T Cell Intrinsic Responses to TGFβ Regulates Allergen Sensitization By Modulating Type 2 T Follicular Helper Cell Development. T Cell Intrinsic Responses to TGFβ Regulates Allergen Sensitization By Modulating Type 2 T Follicular Helper Cell Development

Immunoglobulin E (IgE) is a potent mediator of allergic diseases, but the mechanisms that regulate IgE responses to innocuous environmental and food antigens remain unclear. Patients with Loeys-Dietz syndrome (LDS) who have mutations in genes encoding the TGFβ receptor are predisposed to IgE-mediated disorders. Here, using patient samples and a mouse model, we demonstrate that LDS mutations lead to reduced canonical TGFβ signaling, elevated total and allergen-specific IgE, increased type 2 follicular helper T cells (Tfh2), and exaggerated germinal center activity that was not prevented by the presence of wild type T regulatory cells. T cell intrinsic defects in LDS mice resulted in spontaneous sensitization to orally administered OVA, and inhibition of mammalian target of rapamycin (mTOR) prevented the exaggerated Tfh and IgE responses to OVA. Thus, TGFβ limits human and mouse Tfh2 cell development via the phosphatidylinositol-3-OH kinase gamma (PI3Kg)/AKT/mTOR pathway, and disruption of this pathway promotes allergic inflammation. Overall design: The M318R allele previously identified in LDS patients was knocked into the mouse Tgfbr1 locus resulting in heterozygous TgfbrIWT/mut mice. Eight week old mice were orally gavaged once a week for 4 weeks with 5mg crude peanut extract and 10g Cholera Toxin from Vibrio cholera (Sigma) in 200uL water. One week after the last gavage, mice were bled from the submandibular cheek vein. OTII cells were purified from lymph nodes and spleen from OTII+/+RAG-/- mice that were WT or TgfbrIWT/mut, CD4+ bead purified (Miltenyi Biotec), and injected i.v. into CD45.1 WT mice. Hosts were fed water with 1.5% endotoxin-free OVA (Sigma Aldrich) for 7 days post-injection with normal mouse chow. In the rapamycin treatment experiments, mice were orally gavaged daily with vehicle or 125g rapamycin dissolved in DMSO and Kolliphor (Sigma) in a 3:1 ratio and then dissolved in water in a 1:25 ratio, on days 3-7. On day 8, the mesenteric lymph node and Peyer’s patches were collected, and smashed through 70M filters to make single-cell suspensions.

免疫球蛋白E（Immunoglobulin E, IgE）是过敏性疾病的强效介质，但调控IgE针对无害环境与食物抗原应答的分子机制至今仍未明确。携带转化生长因子β（transforming growth factor β, TGFβ）受体编码基因突变的洛伊茨-迪茨综合征（Loeys-Dietz Syndrome, LDS）患者，易罹患IgE介导的疾病。本研究借助患者样本与小鼠模型，证实LDS突变可导致经典TGFβ信号通路活性降低、总IgE与过敏原特异性IgE水平升高、2型滤泡辅助性T细胞（type 2 follicular helper T cells, Tfh2）数量增多，以及生发中心反应过度活化——且该效应无法被野生型调节性T细胞（T regulatory cells, Tregs）所抑制。LDS小鼠的T细胞固有缺陷可诱发其对经口给予的卵清蛋白（ovalbumin, OVA）发生自发致敏，而哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin, mTOR）抑制剂可阻断OVA诱导的过度Tfh与IgE应答。综上，TGFβ可通过磷脂酰肌醇-3-羟基激酶γ（phosphatidylinositol-3-OH kinase gamma, PI3Kγ）/AKT/mTOR通路限制人与小鼠的Tfh2细胞发育，该通路的功能失调会促进过敏性炎症。总体实验设计：将此前在LDS患者中鉴定到的M318R等位基因敲入小鼠Tgfbr1基因座，构建得到杂合子Tgfbr1^WT/mut小鼠。8周龄小鼠每周经口灌胃一次，连续4周，每次给予5mg粗制花生提取物与10μg霍乱弧菌来源的霍乱毒素（Sigma），溶于200μL去离子水中。末次灌胃后1周，通过颌下颊静脉采集小鼠外周血。从基因型为野生型或Tgfbr1^WT/mut的OTII+/+RAG-/-小鼠的淋巴结与脾脏中纯化OTII细胞，采用磁珠分选（Miltenyi Biotec）富集CD4+ T细胞后，经尾静脉注射至CD45.1野生型小鼠体内。受体小鼠在细胞注射后，喂食添加1.5%无内毒素卵清蛋白（Sigma Aldrich）的饮用水，并配合正常小鼠饲料，持续7天。雷帕霉素处理实验中，小鼠于第3至7天每日经口灌胃溶剂对照，或125μg雷帕霉素（以3:1比例溶于二甲基亚砜与Kolliphor，再以1:25比例溶于去离子水中）。第8天采集肠系膜淋巴结与派尔集合淋巴结，通过70μm滤膜研磨制备单细胞悬液。