “Materials and methods, Immunization.” After 7 to 12 months, CD8⁺ cells were purified from spleen and BM, labeled with CFSE, and cultured (5 × 10⁴ cells/well) for 7 days in the presence of graded concentrations of IL-15 and IL-7, each tested alone or in combination with IL-21. (A,B) CD8-cell purification and CD45.1 staining. Typical cytometric profiles of total CD8⁺ cells purified from spleen and BM (A) and of CD45.1 staining within CD8⁺ cells (B). The scales on x-axis and y-axis are logarithmic, in arbitrary units. Numbers indicate the percentages in the gated region, showing the purity of CD8 cell preparation (A) and the frequency of CD45.1⁺ OT-I cells within CD8 cells (B). All CD45.1⁺CD8⁺ cells were CD45.2⁻ and OVA-tetramer⁺. (C,D) Analysis of CD45.1⁺-cell proliferation by CFSE labeling. After 7 days of culture with IL-15, IL-7, and IL-21, cells were analyzed by flow cytometry and the percentage of divided cells, those with decreased CFSE fluorescence intensity, was determined within CD45.1⁺ cells. For each organ, the marker was set based on CFSE intensity of unstimulated cells. Example of CFSE staining profiles of spleen and BM CD45.1⁺ CD8 cells in response to IL-15 and IL-7 (each at 25 ng/mL), with or without IL-21 (at 50 ng/mL) (C) and comparison of spleen and BM CD45.1⁺ CD8-cell proliferative response to graded concentrations of IL-15 and IL-7, each tested alone or in combination with 50 ng/mL of IL-21 (D). In (C), the scale on x-axis is logarithmic, in arbitrary units, and that on y-axis is linear (maximum value 20, except for IL-15+IL-21 panels, which have a maximum value of 80). The unstimulated cell profiles are shown with gray lines. The numbers represent the percentages of cells in the marked region. One representative experiment is shown of 3. For each experiment, cells were pooled from 4 to 5 mice. Differences between BM and spleen were similar in the 3 experiments and not statistically significant (average difference never > 20% for any cytokine concentration).