American Society of Hematology

Figure 4

$Figure 4. STAT3 regulates NK cell–dependent IFN-γ production by directly binding to the IFN-γ promoter. (A) Splenocytes from Stat3fl/fl and Stat3Δ/ΔNcr1-iCreTg mice were analyzed directly ex vivo or after 4 hours stimulation with IL-2 + IL-12. Cells were stained for CD3 and NKp46 (CD3−NKp46+) followed by fixation, permeabilization, and intracellular staining of T-bet and IFN-γ. Levels were analyzed by flow cytometry. % IFN-γ+ NK cells and mean fluorescence intensities (MFI) of T-bet are depicted in representative FACS plots. Statistics are included in supplemental Figure 4D. (B) Splenocytes from Stat3fl/fl and Stat3Δ/ΔNcr1-iCreTg mice were analyzed directly ex vivo or after stimulation for 4 or 8 hours with IL-2 + IL-12. Intracellular IFN-γ expression of NK cells were analyzed by flow cytometry. Stat3Δ/ΔNcr1-iCreTg NK cells show a decreased IFN-γ production after 4 hours that is no longer detectable after 8 hours. Data represent mean ± SEM of 3 independent experiments (n ≥ 13 in total). (C) Primary IL-2 cultured NK cells were stimulated for 30 minutes with IL-2 or IL-2 + IL-12. The reaction was stopped by addition of formaldehyde. Chromatin immunoprecipitation was performed using an anti-STAT3 antibody and n-fold enrichment was calculated relative to the expression of a negative region (“CD19 down”). (D) NK cells of Stat3fl/fl and Stat3Δ/ΔNcr1-iCreTg mice were MACS-purified and FACS-sorted (CD3−NK1.1+) and stimulated with IL-2 and IL-12 for the indicated time. Cell lysates were used for western blot analysis of pSTAT5-Y694, STAT5a/b, STAT3, pSTAT4-Y693, and STAT4. β-actin was used as loading control. Stat3Δ/ΔNcr1-iCreTg NK cells show an increase in STAT4 and STAT5 activation compared with Stat3fl/fl controls. (E) Splenocytes from Stat3fl/fl and Stat3Δ/ΔNcr1-iCreTg mice were stimulated with anti-NK1.1 (PK136) for 4 or 8 hours before staining for CD3 and NKp46 (CD3−NKp46+) followed by fixation, permeabilization, and intracellular staining and detection of IFN-γ expression by flow cytometry. (F) Splenocytes from Stat3fl/fl and Stat3Δ/ΔNcr1-iCreTg mice were stimulated with anti-NK1.1 (PK136) for 6 hours before staining for CD3, NKp46, and Ly49C/I. Cells were fixed and permeabilized, and the intracellular IFN-γ expression of the Ly49C/I negative and positive NK cell fraction (CD3−NKp46+ cells) was analyzed. Statistics are included in supplemental Figure 4A.$

STAT3 regulates NK cell–dependent IFN-γ production by directly binding to the IFN-γ promoter. (A) Splenocytes from Stat3^fl/fl and Stat3^Δ/ΔNcr1-iCreTg mice were analyzed directly ex vivo or after 4 hours stimulation with IL-2 + IL-12. Cells were stained for CD3 and NKp46 (CD3⁻NKp46⁺) followed by fixation, permeabilization, and intracellular staining of T-bet and IFN-γ. Levels were analyzed by flow cytometry. % IFN-γ⁺ NK cells and mean fluorescence intensities (MFI) of T-bet are depicted in representative FACS plots. Statistics are included in supplemental Figure 4D. (B) Splenocytes from Stat3^fl/fl and Stat3^Δ/ΔNcr1-iCreTg mice were analyzed directly ex vivo or after stimulation for 4 or 8 hours with IL-2 + IL-12. Intracellular IFN-γ expression of NK cells were analyzed by flow cytometry. Stat3^Δ/ΔNcr1-iCreTg NK cells show a decreased IFN-γ production after 4 hours that is no longer detectable after 8 hours. Data represent mean ± SEM of 3 independent experiments (n ≥ 13 in total). (C) Primary IL-2 cultured NK cells were stimulated for 30 minutes with IL-2 or IL-2 + IL-12. The reaction was stopped by addition of formaldehyde. Chromatin immunoprecipitation was performed using an anti-STAT3 antibody and n-fold enrichment was calculated relative to the expression of a negative region (“CD19 down”). (D) NK cells of Stat3^fl/fl and Stat3^Δ/ΔNcr1-iCreTg mice were MACS-purified and FACS-sorted (CD3⁻NK1.1⁺) and stimulated with IL-2 and IL-12 for the indicated time. Cell lysates were used for western blot analysis of pSTAT5-Y694, STAT5a/b, STAT3, pSTAT4-Y693, and STAT4. β-actin was used as loading control. Stat3^Δ/ΔNcr1-iCreTg NK cells show an increase in STAT4 and STAT5 activation compared with Stat3^fl/fl controls. (E) Splenocytes from Stat3^fl/fl and Stat3^Δ/ΔNcr1-iCreTg mice were stimulated with anti-NK1.1 (PK136) for 4 or 8 hours before staining for CD3 and NKp46 (CD3⁻NKp46⁺) followed by fixation, permeabilization, and intracellular staining and detection of IFN-γ expression by flow cytometry. (F) Splenocytes from Stat3^fl/fl and Stat3^Δ/ΔNcr1-iCreTg mice were stimulated with anti-NK1.1 (PK136) for 6 hours before staining for CD3, NKp46, and Ly49C/I. Cells were fixed and permeabilized, and the intracellular IFN-γ expression of the Ly49C/I negative and positive NK cell fraction (CD3⁻NKp46⁺ cells) was analyzed. Statistics are included in supplemental Figure 4A.

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