GM-CSF can prevent T1D in NOD mice, experimental autoimmune thyroiditis (EAT) in CBA mice and autoimmune myasthenia gravis (EAMG) in C57Bl/6 mice. Antigen presentation by GM-CSF-induced tolerogenic DCs increased Tregs, which suppressed Teff cell responses through enhanced IL-10 production. Bone marrow precursors differentiated in the presence of GM-CSF (G-BMDCs) were able to selectively expand Tregs in co-cultures with naive CD4+ T cells while spleen derived DCs (SpDCs) failed to do so. Studies using MHC class-II-/- G-BMDCs indicated that G-BMDC-T cell contact via OX40L-OX40 interaction and exogenous IL-2 were required, while TCR signaling was not required for Treg proliferation. Co-culture of T cells and SpDCs supplemented with an OX40 agonist did not lead to Treg expansion and suggested that other molecular interactions may be required. We found higher levels of Jagged-1 (Jag-1) on G-BMDCs relative to SpDCs, and higher levels of Notch3 in Foxp3+T cells relative to Ffox3- T cells. Blockade of either Jag-1 or Notch3, using blocking antibodies, inhibited Treg expansion. Adoptive transfer of only OX40L+Jag-1+ G-BMDCs into recipient mice led to in vivo Treg expansion and disease suppression, and indicated that concurrent signaling via OX40 and Notch3 expressed on Tregs was essential for the TCR- independent G-BMDC-induced Treg expansion. Recent studies by others suggest that in the absence of TCR stimulation, OX40 can form a signalosome involving PKC-? and promote T-cell proliferation through NF-kB activation. Further, Notch3 has been shown to co-operatively regulate FOXP3 expression through PKC-? activation. Thus based on our current findings, we hypothesize that OX40L-OX40 and Jag-1-Notch3 co-signaling in Tregs can activate PKC-?, which can bind to OX40 signalosome and activate NF-kB to cause Treg expansion and suppress T1D in NOD mice.
Aims are:
Aim -1: To establish the sufficiency of OX40L-OX40 and Jag-1-Notch-3 co-signaling in the ex vivo expansion of Tregs by G-BMDCs.
Aim -2: To elucidate the molecular mechanism of TCR-independent but Notch3-OX40 mediated signaling dependent Treg proliferation.
Aim -3: To determine the therapeutic utility of G-BMDC membrane bound and soluble OX40L and Jag-1 in suppressing T1D in NOD mice and in ex vivo expansion of functional human Tregs. G-BMDC-induced, TCR independent, Treg, but not Teff, proliferation has profound implications for developing effective therapy for human autoimmune diseases. In this study, we will determine the critical requirement (aim-1) and the underlying mechanism (aim-2) of co-signaling by Jag-1 and OX40L in Treg proliferation. Further, we will test if ex vivo generated G-BMDCs or Tregs, or soluble Jag-1 and OX40L, can suppress T1D in NOD mice (aim-3). Finally, we will test if human G-BMDCs can similarly expand functional human Tregs.

Public Health Relevance

A large number of chronic human diseases are autoimmune diseases with a very high associated morbidity. More recently, regulatory T cells (Tregs) that our body naturally produces have been shown to suppress different autoimmune diseases in animals without any harmful side effects. However, generating large numbers of Tregs without loss of their function has remained a challenge. Our laboratory has discovered that bone-marrow derived dendritic cells differentiated in the presence of GM-CSF (G-BMDCs) can cause Treg proliferation without TCR mediated signaling. We have discovered that signaling induced by OX40L and Jagged-1, uniquely expressed on the surface of G-BMDCS, are essential for Treg proliferation. In this proposal, we will study the critical interactions of OX40L and Jagged-1 with their cognate receptors, OX40 and Notch3, expressed on Tregs. We will explore how signaling from OX40 and Notch3 contribute to BMDC induced Treg expansion. Further, we will optimize methods to expand large numbers of Tregs ex vivo and evaluate their therapeutic potential in the NOD model of type 1 diabetes. Additionally, we will use very similar approach to generate G-BMDCs using human bone marrow cells and GM-CSF, and co-culture them with human CD4+ T cells to expand functional Tregs ex vivo. Since Tregs can be used to treat other autoimmune diseases as well, the proposed project is of high clinical relevance.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI107516-03
Application #
9108851
Study Section
Hypersensitivity, Autoimmune, and Immune-mediated Diseases Study Section (HAI)
Program Officer
Bourcier, Katarzyna
Project Start
2014-08-01
Project End
2018-07-31
Budget Start
2016-08-01
Budget End
2017-07-31
Support Year
3
Fiscal Year
2016
Total Cost
Indirect Cost
Name
University of Illinois at Chicago
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
098987217
City
Chicago
State
IL
Country
United States
Zip Code
60612
Kumar, Prabhakaran; Marinelarena, Alejandra; Raghunathan, Divya et al. (2018) Critical role of OX40 signaling in the TCR-independent phase of human and murine thymic Treg generation. Cell Mol Immunol :
Kumar, Prabhakaran; Saini, Shikha; Khan, Saad et al. (2018) Restoring self-tolerance in autoimmune diseases by enhancing regulatory T-cells. Cell Immunol :
Kumar, Prabhakaran; Bhattacharya, Palash; Prabhakar, Bellur S (2018) A comprehensive review on the role of co-signaling receptors and Treg homeostasis in autoimmunity and tumor immunity. J Autoimmun 95:77-99
Danikowski, K M; Jayaraman, S; Prabhakar, B S (2017) Regulatory T cells in multiple sclerosis and myasthenia gravis. J Neuroinflammation 14:117
Alharshawi, Khaled; Marinelarena, Alejandra; Kumar, Prabhakaran et al. (2017) PKC-? is dispensable for OX40L-induced TCR-independent Treg proliferation but contributes by enabling IL-2 production from effector T-cells. Sci Rep 7:6594
Kumar, Prabhakaran; Alharshawi, Khaled; Bhattacharya, Palash et al. (2017) Soluble OX40L and JAG1 Induce Selective Proliferation of Functional Regulatory T-Cells Independent of canonical TCR signaling. Sci Rep 7:39751
Haddad, Christine S; Bhattacharya, Palash; Alharshawi, Khaled et al. (2016) Age-dependent divergent effects of OX40L treatment on the development of diabetes in NOD mice. Autoimmunity 49:298-311
Bhattacharya, Palash; Thiruppathi, Muthusamy; Elshabrawy, Hatem A et al. (2015) GM-CSF: An immune modulatory cytokine that can suppress autoimmunity. Cytokine 75:261-71
Bhattacharya, Palash; Budnick, Isadore; Singh, Medha et al. (2015) Dual Role of GM-CSF as a Pro-Inflammatory and a Regulatory Cytokine: Implications for Immune Therapy. J Interferon Cytokine Res 35:585-99
Kong, Sinyi; Dong, Hongxin; Song, Jianxun et al. (2015) Deleted in Breast Cancer 1 Suppresses B Cell Activation through RelB and Is Regulated by IKK? Phosphorylation. J Immunol 195:3685-93