Immune Mechanisms in Ocular Graft versus Host Disease

摘要 异基因造血干细胞移植（aHSCT）已成为治疗造血干细胞移植的标准护理。 几种危及生命的恶性血液病、免疫缺陷和遗传病。可惜作为 这些患者的生存率提高，生活质量受到移植物抗宿主病的负面影响 （GVHD）。GVHD是一种复杂的多器官疾病，由供体同种异体反应性T细胞的免疫攻击引起。 细胞，导致损害胃肠道，肝脏，皮肤和眼睛。眼部GVHD（oGVHD）发生在>60%的 患有GVHD的患者，由于泪腺和结膜损伤导致干眼症， 角膜病变和角膜穿孔。尽管在GVHD患者中眼睛受累的频率很高， 关于导致角结膜炎的oGVHD的潜在免疫机制已知 干燥不幸的是，这些患者的眼科护理仅限于姑息治疗和抗- 炎症药物具有有限的作用机制和功效。我们最近的令人兴奋的发现， 分析，支持选择的基因，包括细胞因子，抗原呈递/ MHC，T细胞和TNF TNFRSF超家族（TNFRSF）途径被差异调节并参与影响结膜的oGVHD 和泪腺（Conj+LaGL）。在这个应用中，我们继续使用临床前模型来理解为什么 眼是GVHD靶组织，并提出开发新的翻译靶向免疫疗法， 预防和治疗oGVHD。本文的实验将研究CD 4和CD 8 Teff介导的对Conj+LaGL的损伤 (Aim 1）。我们将利用我们的TCR转基因（Tg）和新开发的双报告基因（B6-nur 77 GFPFoxP 3RFP） 小鼠以了解CD 4和CD 8 Teff和Teff的局部活化。使用scRNAseq和质谱细胞术成像， 研究将提供对接受同种异体抗原刺激的局部T细胞的了解。接下来，我们将审问 第一次，造血和非造血抗原呈递细胞（APC）途径在oGVHD（目的2）。我们 将应用结合TCR Tg供体与造血嵌合体的体内模型， Conj+LaGL损伤中的直接和间接抗原呈递途径。这些研究将推动 利用生物试剂和表观遗传调控制定新的预防和治疗策略 aHSCT可以转化为患者（目标3）。值得注意的是，我们的2-受体通路策略使用一种新的 靶向TNFRSF 25和IL-2的融合蛋白（TL 1A-Ig），其靶向CD 25，局部扩增眼部TlR。 由于布罗莫结构域蛋白在调节炎症基因转录中具有核心作用，我们提出， 使用这些蛋白质的抑制剂（BETi），作为局部眼部制剂施用，通过浸润 和实质Conj+LaGL细胞群。此外，我们将产生一种新的oGVHD治疗方法， 通过将我们的局部2-通路Treg扩增策略与这种表观遗传调节相结合。我们的整体目标 目的是检验免疫介导的Conj+LaGL损伤是干燥症发生的基础这一假设， 可以通过靶向免疫治疗来预防。

Abstract Allogeneic hematopoietic stem cell transplantation (aHSCT) has become the standard of care for treatment of several life-threatening hematologic malignancies, immunodeficiency and genetic diseases. Unfortunately, as the survival rate of these patients is improved, the quality of life is negatively impacted by Graft vs Host Disease (GVHD). GVHD is a complex, multi-organ disorder arising from immunological attack by donor allo-reactive T cells that results in damage to the GI tract, liver, skin and the eye. Ocular GVHD (oGVHD) occurs in >60% of patients with GVHD and can threaten vision due to lacrimal gland and conjunctival damage leading to dry eye, keratopathy and corneal perforation. Despite the high frequency of eye involvement in GVHD patients, little is known regarding the underlying immune mechanisms responsible for oGVHD that lead to keratoconjunctivitis sicca. Unfortunately, the ophthalmic care of these patients is restricted to palliative therapies and anti- inflammatory drugs with limited mechanisms of action and efficacy. Our recent exciting findings using nanostring analysis, support the notion that selected genes including cytokines, antigen presenting / MHC, T cell, and TNF superfamily (TNFRSF) pathways are differentially regulated and involved in oGVHD affecting the conjunctiva and lacrimal gland (Conj+LaGL). In this application, we continue to use pre-clinical models to understand why the eye is a target tissue in GVHD and propose to develop new translational targeted immunotherapies to locally prevent and treat oGVHD. Experiments here will investigate CD4 and CD8 Teff mediated damage to Conj+LaGL (Aim 1). We will utilize our TCR transgenic (Tg) and newly developed double reporter (B6-nur77GFPFoxP3RFP) mice to understand local activation of CD4 and CD8 Teff & Tregs. Using scRNAseq and mass cytometry imaging, studies will provide insights into local T cells receiving alloantigen stimulation. Next, we will interrogate for the first time, hematopoietic and non-hematopoietic antigen presenting cell (APC) pathways in oGVHD (Aim 2). We will apply in vivo models combining TCR Tg donors with hematopoietic chimeras to enable precise interrogation of direct and indirect antigen presentation pathways in the Conj+LaGL damage. These studies will drive development of new strategies using biological reagents and epigenetic regulation for prevention and treatment of aHSCT that can be translated to patients (Aim 3). Notably, our 2-receptor pathway strategy using a novel fusion protein (TL1A-Ig) targeting TNFRSF25 and IL-2, which targets CD25 locally expands ocular Tregs. Because bromodomain proteins have a central role in regulating transcription of inflammatory genes, we propose to use inhibitors of these proteins (BETi), applied as a local ocular formulation to suppress cytokines by infiltrating and parenchymal Conj+LaGL cell populations. Furthermore, we will generate a new oGVHD treatment approach by combining our local 2-pathway Treg expansion strategy with this epigenetic regulation. Our overall objective is to test the hypothesis that immune mediated damage to the Conj+LaGL underlie development of sicca which can be prevented by targeted immune therapy.