G-BMDC-induced Treg mediated suppression of Type 1 Diabetes in NOD mice

University Of Illinois At Chicago, Chicago IL

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Abstract

DESCRIPTION (provided by applicant): 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.