PKC-ϴ is Dispensable for G-BMDC-Induced TCR-Independent Treg Proliferation

GM-CSF-induced bone marrow derived dendritic cells (G-BMDCs) have been shown by our lab to be able to selectively cause Treg proliferation when co-cultured with CD4+ T cells. This Treg proliferation was shown to be TCR-independent but OX40L/OX40-dependent. In this thesis work we studied the signaling involved in the G-BMDC-induced Treg proliferation. Firstly, we confirmed the critical role of OX40L/OX40 signaling in Treg expansion by using CD4+ T cells from OX40 deficient mice, which failed to proliferate significantly. Because PKC-ϴ was shown to play an important role downstream of OX40 signaling in conventional T cells in the absence of TCR stimulation, we investigated the role of PKC-ϴ in G-BMDC-induced Treg expansion. Interestingly, CD4+ T cells from PKC-ϴ deficient mice, upon co-culture with WT G-BMDCs, showed impaired Treg proliferation. However, supplementation of the co-culture with exogenous IL-2 restored Treg proliferation, suggesting that G-BMDC-induced Treg proliferation per se was PKC-ϴ-independent. Our data further suggested that PKC-ϴ is likely required for optimum IL-2 production by T effector cells. Finally, our data suggested that OX40 mediated downstream signaling in Tregs likely involves TRAF1. Therefore, OX40L mediated TCR-independent Treg proliferation could be an effective means of selectively expanding Treg as a potential therapeutic for autoimmune disease including T1D

PKC-ѳ is dispensable for OX40L-induced TCR-independent Treg proliferation but contributes by enabling IL-2 production from effector T-cells.

We have previously shown that OX40L/OX40 interaction is critical for TCR-independent selective proliferation of Foxp3+ Tregs, but not Foxp3- effector T-cells (Teff), when CD4+ T-cells are co-cultured with GM-CSF derived bone marrow dendritic cells (G-BMDCs). Events downstream of OX40L/OX40 interaction in Tregs responsible for this novel mechanism are not understood. Earlier, OX40L/OX40 interaction has been shown to stimulate CD4+ T-cells through the formation of a signalosome involving TRAF2/PKC-Ѳ leading to NF-kB activation. In this study, using CD4+ T-cells from WT and OX40-/- mice we first established that OX40 mediated activation of NF-kB was critical for this Treg proliferation. Although CD4+ T-cells from PKC-Ѳ-/- mice were also defective in G-BMDC induced Treg proliferation ex vivo, this defect could be readily corrected by adding exogenous IL-2 to the co-cultures. Furthermore, by treating WT, OX40-/-, and PKC-Ѳ-/- mice with soluble OX40L we established that OX40L/OX40 interaction was required and sufficient to induce Treg proliferation in vivo independent of PKC-Ѳ status. Although PKC-Ѳ is dispensable for TCR-independent Treg proliferation per se, it is essential for optimum IL-2 production by Teff cells. Finally, our findings suggest that OX40L binding to OX40 likely results in recruitment of TRAF1 for downstream signalling

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

Critical role of OX40 signaling in the TCR-independent phase of human and murine thymic Treg generation

Regulatory T cells (Tregs) play a pivotal role in immune-tolerance, and loss of Treg function can lead to the development of autoimmunity. Natural Tregs generated in the thymus substantially contribute to the Treg pool in the periphery, where they suppress self-reactive effector T cells (Teff) responses. Recently, we showed that OX40L (TNFSF4) is able to drive selective proliferation of peripheral Tregs independent of canonical antigen presentation (CAP-independent) in the presence of low-dose IL-2. Therefore, we hypothesized that OX40 signaling might be integral to the TCR-independent phase of murine and human thymic Treg (tTreg) development. Development of tTregs is a two-step process: Strong T-cell receptor (TCR) signals in combination with co-signals from the TNFRSF members facilitate tTreg precursor selection, followed by a TCR-independent phase of tTreg development in which their maturation is driven by IL-2. Therefore, we investigated whether OX40 signaling could also play a critical role in the TCR-independent phase of tTreg development. OX40-/- mice had significantly reduced numbers of CD25-Foxp3low tTreg precursors and CD25+Foxp3+ mature tTregs, while OX40L treatment of WT mice induced significant proliferation of these cell subsets. Relative to tTeff cells, OX40 was expressed at higher levels in both murine and human tTreg precursors and mature tTregs. In ex vivo cultures, OX40L increased tTreg maturation and induced CAP-independent proliferation of both murine and human tTregs, which was mediated through the activation of AKT-mTOR signaling. These novel findings show an evolutionarily conserved role for OX40 signaling in tTreg development and proliferation, and might enable the development of novel strategies to increase Tregs and suppress autoimmunity