Enhancing Treg Therapeutic Efficacy in GVHD

描述(申请人提供)：胸腺来源的CD4+25+FoxP3+调节性T细胞(Treg)需要抑制自身免疫和限制外来抗原反应。我们的体外扩增人Tregs的第一阶段剂量递增试验证明在显著降低急性移植物抗宿主病(GVHD)的发生率方面是有效的。统一疗效的两个主要限制被确定：问题1(实现最佳抑制)：抑制百分比是可变的。问题2(保持适当的Treg：T效应比)：1：5.5的比例远低于预防小鼠GVHD所需的E1：1的理想比例；到移植后2周，没有检测到Treg。目的1将重点放在问题1上。PKC-Q协调免疫功能，导致其移动到T效应器而不是Tregs的免疫突触(T细胞和抗原提呈细胞的直接接触点)。PKC-Q基因缺陷的小鼠效应物不能引起移植物抗宿主病，但能保留抗肿瘤和抗病原体的反应。经小分子PKC-Q抑制剂处理的Tregs抑制功能增强，对小鼠结肠炎有更好的抑制作用。在目标1中，针对问题1，我们将在体外或体内使用小分子PKC-Q抑制剂来验证这样的假设，即鼠和人的Treg抑制GVHD的能力受到PKC-Q进入免疫突触的限制。在目标2中，聚焦于问题2，我们将检验一个假设，即持续的IL-2R信号通路将提供一条PKC-Q独立的途径来扩大Treg和抑制GVHD。我们将使用3种策略来改善Treg介导的GVHD抑制：(A)给予IL-2/抗IL2抗体复合体或低剂量IL-2以优先扩大小鼠和人类Treg与T细胞效应器；(B)在小鼠和人Treg中结构性表达STAT5b，基于我们的发现，结构性STAT5b促进小鼠Treg的生成、增殖和功能，包括GVHD抑制，绕过对IL-2的需要，体内存在的限量；以及(C)使用GSK-3b抑制剂在体外或 在人类Treg中表达稳定的b-catenin，因为稳定的b-catenin减少了Treg对IL-2的依赖，并显著提高了小鼠Treg的存活率和预防结肠炎的有效性。在目标3中，我们将从目标1和目标2中的研究中选择最好的1-2方法，用于在临床前GVHD模型中测试移植后保存有益的免疫反应。由于开发了目标1和2的首选方法，不屈服于GVHD致死性的长期嵌合体将受到白血病细胞或细菌或病毒病原体的挑战，以量化免疫系统对移植后危及生命的并发症的反应程度。

DESCRIPTION (provided by applicant): Thymic-derived CD4+25+FoxP3+ regulatory T cells (Tregs) are required to suppress autoimmunity and limit foreign antigen responses. Our first-in-human phase I dose escalation trial of ex vivo expanded human Tregs proved efficacious in significantly reducing the incidence of acute graft-vs-host disease (GVHD). Two major limitations to uniform efficacy were identified: Problem 1 (achieving optimal suppression): The %suppression was variable. Problem 2 (maintaining adequate Treg:Teffector ratios): Ratios of 1:5.5 achieved were well below the desired ratios of e1:1 need for GVHD prevention in mice; no Tregs were detected by 2 weeks post-transfer. Aim 1 will focus on Problem 1. PKC-q coordinates immune function, which results in its movement to the immunological synapse (direct contact point between T cell and antigen-presenting cell) in Teffectors but not Tregs. PKC-q deficient murine Teffectors are unable to cause GVHD but can retain anti-tumor and anti- pathogen responses. Tregs treated with a small molecule PKC-q inhibitor had augmented suppressor function and were more effective in suppressing murine colitis. In aim 1, focused on Problem 1, we will use small molecule PKC-q inhibitors in vitro or in vivo to test the hypotheses that murine and human Treg potency for GVHD suppression is limited by movement of PKC-q into the immunological synapse. In aim 2, focused on Problem 2, we will test the hypothesis that sustained IL-2R signaling pathways will provide a PKC- q independent route to Treg expansion and GVHD suppression. We will use 3 strategies to improve Treg mediated GVHD suppression: (a) administer IL-2/anti-IL2 antibody complexes or low dose IL-2 to preferentially expand murine and human Tregs vs Teffectors; (b) Constitutively express STAT5b in murine and human Tregs based upon our findings that constitutive STAT5b promotes murine Treg generation, proliferation and function including GVHD suppression, bypassing the need for IL-2, present in limiting amounts in vivo; and (c) increase b-catenin signaling using a GSK-3b inhibitor in vitro or expressing a stabilized b-catenin in human Tregs since stabilized b-catenin reduces Treg dependency upon IL-2 and provides a marked enhancement of murine Treg survival and efficacy in preventing colitis. In aim 3, we will select the best 1-2 approaches, derived from studies in aims 1 and 2, for testing in preclinical GVHD models for the preservation of beneficial immune responses later post-transplant. Long-term chimeras that do not succumb to GVHD lethality, as a result of preferred approaches developed aims 1 and 2, will be challenged with leukemia cells or bacterial or viral pathogens to quantify the extent to which the immune system can respond to life-threatening complications post-transplant.