The role of regulatory T cells in biliary atresia

描述（申请人提供）：胆道闭锁（BA）是一种进行性肝外胆道纤维炎性梗阻，是全世界儿童肝移植最常见的适应症。它仅局限于新生儿早期，轮状病毒（RRV）感染仅在小鼠出生后3天内注射时引起BA。我们已经报道，在这段时间内，肝脏中没有调节性T细胞（Tregs），但在RRV攻击后，在RRV诱导的BA中保护的老年小鼠中，调节性T细胞（Tregs）迅速出现。在初步研究中，我们已经证明，在接种RRV之前过继转移含有treg的CD4+细胞限制了NK和CD8淋巴细胞的肝脏扩张，并减弱了BA表型，这可以通过降低血清胆红素水平和减轻炎症性胆管阻塞来证明。在机制上，这些变化与B7共刺激分子对肝髓树突状细胞的下调有关。缺乏趋化因子受体CXCR3的CD4细胞过继转移实验表明Tregs的局部定位在控制肝脏免疫应答中起关键作用。在BA婴儿的样本中，我们发现与没有肝损伤的年龄匹配的对照组相比，循环t淋巴细胞中促炎细胞因子的产生增加，肝髓树突状细胞上B7分子的上调，循环treg缺乏CXCR3表达的频率增加。基于这些数据，我们提出Tregs通过局部控制效应淋巴细胞激活来保护新生儿胆管损伤的总体假设。这一假设将在三个重叠的特定目标中得到验证：1)确定Treg抑制胆管损伤的机制，2)确定BA中Treg的免疫调节途径，以及3)阐明Treg运输到BA炎症部位的分子决定因素。对于目的1，将在小鼠BA中检测Treg/CD4+细胞过继转移对NK细胞毒性活性和t淋巴细胞分化的影响，并将在BA婴儿和健康对照中研究Treg抑制循环t淋巴细胞中细胞因子产生的作用。在目的2中，树突状细胞TGF？和IL10作为Treg抑制的细胞和细胞因子介质将通过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刺激能力的共培养试验来研究。BA婴儿肝脏树突状细胞b7分子上调的初步结果将在更大的队列中得到验证。对于目的3，CXCR3对treg迁移到肝脏的影响将在过继性转移实验中阐明，并通过确定BA受试者和对照组循环和肝脏treg的功能特征。总的来说，在小鼠和BA婴儿组织中的补充研究将确定Tregs控制疾病发病机制的关键调控途径，指导未来的治疗方法以阻断胆道损伤的进展，并改善儿童的长期预后。

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.