Role of OX -Notch Signaling in Thymic Treg Development and Homeostasis

Regulatory T cells (Tregs) plays a crucial role in immune tolerance and diminution of their homeostatic balance causes autoimmunity. Tregs are generated in the thymus (Natural Tregs) and contribute a major portion of the peripheral Treg pool where they suppress self-reactive effector T-cell response. Thymic Treg development occurs in two phases i) In TCR-dependent phase where thymocytes expressing high-affinity T-cell Receptors (TCR) give rise to CD4+CD25-FoxP3low and CD4+CD25+FoxP3- Treg progenitors, ii) In the second TCR-independent phase thymic Treg progenitors gain FoxP3 expression through an IL-2 dependent STAT-5 activation and become mature Tregs. In addition, proliferation of these Tregs in the thymus also contribute to Treg homeostasis. Recently, we have shown that OX40L (TNF superfamily ligand 4) and Jagged-1 (Notch family ligand) can induce TCR-independent proliferation of peripheral Tregs. Therefore, we hypothesized that OX40L and Jagged1 treatment can induce Treg proliferation in a TCR-independent manner that contributes to the thymic Treg homeostasis.

We found that OX40L and Jagged-1 treatment increased thymic Treg numbers in C57BL/6 wild-type (WT) mice in vivo but not in OX40-/- and Notch3-/- mice indicating the crucial role played by these cognate receptors in OX40L and Jagged1 induced augmentation of thymic Tregs. Moreover, using Ki67 (proliferation marker) and Bcl-2 (pro-survival factor) staining, we found that OX40L and Jagged1 treatment increased proliferation rather than survival of Tregs. Since earlier studies have identified a role for TNFRSF signaling in thymic Treg maturation from precursors. We analyzed whether OX40L and Jagged1 treatment can regulate thymic Treg precursor differentiation and maturation and found that OX40L and Jaggaed1 treatment significantly increased the frequencies of CD4+CD25+FoxP3- and CD4+CD25-FoxP3low Treg progenitors in the thymus

Furthermore, dose response studies in ex vivo Treg proliferation assays revealed that lower concentration of Jagged1 synergistically facilitated thymic Treg proliferation while higher concentration inhibited the same at standard doses of OX40L. Next, we characterized the suppressive phenotype of OX40L and Jagged1 expanded Tregs by analyzing the expression of Treg functional and lineage stability markers such as CTLA-4, CD39, Helios, EOS, and GARP and found that expanded Tregs retained their stable and suppressive phenotype. To further test if OX40L and Jagged1 treatment can expand functional Tregs in autoimmune conditions, we immunized CBA/j mice with porcine thyroglobulin and CFA to induce experimental autoimmune thyroiditis (EAT) and treated mice with OX40L and Jagged1. Our results showed that OX40L and Jagged1 treatment expanded Tregs in EAT mice.

However, we did not find a significant reduction in the levels of autoantibodies upon OX40L and Jagged1 treatment despite increase in Tregs. Taken together, we have identified the mechanism by which OX40L and Jagged1 treatment expanded thymic Tregs in vivo and ex vivo and characterized the phenotype of expanded Tregs. However, further studies are needed to optimize the time and dose of OX40L and Jagged1 to attain desirable protective effect against EAT and other autoimmune disease.

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