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

描述（由申请人提供）：我们最近在NOD和B6小鼠的调节性t细胞中发现tyrphostin AG490通过IL-2-JAK-Stat5信号通路阻断Stat5的磷酸化。我们还报道了NOD小鼠中Stat5B与DNA结合亲和力较弱是由于该基因DNA结合域的点突变，并证明该突变是NOD小鼠中唯一的突变。在这些研究过程中，我们使用了一种JAK激酶抑制剂tyrphostin AG490来阻断IL-2刺激天然调节性T细胞中Stat5B的激活。令人震惊的是，CD4的体外治疗？携带AG490的Foxp3- t细胞将这个群体转化为CD4？包括NOD在内的几种不同小鼠品系的t细胞。我们的初步数据表明CD4?？经AG490转化的T细胞对TCR刺激无能并抑制CD4？Foxp3- T细胞的体外培养。与性别和年龄匹配的假药NOD对照组小鼠（n=9）相比，腹膜间注射AG490可显著延迟NOD小鼠的糖尿病发病（p<0.004, n=8）。这是我们关于通过tyrphostin AG490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)通过转基因NOD的CD4 t细胞与糖尿病源性脾细胞过继共转移预防/延迟1型糖尿病的发生。2)通过NOD中AG490介导的NOD胰岛抗原特异性t细胞重编程的糖尿病源性脾细胞过继性共转移预防/延迟1型糖尿病的发病。scid小鼠。总之，我们报告了一种新的方法，用于体外衍生大量调节性t细胞，并提出了预防NOD小鼠T1D的方法。与其他已发表的方法相比，我们用AG490生成调节性t细胞的方法价格低廉，而且速度非常快。该方法可能为在体外制造大量调节性t细胞提供了一种方法，可用于基于细胞的T1D预防和抑制胰岛异体移植排斥反应的治疗。项目相关性-据我们所知，我们是第一个描述typhostin AG490在小鼠中的免疫调节特性的人。利用tyrphsotin AG490对常规t细胞进行重编程，从而产生抗原特异性调节性t细胞是一个非常有趣的研究方向。我们关于tyrphostin AG490产生调节性t细胞的发现是高度创新和新颖的，具有很大的潜力转化为临床和延迟/预防自身免疫性疾病和/或预防同种异体移植排斥反应。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。我们非常热衷于通过本提案中描述的方法改进调节性t细胞的制造，通过资助这项拨款申请，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.