Generating regulatory T-cells by a JAK-STAT5 kinase inhibiotr: A noval approach t

描述（由申请人提供）：我们最近已经表明，酪氨酸磷酸化抑制剂AG 490通过NOD和B6小鼠中调节性T细胞中的IL-2-JAK-Stat 5信号传导途径阻断Stat 5的磷酸化。我们还报道了NOD小鼠Stat 5 B与DNA结合亲和力减弱的原因是该基因DNA结合结构域的点突变，并证明该突变是NOD小鼠中唯一的突变。在这些研究过程中，我们使用酪氨酸磷酸化抑制剂AG 490（一种JAK激酶抑制剂）阻断天然调节性T细胞中IL-2刺激对Stat 5 B的激活.令人惊讶的是，体外治疗的CD 4？Foxp 3-T细胞与AG 490转换成CD 4？包括NOD在内的几种不同小鼠品系中的T细胞。我们的初步数据表明，CD 4？？由AG 490转化的T细胞对TCR刺激无反应性并抑制CD 4？体外Foxp 3- T细胞。腹腔注射AG 490显著延迟NOD小鼠糖尿病的发病（p<0.004，n=8）与性别和年龄匹配的假治疗NOD对照小鼠（n=9）相比。这是我们关于通过tyrphostin AG 490产生调节性T细胞的新发现的后续研究，我们提出了两个具体目标; 1）通过在免疫缺陷NOD.scid小鼠中过继性共转移致糖尿病脾细胞与经AG 490重编程的转基因NOD.BDC2.5小鼠的CD 4 T细胞来预防/延迟1型糖尿病的发作，以及，2）通过在NOD.scid小鼠中过继共转移致糖尿病脾细胞与由AG 490进行的NOD胰岛抗原特异性T细胞重编程来预防/延迟1型糖尿病的发作。总之，我们报告了一种用于体外衍生大量调节性T细胞的新方法，并提出预防NOD小鼠中的T1 D。我们的方法来产生调节性T细胞的AG 490是廉价的，是非常快的，与其他已公布的方法相比。这种方法可以提供一种方法，用于制造大量的调节性T细胞在体外，可用于基于细胞的治疗T1 D的预防和抑制胰岛同种异体排斥。项目相关性-据我们所知，我们是第一个描述免疫调节特性的tyrphostin AG 490在小鼠。通过用酪氨酸蛋白AG 490重编程常规T细胞来产生抗原特异性调节性T细胞是非常令人感兴趣的。我们关于通过tyrphostin AG 490产生调节性T细胞的发现是高度创新和新颖的，并且具有转化为临床和延迟/预防自身免疫性疾病和/或预防同种异体移植物排斥的巨大潜力。我们的方法非常便宜，快速，可以在任何普通免疫学实验室进行。我们有极大的热情通过本提案中描述的方法来改善调节性T细胞的制造，并且通过资助该资助申请，我们将有机会产生令人信服的体外和体内数据，并达到适合临床前研究的水平。

DESCRIPTION (provided by applicant): We have recently shown that tyrphostin AG490 blocked phosphorylation of Stat5 via the IL-2-JAK-Stat5 signaling pathway in regulatory T-cells in NOD and B6 mice. We have also reported that weaker DNA binding affinity of Stat5B to the DNA in NOD mouse is due to a point mutation in DNA binding domain in this gene and demonstrated that this mutation is a unique mutation in NOD mouse. In the course of these studies, we used tyrphostin AG490, a JAK kinase inhibitor, to block activation of Stat5B by IL-2 stimulation in natural regulatory T- cells. Stunningly, in-vitro treatment of CD4?Foxp3- T-cells with AG490 converted this population into CD4? T-cells in several different mouse strains including NOD. Our preliminary data indicate that CD4?? T cells converted by AG490 are anergic to TCR stimulation and suppress proliferation of CD4? Foxp3- T cells in vitro. Interperitoneal injection of AG490 significantly delayed onset of diabetes in NOD mice (p<0.004, n=8) when compared with sex and age matched sham treated NOD control mice (n=9).This is a follow- up study of our novel finding regarding generating regulatory T-cells by tyrphostin AG490 and we propose two specific aims; 1) prevention/delay onset of type 1 diabetes by adoptive co-transfer of diabetogenic splenocytes with CD4 T-cells of transgenic NOD.BDC2.5 mice reprograms by AG490 in immunodeficient NOD.scid mice and, 2) prevention/delay onset of type 1 diabetes by adoptive co-transfer of diabetogenic splenocytes with NOD islet antigens-specific T-cell reprograms by AG490 in NOD.scid mice. In summary, we report a novel approach for the in- vitro derivation of large numbers of regulatory T-cells and propose to prevent T1D in NOD mouse. Our method to generate regulatory T-cells by AG490 is inexpensive and is very quick when compared with other published methods. This method may provide an approach for manufacturing a large number of regulatory T-cells in- vitro that can be used in cell-based therapies of T1D prevention and suppression of islet allograft rejection.Project relevance- To the best of our knowledge we are the first to describe immuno-regulatory properties of tyrphostin AG490 in mouse. Generating antigen-specific regulatory T-cells by reprogramming conventional T-cells by tyrphsotin AG490 is of great interest. Our finding regarding generating regulatory T-cells by tyrphostin AG490 is highly innovative and novel and has a great potential to translate into clinic and delay/prevent autoimmune diseases and/or to prevent rejection of allograft transplant. Our method is very inexpensive and is quick and can be performed in any general immunology laboratories. We have a great enthusiasm to improve manufacturing of regulatory T-cells by the method describe in this proposal and by funding this grant application we will have this opportunity to generate convincing in-vitro and in-vivo data and reach to a level suitable for a pre- clinical study.