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

1型糖尿病(T1D)是由产生胰岛素的胰岛β细胞的自身免疫破坏引起的。T1D 治疗方法在人体临床试验中已基本失败，因此迫切需要以新型免疫为靶点。 小路。我们有令人兴奋的数据表明，TLR4/MD2激动型抗体(TLR4-Ab)永久逆转 在急性糖尿病非肥胖糖尿病(NOD)小鼠中，T1D的有效率为71%，临床有效率为90%。在……里面 我们最近的糖尿病论文表明，TLR4-Ab可以动员和激活髓系抑制细胞 (MDSC)体内，过继转移时抑制T细胞并改善急性T1D。我们展示了TLR4- 与经典的TLR4激动剂脂多糖(不能)不同，AB诱导TLR4/MD-2在内吞体内隔离 逆转T1D)；然而，TLR4-Ab逆转T1D的机制尚不清楚。我们现在有了 产生了抗人TLR4抗体，使我们能够将这些发现应用于人类T1D。我们的联合 自身免疫/T1D(Ridgway)、分子生物物理学/免疫学(HERR)和人类T1D(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.T1D逆转机制 TLR4-Ab诱导的MDSCs。TLR4-Ab内体隔离可能通过诱导信号传导延长 TLR4介导的TRIF途径，保护免受T1D(2015，Chervonsky等人)。我们证明了这种固定化 将TLR4-Ab放在平板上，防止内体隔离，消除了TLR4信号。因此，我们 假设TLR4-Ab诱导的TLR4/MD2内体隔离导致持续的TRIF内体 诱导APC经历MDSC成熟、诱导免疫调节和逆转T1D的信号。 特异性目的2.Fc结构在TLR4-Ab逆转T1D和细胞抑制中的机制作用而当 抗体F(Ab)结构决定特异性，Fc区通过受体发挥抗体功能 结合和糖基化。TLR4-Ab是一种IgG3亚型，铰链区最长， 糖基化位点。IgG3抗体还会形成冷球蛋白，影响亲和力和内化。我们的预赛 数据显示，与全长相比，TLR4-AbF(Ab)和F(ab‘)2片段诱导的NFκB信号减弱 TLR4-Ab.这表明Fc结构在TLR4-Ab功能中起着关键作用。因此，我们假设 TLR4-Ab的IgG3 Fc部分对其耐受功能至关重要，我们将用免疫学和 分子生物物理方法。具体目标3.测试一组新型人类的治疗效果 人T1D APC上的抗TLR4抗体由于TLR4/MD2途径在进化上高度保守， 我们研制了激动型人重组TLR4-Ab(hTLR4-Ab)。我们在这里展示了这些hTLR4-Abs 绑定并激活TLR4/MD2。我们的假设是hTLR4-Ab治疗将从髓系诱导MDSCs 并且这些huMDSCs将抑制人类T细胞的增殖和激活。这些研究将 描述TLR4-Ab治疗逆转急性T1D的新的天然免疫机制，以及我们的 人类新型TLR4抗体的研究是向人类T1D转化的第一步。

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.