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. 2023 Sep 22;14:1257652. doi: 10.3389/fimmu.2023.1257652

Figure 8.

Figure 8

In vivo activity of attenuated IL-2 muteins in a mouse inflammatory skin disease model. (A) Study design. Female Balb/c mice were shaved on the back on day -1 and injected with IL-2 at 25 μg per mouse by s.c. Starting day 0, Aldara cream (3.125 mg per dose) was applied daily on the back and the ear for 5 doses and the study was terminated on d5. Blood was collected on day 3 post IL-2 dosing (d2 of disease induction) for flow analysis for T and NK cell response to IL-2. n=6 per group. (B) Treg, NK, and Tcon as percentage of CD45+ cells on day 3 blood. Each dot represents an animal, shown are the mean of the group and the error bars indicate SEM. One-way ANOVA analysis with Tukey’s multiple comparisons test, ****p < 0.0001, **p < 0.01, ns, not significant. Naive animals were shaved and handled the same way as disease animals, but they were not dosed with IL-2 or treated with Aldara cream. (C) eTreg marker expression in Tregs on d3 post IL-2 dosing in blood. GITR, ICOS, and KLRG1 expression was determined by flow cytometric analysis and the expression level was summarized as gMFI in total Treg population for each treatment group. One-way ANOVA with Tukey’s multiple comparisons test, ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05. (D) T and NK cell composition in the skin on day 5 at take down. The breakdown of total T and NK cells, shown as the percentages of Treg, CD8 T, Tcon (Foxp3- CD4 T), NK, and γδ T cells of CD45+ live cells in the skin on day 5, are summarized for the different treatment and disease groups. (E) The percentages of γδ T and NK cells among CD45+ live cells in the skin on day 5. The data in (D) are presented individually for γδ T and NK cells. One-way ANOVA with Tukey’s multiple comparisons test, ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05. For (D, E), n=3 per group. Each symbol represents an animal.