The ultimate goal--to radically improve graft survival without toxic side effects--must be achieved through induction of transplantation tolerance. However, tolerance induction requires initial deletion of donor-specific T and possibly B cell clones and subsequent generation of regulation to maintain tolerance. So far, no therapy induces controllable and selective deletion of donor-specific T and B cells without risking increased morbidity or mortality. Therefore, we will test two new families of apoptosis-indueing agents: 1) inducing single DNA breaks doxorubicin analogues a(nnamaycin; ANA, WP744, WP796, and WP853); and 2) selective Janus tyrosine kin ase (Jak)3 inhibitors (NC1153, WP938, WP988 and WP979). We already showed that WP744 induces apoptosis of only activated but not non-activated T cells; combination of WP744 with CD40Ligand (L) monoclonal antibody (mAb) induced tolerance to heart allografts. Similarly, NC 1153 alone induced T cell apoptosis resulting in tolerance to kidney allografts and in combination with CTL4-Ig to heart allografts. We will use unique models: T cells (but not B cells) from stimulators and activators of transcription (Stat)5a/b-deficient mice enter apoptosis after activation; whereas T cells from Stat4 and Stat6-deficient mice develop into interleukin (IL)-2-producing T helper (Th) 1 and IL-4-producing Th2, respectively; T cells from anti-apototic Bcl-2 gene over-expressing transgenic (Tg) mice display resistance to apoptosis. Using these mice we will examine the role of Stats in Bcl-2-dependent sensitivity or resistance to apoptosis in non-activated, activated and memory T cells following treatment with apoptosis-inducing agents. Apoptosis will be assessed by [a] visualization of karyolytic nuclear degeneration, [b] enzymatic detection of TdT-positive DNA degradation, [c] automated cytometric detection of annexin-V translocation, [d] expression of pro- versus antiapoptotic mRNAs in gene microarray/real-time PCR, and re] expression of Bcl-2 and Bcl-xL proteins by Western blot. In vivo, donor-specific T cell clones will be quantified by cytokine production by real-time PCR (IL-2, IL-4, IL10 and IFN-gamma) and RNase protection assay. Since T regulatory (Treg) cells are characterized as """"""""memory"""""""" T helper 2- type CD4+/CD25 + (Th2reg) cells, we plan to explore the IL-4/Stat6-regulated apotosis-resistance mechanism. The new agents may provide potent and save method for deletion of donor-specific lymphocytes for tolerance.
