Ponesimod treatment affects autoantigen spreading.

<p>Spleen and pancreatic lymph node (PLN) cells were harvested either from control diabetic NOD mice (n = 6) or from 35-week-old diabetes-free NOD mice treated with ponesimod since 6 weeks of age (n = 8). Production of IFNγ was measured by ELISPOT after incubation with PI-B_15–23, GAD_206–220 or IGRP_206–214 peptides. Results are expressed as mean ± SD of spot-forming units (SFU)/10⁶ cells.</p

Migration and proliferation of CD4⁺BDC2.5 T cells in pancreatic tissues and lymph nodes of ponesimod-treated NOD mice.

<p>Six-week-old NOD mice were treated with oral ponesimod for 12 consecutive weeks. Eight million CFSE-labeled CD4⁺BDC2.5⁺ T cells were then infused in the treated mice. Recipients were sacrificed on day 7 post-transfer and cell suspensions were recovered from pancreatic islets and lymph nodes and analyzed by flow cytometry. (A) Tet/p79 tetramer staining (MHC class II tetramer carrying a BDC2.5 T cell-specific peptide p79) in the CD4⁺ T cell gate. (B) Proliferation of BDC2.5 T cells measured by CFSE staining in the CD4⁺Tet/p79⁺ T cell gate. (C) Expression of CD69, CD44 and CD62L by proliferating BDC2.5 T cells (CD4⁺Tet/p79⁺ T cell gate). The results shown are representative of three independent experiments.</p

Cytokine production.

<p>Spleen CD4⁺ T cells (A) or total pancreatic lymph node cells (B) were isolated from age-matched NOD mice orally treated or not with ponesimod. Administration started at 6 weeks of age. IL-4, IL-10, IFNγ and TNFα production was measured by ELISA after an <i>in vitro</i> 48 h stimulation with CD3 antibodies (5 µg/ml).</p

T cells from ponesimod-treated NOD mice exhibit an activated phenotype.

<p>Staining with CD44, CD62L, CD25 or CD69 antibodies was performed on spleen or pancreatic lymph node cells recovered from NOD mice treated with ponesimod from 6 to 18 weeks of age. Eighteen-week-old untreated mice were used as controls. (A) Analysis within the CD4⁺ T cell gate. (B) Analysis within the CD8⁺ T cell gate.</p

Oral administration of ponesimod can reverse established diabetes and combination with CD3-specific antibodies restores self-tolerance.

<p>(A) Recently diagnosed diabetic NOD mice were treated with oral ponesimod (n = 23) and diabetes remission was evaluated by glycosuria and glycemia measurements. Durable remission occurred in 57% of mice. (B) Administration of CD3-specific monoclonal antibodies (50 µg/day i.v. for 5 consecutive days) was performed in NOD mice that showed remission upon continuous ponesimod treatment (n = 6). Oral ponesimod was withdrawn at the end of CD3 antibody treatment. All mice that received ponesimod and CD3 antibody treatment showed durable disease remission. In contrast, all mice treated with ponesimod alone showed disease relapse by 1 week following ponesimod withdrawal (**p = 0.003). (C) NOD mice showing sustained diabetes remission after combined treatment with ponesimod and CD3-specific antibodies were treated with streptozotocin (225 mg/kg) to destroy all endogenous β-cells and artificially induce diabetes. Three days later, they were transplanted under the kidney capsule with 500 syngeneic pancreatic islets isolated from <i>RAG^−/−</i> NOD mice (n = 3). Untreated overtly diabetic NOD mice were used as controls (n = 3). Survival of syngeneic islets was significantly prolonged in the ponesimod/CD3 antibody-treated NOD mice as compared to untreated NOD recipients. (D) Spleen cells were harvested from NOD mice having received combined treatment with ponesimod and CD3 antibodies and transplanted with syngeneic islets. Production of IFNγ was measured at the time of rejection by ELISPOT after a 20 hrs-incubation of total splenocytes with IGRP_206–214 peptides or control viral peptides (EBV, CMV, HIV). Results are expressed as mean ± SD of spot-forming units (SFU)/10⁶ cells and represent the average response of 3 mice/group.</p

Chronic oral administration of the selective S1P₁ receptor modulator ponesimod protects from diabetes development.

<p>(A) Ponesimod was administered to 6- (n = 23), 10- (n = 16), 13- (n = 16) or 16-week old (n = 15) NOD mice. Treatment was continuous and diabetes occurrence was monitored up to 36 weeks of age. (B) Histological evaluation of insulitis i.e. pancreatic islet infiltration by mononuclear cells. Pancreata were recovered from untreated or ponesimod-treated NOD mice at different time-points (beginning of treatment: 6 weeks of age). Paraffin-embedded pancreas sections were stained with eosin/hematoxylin to score insulitis. (C) Monitoring of diabetes development after withdrawal of ponesimod treatment in 35-week-old diabetes-free NOD mice treated since 6 (n = 13) or 10 (n = 5) weeks of age.</p

Lymphocyte subsets in peripheral blood and lymphoid organs of ponesimod-treated NOD mice.

<p>(A) Six-week-old NOD mice were treated with ponesimod for 24 h and T and B cell proportions were evaluated in peripheral blood (PBL), spleen and pancreatic lymph nodes (PLN) of 3 individual mice. (B) Ponesimod was administered to six-week-old NOD mice. Peripheral blood, spleen and pancreatic lymph nodes were recovered at 2, 4, 7, 9 or 12 weeks to monitor proportions of T cell subsets and B cells (n = 3 for all time-points). Results are expressed as % as compared to untreated age-matched NOD mice (**p<0.005). (C) B cells purified from pancreatic lymph nodes of 18-week-old ponesimod-treated NOD mice (12 weeks of treatment) were stained with antibodies against CD21, CD23 and CD24 (bold line). B cells from age-matched untreated NOD mice were used as controls (thin line).</p

Protected mice still harbor diabetogenic effectors.

<p>Adoptive transfer experiments: <i>RAG^−/−</i> NOD mice were injected i.v. with 10⁷ splenocytes (A) or 10⁶ pancreatic lymph nodes cells (B) recovered either from untreated overtly diabetic NOD mice or from 35-week-old diabetes-free ponesimod-treated NOD mice (beginning of treatment at 6, 10 or 16 weeks of age).</p

Antibiotics in Early Life Alter the Gut Microbiome and Increase Disease Incidence in a Spontaneous Mouse Model of Autoimmune Insulin-Dependent Diabetes

<div><p>Insulin-dependent or type 1 diabetes is a prototypic autoimmune disease whose incidence steadily increased over the past decades in industrialized countries. Recent evidence suggests the importance of the gut microbiota to explain this trend. Here, non-obese diabetic (NOD) mice that spontaneously develop autoimmune type 1 diabetes were treated with different antibiotics to explore the influence of a targeted intestinal dysbiosis in the progression of the disease. A mixture of wide spectrum antibiotics (i.e. streptomycin, colistin and ampicillin) or vancomycin alone were administered orally from the moment of conception, treating breeding pairs, and during the postnatal and adult life until the end of follow-up at 40 weeks. Diabetes incidence significantly and similarly increased in male mice following treatment with these two antibiotic regimens. In NOD females a slight yet not significant trend towards an increase in disease incidence was observed. Changes in gut microbiota composition were assessed by sequencing the V3 region of bacterial 16S rRNA genes. Administration of the antibiotic mixture resulted in near complete ablation of the gut microbiota. Vancomycin treatment led to increased <i>Escherichia</i>, <i>Lactobacillus</i> and <i>Sutterella</i> genera and decreased members of the <i>Clostridiales</i> order and <i>Lachnospiraceae</i>, <i>Prevotellaceae</i> and <i>Rikenellaceae</i> families, as compared to control mice. Massive elimination of IL-17-producing cells, both CD4+TCRαβ+ and TCRγδ+ T cells was observed in the lamina propria of the ileum and the colon of vancomycin-treated mice. These results show that a directed even partial ablation of the gut microbiota, as induced by vancomycin, significantly increases type 1 diabetes incidence in male NOD mice thus prompting for caution in the use of antibiotics in pregnant women and newborns.</p></div

Immune cells in the intestinal lamina propria and spleen.

<p><b>(a)</b> Percentages of IFNγ and IL-17-producing T cells (upon 6-hr stimulation with PMA/ionomycin) among CD45+CD4+TCRαβ+ and CD45+TCRγδ+ T cells isolated from the spleen and the lamina propria of the colon and ileum of 10 week-old vancomycin-treated mice (black bars) <i>versus</i> controls (open bars). The data in males (upper panels) and in females (lower panels) are shown. Five animals per group were analyzed. Significant differences are indicated as follows: * p<0.05; ** p<0.01. <b>(b)</b> Representative dot plots of intracytoplasmic staining to detect IFNγ and IL-17-producing T cells among CD45+CD4+TCRαβ+ or CD45+TCRγδ+ T cells. <b>(c)</b> Percentages of Foxp3+ cells among CD4+CD45+ T cells and of CD62L+ cells and Helios+ cells among CD45+CD4+FoxP3+ lymphocytes in 10-week-old NOD males (upper panels) and females (lower panels) treated with vancomycin (black bars) or controls (open bars). Significant differences are indicated as follows: * p<0.05; ** p<0.01.</p