Fig. 8: IL2 + IL12 immunotherapy operates through a parallel IL-2-FOXO1/IL-12-STAT4-mediated mechanism. | Cell Death & Disease

Fig. 8: IL2 + IL12 immunotherapy operates through a parallel IL-2-FOXO1/IL-12-STAT4-mediated mechanism.

From: The transcription factor ZEB2 mediates the antitumor efficacy of tumor-infiltrating lymphocytes in non–small cell lung cancer

Fig. 8: IL2 + IL12 immunotherapy operates through a parallel IL-2-FOXO1/IL-12-STAT4-mediated mechanism.

a Schematic of the proposed parallel IL-2-Foxo1/IL-12-Stat4-mediated mechanism. b Naïve CD8+ cells isolated from WT mice, constitutive nuclear-localized Foxo1-triple alanine phosphorylation mutant mice (Foxo1-AAA), and phosphorylation-defective Stat4 mutant mice (Stat4Y693A) were activated ex vivo with anti-CD28 and anti-CD3 antibodies and treated with either vehicle control, IL-2-MSA, IL-12-MSA, or the combination for three days. c Western blotting analysis of Jak1/Akt signaling and Foxo1S256 phosphorylation as well as phosphorylation of Jak2Y1007 and Stat4Y694. Full, uncropped Western blots are provided in the Supplementary Material. d qPCR analysis of Tbx21 mRNA expression and Zeb2 mRNA expression. e Schematic of the murine serum albumin (MSA)-cytokine fusion study. WT, Foxo1-AAA, or Stat4Y693A mice inoculated with KP.SIY lung tumors received either vehicle control, IL-2-MSA, IL-12-MSA, or the combination on day 7 post-tumor inoculation. f Quantification of the percentage of tumor area per lung lobe assessed on day 21 post-tumor inoculation. g Survival analysis of WT, Foxo1-AAA, or Stat4Y693A mice inoculated with KP.SIY lung tumors that received either vehicle control, IL-2-MSA, or IL-2-MSA + IL-12-MSA. h–j Analysis of (h) GzmB, i TNF-α, and j IFN-γ expression on endogenous SIY-reactive CD8+ cells in KP.SIY tumor-bearing mice on day 10 after tumor inoculation. k IFN-γ ELISpot assay of IFN-γ producing splenocytes from KP.SIY tumor-bearing mice on day 10 post-tumor inoculation. l To assay in vivo cytotoxicity, mice inoculated with KP.SIY lung tumors were challenged intravenously with SIY-pulsed CFSElo or unpulsed CFSEhi splenocytes on day 10 post-tumor inoculation. Four hours following splenocyte challenge, mouse spleens were analyzed. Data depicts means ± SDs or medians ± ranges from n = 12 mice/group. *P < 0.05; **P < 0.01 [d, f, h–l two-way ANOVA, g log-rank test]. The figure is created using elements from BioRender.com.

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