The IL-2 SYNTHORIN molecule promotes functionally adapted Tregs in a preclinical model of type 1 diabetes

Abstract

Deficits in IL-2 signaling can precipitate autoimmunity by altering the function and survival of FoxP3+ regulatory T cells (Tregs) while high concentrations of IL-2 fuel inflammatory responses. Recently, we showed that the non-beta IL-2 SYNTHORIN molecule SAR444336 (SAR'336) can bypass the induction of autoimmune and inflammatory responses by increasing its reliance on IL-2 receptor α chain subunit (CD25) to provide a bona fide IL-2 signal selectively to Tregs, making it an attractive approach for the control of autoimmunity. In this report, we further demonstrate that SAR'336 can support non-beta IL-2 signaling in murine Tregs and limit NK and CD8+ T cells' proliferation and function. Using a murine model of spontaneous type 1 diabetes, we showed that the administration of SAR'336 slows the development of disease in mice by decreasing the degree of insulitis through the expansion of antigen-specific Tregs over Th1 cells in pancreatic islets. Specifically, SAR'336 promoted the differentiation of IL-33-responsive (ST2+), IL-10-producing GATA3+ Tregs over other Treg subsets in the pancreas, demonstrating the ability of this molecule to further orchestrate Treg adaptation. These results offer insight into the capacity of SAR'336 to generate highly specialized, tissue-localized Tregs that promote restoration of homeostasis during ongoing autoimmune disease.

Keywords: Autoimmune diseases; Autoimmunity; Cytokines; T cells; Therapeutics.

Figure 1. A pegylated IL-2 mutein targeting the CD25/STAT5 signaling pathway promotes specific Tregs’ expansion and Treg-associated gene expression.

(A–C) Geometric mean fluorescence intensity (MFI) of phosphorylated STAT5 (pSTAT5) in FoxP3⁺CD4⁺CD3⁺ T cells (Tregs), NK1.1⁺CD3^– cells (NK cells), and CD8⁺CD3⁺ T cells (CD8⁺ T cells) isolated from C57BL/6 mice and exposed to increasing concentrations of rhIL-2 (A) or SAR’336 (B) for 45 minutes. Data representative of more than 1 study. (C) Representative flow cytometry of the gating strategy for the identification of murine CD25^hi and CD25^lo FoxP3⁺ Tregs at 45 minutes. (D) Effect of the concentration (pg/mL) of rhIL-2 or SAR’336 on the geometric MFI of p-STAT5 expression in total FoxP3⁺, CD25^hiFoxP3⁺, and CD25^loFoxP3⁺ Tregs. The red line represents the average of FoxP3⁺ T cells in the presence of rhIL-2, and the green line represents the average of FoxP3⁺ T cells in the presence of SAR’336. (E–H) Murine CD4⁺GFP⁺ Tregs from the spleens of B6.FoxP3^GFPki mice were purified and activated by plated α-CD3 and α-CD28 for 72 hours in the presence of rhIL-2 or SAR’336. (E) Representative histogram of the expression of Ki-67 at 72 hours. (F) Effect of increasing dose of SAR’336 and rhIL-2 on the frequency of Ki-67⁺ among live CD4⁺FoxP3⁺ cells at 72 hours. (G) Effect of increasing doses in the total number of live Tregs at 72 hours. Two-way ANOVA. (H) Percentage increase in geometric MFI of Helios, FoxP3, and CD25 in the presence of 1 μg/mL of each cytokine/mutein over the medium alone (0 μg/mL). (Mean MFI of the experiment/mean MFI of medium alone) (n = 3 per experiment, 3 individual experiments). Two-way ANOVA. (I) CD4⁺GFP⁺ Tregs were cocultured with CellTrace Violet–labeled (CTV-labeled) CD4⁺GFP^– Teffs in the presence of MitoC-treated antigen-presenting cells (APCs) (CD4^– fraction) and soluble α-CD3 (1 μg/mL) with 1 μg/mL of rhIL-2 or SAR’336 for 72 hours. Percentage increase in the number of Tregs relative to medium: (# cells in rhIL-2 or SAR’336/mean # in medium) × 100. Compiled results of 3 distinct experiments with triplicates. One-way ANOVA. Tukey’s correction. ***P < 0.01.

Figure 2. SAR’336 promotes the rapid and specific expansion of CD4⁺FoxP3⁺ T cells.

(A) Female NOD and NOD BDC2.5 mice were administered 0.3 mg/kg of SAR’336 or the vehicle (Veh) s.c., and cells from the blood (PBMCs), the spleen, inguinal and axillary LN (pLN), and the pancreas were collected at day 2 and 4 after injection. (n = 4–5/group.) (B) Frequency of FoxP3⁺ among CD4⁺ T cells isolated in each organ at days 2 and 4 after injection. (C) Pie charts representing the mean frequency of conventional CD4⁺ T cells (Tconv), Tregs, CD8⁺ T cells, and NK and B cells as parts of whole cells collected from the pancreas of NOD mice at day 4 postinjection. Two-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001. (D and E) BDC2.5⁺CD4⁺ T cells were isolated, and 5 × 10⁶ cells were adoptively transferred (i.v.) into NOD mice before the administration of 0.01, 0.1, and 0.3 mg/kg of SAR’336 or Veh s.c. (n = 4–5/group). (E) Number of BDC2.5 tetramer (Tet⁺) CD4⁺FoxP3^– T cells in the spleen. (F) Number of CD4⁺FoxP3⁺Tet⁺ cells in the spleen. (G) Number of NKp46⁺CD3^– NK cells in the spleen at day 4 after injection. One-way ANOVA. Tukey’s correction. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3. SAR’336 minimizes cytotoxic responses and expands IL-10–producing, antigen-specific Tregs in the pancreas.

(A) CD4⁺ T cells were isolated from female NOD BDC2.5 mice, and 3 × 10⁵ cells were adoptively transferred i.v. into female NOD mice. A total of 0.03 mg/kg of SAR’336 was administered s.c. twice a week up to 21 days. Lymphocytes from the spleen, pancreatic LNs (panLN), and pancreas were collected at day 7 (n = 7–8/group) and day 21 (n = 3–5/group). Data compiled from 2 distinct experiments. (B) Representative histology slide of the pancreas at day 21 (hematoxylin and eosin). Black arrows show β-islet infiltration while red arrows point to perivascular infiltration of immune cells. (C) Histology score at day 21 posttransfer. Adapted from Papaccio et al. 2000 (86). 1 = infiltrates in small foci at the islet periphery; 2 = infiltrates surrounding the islets (peri-insulitis); 3 = intraislet infiltration < 50% of the islet, without islet derangement; 4 = extensive infiltration over 50% of the islet, cell destruction, and prominent cytoarchitectural derangement; 5 = complete islet atrophy and β cell loss. (D) Total number of cells isolated from the pancreas at day 7 and 21. (E) Number of IFN-γ–producing CD3⁺CD8β⁺ T cells (CD8⁺ T cells) in the pancreas. (F) Number of IFN-γ–producing CD3⁺CD4⁺ T cells in distinct organs at day 21 after transfer. (G) Representative flow cytometry of the expression of FoxP3 and Vβ4 in the pancreas at day 21 after transfer. (H) Frequency of FoxP3⁺CD4⁺ T cells among Vβ4⁺ and Vβ4^– T cells at day 21 after transfer. (I) Number of FoxP3⁺CD4⁺ T cells in the pancreatic lymph node at day 7 and 21 after transfer. (J) Frequency of IL-10–producing CD4⁺FoxP3⁺ Tregs in the pancreas (Pan), pancreatic LNs (panLN), and spleen (Spl) at day 21 after transfer. One way-ANOVA. Tukey’s correction. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4. SAR’336 promotes the migration of antigen-specific, IL-10⁺ Tregs.

(A–C) Splenic CD4⁺GFP^– Teffs were CTV-labeled and activated in the presence of APCs and soluble anti-CD3 (1 μg/mL) with 5 ng/mL murine TGF-β and 1 μg/mL of SAR’336 for 72 hours. Representative flow cytometry of CTV and FoxP3-GFP expression. Representative of 2 distinct experiments. (B) Frequency of FoxP3⁺ among total live CD4⁺ cells. (C) Geometric MFI of FoxP3 among total CD4⁺ T cells. Seventy-two hours. Two-way ANOVA. ***P < 0.001. (D–F) CD4⁺GFP^– (Treg depleted) or total CD4⁺ T cells were isolated from female BDC2.5 FoxP3^GFPki NOD mice, and 3 × 10⁵ cells were adoptively transferred i.v. into female NOD mice. At days 0 and 3 after transfer, 0.03 mg/kg of SAR’336 was administered s.c. One-way ANOVA. Tukey’s correction. *P < 0.05, **P < 0.01.

Figure 5. SAR’336 expands Helios⁺ST2⁺ Tregs in the pancreas.

(A) Representative flow cytometry plot of IL-10 and Helios expression among CD4⁺FoxP3⁺ T cells in the pancreas of vehicle- (left) and SAR’336-treated (right) mice at day 21. (B) Intracellular expression of master transcription factors RORγT (red), T-bet (gray), and GATA3 (green) in CD4⁺FoxP3⁺ T cells isolated from the pancreas at day 21 after adoptive transfer. Individual Student’s t test between markers. **P < 0.01; ***P < 0.001; ^###P < 0.001. (C) Representative flow cytometry plot of the expression of GATA3 and ST2 in CD3⁺CD4⁺FoxP3⁺ T cells isolated from the pancreas of vehicle- (veh) or SAR’336-treated mice at day 21. (D) Differential expression of ST2, GATA3, T-bet, RORγT, Helios, FoxP3, and IL-18R1 on CD4⁺FoxP3⁺ Tregs (gMFI/mean gMFI in both groups) from the pancreas at day 7. (n = 7–8/group.) Two-way ANOVA. *P < 0.05; **P < 0.01. (E) Representative flow cytometry of Helios and ST2 expression on pancreas-isolated CD4⁺FoxP3⁺ T cells at day 7 after transfer and frequency of Helios⁺ Tregs in the pancreas, panLN, and spleen at day 7 after transfer. Two-way ANOVA. ***P < 0.001. (F) Frequency of ST2⁺ among FoxP3⁺ Tregs in the pancreas, panLN, and spleen at day 7 after transfer. Two-way ANOVA. ***P < 0.001. (G) Frequency of IL-18R⁺ among FoxP3⁺ Tregs in the pancreas, panLN, and spleen at day 7 after transfer. Two-way ANOVA. *P < 0.05.