The role of regulatory T cells in biliary atresia

描述（由申请方提供）：胆道闭锁（BA）是一种肝外胆道系统的进行性纤维炎性阻塞，是全球儿科肝移植的最常见适应症。它仅限于新生儿早期，轮状病毒（RRV）感染仅在生命的前3天注射时引起小鼠BA。我们已经报道，在这段时间内，调节性T细胞（T细胞）在肝脏中不存在，但在RRV激发后，在免受RRV诱导的BA的老年小鼠中迅速出现。在初步研究中，我们已经表明，过继转移含Treg的CD 4+细胞RRV接种前限制NK和CD 8淋巴细胞的肝扩增和衰减的BA表型，证明了较低的血清胆红素水平和减少炎性胆管梗阻。从机制上讲，这些变化与肝髓样树突状细胞上B7共刺激分子的下调有关。用缺乏趋化因子受体CXCR 3的CD 4细胞进行的连续转移实验表明，TcR在控制肝脏免疫应答中的局部定位具有关键作用。在患有BA的婴儿的样品中，我们发现与没有肝损伤的年龄匹配对照相比，循环T淋巴细胞中促炎细胞因子的产生增加，肝髓样树突状细胞上B7分子的上调，以及缺乏CXCR 3表达的循环T淋巴细胞的频率增加的趋势。基于这些数据，我们提出了总体假设，即THBE通过局部控制效应淋巴细胞活化来保护新生儿胆管损伤。该假设将在三个重叠的特定目的中进行测试：1）确定TdR抑制胆管损伤的机制，2）鉴定BA中TdR的免疫调节途径，和3）阐明Treg运输至BA中炎症部位的分子决定因素。对于目的1，将在鼠BA中检查Treg/CD 4+细胞的过继转移对NK细胞的细胞毒性活性和T淋巴细胞的分化的影响，并将在患有BA的婴儿和健康对照中研究Treg对循环T淋巴细胞中细胞因子产生的抑制。目的2，树突状细胞，TGF？和IL 10作为Treg抑制的细胞和细胞因子介导物将使用敲除和报告基因转基因小鼠、细胞移植实验、细胞因子中和抗体和测量树突状细胞的刺激能力的共培养测定来研究。BA婴儿肝脏树突状细胞上B7分子上调的初步结果将在更大的队列中得到验证。对于目标3，将在过继转移实验中并通过确定BA受试者和对照中循环和肝Treg的功能概况来阐明CXCR 3对Treg向肝脏迁移的影响。总的来说，在小鼠和BA婴儿组织中进行的补充研究将确定关键的调控途径，TdR通过这些途径控制疾病的发病机制，指导未来的治疗以阻断胆道损伤的进展，并改善儿童的长期结局。

DESCRIPTION (provided by applicant): Biliary atresia (BA) is a progressive fibroinflammatory obstruction of the extrahepatic biliary tree and the most common indication for pediatric liver transplantation worldwide. It is uniquely restricted to the early neonatal period, and rotavirus (RRV) infection only causes BA in mice when injected during the first 3 days of life. We have reported that regulatory T cells (Tregs) are absent in the liver during this time period, but emerge rapidly following RRV challenge in older mice protected from RRV induced BA. In preliminary studies, we have shown that adoptive transfer of Treg-containing CD4+ cells prior to RRV inoculation constrained hepatic expansion of NK and CD8 lymphocytes and attenuated the BA phenotype, as evidenced by lower serum bilirubin levels and reduced inflammatory bile duct obstruction. Mechanistically, these changes were linked to down-regulation of B7 costimulatory molecules on hepatic myeloid dendritic cells. Adoptive transfer experiments with CD4 cells lacking the chemokine receptor CXCR3 indicated a critical role for local positioning of Tregs in control of hepatic immune responses. In samples of infants with BA, we found trends towards increased production of pro-inflammatory cytokines in circulating T-lymphocytes, up-regulation of B7 molecules on hepatic myeloid dendritic cells, and increased frequency of circulating Tregs lacking expression of CXCR3 compared with age- matched controls without liver injury. Based on these data we propose the overarching hypothesis that Tregs protect from neonatal bile duct injury through local control of effector lymphocyte activation. This hypothesis will be tested in three overlapping specific aims: 1) to determine the mechanisms by which Tregs constrain bile duct injury, 2) to identify pathways of immune regulation by Tregs in BA, and 3) to elucidate molecular determinants for Treg trafficking to the site of inflammation in BA. For aim 1, the effects of adoptive transfer of Treg/CD4+ cells on cytotoxic activity of NK cells and differentiation of T-lymphocytes will be examined in murine BA, and Treg suppression of cytokine production in circulating T-lymphocytes will be studied in infants with BA and in healthy controls. For aim 2, the roles of dendritic cells, TGF? and IL10 as cellular and cytokine mediators of Treg suppression will be investigated using knockout and reporter transgenic mice, cell transplantation experiments, cytokine neutralizing antibodies, and co-culture assays measuring the stimulatory capacity of dendritic cells. Preliminary results of up-regulation of B7-molecules on hepatic dendritic cells in infants with BA will be validated in a larger cohort. For aim 3, the influence of CXCR3 on Treg-migration to the liver will be elucidated in adoptive transfer experiments and by determining the functional profile of circulating and hepatic Tregs in BA subjects and controls. Collectively, the complementary studies in mice and tissue of infants with BA will identify key regulatory pathways by which Tregs control pathogenesis of disease, guide future therapies to block the progression of biliary injury, and improve long term outcomes in children.