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

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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.