Deletion of T and B Cells to Induce Tolerance

删除 T 和 B 细胞以诱导耐受

• 批准号: 6913679
• 负责人: Stanislaw M Stepkowski
• 金额: $ 27.03万
• 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/6913679
• 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.

最终的目标-从根本上提高移植物的存活率而没有毒副作用-必须通过诱导移植耐受来实现。然而，耐受性诱导需要供体特异性T细胞克隆和可能的B细胞克隆的初始缺失以及随后产生的调节以维持耐受性。到目前为止，还没有一种治疗方法能诱导供体特异性T和B细胞的可控和选择性缺失，而不增加发病率或死亡率。 因此，我们将测试两个新的家族的凋亡诱导剂：1）诱导单个DNA断裂阿霉素类似物a（nnamaycin; ANA、WP 744、WP 796和WP 853）;和2）选择性Janus酪氨酸激酶（Jak）3抑制剂（NC 1153、WP 938、WP 988和WP 979）。我们已经表明，WP 744仅诱导活化的T细胞凋亡，而不诱导非活化的T细胞凋亡; WP 744与CD 40配体（L）单克隆抗体（mAb）的组合诱导对CD 40配体（L）的耐受。 心脏移植类似地，单独的NC 1153诱导T细胞凋亡，导致对肾同种异体移植物的耐受，并且与CTL 4-IG组合诱导对心脏同种异体移植物的耐受。我们将使用独特的模型：来自转录刺激因子和激活因子（Stat）5a/b缺陷小鼠的T细胞（但不是B细胞）在激活后进入细胞凋亡;而来自Stat 4和Stat 6缺陷小鼠的T细胞分别发育为产生白细胞介素（IL）-2的辅助性T细胞（Th）1和产生IL-4的Th 2;来自抗凋亡Bcl-2基因过表达转基因（Tg）小鼠的T细胞表现出抗凋亡性 凋亡使用这些小鼠，我们将检查的Bcl-2依赖性的敏感性或抗凋亡的非活化，活化和记忆T细胞与凋亡诱导剂治疗后的Stats的作用。将通过[a]可见溶核性核变性，[B]酶促检测TdT阳性DNA降解，[c]自动化细胞计数检测膜联蛋白-V易位，[d]基因微阵列/实时PCR中促凋亡mRNA与抗凋亡mRNA的表达，以及通过Western blot检测Bcl-2和Bcl-xL蛋白的再表达来评估细胞凋亡。 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、IL 10和IFN-γ）和RNA酶保护测定的细胞因子产生来定量。由于调节性T细胞（Treg）的特征是“记忆性”辅助性T细胞2型CD 4 +/CD 25+（Th 2 reg）细胞，因此我们计划探索IL-4/Stat 6调节的抗凋亡机制。这些新的药物可能为消除供者特异性淋巴细胞的耐受性提供有效和安全的方法。