Reversal of T1D and Rapid Reduction of β-Cell Stress by IL-10–Dependent MDSCs Induced by TLR4-Ab

Type 1 diabetes (T1D) is caused by the autoimmune destruction of insulin-producing pancreatic β-cells. We published that an IgG3 Toll-like receptor 4 (TLR4)/myeloid differentiation factor 2 (MD2) agonistic antibody (TLR4-Ab) reversed acute T1D in NOD mice by induction of myeloid-derived suppressor cells (MDSCs), but the mechanism of MDSC induction was unclear. Here we show that TLR4-Ab reversal of acute T1D induced profound intraislet immune and endocrine changes in single-cell RNA sequencing compared with controls. Islet-infiltrating S100A9+ cells significantly upregulated 18 genes associated with MDSCs, including Nos2, the gene responsible for inducible nitric oxide synthase (iNOS) production. TLR4-Ab treatment induced significant changes in β-cell gene expression, with markedly reduced endoplasmic reticulum stress and reduced unfolded protein response. Bone marrow (BM) cells cultured with TLR4-Ab, but not granulocyte macrophage–colony-stimulating factor or lipopolysaccharide, induced MDSCs that suppressed T cells, and blockade of iNOS activity eliminated T-cell suppression. Blockade of interleukin 10 (IL-10) signaling during BM differentiation prevented T-cell suppression induced by TLR4-Ab–MDSC, demonstrating a central role for IL-10 in TLR4-Ab–mediated MDSC development. Reporter cell assays demonstrated predominant endosomal signaling with TLR4-Ab, and soluble (not plate-bound) TLR4-Ab was necessary for MDSC induction. TLR4-Ab treatment of T1D therefore induces an IL-10–dependent immunoregulatory MDSC response, which significantly downregulates β-cell stress. Article Highlights Therapy with IgG3 Toll-like receptor 4 (TLR4)/myeloid differentiation factor 2 (MD2) agonistic antibody (TLR4-Ab) is a novel approach to type 1 diabetes (T1D) immunotherapy that targets the innate immune system by inducing immunoregulatory myeloid-derived suppressor cells (MDSCs). We sought to define the mechanisms by which TLR4-Ab induces MDSCs and how they reverse T1D. TLR4-Ab induction of MDSCs requires interleukin 10 signaling in the bone marrow, and MDSCs mediate suppression via inducible nitric oxide synthase. Induction of immunoregulatory MDSCs results in profound downregulation of β-cell endoplasmic reticulum stress and unfolded protein response associated with acute T1D. Establishing mechanisms by which TLR4-Ab reverses acute T1D defines new therapeutic pathways for human T1D that target innate immunity to restore immune tolerance.