![Figure 2. Restored macrophage functionality upon Lv.SFFV.Ifnγr1 transduction of Ifnγr1−/− cells. (A) Representative histograms depicting major histocompatibility class II (MHC-II) upregulation on IFN-γ-stimulated vs unstimulated (unstim) WT, Ifnγr1−/−, and Lv.SFFV.Ifnγr1-transduced macrophages. Bar graphs depict summarized results of 3 independent experiments (two-way analysis of variance [ANOVA] using Sidak’s multiple comparisons post hoc testing. (B) Representative histograms depict CD86 upregulation on IFN-γ-stimulated vs unstimulated WT, Ifnγr1−/−, and Lv.SFFV.Ifnγr1-transduced macrophages. Bar graphs depict summarized results of 3 independent experiments (two-way ANOVA using Sidak’s multiple comparisons post hoc testing). (C) IFN-γ internalization of WT, Ifnγr1−/−, and Lv.SFFV.Ifnγr1-transduced macrophages over 24 hours (normalized to 0 hours; n = 4; two-way ANOVA using Tukey’s multiple comparisons post hoc testing). (D) Phosphorylation of STAT1 (pSTAT1) in unstimulated (–) and IFN-γ-stimulated (+) WT, Ifnγr1−/−, and Lv.SFFV.Ifnγr1-transduced macrophages compared with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) loading control. (E) Relative messenger RNA expression of Irf1, Nos2, and Ido in IFN-γ–stimulated WT, Ifnγr1−/−, and Lv.SFFV.Ifnγr1-transduced macrophages normalized to unstimulated samples (Irf1, n = 4; Nos2 and Ido, n = 3 each). (F) Induction of ovalbumin (OVA)-specific T-cell proliferation by WT, Ifnγr1−/−, and Lv.SFFV.Ifnγr1-transduced macrophages upon stimulation with IFN-γ or IFN-γ and OVA. Bars depict the percentage of eFluor670low CD4+ T cells (n = 3; two-way ANOVA using Bonferroni’s multiple comparisons post hoc testing). (G) Bacterial burden of M avium in macrophages 0 hours and 24 hours after infection (n = 3; two-way ANOVA using Sidak’s multiple comparisons post hoc testing). (H) Bacterial burden of Bacille Calmette-Guérin colonies in macrophages 8 days after infection (normalized to uptake value 4 hours after infection; n = 3; one-way ANOVA using Dunnett’s multiple comparisons post hoc testing). *P ≤ .05; **P ≤ .01; ***P ≤ .001; ****P ≤ .0001. For all bars, mean and standard error of the mean (SEM) are shown. ns, not significant.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/131/5/10.1182_blood-2017-10-812859/4/blood812859f2.jpeg?Expires=1723725028&Signature=bicDHiyRofzBAaX1hzeQ~vf41b5JpI~SDuKOS46nv-J5xDm8pTrwYmMXBSEPCk3JVmQTweaDXCTwqM2J0~wzFEUSbwrFb0oCrXNFt~UlToX33OpD4M3V304n-c1-nRRVXYZhuxWEUAjuTi5b30xcYnyk0LAWCwVhR0QlEKDtbczQAqcQNL4OZiDoOtvWdwxWPuTF6X~SGZebq9emcelGZ91wbmw1xzXL-BBeAC9jAgOg8Zni2Eg3KuDp9TLZkbHChvGFLv7xQwrgMvaA8JSCO-OjFVmvfOnbRH~h~-xSzz9DHbpDNEZDIpFbGdQbBmNF-NMVJ5pFWnX0n3dmAjTr3Q__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Restored macrophage functionality upon Lv.SFFV.Ifnγr1 transduction of Ifnγr1−/−cells. (A) Representative histograms depicting major histocompatibility class II (MHC-II) upregulation on IFN-γ-stimulated vs unstimulated (unstim) WT, Ifnγr1−/−, and Lv.SFFV.Ifnγr1-transduced macrophages. Bar graphs depict summarized results of 3 independent experiments (two-way analysis of variance [ANOVA] using Sidak’s multiple comparisons post hoc testing. (B) Representative histograms depict CD86 upregulation on IFN-γ-stimulated vs unstimulated WT, Ifnγr1−/−, and Lv.SFFV.Ifnγr1-transduced macrophages. Bar graphs depict summarized results of 3 independent experiments (two-way ANOVA using Sidak’s multiple comparisons post hoc testing). (C) IFN-γ internalization of WT, Ifnγr1−/−, and Lv.SFFV.Ifnγr1-transduced macrophages over 24 hours (normalized to 0 hours; n = 4; two-way ANOVA using Tukey’s multiple comparisons post hoc testing). (D) Phosphorylation of STAT1 (pSTAT1) in unstimulated (–) and IFN-γ-stimulated (+) WT, Ifnγr1−/−, and Lv.SFFV.Ifnγr1-transduced macrophages compared with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) loading control. (E) Relative messenger RNA expression of Irf1, Nos2, and Ido in IFN-γ–stimulated WT, Ifnγr1−/−, and Lv.SFFV.Ifnγr1-transduced macrophages normalized to unstimulated samples (Irf1, n = 4; Nos2 and Ido, n = 3 each). (F) Induction of ovalbumin (OVA)-specific T-cell proliferation by WT, Ifnγr1−/−, and Lv.SFFV.Ifnγr1-transduced macrophages upon stimulation with IFN-γ or IFN-γ and OVA. Bars depict the percentage of eFluor670low CD4+ T cells (n = 3; two-way ANOVA using Bonferroni’s multiple comparisons post hoc testing). (G) Bacterial burden of M avium in macrophages 0 hours and 24 hours after infection (n = 3; two-way ANOVA using Sidak’s multiple comparisons post hoc testing). (H) Bacterial burden of Bacille Calmette-Guérin colonies in macrophages 8 days after infection (normalized to uptake value 4 hours after infection; n = 3; one-way ANOVA using Dunnett’s multiple comparisons post hoc testing). *P ≤ .05; **P ≤ .01; ***P ≤ .001; ****P ≤ .0001. For all bars, mean and standard error of the mean (SEM) are shown. ns, not significant.
