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Fig. 6. CD8⁺ T cells from wild-type (WT) and IL-15Ra(-/-) mice were stained with anti-IL-15Ra(R & D Systems), and then stained with mouse anti-goat IgG-FITC. Cells were analyzed by flow cytometry.

Fig. 7. Positively purified (Miltenyi Biotec, Auburn, CA) spleen CD8⁺ T cells from mice immunized with vPE16 ± IL-15 were labeled with 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) and plated at 5 × 10⁴ cells per well in a 96-well flat-bottomed microtiter plate (Costar). Irradiated (3,000 rads) syngeneic splenocytes pulsed with different concentrations of peptides were added at 1 × 10⁵ cells per well. On day 5, cell proliferation was measured by a flow cytometric method quantifying the number of cell divisions by the dilution of CFSE with each cell division. These experiments all represent bulk ex vivo populations from immunized animals, not lines or clones, but as cells in the histograms are gated on anti-CD8 and tetramer-positive T cells, all of the cells are specific for the antigen.

Fig. 8. Antigen-specific lytic activity of preparatively sorted CD8⁺ CTLs was measured by a 5-h ⁵¹Cr release assay against target cells with low density of antigen (pulsed at 0.001 m M) to measure high-avidity CTL activity as described.

Fig. 9. Both high, grown with 0.001 m M, and low, grown with 1.0 m M P18-I10, avidity CTL lines were labeled with CFSE and cultured in the media containing 20 ng/ml IL-15, and the percent of proliferating cells was analyzed by flow cytometry measurements of the dilution of CFSE on days 2, 4, and 6. Because CFSE staining counts individual cells that have proliferated different numbers of divisions, it cannot be biased by rapid proliferation of a small subset of cells. Only high-avidity CD8⁺ CTLs vigorously responded to IL-15 by CFSE staining (as well as by [³H]thymidine incorporation; data not shown).

Fig. 10. Spleen CD8⁺ T cells from the immunized mice were restimulated with 0.001 mM for 1 week. Cells were stained with anti-CD8a, tetramer, and CD8b. Data show the levels of CD8bexpression of the cells gated on anti-CD8aand tetramer-positive cells. The numbers in the histograms represent geometric mean fluorescence intensity ± SEM of four individual mice in each group.