Data Sheet 1_Development of a humanized mouse model of graft-versus-host disease to assess human regulatory T cell function.docx

IntroductionRegulatory T cells (Treg)-based therapies are increasingly used for treating autoimmune or graft-versus-host (GVHD) disease. Given their low frequency, several approaches aimed at amplifying them or at increasing their immunosuppressive activities are currently investigated. Unfortunately, the impact of these strategies on human Treg function has remained difficult to assess in vivo. Here, we report the development of a novel humanized mouse model of allogeneic and xenogeneic GVHD intended to characterize the immunosuppressive activity of human Treg in vivo.
MethodsIn this model, GVHD is induced by injecting CD25-depleted HLA-A2− peripheral blood mononuclear cells (PBMC) into irradiated NSG-HLA-A2-HHD mice. The CD25+ (Treg) fraction is maintained in vitro for 48h during which Treg-promoting treatments can be tested before infusion into mice. We took advantage of this model to investigate whether tumor necrosis factor alpha (TNF-α) priming of Treg would increase their suppressive function and improve their ability at preventing xenogeneic GVHD, after assessing the effect of TNF-α on Treg in vitro.
ResultsIn vitro, single cell RNAseq analyses showed that eleven Hallmark pathways, including Interferon response, IL-6-JAK-STAT3 signaling, mTORC1 signaling, and TNF-α signaling via NF-κB, were significantly upregulated in Treg following TNF-α priming. In vivo, Treg infusion resulted in higher Treg levels, lower counts of human cells, lower conventional CD4+ (Tconv) and CD8+ T-cell counts, lower KI67 and HLA-DR expression by Tconv and lower Granzyme B expression by CD8+ T-cells in peripheral blood. No significant impact of TNF-α priming of Treg on survival was observed.
DiscussionThese results emphasize the importance of reliable techniques to assess Treg in vivo as efficient methods to activate them in vitro do not always result in an enhanced function in the in vivo setting. In summary, we present here the development of a novel humanized model of GVHD designed to evaluate the in vivo functionality of human Treg. Taking advantage of that model, we observed that TNF-α priming of human Treg did not increase their suppressive activity in vivo.

引言
基于调节性T细胞（Regulatory T cells, Treg）的疗法在自身免疫病或移植物抗宿主病（Graft-versus-Host Disease, GVHD）的治疗中应用日益广泛。鉴于Treg的体内天然丰度较低，当前已有多种旨在扩增Treg群体或增强其免疫抑制活性的策略被研究。遗憾的是，这类策略对人Treg功能的体内影响仍难以精准评估。本研究构建了一种新型同种异体及异种GVHD人源化小鼠模型，用于体内表征人Treg的免疫抑制活性。

方法
本模型通过向辐照后的NSG-HLA-A2-HHD小鼠注射CD25耗竭的HLA-A2阴性外周血单个核细胞（PBMC）诱导GVHD。将CD25阳性（Treg）组分在体外培养48小时，期间可先测试促Treg处理策略，随后再输注给小鼠。本研究先在体外评估了肿瘤坏死因子α（TNF-α）对Treg的作用，随后利用该模型探究Treg经TNF-α预激活后，是否可增强其免疫抑制功能，提升其预防异种GVHD的能力。

结果
体外实验中，单细胞RNA测序（single cell RNAseq）分析显示，经TNF-α预激活后的Treg中，共11条特征通路显著上调，包括干扰素应答、IL-6-JAK-STAT3信号通路、mTORC1信号通路以及经NF-κB介导的TNF-α信号通路。体内实验中，Treg输注可使小鼠外周血中Treg水平升高，人类细胞总数降低，常规CD4阳性（Tconv）及CD8阳性T细胞计数减少；同时常规CD4阳性T细胞的Ki67与HLA-DR表达水平降低，CD8阳性T细胞的颗粒酶B表达水平也有所下降。未观察到Treg经TNF-α预激活对小鼠存活率产生显著影响。

讨论
本研究结果凸显了可靠的体内Treg评估技术的重要性——因为体外激活Treg的高效方法，未必总能在体内环境中增强其功能。综上，本研究构建了一种新型GVHD人源化模型，用于评估人Treg的体内功能。利用该模型，我们发现人Treg经TNF-α预激活后，并未在体内增强其免疫抑制活性。