Notch ligand delta-like 4 blockade alleviates experimental autoimmune encephalomyelitis by promoting regulatory T cell development

Abstract

Notch signaling pathway plays an important role in T cell differentiation. Delta-like ligand (Dll)4, one of five known Notch ligands, has been implicated in regulating Th2 cell differentiation in animal models of human diseases. However, the role of Dll4 in Th1/Th17-mediated autoimmune diseases remains largely unknown. Using an anti-Dll4 blocking mAb, we show that neutralizing Dll4 during the induction phase of experimental autoimmune encephalomyelitis in C57BL/6 mice significantly increased the pool of CD4(+)Foxp3(+) regulatory T cells (Treg) in the periphery and in the CNS, and decreased the severity of clinical disease and CNS inflammation. Dll4 blockade promoted induction of myelin-specific Th2/Treg immune responses and impaired Th1/Th17 responses compared with IgG-treated mice. In vitro, we show that signaling with recombinant Dll4 inhibits the TGF-β-induced Treg development, and inhibits Janus kinase 3-induced STAT5 phosphorylation, a transcription factor known to play a key role in Foxp3 expression and maintenance. Depletion of natural Treg using anti-CD25 Ab reversed the protective effects of anti-Dll4 Ab. These findings outline a novel role for Dll4-Notch signaling in regulating Treg development in EAE, making it an encouraging target for Treg-mediated immunotherapy in autoimmune diseases, such as multiple sclerosis.

FIGURE 1

Dll4 blockade during EAE induction phase decreases disease severity and CNS inflammation. A, Clinical scores of C57BL/6 mice immunized with MOG(35–55)/CFA and treated with anti-Dll4 blocking Ab or control IgG every other day and for a total of five injections (arrows) starting on the day of the immunization. Results are representative of three independent experiments with five mice per group. *p < 0.05. B, Histopathology of H&E-stained spinal cord sections of mice described in A on DPI 14. Boxed area in top row is enlarged below. Original magnification ×10 (top row) and ×40 (bottom row). Results are representative of two independent experiments with two mice per group.

FIGURE 2

Dll4 blockade in EAE regulates T cell differentiation. EAE was induced in C57BL/6 mice immunized with MOG(35–55)/CFA and treated with anti-Dll4 blocking Ab or control IgG every other day and for a total of five injections starting on the day of the immunization. Flow cytometry plots from individual mice from each group of splenocytes (A) on day 10 (preclinical disease) and spinal cord-infiltrating cells (B) on day 14 (after disease onset). Results are representative of three independent experiments with five mice per group. C, Enumeration of cytokine-producing cells by ELISPOT collected on day 10 postimmunization and then cultured in triplicates in the presence of various MOG(35–55) concentrations (0–60 mg/ml). Results are representative of two independent experiments with five mice per group (means ± SEM). *p < 0.05, **p < 0.005, ***p < 0.0005 by Student t test.

FIGURE 3

Dll4-mediated signaling regulates Treg expansion in vivo and in vitro. EAE was induced in C57BL/6 mice immunized with MOG(35–55)/CFA and treated with anti-DLL4 blocking Ab or control IgG every other day and for a total of five injections (arrows) starting on the day of the immunization. A, Flow cytometry plots from individual mice from each group for CD4 and Foxp3 staining of splenocytes or lymph node cells on DPI 10 (preclinical disease) and spinal cord cells on DPI 14 (peak of disease). Results are representative of three independent experiments with five mice per group. B, Statistical analysis of staining described in A. *p < 0.05, **p < 0.005 by unpaired t test. CD4⁺Foxp3⁻ T cells (C) or total CD4⁺ T cells (D) were isolated from naive Foxp3.GFP.KI mice and stimulated in vitro with plate-bound anti-CD3 and anti-CD28 (1 µg/ml) in the presence of plate-bound rDll4 or control IgG (2 ug/ml). Optimal dose TGF-β (3 ng/ml) or IL-2 (20 ng/ml) was added to the culture medium when indicated and then supplemented to the culture medium on day 2 of culture. Cells were washed and stained for CD4 and Foxp3 on day 4 of culture. Results are representative of three independent experiments.

FIGURE 4

Dll4 signaling inhibits JAK3/STAT5 activation. Naive CD4⁺ cells were stimulated with anti-CD3 and anti-CD28 (1 µg/ml) in the presence of rDll4 or control IgG (2 µg/ml) for 10, 20, 30, and 60 min. Nonstimulated cells were used as control (Ct). Cell lysates were prepared, and Western blotting with anti–phospho-STAT5 was performed. The same blots were stripped and reprobed with anti–phospho-JAK3 or anti-mouse β-actin (used as the protein-loading control). Results are representative of three independent experiments.

FIGURE 5

Anti-Dll4–mediated suppression of EAE is Treg dependent. A, Naive C57BL/6 mice received 250 µg anti-CD25 mAb (open symbols) or not (filled symbols) i.p. on days −3 and −1 before EAE induction. Mice were immunized with MOG(35–55)/CFA and treated with anti-Dll4 mAb (circles) or control IgG (squares) on the day of the immunization and on days 2, 4, 6, and 8 after immunization. Clinical EAE scores are representative of two independent experiments with five mice per group. B, Flow cytometric profile of CD4⁺ cells from splenocytes of mice described in A prior to immunization. Results are representative of two independent experiments with three mice per group.