Enhancing Treg Therapeutic Efficacy in GVHD

翻译后摘要：这种更新继续我们的优化调节性T细胞（Treg）治疗，创造移植 耐受性，以防止移植物抗宿主病（GVHD）。我们的第一个人体I期CD 4胸腺Treg（tTreg）和CD 4 诱导性Treg（iTreg）试验显示GVHD减少但不消除。重要的是，复发率是 没有改善和减少GVHD但没有复发是不充分的结果。在这次更新中，我们将建立 基于我们激动人心的发现，TcB可以获得超级抑制功能，并通过整合抗CD 19 scFv， CAR（CAR 19）增加其杀伤效力。在其他研究中，我们开发了高度抑制性CAR 19 CD 8 具有内在杀伤机制的疫苗。比较这些细胞类型单独或联合应用的相对功效， 联合使用，将使我们能够启动临床试验，使用一种产品同时抑制GVHD， 矛盾地杀死淋巴瘤，解决了2个主要的alloHCT局限性。多样性中的共性 方法和Treg亚群获得超级抑制功能的原因是存在显著的代谢异常。 方面的影响.我们发现，靶向选定的细胞表面或细胞内蛋白质导致线粒体融合， 高氧化磷酸化（OXPHOS）和超抑制因子TdR。极性代谢物分析指出， 关键KEGG途径[丝氨酸-甘氨酸-一碳网络]，由甲硫氨酸和叶酸循环连接，反式- 硫化谷胱甘肽和丙氨酸-天冬氨酸-L-谷氨酸。最终结果是一碳（1C）甲基、甲酰基或 甲基转移用于生物合成、控制基因表达和破坏性氧化还原反应。CAR19 表达对hCD 19+靶具有高度抑制性和细胞溶解性的CD 4 tT细胞和人CD 8 tT细胞将 在GVHD/GVL模型中进行研究。值得注意的是，尽管活化的CAR 19 CTL可引起严重的细胞因子释放， 对于CRS综合征，我们的初步数据表明CAR 19 CD 4 tT细胞产生低炎性细胞因子， 不会导致CRS。目的1A验证TdR的代谢重编程依赖于嘌呤的假设 合成、线粒体融合和OXPHOS以及用于氧化还原平衡的谷胱甘肽。Aim 1B利用Tibet 4-1BB vs CD 28和CD 2）信号传导结构域，影响代谢重编程和Treg 最佳GVHD/GVL的持久性。目的2关注这些Treg亚群使用的杀伤机制 来溶解目标为此，我们最近发现了CTL超分子攻击颗粒（SMAPs）作为一种新的 更持久的杀戮机制。SMAPs是含有颗粒酶B的细胞毒性核-壳颗粒， 穿孔素，并在触发免疫突触（IS）后分泌，IS是T细胞的接触信号点 APC的。识别用于SMAP释放的整合IS信号将提供对Treg杀伤机制的见解， 援助目标1.模拟IS的支持脂质双层将评估CD 2作为IS信号积分器/放大器， 通过分析双CAR T细胞来杀死。我们的中心假设是，在aim 1中，T细胞被重新编程为1C-， 嘌呤和谷胱甘肽代谢增强了抑制和SMAP释放，由CD 2 整合和放大的IS信号和最佳的GVHD/GVL。

Abstract: This renewal continues our optimization of regulatory T cell (Treg) therapy for creating transplant tolerance to prevent graft-vs-host disease (GVHD). Our first-in-human phase I CD4 thymic Treg (tTreg) and CD4 inducible Treg (iTreg) trials that showed reduction but not elimination of GVHD. Importantly, relapse rates were not improved and decreasing GVHD but not relapse is an insufficient outcome. In this renewal, we will build on our exciting finding that Tregs can acquire super-suppressor function and by incorporating an anti-CD19 scFv CAR (CAR19) increase their potency of killing. In other studies, we developed highly suppressive CAR19 CD8 iTregs that have an intrinsic killing mechanism. Comparing the relative efficacy of these cell types, alone or in combination, will allow us to initiate clinical trials using one product simultaneously suppress GVHD and paradoxically kill lymphoma, solving the 2 major alloHCT limitations. A commonality in the diverse approaches and Treg subsets that acquire super-suppressor function is the presence of a striking metabolic effects. We discovered that targeting selected cell surface or intracellular proteins led to mitochondria fusion, high oxidative phosphorylation (OXPHOS) and super-suppressor Tregs. Polar metabolite analytics pointed to key KEGG pathways [serine-glycine-one carbon network], linked by methionine and folate cycles, trans- sulfuration for glutathione, and alanine-aspartate-L-glutamate. A net result is one-carbon (1C) methyl, formyl, or methyl group transfer for biosynthesis, control of gene expression and damaging redox reactions. CAR19 expressing CD4 tTregs and human CD8 iTregs that are highly suppressive and cytolytic to hCD19+ targets will be studied in GVHD/GVL models. Notably, whereas activated CAR19 CTLs can cause severe cytokine release syndrome (CRS), our preliminary data indicate that CAR19 CD4 tTregs produce low inflammatory cytokines and do not cause CRS. Aim 1A tests the hypothesis that metabolic reprogramming of Tregs depends on purine synthesis, mitochondria fusion and OXPHOS, and glutathione for redox balance. Aim 1B exploits Tregs with 4-1BB vs CD28 and CD2) signaling domains that affect metabolic reprogramming and Treg persistence for optimal GVHD/GVL. Aim 2 is focused on the killing mechanisms that these Treg subsets use to lyse targets. Toward that end, we recently discovered CTL supramolecular attack particles (SMAPs) as a new mechanism for more sustained killing. SMAPs are cytotoxic core-shell particles that contain granzyme B and perforin and are secreted after triggering the immunological synapse (IS), the contact signaling point for T cells and APC. Identifying integrated IS signals for SMAP release will provide insights into Treg killing mechanisms, aiding Aim 1. Supported lipid bilayers simulating the IS will assess CD2 as an IS signal integrator/amplifier of killing by analyzing dual CAR Tregs. Our central hypotheses are that Tregs reprogrammed in aim 1 for 1C-, purine- and glutathione- metabolism have enhanced suppression and SMAP release, triggered by CD2 integrated and amplified IS signals and optimal GVHD/GVL.