Supporting Figures

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Supporting Figure 6
Supporting Figure 7
Supporting Figure 8
Supporting Figure 9

Fig. 6. Survival of IL-15 in vitro. (A) Purified carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled MP CD8⁺ T cells were added to cultures containing IL-15 alone at 5 ng/ml (Upper, gray), 100 ng/ml (Lower), or 5 ng/ml of IL-15 plus 1 mg/ml IL-15Ra-Fc (s, soluble; Upper, solid line). These cultures were either freshly prepared (fresh) or were left for 48 h at 37°C (48 h preculture) before the addition of T cells. (B) The same as A except that T cells were cultured with supernatants taken from the "48 h precultures" (supernatant) vs. the latter cultures, which had been emptied of supernatant without washing (well-bottom). Interpretation: The experiment shows that the biological activity of IL-15 (cultured alone) did not decline significantly during culture for 48 h at 37°C, thus making it unlikely that sIL-15Ra-Fc acted simply by prolonging the half-life of IL-15. Furthermore, the absence of proliferation of cells transferred to the emptied wells (well-bottom) suggests that the enhancing activity of the soluble receptor did not reflect cross-linked presentation of IL-15 bound via the receptors to the plastic bottom of the well.

Fig. 7. Human sIL-15Ra-Fc enhances the response of mouse memory-phenotype (MP) CD8⁺ cells to both mouse and human IL-15. CFSE-labeled purified MP CD8⁺ T cells were cultured at 5 × 10⁴ cells per well with either 100 ng/ml of human IL-15 or 5 ng/ml of murine IL-15. As indicated, 1 mg/ml of human sIL-15Ra-Fc was added to the cultures. CFSE dilution was assessed after 3 days of culture. Note that, in direct contrast to CTLL (which express IL-15Ra plus bg_c), mouse MP CD8⁺ cells respond better to mouse IL-15 than to human IL-15 (1).

1. Eisenman, J., Ahdieh, M., Beers, C., Brasel, K., Kennedy, M. K., Le, T., Bonnert, T. P., Paxton, R. J. & Park, L. S. (2002) Cytokine 20, 121–129.

Fig. 8. Soluble IL-15/sIL-15Ra-Fc complexes have a longer lifespan in vivo than free IL-15. Normal B6 mice were injected with IL-15 (5 mg) or IL-15 (1 mg) plus sIL-15Ra-Fc (5 mg) at 18 h, 4.5 h, 1.5 h, or at 30 min before, or immediately after (0 h), adoptive transfer of CFSE-labeled MP CD8⁺ T cells. At 42 hours after adoptive transfer, spleens were harvested, and the percentage of maximal proliferation was calculated based on proliferation at time point 0 h being 100%. Each time point represents two mice for IL-15 and two mice for IL-15 plus sIL-15Ra-Fc.

Fig. 9. Blocking effects of sIL-15Ra-Fc for responses to mouse vs. human IL-15. (A–C) CTLL-2 cells were cultured for 2 days with murine IL-15 (A), human IL-15 (B), or murine IL-2 (C). As indicated, cultures were supplemented with 1 mg/ml of murine sIL-15Ra-Fc. [³H]thymidine is added during the last 24 h of culture. The data show mean levels of [³H]thymidine incorporation (±SD) for triplicate cultures. (D) One million OT-1 cells (Thy1.1 congenic) were adoptively transferred i.v. into B6 recipient mice on day –1. On day 0, mice were vaccinated with 1 million SIINFEKL-pulsed dendritic cells i.v. On days 1–7, recipient mice were given daily i.p. injections of PBS, sIL-15Ra-Fc alone (5 mg), IL-15 alone (1 mg), or sIL-15Ra-Fc plus IL-15 (5 mg and 1 mg, respectively). On day 8, spleens were harvested, counted, and evaluated by flow cytometric analysis for donor OT-1 cells. The data show a fold increase of absolute numbers of OT-1 cells relative to vaccination without cytokine or receptor treatment. All data are representative of at least two independent experiments.