Mechanistic and Therapeutic Role of the TLR4 Signaling Pathway in Type 1 Diabetes

1型糖尿病（T1 D）是由产生胰岛素的胰腺β细胞的自身免疫性破坏引起的。T1d 治疗在人类临床试验中大部分失败，因此迫切需要靶向新的免疫治疗。 途径。我们有令人兴奋的数据表明，TLR 4/MD 2激动性抗体（TLR 4-Ab）可以永久逆转 在急性糖尿病非肥胖糖尿病（NOD）小鼠中，T1 D的发生率为71%，并且在90%中具有显著的临床效果。在 在我们最近的糖尿病论文中，我们发现TLR 4-Ab可以动员和激活髓源性抑制细胞， （MDSC），其在过继转移后抑制T细胞并改善急性T1 D。我们发现TLR 4- Ab诱导TLR 4/MD-2在内体中隔离，不像典型的TLR 4激动剂LPS（其不能 逆转T1 D）;然而，TLR 4-Ab逆转T1 D的机制仍不清楚。我们现在已经 产生了抗人类TLR 4抗体，使我们能够将这些发现应用于人类T1 D。我们的联合 在自身免疫/T1 D（Ridgway），分子生物物理学/免疫学（Herr）和人类T1 D（Dolan）方面的专业知识是 非常适合进行这些研究。我们将追求三个目标：具体目标1。T1 D逆转机制 通过TLR 4-Ab诱导的MDSC。TLR 4-Ab内体隔离可以通过TLR 4-Ab内体隔离诱导延长的信号传导。 TLR 4介导的TRIF通路，其保护免受T1 D（2015，Chervonsky等人）。我们发现， 在板上的TLR 4-Ab，其防止内体螯合，消除了TLR 4信号传导。所以我们 假设TLR 4-Ab诱导的TLR 4/MD 2内体隔离引起持续的TRIF内体 它通过诱导APC经历MDSC成熟的信号传导，诱导免疫调节和逆转T1 D。 具体目标2。Fc结构在TLR 4-Ab逆转T1 D和细胞抑制中的机制作用。而 抗体F（ab）结构决定特异性，Fc区通过受体参与抗体功能 结合和糖基化。TLR 4-Ab是IgG 3同种型，其具有最长的铰链区并且增加了 糖基化位点IgG 3抗体还形成cryobulins，影响亲合力和内化。我们的初步 数据显示，与全长相比，TLR 4-Ab F（ab）和F（ab '）2片段引起NFκB信号传导减少 TLR 4抗体这表明Fc结构在TLR 4-Ab功能中的关键作用。因此，我们假设 TLR 4-Ab的IgG 3 Fc部分对其耐受功能至关重要，我们将用免疫学和免疫学方法对此进行测试。 分子生物物理方法。具体目标3。测试一组新的人类 人T1 D APC上的抗TLR 4抗体由于TLR 4/MD 2途径在进化上是高度保守的， 我们已经开发了激动性人重组TLR 4-Ab（hTLR 4-Ab）。我们发现这些hTLR 4抗体 结合并激活TLR 4/MD 2。我们的假设是hTLR 4-Ab处理将诱导骨髓间充质干细胞从骨髓中分化出来， 这些huMDSC将抑制人T细胞增殖和活化。这些研究将 描述了TLR 4-Ab治疗可以逆转急性T1 D的新的先天免疫机制， 对新型人TLR 4抗体的研究是翻译成人T1 D的第一步。

Type 1 Diabetes (T1D) is caused by autoimmune destruction of insulin-producing pancreatic beta cells. T1D therapies have largely failed in human clinical trials and thus an urgent need exists for targeting novel immune pathways. We have exciting data showing that a TLR4/MD2 agonistic antibody (TLR4-Ab) permanently reversed T1D in 71%, and had a significant clinical effect in 90%, of acutely diabetic non-obese diabetic (NOD) mice. In our recent Diabetes paper, we showed that TLR4-Ab can mobilize and activate myeloid-derived suppressor cells (MDSC) in vivo, that suppress T cells and ameliorate acute T1D upon adoptive transfer. We showed that TLR4- Ab induces TLR4/MD-2 sequestration in endosomes, unlike the canonical TLR4 agonist LPS (which cannot reverse T1D); however, the mechanism by which TLR4-Ab reverses T1D remains unclear. We have now produced anti-human TLR4 antibodies, allowing us to apply these findings to human T1D. Our combined expertise in autoimmunity/T1D (Ridgway), molecular biophysics/Immunology (Herr) and human T1D (Dolan) is well suited to perform these studies. We will pursue three aims: Specific Aim 1. Mechanism of reversal of T1D by TLR4-Ab-induced MDSCs. TLR4-Ab endosomal sequestration may induce prolonged signaling via the TLR4-mediated TRIF pathway, which protects from T1D (2015, Chervonsky et al.). We show that immobilization of TLR4-Ab on a plate, which prevents endosomal sequestration, eliminated TLR4 signaling. Therefore, we hypothesize that TLR4-Ab-induced TLR4/MD2 endosomal sequestration causes sustained TRIF endosomal signaling that induces APCs to undergo MDSC maturation, inducing immune regulation and reversing T1D. Specific Aim 2. Mechanistic role of Fc structure in TLR4-Ab reversal of T1D and cell suppression. While antibody F(ab) structure determines specificity, the Fc region contributes to antibody function through receptor binding and glycosylation. TLR4-Ab is an IgG3 isotype, which has the longest hinge region and increased glycosylation sites. IgG3 antibodies also form cryoglobulins, impacting avidity and internalization. Our preliminary data show that TLR4-Ab F(ab) and F(ab’)2 fragments elicit decreased NFκB signaling compared to full-length TLR4-Ab. This shows a critical role for Fc structure in TLR4-Ab function. Therefore, we hypothesize that the IgG3 Fc portion of the TLR4-Ab is critical to its tolerizing function and we will test this with immunological and molecular biophysical approaches. Specific Aim 3. Testing therapeutic effects of a novel panel of human anti-TLR4 antibodies on human T1D APCs Since the TLR4/MD2 pathway is strongly evolutionarily conserved, we have developed agonistic human recombinant TLR4-Ab (hTLR4-Ab). We show here that these hTLR4-Abs bind to, and activate TLR4/MD2. Our hypothesis is that hTLR4-Ab treatment will induce MDSCs from myeloid precursors and that these huMDSCs will suppress human T-cell proliferation and activation. These studies will characterize novel innate immune mechanisms by which TLR4-Ab treatment can reverse acute T1D, and our studies on the novel human TLR4 antibodies are the first step in translation to human T1D.