Deletion of T and B Cells to Induce Tolerance

最终目标--从根本上提高移植物的存活率而不产生毒副作用--必须通过诱导移植耐受来实现。然而，耐受性诱导需要最初删除供体特异性T细胞克隆和可能的B细胞克隆，并随后产生调节以维持耐受性。到目前为止，还没有一种治疗方法可以在不增加发病率或死亡率的情况下诱导供者特异性T和B细胞的可控和选择性缺失。 因此，我们将测试两个新的凋亡诱导剂家族：1)诱导单个DNA断裂阿霉素类似物a(那那霉素；ANA，WP744，WP796和WP853)；2)选择性Janus酪氨酸酶(JAK)3抑制剂(NC1153，WP938，WP988和WP979)。我们已经证明WP744只诱导激活的T细胞而不诱导非激活的T细胞凋亡；WP744与CD40配体(L)单抗(MAb)联合诱导耐受 同种异体心脏移植同样，NC1153单独诱导T细胞凋亡，导致对肾移植的耐受，并与CTL4-Ig联合应用于心脏移植。我们将使用独特的模型：来自刺激和转录激活因子(STAT)5a/b缺陷小鼠的T细胞(而不是B细胞)在激活后进入凋亡；而来自STAT4和Stat6缺陷小鼠的T细胞分别发育为产生IL-2的T辅助细胞(Th)1和产生IL-4的Th2；来自反细胞凋亡的过度表达的转基因(TG)小鼠的T细胞表现出对 细胞凋亡。利用这些小鼠，我们将研究Stats在非激活、激活和记忆T细胞中对Bcl2依赖的敏感性或抗凋亡中的作用，这些T细胞经过凋亡诱导剂处理后。通过[a]显示核变性，[b]酶法检测TdT阳性的DNA降解，[c]自动细胞仪检测Annexin-V易位，[d]在基因微阵列/实时定量聚合酶链式反应中表达促和抗凋亡的mRNAs，以及用Western重新表达Bcl2和Bclxl蛋白，来评估细胞凋亡。 污点。在体内，供者特异性T细胞克隆将通过实时定量聚合酶链式反应(IL-2、IL-4、IL-10和干扰素-γ)和核糖核酸酶保护试验来量化细胞因子的产生。由于T调节(Treg)细胞是“记忆性”的辅助性T细胞2型(Th2reg)细胞，我们计划探索IL-4/Stat6调节的细胞凋亡抵抗机制。新试剂可能为耐受供者特异性淋巴细胞的缺失提供有效的保存方法。

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.