Soluble RAGE Prevents Type 1 Diabetes Expanding Functional Regulatory T Cells

Abstract

Type 1 diabetes is an autoimmune disease with no cure, where clinical translation of promising therapeutics has been hampered by the reproducibility crisis. Here, short-term administration of an antagonist to the receptor for advanced glycation end products (sRAGE) protected against murine diabetes at two independent research centers. Treatment with sRAGE increased regulatory T cells (Tregs) within the islets, pancreatic lymph nodes, and spleen, increasing islet insulin expression and function. Diabetes protection was abrogated by Treg depletion and shown to be dependent on antagonizing RAGE with use of knockout mice. Human Tregs treated with a RAGE ligand downregulated genes for suppression, migration, and Treg homeostasis (FOXP3, IL7R, TIGIT, JAK1, STAT3, STAT5b, CCR4). Loss of suppressive function was reversed by sRAGE, where Tregs increased proliferation and suppressed conventional T-cell division, confirming that sRAGE expands functional human Tregs. These results highlight sRAGE as an attractive treatment to prevent diabetes, showing efficacy and reproducibility at multiple research centers and in human T cells.

Trial registration: ClinicalTrials.gov NCT01030861 NCT01773707.

Figure 1

Treatment with sRAGE provides lasting protection against autoimmune diabetes in an international multisite preclinical trial. A: NOD/ShiLt mice were administered vehicle at site 1 or were untreated at site 2 (black bars/circles) or treated with 25 µg sRAGE twice daily (red bars/triangles) or 100 µg sRAGE once daily (brown bars/triangles) from days 50–64 of life. B: Autoimmune diabetes incidence. Site 1, three independent experiments, n = 23/group; site 2, one independent experiment, n = 14–20/group. C: Nonfasting blood glucose concentrations shown as linear regression ± 95% CI (left) and residuals representing variability of blood glucose levels from the regression line (right). D–F: Pancreatic islet infiltration. D: Islet infiltration index (0 indicates no infiltration; to 1 indicates >75% infiltration). E: Degree of islet infiltration (grade 0, none; grade 1, peri-infiltration; grade 2, <25% infiltration; grade 3, 25–75% infiltration; grade 4, >75% infiltration). F: Representative hematoxylin-eosin photomicrographs (n = 7–33 sections/group from n = 4–7 mice/group; scale bar = 40 µm). G: Islet count normalized by tissue area. Column graphs are shown as median (IQR), with analysis with two-tailed Mann-Whitney U test. Box-and-whisker plot variances were analyzed with F test. Degree of insulitis is shown as mean and proportions analyzed with Fischer exact test. Diabetes incidence is shown with Kaplan-Meier survival curves and was analyzed with log-rank test. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2

T_regs are a nonredundant mechanism of action for sRAGE treatment. A–F: Flow cytometry quantification of T_reg-to-T_eff ratios on day 64 (A–C) and day 225 (D–F) in NOD/ShiLt mice (n = 4–13/group). G: In an adoptive transfer model of autoimmune diabetes, splenocytes from diabetic NOD/ShiLt donors were adoptively transferred into NOD/SCID recipients. Recipient mice were treated with vehicle or 25 µg sRAGE twice daily for 14 days and isotype control or anti-folate receptor 4 (FR4) monoclonal antibodies (mAb) at 3, 7, 10, and 14 days. Diabetes incidence (three independent experiments, n = 16 mice/group) (H) and flow cytometry quantification of T_reg frequencies (n = 16 mice/group) (I and J). I: Quantification of frequencies. J: Representative dot plots. Column graphs are shown as median (IQR), with analysis with two-tailed Mann-Whitney U test. Diabetes incidence is shown with Kaplan-Meier survival curves and was analyzed with log-rank test. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3

RAGE is required for the modulation of T_reg-to-T_eff ratios by sRAGE treatment. A and B: Flow cytometry quantification of RAGE expression on CD4⁺CD8⁻CD25⁺FoxP3⁺ T_regs in a mouse model of autoimmune diabetes (NOD/ShiLt) and C57BL/6 mice on day 50 of life. A: Representative histograms. B: Proportion of RAGE⁺ T_regs (n = 4–8 mice/tissue). C: Wild-type and RAGE KO C57BL/6 mice were administered vehicle (Veh) (black bars/circles) or treated with 25 µg sRAGE twice daily (red bars/triangles) on day 50–64 of life. D and E: T_reg-to-T_eff ratios on day 64 in wild-type (WT) (D) and RAGE KO (E) mice (n = 8 mice/group). F–H: TIGIT, KLRG1, CD62L, CD44, and Ki67 expression. F: Representative histograms and gating strategies. G: Wild-type mice. H: RAGE KO mice. Column graphs are shown as means ± SD, with analysis with two-tailed Student t test. *P<0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. mAb, monoclonal antibody; NS, not significant; PE, phycoerythrin.

Figure 4

Improvements in islet-infiltrating T_regs, insulin expression, and oral glucose tolerance following sRAGE treatment in NOD/ShiLt mice. A–D: Multiplexed immunofluorescence staining and quantification of CD3, CD4, FoxP3, insulin, and DAPI (n = 10–20 sections/mouse from n = 6 mice/group). Inset images are ×2 magnified. A: Proportion of islet-infiltrating CD3⁺CD4⁺FoxP3⁺ T_regs. B: Insulin area per field of view (FOV). C: Representative photomicrographs. Bar, 40 µm. D: Correlation analyses of CD3⁺CD4⁺FoxP3⁺ T_reg proportions and insulin area (shaded bars are Kernel plots showing variable distribution). E–I: OGTTs on day 80 (n = 15/group) and 225 (n = 3–6/group) of life. E: Day 80 AUC_insulin. F: Day 80 AUC_glucose–to–AUC_insulin ratios. G: Day 225 blood glucose concentrations. H: Day 225 AUC_glucose. I: Day 225 AUC_glucose–to–AUC_insulin ratio. Data shown as means ± SD and were analyzed with two-tailed unpaired or paired Student t tests. Correlation analyses were performed with Spearman test. *P < 0.05 between groups; **P < 0.01 between groups; ****P < 0.0001 between groups; §P < 0.05 vs. 0 min within the same group; ¶P < 0.05 vs. 15- and 30-min time points within the same group. Veh, vehicle.

Figure 5

Human nT_regs bind AGEs in a RAGE-dependent manner and proliferate more in coculture when treated with sRAGE. A: Human CD3⁺CD4⁺CD25⁺CD127^lo/− nT_regs were incubated with AGE-modified HSA (AGE-HSA or AGE) or unmodified HSA (HSA)—both labelled with Alexa Fluor 488 (AF488). Anti-RAGE antibody (Ab) was added to the culture as indicated. Bar, 10 µm. n = 3 donors/group. B–D: CFSE-labeled CD3⁺CD4⁺CD25⁺CD127^lo/− nT_regs and CellTrace Violet–labeled CD3⁺CD4⁺CD25⁻ T_convs were stimulated in 3-day coculture at a 1:1 ratio containing anti-CD3/CD28 MACSiBeads (1:10 bead:cell ratio). B: Representative histograms (unstimulated controls presented as semi-transparent peaks). C–D: Proliferation indices of nT_regs and T_convs when administered vehicle or 50 µg sRAGE daily (n = 9/group), as well as sRAGE in addition to PI3K-Akt-mTOR pathway inhibitor wortmannin or triciribine (n = 3/group). E and F: CFSE-labeled CD3⁺CD4⁺CD25⁺CD127^lo/− nT_regs were stimulated in 3-day monoculture containing anti-CD3/CD28 MACSiBeads (1:20 bead:cell) and 200 IU/mL IL-2. E: Representative histograms. F: Percent change in CFSE mean fluorescence intensity (MFI) (n = 4/group). Data are shown as mean ± SD, with analysis with paired two-tailed Student t tests. §P < 0.05 vs. all previous time points. ¶P < 0.05 vs. 0, 5, 10, and 15 min. *P < 0.05; ***P < 0.001; ****P < 0.0001. AU, arbitrary units; Veh, vehicle.

Figure 6

AGE treatment of human nT_regs downregulates key genes for suppressive function and manipulates signaling cascades upstream and downstream of FOXP3. A: PCA with the top five variable loadings shown as arrows. B: Volcano plot visualization for changes in gene expression (number of differentially expressed genes shown in top corners). C: Pathway enrichment analysis with use of the Reactome and Gene Ontology databases. D: Upstream regulator predictions with use of Ingenuity Pathway Analysis (161 significant regulators were identified). E: Network analysis with FOXP3 as the focus node. Neighboring molecules are connected to FOXP3 with bolded blue lines. All other molecules are two to three nodes adjacent to FOXP3. Direct relationships are shown as solid lines, and indirect relationships are shown as dashed lines. n = 3/group. P values were corrected with false discovery rate or Bonferroni adjustment. PC1, principal component 1.