Immune Mechanisms in Ocular Graft versus Host Disease

摘要 异基因造血干细胞移植(AHSCT)已成为治疗急性髓细胞白血病的标准治疗方法。 几种危及生命的血液系统恶性肿瘤、免疫缺陷和遗传病。不幸的是，由于 移植物抗宿主病提高了患者的存活率，影响了患者的生活质量 (GVHD)。GVHD是一种复杂的多器官疾病，由供者同种异体反应性T细胞免疫攻击引起 对胃肠道、肝脏、皮肤和眼睛造成损害的细胞。眼部移植物抗宿主病(OGVHD)发生在 患有移植物抗宿主病的患者会因泪腺和结膜损伤导致干眼而威胁视力， 角膜病变和角膜穿孔。尽管GVHD患者眼睛受累的频率很高，但很少有 已知导致角结膜炎的oGVHD的潜在免疫机制 干酪。不幸的是，这些患者的眼科护理仅限于姑息治疗和抗肿瘤治疗。 作用机制和疗效有限的炎症性药物。我们使用纳米线的最新令人兴奋的发现 分析，支持选择的基因包括细胞因子、抗原提呈/MHC、T细胞和肿瘤坏死因子 超家族(TNFRSF)通路在影响结膜的oGVHD中有不同的调节和参与 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 眼睛是GVHD的靶组织，并建议开发新的翻译靶向免疫疗法来局部 预防和治疗oGVHD。这里的实验将研究CD4和CD8 Tef介导的Conj+LaGL的损伤 (目标1)。我们将利用我们的TCR转基因(TG)和新开发的双报告(B6-Nur77GFPFoxP3RFP) 使小鼠了解局部激活的CD4和CD8 T细胞和T细胞。使用scRNAseq和质量细胞仪成像， 研究将为局部T细胞接受同种异体抗原刺激提供洞察力。接下来，我们将审问 首次在oGVHD中发现了造血系和非造血系抗原提呈细胞(APC)通路(目标2)。我们 将应用体内模型，结合TCR TG捐赠者和造血嵌合体，以实现精确的讯问 Conj+LaGL损伤中直接和间接抗原提呈途径的研究。这些研究将推动 利用生物试剂和表观遗传调控进行防治的新策略的开发 可转译给患者的aHSCT(目标3)。值得注意的是，我们的2受体途径策略使用了一种新的 靶向TNFRSF25和IL-2的融合蛋白(TL1A-Ig)局部靶向CD25扩大眼球。 由于溴结构域蛋白在调控炎症基因转录中起着核心作用，我们建议 使用这些蛋白的抑制物(Beti)，作为局部眼部制剂，通过渗透抑制细胞因子 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扩展策略与这种表观遗传调控相结合。我们的总体目标 是为了检验一种假设，即免疫介导的Conj+LaGL损伤是SCCA发生的基础 可以通过靶向免疫疗法来预防。

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.