Figure 4
Figure 4. Development of eosinophils from myeloid progenitors is inhibited by IFN-γ. (A) Absolute numbers of total T cells and T-cell subsets in the bone marrow (1 femur and 1 tibia) of WT and CD70TG mice. EM indicates effector memory; and CM, central memory. (B) Intracellular expression of IFN-γ in CD3+CD4+ gated and CD3+CD8+ gated T cells from bone marrow of WT and CD70TG mice on phorbol myristate acetate and ionomycin stimulation (percentages are shown). (C) Expression of IFN-γR1 on CMPs and GMPs of WT (thin line) and CD70TG mice (bold line). Filled graph shows fluorescence-minus-one control staining. (D) Expression of IFN-γR1 on CMPs and GMPs as the average geometric mean fluorescence intensity (GeoMFI). (E) Number of eosinophils generated from 5.0 × 103 WT CMPs after 9 days of culture with SCF and IL-5 with or without addition of various concentrations of IFN-γ. (F) Number of cells generated from 2.5 × 103 WT CMPs after 9 days of culture with SCF, IL-5, and GM-CSF with or without addition of various concentrations of IFN-γ. (G) Representative plot of staining for CD34+ IL-5Rα+ EoPs gated on the Lineage−CD16/32+c-Kitlow compartment after a 3-day culture of GMPs in the presence of SCF, IL-3, IL-5, and GM-CSF with or without the addition of IFN-γ (data indicate percentage of EoPs from total cells). (H) Flow cytometric analysis of IL-5Rα expression on TF1 cells cultured with IL-5 in the presence (thick line) or absence (thin line) of IFN-γ. Isotype control is shown in shaded gray. (I-K) Human CD34+ progenitor cells were cultured for 3 days in the presence of SCF, FLT-3, GM-CSF, IL-3, and IL-5 followed by culture for 15 days in IL-3 and IL-5. Expansion of progenitor cells at day 5 (I) and day 18 (J) in cultures in the presence of IFN-γ, expressed as percentage of expansion in the cultures without IFN-γ. (K) Percentage of differentiated eosinophils after 18 days of culture analyzed after May-Grünwald Giemsa staining of cytospins. (A,D) Data are the mean of 3 mice per group. (E-F) Data are mean ± SD for 2 cultures per condition. (H) Data are mean ± SD for 4 cultures per condition. (A-H) Data are representative of at least 2 independent experiments. (I-K) Data are the mean of 3 independent experiments from different donors. Data are mean ± SD. *P < .05; **P < .01; ***P < .001.

Development of eosinophils from myeloid progenitors is inhibited by IFN-γ. (A) Absolute numbers of total T cells and T-cell subsets in the bone marrow (1 femur and 1 tibia) of WT and CD70TG mice. EM indicates effector memory; and CM, central memory. (B) Intracellular expression of IFN-γ in CD3+CD4+ gated and CD3+CD8+ gated T cells from bone marrow of WT and CD70TG mice on phorbol myristate acetate and ionomycin stimulation (percentages are shown). (C) Expression of IFN-γR1 on CMPs and GMPs of WT (thin line) and CD70TG mice (bold line). Filled graph shows fluorescence-minus-one control staining. (D) Expression of IFN-γR1 on CMPs and GMPs as the average geometric mean fluorescence intensity (GeoMFI). (E) Number of eosinophils generated from 5.0 × 103 WT CMPs after 9 days of culture with SCF and IL-5 with or without addition of various concentrations of IFN-γ. (F) Number of cells generated from 2.5 × 103 WT CMPs after 9 days of culture with SCF, IL-5, and GM-CSF with or without addition of various concentrations of IFN-γ. (G) Representative plot of staining for CD34+ IL-5Rα+ EoPs gated on the Lineage−CD16/32+c-Kitlow compartment after a 3-day culture of GMPs in the presence of SCF, IL-3, IL-5, and GM-CSF with or without the addition of IFN-γ (data indicate percentage of EoPs from total cells). (H) Flow cytometric analysis of IL-5Rα expression on TF1 cells cultured with IL-5 in the presence (thick line) or absence (thin line) of IFN-γ. Isotype control is shown in shaded gray. (I-K) Human CD34+ progenitor cells were cultured for 3 days in the presence of SCF, FLT-3, GM-CSF, IL-3, and IL-5 followed by culture for 15 days in IL-3 and IL-5. Expansion of progenitor cells at day 5 (I) and day 18 (J) in cultures in the presence of IFN-γ, expressed as percentage of expansion in the cultures without IFN-γ. (K) Percentage of differentiated eosinophils after 18 days of culture analyzed after May-Grünwald Giemsa staining of cytospins. (A,D) Data are the mean of 3 mice per group. (E-F) Data are mean ± SD for 2 cultures per condition. (H) Data are mean ± SD for 4 cultures per condition. (A-H) Data are representative of at least 2 independent experiments. (I-K) Data are the mean of 3 independent experiments from different donors. Data are mean ± SD. *P < .05; **P < .01; ***P < .001.

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