. Type 1 diabetes mellitus (T1DM) is a chronic autoimmune disease caused by the T-cell mediated destructionofthepancreaticinsulin-producingb?-cells(1,2).Currentstandardoftreatmentislimitedtolife-long insulin therapy (1, 2). In previous studies, we have reported the treatment of mice with granulocyte macrophage colony-stimulating factor (GM-CSF) prevented the development of T1DM in non-obese diabetic (NOD) mice through the mobilization of a specific subset of dendritic cells (DCs) that could stimulate the expansion of regulatory T cells (Tregs) in vivo (11, 12). Similarly, DCs generated from bone marrow (BM) precursor cells cultured with GM-CSF (G-BMDCs) were capable of expanding Tregs ex vivo in a contact- dependent manner, independent of TCR activation (13). Furthermore, we determined OX40L was one of the criticalsurfaceboundmoleculesonG-BMDCsthatfacilitatedthisphenomenonofTregexpansionsuggesting OX40L+ DCs may play a role in physiological Treg homeostasis (13).
The aims of this research project is to definethisOX40L+tolerogenicDCsubsetinducedbyGM-CSFexvivo,identifythephysiologicalcounterpartin vivo, confirm the functionally suppressive capacity of DC-expanded Tregs, and determine the therapeutic potential of tolerogenic DCs in the treatment of T1DM. CD11c+OX40L+G-BMDCs will be generated ex vivo from bone-marrow precursor cells isolated from NOD mice and characterized for the expression of various subset-specificDCsurfacemarkers,chemokinereceptors,andlineagespecificmarkers.Subsequently,NOD mice treated with GM-CSF will be analyzed for tissue compartmentalization of OX40L+CD11c+ DCs, and this specificDCsubsetwillalsobeanalyzedforthevarioussurfacemarkersasstatedinthepreviousexperiment. Furthermore,wewillalsoisolateOX40L+CD11c+DCsfromGM-CSFtreatedNODmiceandassessthedegree of DC-stimulated Treg expansion. We will assess the suppressive function of the DC-expanded Tregs by analyzing various markers highly implicated in Treg suppressive function and performing Treg suppressive assaystounequivocallyconfirmtheimmunoregulatoryfunctionoftheseDC-expandedTregs.Oncewehave established and identified a distinct subset of tolerogenic DCs ex vivo and in vivo, OX40L+CD11c+G-BMDCs generated ex vivo will be adoptively transferred into pre-diabetic NOD mice and T1DM suppression will be monitoredtoconfirmthetolerogenicphenotypeoftheseOX40L+CD11c+G-BMDCs.Lymphocytesfromtreated NODmicewillbeanalyzedforTregexpressionandcytokinesecretiontofurtherelucidateT1DMsuppression is mediated through DC-induced expansion of functionally suppressive Tregs. Additionally, Tregs will be isolated from OX40L+CD11c+G-BMDCs recipients and will be further tested for the ability to suppress the manifestation of T1DM by CD4+ T-cell co-transfer to histocompatible, immunodeficient NOD.scid mice. The valuable knowledge gained from these experiments will provide new insights that could lead to the developmentofmoreeffectivetherapeuticstrategiesofexpandingTregsinvivointhetreatmentofT1DM.
We previously reported the treatment of mice with granulocyte macrophage colony-stimulating factor (GM- CSF) prevented the development of T1DM in non-obese diabetic (NOD) mice through the mobilization of a specificsubsetofdendriticcells(DCs)thatcouldstimulatetheexpansionofregulatoryT-cells(Tregs)invivo and suppress autoimmune disease (11, 12). We found GM-CSF derived-DCs, generated ex vivo from bone marrow (BM) precursor cells cultured with GM-CSF (G-BMDCs), were able to cause robust proliferation of Tregs independent of TCR activation, however, this expansion was dependent on the DC-surface bound moleculeOX40L,amemberofthetumornecrosisfactorsuperfamilywithco-stimulatoryfunctionssuggestinga novel role of OX40L+ DCs in physiological Treg homeostasis (13, 14). The aims of this research project will contribute to identifying this novel tolerogenic OX40L+ DC subset and, most importantly, determining the therapeuticpotentialofthistolerogenicDCsubsetinthetreatmentofT1DM.
