Table_2_Activation of the DR3-TL1A Axis in Donor Mice Leads to Regulatory T Cell Expansion and Activation With Reduction in Graft-Versus-Host Disease.xlsx

Death receptor 3 (DR3) is a tumor necrosis factor receptor superfamily member (TNFRSF25), which is minimally expressed on resting conventional T cells (though readily inducible upon cell activation), yet highly expressed on resting FoxP3+ regulatory T cells (Treg). We recently demonstrated that activation of DR3 with an agonistic antibody (4C12) leads to selective expansion and activation of Treg in healthy mice and suppression of graft-versus-host disease (GVHD) in recipient mice when donor mice are treated. However, given the long antibody half-life and concomitant safety concerns, along with the lack of a humanized agonistic antibody to DR3, both human and murine fusion proteins incorporating the natural DR3 ligand TL1A (TL1A-Ig) have been developed. Herein, we show that DR3 activation with 4C12 or with TL1A-Ig, with or without the addition of low dose IL-2 to the treatment regimen, led to a significant expansion of murine Treg in spleen, lymph nodes, and peripheral blood. Bioluminescent imaging revealed peak Treg expansion around day 7–8, with return to near baseline after 2–3 weeks. In addition to expansion, all DR3 agonist treatment regimens led to increased activation of Tregs, with significant upregulation of the activation markers ICOS, KLRG-1, PD-1, and CD103, and the proliferation marker Ki-67. The near absence of activated Treg populations in control treated spleens was also detected on tSNE analysis of flow cytometry data. Subtly different patterns of splenic Treg activation by the different DR3 agonists were noted in both tSNE analysis of flow cytometry data and RNA-sequencing analysis. However, upregulation of gene transcripts which play important roles in cell proliferation, trafficking, activation, and effector function were observed regardless of the DR3 agonist treatment regimen used. In the major MHC-mismatch model of hematopoietic cell transplantation, DR3 agonist-mediated expansion and activation of Tregs in donor mice led to a significant improvement in GVHD in recipient mice. These data provide important preclinical information regarding the outcome of DR3 activation with an agonistic antibody or natural ligand and provide insight into the therapeutic use of this approach to reduce GVHD in recipients and improve outcomes of hematopoietic cell transplantation.

死亡受体3（Death receptor 3, DR3）属于肿瘤坏死因子受体超家族成员25（TNF receptor superfamily member 25, TNFRSF25），在静息常规T细胞中呈低表达（但其表达可在细胞活化后快速诱导），而在静息FoxP3阳性调节性T细胞（regulatory T cell, Treg）中则高表达。我们近期的研究证实，使用激动性抗体（agonistic antibody, 4C12）激活DR3，可在健康小鼠中实现Treg的选择性扩增与活化；若对供体小鼠实施该处理，还可减轻受体小鼠的移植物抗宿主病（graft-versus-host disease, GVHD）。然而，由于抗体半衰期较长且伴随安全隐患，同时缺乏针对DR3的人源化激动性抗体，目前已开发出融合天然DR3配体TL1A的人源及鼠源融合蛋白（TL1A-Ig）。本研究结果显示，无论是采用4C12还是TL1A-Ig激活DR3，无论治疗方案中是否添加低剂量IL-2，均可显著扩增小鼠脾脏、淋巴结及外周血中的Treg。生物发光成像结果表明，Treg扩增在第7~8天达到峰值，2~3周后恢复至接近基线水平。除扩增效应外，所有DR3激动剂治疗方案均可增强Treg的活化状态，显著上调活化标志物诱导性共刺激分子（Inducible T-cell costimulator, ICOS）、KLRG-1、程序性死亡受体1（Programmed cell death protein 1, PD-1）及CD103，以及增殖标志物Ki-67的表达。对流式细胞术数据进行t分布邻域嵌入（t-distributed Stochastic Neighbor Embedding, tSNE）分析后发现，对照组小鼠脾脏中几乎不存在活化的Treg群体。通过tSNE分析流式细胞术数据及RNA测序（RNA-sequencing）分析，均观察到不同DR3激动剂对脾脏Treg的活化模式存在细微差异。但无论采用何种DR3激动剂治疗方案，均可观测到参与细胞增殖、趋化、活化及效应功能的基因转录本显著上调。在主要组织相容性复合体（Major Histocompatibility Complex, MHC）不匹配的造血干细胞移植（hematopoietic cell transplantation）模型中，对供体小鼠进行DR3激动剂介导的Treg扩增与活化，可显著改善受体小鼠的GVHD病情。本研究数据为使用激动性抗体或天然配体激活DR3的临床前研究提供了重要参考，同时为该策略用于降低受体GVHD风险、改善造血干细胞移植预后提供了理论依据。