PROJECT SUMMARY – 30 lines of text Type 1 Diabetes (T1D) is a chronic disease caused by autoimmune destruction of insulin-producing pancreatic beta cells, and there is currently no treatment that reverses the disease. Many T1D reversal approaches have failed in human clinical trials and thus an ongoing and urgent need exists for novel therapies targeting new immune pathways. We have exciting data showing that a TLR4/MD2 agonistic antibody (TLR4-Ab) permanently reversed T1D in 71%, and induced a significant clinical effect in 90%, of acutely diabetic non-obese diabetic (NOD) mice. Recently, we showed that TLR4-Ab can mobilize and activate myeloid-derived suppressor cells (MDSC) that suppress T cells and reverse T1D upon adoptive transfer. We showed that TLR4-Ab remains sequestered in endosomes, unlike the TLR4 agonist LPS (which cannot reverse T1D). However, the mechanism by which TLR4-Ab reverses T1D remains unclear. Herein, we propose mechanistic studies to determine the structural and immune basis of TLR4-Ab reversal of T1D. We have also produced anti-human TLR4 antibodies which will allow us to apply these finding to human T1D.We will achieve this in three aims. Specific Aim 1. Mechanism of T cell suppression and reversal of T1D by TLR4-Ab-induced MDSCs. We hypothesize that TLR4-Ab endosomal sequestration causes sustained endosomal signaling that induces MDSC maturation. We will test this by inhibiting endosomal and surface signaling in myeloid cells while treating with TLR4-Ab and testing effects on MDSC phenotypes, T cell suppression, and ability to reverse T1D. Specific Aim 2. Mechanistic role of Fc structure in TLR4-Ab reversal of T1D and cell suppression. We hypothesize that the IgG3 Fc portion of the TLR4-Ab is critical to its tolerizing function. We will test this by assaying TLR4-Ab F(ab), F(ab)2, and deglycosylated TLR4-Ab in functional assays and on T1D. We will switch the TLR4-Ab Fc from IgG3 to IgG4 subclass to definitively confirm whether IgG3 is required for endosomal sequestration, MDSC formation, T cell suppression and T1D reversal. Specific Aim 3. Characterization of a novel panel of human anti-TLR4 antibodies. We have developed agonistic human recombinant TLR4-Abs (hTLR4-Ab). We show here that these hTLR4-Abs bind TLR4-MD2 and can activate the NF-KB signaling pathway. Our hypothesis is that human TLR4-Ab treatment will induce MDSCs from myeloid cells and that these huMDSCs will suppress human T-cell proliferation and activation. Our studies will characterize a novel innate immune pathway by which TLR4- Ab can reverse acute T1D, and begin to translate these findings to human T1D. This proposal describes a thorough training plan to support my scientific and professional development as an Immunologist.