Article Figures & Data
Figures
- FIGURE 1.
IL-12 + IL-18-induced proliferation of B6 (+/+) and B6 (nu/nu) splenic populations depleted of B cells and APC. Spleen cells from normal B6 mice (+/+) or athymic B6 mice (nu/nu) were depleted of B cells and Ia+ APC as described in Materials and Methods. These two types of spleen cells were cultured with rIL-12 and/or rIL-18 for various days in 96-well microculture plates at a density of 4 × 104 cells/well (A) or for 4 days in 24-well culture plates at a density of 2.5 × 105 cells/well. [3H]TdR uptake was determined after an 8-h pulse with 20 kBq/well [3H]TdR. Data are representative of three similar experiments (A). Cells harvested from 24-well culture plates were stained doubly for CD4 and CD8. The proportion of CD4−CD8− cells is shown. Data are representative of two similar analyses (B).
- FIGURE 2.
Generation of NK1.1+CD3− or NK1.1−CD3− cells from a CD4−CD8−sIg−Ia− splenic population depending on the type of cytokine combinations used. B6 spleen cells were depleted of B cells, APC and CD4+/CD8+ T cells. The resultant population (CD4−CD8−sIg−Ia−) was stained doubly for CD4 and CD8 or for NK1.1 and CD3 (left). Portions of these cells were stimulated with IL-12 + IL-18 or IL-2 + IL-18 (IL-12, 250 pg/ml; IL-18, 100 ng/ml; IL-2, 100 U/ml) for 4 days. Cells harvested were stained doubly for NK1.1 and CD3 (right). Data are representative of four similar experiments.
- FIGURE 3.
Proliferation of a splenic CD4−CD8−sIg−Ia− population induced by stimulation with either IL-12 + IL-18 or IL-2 + IL-18. A CD4−CD8−sIg−Ia− splenic population was stimulated with different cytokines, alone or in combination for various days. Data are representative of three similar experiments.
- FIGURE 4.
Phenotypes of blasts generated from a splenic CD4−CD8−sIg−Ia− population following stimulation with IL-12 + IL-18 or IL-2 + IL-18. Blasts generated from a splenic CD4−CD8−sIg−Ia− population 5 days after stimulation with IL-12 + IL-18 or IL-2 + IL-18 were stained with various Abs as described in Materials and Methods. The results are representative of two similar experiments.
- FIGURE 5.
NK1.1 phenotype conversion induced by a change in the stimulatory cytokine combinations. A CD4−CD8−sIg−Ia− population was stimulated with either IL-12 + IL-18 or IL-2 + IL-18 for 3 days. Portions of cells harvested were stained doubly for NK1.1 and CD3. The rest of cells were again stimulated with the homologous or alternative set of cytokine combinations for an additional 3 days. Cells harvested were stained for NK1.1 and CD3. The numbers on the figures are the percentages of cells stained by each of the reagents. The results are representative of three similar experiments.
- FIGURE 6.
Generation of both NK1.1+CD3− and NK1.1−CD3− blasts from resting NK1.1+CD3− cells in the original splenic population. NK1.1+CD3− and NK1.1−CD3− cells present in a splenic CD4−CD8−sIg−Ia− population were isolated using a cell sorter. These two subsets of cells (2.5 × 105 cells/well) were stimulated with either IL-12 + IL-18 or IL-2 + IL-18 for 4 days in 24-well culture plates. Cells (1 × 105 cells/well) harvested from the former type of stimulation cultures were restimulated with the two sets of cytokine combinations for an additional 3 days. Cells (1 × 105 cells/well) harvested from the latter type of stimulation cultures were again subjected to cell sorting. NK1.1+CD3− and NK1.1−CD3− cells were isolated, and restimulated with two sets of cytokine combinations. The numbers of blasts recovered on day 7 from 1 × 105 day 4 blasts are shown on each panel (× 10−5). The purity of each blast population obtained by cell sorting was >98%. The results are representative of two similar experiments.
- FIGURE 7.
Stimulation of [3H]TdR uptake by IL-12 + IL-18- and IL-2 + IL-18-induced blasts following reexposure to various cytokines. A CD4−CD8−sIg−Ia− population (2.5 × 105 cells/well in 24-well culture plates) was stimulated with IL-12 + IL-18 or IL-2 + IL-18 for 3 or 4 days. Blasts harvested (1 × 104 cells/well in microculture plates) were restimulated with various cytokines, alone or in combination, for an additional 1 to 4 days. Data are representative of three similar experiments.
- FIGURE 8.
Strikingly high levels of IFN-γ production by IL-12 + IL-18-induced blasts following restimulation with IL-12 + IL-18. A CD4−CD8−sIg−Ia− population was stimulated with IL-12 + IL-18 or IL-2 + IL-18 in 24-well culture plates. Three- or 4-day blasts (1 × 105 cells/well) were restimulated with various cytokines alone or in combination in 24-well culture plates. Culture SNs were examined for IFN-γ concentrations by ELISA. Data are representative of three similar experiments.
- FIGURE 9.
IFN-γ production by a freshly isolated CD4−CD8−sIg−Ia− population following stimulation with IL-12 + IL-18. A CD4−CD8−sIg−Ia− population freshly prepared from spleen cells was stimulated with either IL-12 + IL-18 or IL-2 + IL-18 for the indicated days. Culture SNs were assessed for IFN-γ production.
- FIGURE 10.
Potent NK killing by blasts induced by stimulation with IL-12 + IL-18 or IL-2 + IL-18. A CD4−CD8−sIg−Ia− population was stimulated with IL-12 + IL-18 or IL-2 + IL-18 for 4 days. Cells harvested (effectors) were cultured with various types of tumor target cells labeled with 51Cr at the E:T ratios indicated. YAC-1, A/Sn (H-2a)-derived lymphoma; EL-4, B6 (H-2b)-derived lymphoma; P815, DBA-2 (H-2d)-derived mastocytoma; SP2/0, BALB/c (H-2d)-derived lymphoma; WR/19L, BALB/c (H-2d)-derived lymphoma as a control for Fas transfectants; W4, Fas transfectants of WR/19L (W4 and WR/19L were kindly provided by Dr. S. Nagata, Osaka University Medical School, Osaka, Japan.).
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