Divergent Effects of OX40L on Regulatory T Cell Phenotype and Function: Implications for Type 1 Diabetes Therapy

Type 1 diabetes mellitus (T1D) is a T-cell mediated disease characterized by destruction of pancreatic β-cells, resulting in life-long dependence on insulin therapy. Unfortunately, most treatment options for T1D are non-specific, not curative and have many side effects. Foxp3+ T regulatory cells (Tregs) play a crucial role in the maintenance of immune homeostasis against self-antigen. Many studies have revealed that Tregs in patients with various autoimmune diseases, including T1D, are diminished in number and/or dysfunctional. Unfortunately, the therapeutic potential of Tregs has not been fully understood as the risk of effector T cell contamination and the tendency of Tregs to lose their suppressive function upon repeated proliferation remain as central challenges. Recently, we demonstrated a critical role for OX40L/OX40 signaling in Treg expansion by bone marrow-derived dendritic cells (BMDCs). OX40L, expressed on APCs, binds to OX40 on T cells. Signaling through OX40 has been shown to play an important role in effector T cell proliferation and survival. However, its role in Treg biology is still controversial. Thus, in this study, we wanted to further determine the role of OX40L on Treg function and homeostasis in the non-obese diabetic (NOD) mouse, a well-established model for T1D. Our findings suggest that OX40L has divergent effects on Treg phenotype and disease onset depending on the age of the animal. Treatment of 12-week-old NOD mice resulted in rapid onset of hyperglycemia and an inability to expand Tregs in the periphery. In contrast, treatment of 6-week-old NOD mice induced significant expansion of Tregs. Further, our data suggest that OX40L expands Tregs in the thymus. Upon migration to the periphery, expanded Tregs undergo rapid conversion resulting in loss of Foxp3 expression in 12-week-old NOD mice. The inability to expand and sustain Treg survival in the periphery is most likely due to suboptimal IL-2 levels in 12-week-old NOD mice. Co-administration of OX40L/IL- 2 resulted in sustained Treg expansion in the periphery of 12-week-old mice. Thus, we propose that OX40L is required to drive Treg proliferation in the thymus, while IL-2 is required to sustain Treg survival in the periphery of NOD mice

Age-dependent divergent effects of OX40L treatment on the development of diabetes in NOD mice

<p>Earlier, we have shown that GM-CSF derived bone marrow (BM) dendritic cells (G-BMDCs) can expand Foxp3⁺ regulatory T-cells (Tregs) through a TCR-independent, but IL-2 dependent mechanism that required OX40L/OX40 interaction. While some reports have shown suppression of autoimmunity upon treatment with an OX40 agonist, others have shown exacerbation of autoimmune disease instead. To better understand the basis for these differing outcomes, we compared the effects of OX40L treatment in 6-week-old pre-diabetic and 12-week-old near diabetic NOD mice. Upon treatment with OX40L, 6-week-old NOD mice remained normoglycemic and showed a significant increase in Tregs in their spleen and lymph nodes, while 12-week-old NOD mice very rapidly developed hyperglycemia and failed to show Treg increase in spleen or LN. Interestingly, OX40L treatment increased Tregs in the thymus of both age groups. However, it induced Foxp3⁺CD103⁺CD38⁻ stable-phenotype Tregs in the thymus and reduced the frequency of autoreactive Teff cells in 6-week-old mice; while it induced Foxp3⁺CD103⁻CD38⁺ labile-phenotype Tregs in the thymus and increased autoreactive CD4⁺ T cells in the periphery of 12-week-old mice. This increase in autoreactive CD4⁺ T cells was likely due to either a poor suppressive function or conversion of labile Tregs into Teff cells. Using <i>ex vivo</i> cultures, we found that the reduction in Treg numbers in 12-week-old mice was likely due to IL-2 deficit, and their numbers could be increased upon addition of exogenous IL-2. The observed divergent effects of OX40L treatment were likely due to differences in the ability of 6- and 12-week-old NOD mice to produce IL-2.</p