In Vivo Prevention of Murine GVHD

摘要 我们的总体目标是通过先天性淋巴系统预防和治疗移植物抗宿主病(GVHD)。 2型细胞(ILC2)具有抗炎和组织修复功能。我们发现中的宿主ILC2被消除了 通过全身照射或化疗，并在90天内保持耗尽。这一发现是非常重要的 相关，因为外周血激活的ILC2与移植物抗宿主病之间存在负相关。就其本身而言。我们 试图确定补充输注成熟供体ILC2是否可以用来预防小鼠 GVHD。我们首次证明供体ILC2可以通过双调节蛋白预防或部分治疗GVHD (AREG)依赖过程。无论AREG或ILC2与肠道干细胞直接接触(ISC)是否支持小鼠 TBI治疗的小鼠或有机化合物对肠上皮细胞的修复尚不清楚。此外，第三方ILC2输液 也显著降低了小鼠移植物抗宿主病的致死率。出于翻译目的，我们发现ILC2是 相对类固醇耐受。骨髓移植周IL-33在骨髓移植日增加ST2/IL33R ILC2 0，减少GVHD。KO小鼠GVHD加速；骨髓移植前给予IL-33可阻止LLC_2的完全丢失。IL-33 KO受体有低增殖的上皮细胞，ISCs和Paneth细胞减少，隐窝高度变小。 和数字。体外肠道器官培养模型显示IL-33通过 诱导表皮生长因子(EGF)的产生，明显降低脑损伤后的EGF水平。EGF修复了ISC缺陷，发现 ISCs和Paneth细胞之间的肠道修复IL-33/EGF环。供体IL-13 Ko ILC2或宿主IL 13ra Ko小鼠 减少GVHD。ILC2 IL 13支持ST2簇状细胞和杯状细胞。簇状细胞产生IL-25驱动ILC2 生产和生存。脑挫伤后38天内簇状细胞显著减少，而KO受者显著增加 在GVHD中。当给予野生型小鼠时，外源性IL-25显着降低GVHD。Ko Of的后果 簇状细胞(和ILC2s)对供者T细胞的扩增、转运和功能尚不清楚。白介素25和白介素2的作用 尚未对IL 17RB进行检查。我们将讨论ILC2、IL-33和ILC2之间的动态和相互作用 TBI后和GVHD期间宿主肠道细胞(绒毛细胞、ISCs、Paneth细胞)。 目标1将检验这样的假设：由于ILC2 BM生态位的破坏，供体ILC2重新繁殖失败 它支持ILC2。体外Pre-lLC2分化、成熟和扩张±促炎 将研究细胞因子和支持细胞因子的ILC2。GATA3-GFPhi Pre-lLC2/成熟ILC2转移 到致死照射的同种骨髓移植接受者将提供关于不同能力的数据 BM。如果骨髓不能支持Pre-lLC2/lLC2，我们将研究干细胞缺陷小鼠。目标2将测试 假设：Pre-lLC_2/lLC_2及其分泌产物对肠道细胞亚群有直接影响。 来自野生型和带有同基因Tregs和ILC2的IL-33 Ko小鼠的肠道器官培养将测量 类器官的大小、数量和基因表达与增殖、细胞周期调节和特异性 动态平衡或脑外伤后上皮谱系标记物。抗AREG单抗或与AREG Ko共培养 淋巴细胞将以促进上皮再生为特征。目标3将检验这样的假设：簇状 细胞对ILC2的发育和生存至关重要，ILC2通过释放IL-13刺激簇状细胞 释放导致ILC2增殖的IL-25(目标3)。TUFT细胞和ILC2 Ko宿主加速了GVHD。我们 假设IL-25的作用是由于ILC2的直接刺激，或者供体IL-17RB T的作用 细胞。 意义重大。R37扩展阶段的研究将提供有关机制的基本信息 IL-33通过对ST2宿主ILC2和调节性T细胞的作用来降低GVHD的致死率， 两者都会产生AREG，以及发生在ISCs和Paneth细胞之间的EGF/IL-33环，导致 小肠修复术。此外，宿主ILC2在抑制GVHD中的关键作用，BMT后ILC2的性质 在骨髓移植前调理方案后出现的缺陷，并使患者易患移植物抗宿主病， 对绒毛细胞或其产物IL-25的基本要求将被阐明。最后，关键的见解将是 获得了前lLC2生成、肠道迁移或分化的能力。 翻译影响：供者和第三方ILC2输注预防和治疗GVHD的效果 支持我们计划的人类ILC2临床试验，由其他赞助提供资金，这将注入“现成的”第三 派对ILC2治疗类固醇难治性肠道移植物抗宿主病，这预示着预后特别差，在2- 明尼苏达大学和北卡罗来纳大学的机构研究。

Abstract Our overall goals are focus on the prevention and treatment of graft-vs-host disease (GVHD) via innate lymphoid type 2 cell (ILC2) anti-inflammatory and tissue reparative properties. We found that host ILC2s in are eliminated by total body irradiation {TBI} or chemotherapy and remain depleted for at >/=90 days. This finding is highly relevant as there is an inverse correlation between peripheral blood activated ILC2s and GVHD. As such. we sought to determine whether supplemental infusion of mature donor ILC2s could be used to prevent murine GVHD. We showed for the first time that donor ILC2s could prevent or partially treat GVHD in an amphiregulin (AREG) dependent process. Whether AREG or ILC2 direct contact with intestinal stem cells {ISC) supports small intestine epithelial cell repair in TBI treated mice or organoids is unknown. Additionally, third-party ILC2 infusion also significanUy reduced murine GVHD lethality. lmportanUy for translational purposes, we found ILC2s to be relatively steroid resistant. Peri-BMT {bone marrow transplant) IL-33 increased ST2/IL33R+ ILC2s at BMT day 0 and reduced GVHD. Ko mice had accelerated GVHD; IL-33 given pre-BMT prevented the full lLC2 loss. IL-33 ko recipients have hypo-proliferative epithelial cells, reduced ISCs and Paneth cells, and smaller crypt height and numbers. Ex vivo intestine organoid culture modeling revealed that IL-33 coordinated regeneration by inducing epidermal growth factor (EGF), significantly reduced by TBI. EGF restored ISC deficiency, uncovering a gut repair IL-33/EGF loop between ISCs and Paneth cells. Donor IL-13 ko ILC2s or host IL 13Ra ko mice had reduced GVHD. ILC2 IL 13 supports both ST2+ tuft cells and goblet cells. Tuft cells produce IL-25 driving ILC2 production and survival. TBI markedly reduced tuft cells for :!:38 days and ko recipients had a striking increase in GVHD. When given to wildtype mice exogenous IL-25 significantly reduced GVHD. Consequences of ko of tuft cells (and ILC2s) on donor T cell expansion, trafficking and function are unknown. The role of IL-25 and IL 17RB has not been examined. We will address the dynamics and interplay between ILC2, IL-33 and host intestinal cells (tuft cells, ISCs, Paneth cells) after TBI and during GVHD. Aim 1 will test the hypothesis that: Donor ILC2 repopulation fails due to destruction of ILC2 BM niche that supports ILC2s. In vitro pre-lLC2 differentiation, maturation and expansion ± proinflammatory cytokines and ILC2 supporting cytokines will be studied. GATA3-GFPhi pre-lLC2s/mature ILC2s transfer into lethally irradiated congenic BMT recipients will provide data on the differential ability to repopulate the BM. If the BM cannot support pre-lLC2s/lLC2s, we will study stem cell deficient mice. Aim 2 will test the hypothesis that: Pre-lLC2s/lLC2s and their secreted products have direct effects on intestinal cell subsets. Intestinal organoid cultures from wild-type and IL-33 ko mice with syngeneic Tregs and ILC2s will measure organoid size, number, and gene expression related to proliferation, cell cycle regulation, and specific epithelial lineage markers under homeostasis or after TBI. Anti-AREG mAbs or co-cultures with AREG ko lymphocytes will be characterized for promoting epithelial regeneration. Aim 3 will test the hypothesis that: Tuft cells are essential for ILC2 development and survival via an ILC2 release of IL-13 that stimulates tuft cells to release IL-25 that causes ILC2 proliferation (aim 3). Tuft cell and ILC2 ko hosts have accelerated GVHD. We hypothesize that IL-25 effects are due to direct stimulation of ILC2s or alternatively, with donor IL-17RB+ T cells. Significance. Studies in the R37 extension phase will provide fundamental information as to the mechanisms by which peri-BMT IL-33 diminish GVHD lethality via effects on ST2+ host ILC2s and regulatory T cells, both of which produce AREG, and the EGF/IL-33 loop that occurs between ISCs and Paneth cells resulting in small intestine repair. Further, the key role of host ILC2s in subduing GVHD, the nature of post-BMT ILC2 deficiency that occurs after pre-BMT conditioning regimens and predisposes patients to GVHD, and the essential requirement for tuft cells or their product, IL-25 will be elucidate. LasUy, critical insights will be gained as to the inability of pre-lLC2s generation, gut migration or differentiation. Translational Impact: The efficacy of donor and third-party ILC2 infusion in preventing and treating GVHD support our planned human ILC2 clinical trial, funded via other auspices, that will infuse "off-the-shelf' third party ILC2s to treat steroid refractory gut GVHD, that portends a particularly poor prognosis, in a 2- institutional study at the University of Minnesota and UNC-CH.