Fig 2: GITR engagement induces NF-κB pathway signaling in ILC2s. a Volcano plot comparison of whole transcriptome gene expression of sorted WT ILC2s from VAT treated with isotype control or DTA-1 (5 µg/mL) for 24 h in vitro, n = 3. Differentially expressed genes (defined as statistically significant adjusted p-value < 0.05) with changes of at least 1.5 fold-change (FC) are shown in red. Notable differentially expressed genes are labeled. b Heat plot of differentially expressed genes. c Selected genes plotted as the normalized counts in isotype control-treated compared to DTA-1-treated mice. Differentially expressed genes (defined as in a) are shown in red. Gray area indicates region of 1.5 fold-change cutoff or lower change in expression. Shown are expression of cytokine and cytokine receptor genes. d Upregulated (red) and downregulated (green) genes in the NF-κB pathway. e Representative histogram of the expression of NF-κB p65 in isolated ILC2s from mice challenged with IL-33 and cultured in vitro for 24 h with DTA-1 (red) or isotype control (black). The level of isotype-matched stain control is shown as a gray-filled histogram. f Corresponding quantification presented as Mean Fluorescence Intensity of NF-κB p65 with DTA-1 or isotype control. g Network analysis of upregulated (red) and downregulated (green) genes overall significantly predicted to inhibit the apoptosis of leukocytes. h Representative flow cytometry plots of VAT Lin-CD45+IL-7R+ST2+Annexin V+ 7-AAD+ ILC2s from C57/BL6 or GITR-/- mice (n = 5) and corresponding quantitation presented as the percentage of ILC2s. Error bars are the mean ± SEM. Student’s t-test, *p < 0.05, ***p < 0.001

Fig 3: Therapeutic GITR treatment ameliorates established Type 2 Diabetes in Rag2-/- mice. a A cohort of Rag2-/- mice were fed on HFD for 14 weeks. After 8 weeks, mice were either treated with DTA-1 (1 mg/mouse) or isotype control by intraperitoneal injection every four days according to the scheme, n = 8. b Total weight and c fasting blood glucose levels were measured every two weeks for 14 weeks. d Plasma insulin concentration was measured by ELISA. e Glucose tolerance test and f insulin tolerance test were performed in a cohort of mice after 8 weeks of treatment. The corresponding area under the curve was calculated for each group. g Hematoxylin and eosin-stained epididymal adipose tissue sections (×400). Red arrows point to recruited inflammatory cells around adipocytes, scale bars, 100 µm. h Corresponding quantitation presented as the mean adipocyte area. i Weight of the VAT (epididymal adipose tissue) after 8 weeks of treatment. Error bars are the mean ± SEM. Student’s t-test, p < 0.05, **p < 0.01, ***p < 0.001. Mouse image provided with permission from Servier Medical Art

Fig 4: Engagement of GITR protects from the onset of Type 2 Diabetes. a A cohort of C57/BL6 mice fed a normal chow diet (NCD) or a high fat diet (HFD) were either treated with DTA-1 (1 mg/mouse) or isotype control by intraperitoneal injections every four days according to the scheme, n = 8. b Total weight and c fasting blood glucose levels were measured every two weeks for 14 weeks. Glucose tolerance test (d) and insulin tolerance test (e) were performed in a cohort of C57BL/6 mice fed a NCD or a HFD and treated for 14 weeks with or without DTA-1. The corresponding area under the curve was calculated for each group. Time-kinetic qPCR expression of IL-33 (f) and IL-25 (g) on VAT lysates. Error bars are the mean ± SEM. Student’s t-test, **p < 0.01, ***p < 0.001, ns, non-significant. Mouse image provided with permission from Servier Medical Art

Fig 5: GITR is expressed on ILC2s and induces Th2 cytokine secretion in activated ILC2s. a Gating strategy of Lin-CD45+IL-7R+ST2+ ILC2 cells. b Expression of GITR in naïve (white) and IL-33-activated (green) murine white adipose tissue-derived ILC2s compared to the isotype control (gray). Corresponding quantitation of GITR expression shown as MFI+/− SEM, n = 6. c Visceral adipose tissue (VAT) resident ILC2s were isolated from a cohort of naïve C57BL/6 mice and a cohort of activated C57BL/6 mice intraperitoneally challenged with rmIL-33 on 3 consecutive days. Naïve and activated VAT ILC2s were cultured with recombinant mouse (rm) IL-2 and rmIL-7 with DTA-1 (5 µg/mL) or isotype control for 48 h. The levels of IL-5, IL-13, GM-CSF, IL-6, and IL-9 were measured by Luminex on the culture supernatants, n = 6. Error bars are the mean ± SEM. Student’s t-test, ***p < 0.001, ns, non-significant