Deletion of T and B Cells to Induce Tolerance

删除 T 和 B 细胞以诱导耐受

• 批准号: 6813708
• 负责人: Stanislaw M Stepkowski
• 金额: $ 28.25万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6813708
• 关键词: B lymphocyte; BCL2 gene /protein; JAK kinase; T lymphocyte; antineoplastic antibiotics; apoptosis; biological models; chemotherapy; cytokine; doxorubicin; drug screening /evaluation; enzyme inhibitors; gene expression; genetically modified animals; heart transplantation; homologous transplantation; immune tolerance /unresponsiveness; immunopharmacology; laboratory mouse; laboratory rat; leukocyte activation /transformation; leukocyte depletion therapy; nonhuman therapy evaluation; skin transplantation; transcription factor; transplantation immunology

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.

最终目标——从根本上提高移植物存活率且无毒副作用——必须通过诱导移植耐受来实现。然而，耐受性诱导需要首先删除供体特异性 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，还没有一种疗法能够诱导可控且选择性地删除供体特异性 T 细胞和 B 细胞，同时又不会增加发病率或死亡率。 因此，我们将测试两个新的细胞凋亡诱导剂家族：1）诱导单DNA断裂阿霉素类似物a（nnamaycin；ANA、WP744、WP796和WP853）； 2) 选择性 Janus 酪氨酸激酶 (Jak)3 抑制剂（NC1153、WP938、WP988 和 WP979）。我们已经证明 WP744 仅诱导激活的 T 细胞凋亡，而不诱导未激活的 T 细胞凋亡； WP744 与 CD40Ligand (L) 单克隆抗体 (mAb) 的组合诱导耐受 同种异体心脏移植物。同样，NC 1153 单独诱导 T 细胞凋亡，导致对肾同种异体移植物的耐受，与 CTL4-Ig 组合则对心脏同种异体移植物产生耐受。我们将使用独特的模型：来自转录（Stat）5a/b缺陷小鼠的刺激子和激活子的T细胞（但不是B细胞）在激活后进入细胞凋亡；而来自 Stat4 和 Stat6 缺陷小鼠的 T 细胞分别发育为产生白细胞介素 (IL)-2 的 T 辅助细胞 (Th) 1 和产生 IL-4 的 Th2；来自抗凋亡 Bcl-2 基因过表达转基因 (Tg) 小鼠的 T 细胞表现出抗性 细胞凋亡。使用这些小鼠，我们将检查 Stats 在用细胞凋亡诱导剂治疗后，非激活、激活和记忆 T 细胞中 Bcl-2 依赖性敏感性或细胞凋亡抗性中的作用。将通过以下方式评估细胞凋亡：[a] 核溶解核变性的可视化，[b] TdT 阳性 DNA 降解的酶检测，[c] 膜联蛋白-V 易位的自动细胞计数检测，[d] 基因中促凋亡与抗凋亡 mRNA 的表达微阵列/实时 PCR，以及通过 Western 检测 Bcl-2 和 Bcl-xL 蛋白的 re] 表达 印迹。在体内，供体特异性 T 细胞克隆将通过实时 PCR（IL-2、IL-4、IL10 和 IFN-gamma）和 RNase 保护测定的细胞因子产生进行量化。由于调节性 T (Treg) 细胞被称为“记忆”辅助 T 2 型 CD4+/CD25 + (Th2reg) 细胞，因此我们计划探索 IL-4/Stat6 调节的凋亡抵抗机制。新试剂可能为删除供体特异性淋巴细胞以实现耐受提供有效且节省的方法。