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BackgroundIntestinal transplantation (ITx) represents the only curative option for patients with irreversible intestinal failure. Nevertheless, its rejection rate surpasses that of other solid organ transplants due to the heightened immunological load of the gut. Regulatory T-cells (Tregs) are key players in the induction and maintenance of peripheral tolerance, suggesting their potential involvement in modulating host vs. graft responses after ITx. Thus, we investigated the association of Tregs with allograft outcomes in pediatric patients and in an experimental model of small bowel transplantation.MethodsTreg frequency in human samples was analyzed by Flow cytometry (CD4+CD25highCD127-, blood samples) and immunohistochemistry (FoxP3, graft samples). Experimental allogenic-heterotopic small bowel transplantation was performed in rats and animals divided into 3 groups: non-immunosuppressant treatment, rapamycin (2 mg/kg), and tacrolimus (0.6 mg/kg) treatment. Acute cellular rejection (ACR) was diagnosed based on clinical and histological findings, graft gene expression of pro- and anti-inflammatory mediators assessed by RT-qPCR, serum IL-6 and IL-10 levels by Luminex, and Treg frequency analyzed by flow cytometry (CD4+CD25highFoxP3+).ResultsBlood samples from patients undergoing ACR exhibited a significant reduction in the Treg number compared to those with normo-functional grafts. Similarly, a diminished number of FoxP3+ cells was observed in mucosa samples with ACR. In the experimental model, rapamycin-treated animals displayed clinical and histological findings resembling those not receiving immunosuppression treatment. Notably, ACR correlated with a high CD8/CD4 ratio, loss of T-cell chimerism, mRNA upregulation of pro-inflammatory genes and diminished graft Treg frequency. In contrast, tacrolimus treatment prevented ACR and facilitate blood and graft Treg expansion. Remarkably, recipients who achieved Treg expansion within the graft remained free of ACR even after discontinuation of the immunosuppressant treatment and this phenomenon was associated with increased levels of serum IL-10.ConclusionOur clinical and experimental findings underscore the association between Treg frequency and graft rejection after ITx, advocating for strategies that promote their expansion within the gut mucosa to enhance long-term outcomes.

背景：小肠移植（Intestinal transplantation, ITx）是不可逆肠功能衰竭患者唯一的根治性治疗手段。然而，由于肠道免疫负荷较高，其排斥反应发生率高于其他实体器官移植。调节性T细胞（Regulatory T-cells, Tregs）是外周免疫耐受诱导与维持的关键效应细胞，提示其可能参与调控肠移植术后的宿主抗移植物反应。因此，本研究探讨了儿科患者小肠移植术后Treg频率与同种异体移植物结局的关联，并构建小肠移植实验模型开展相关研究。
方法：采用流式细胞术（Flow cytometry，标记方案为CD4+CD25highCD127-，检测血液样本）与免疫组织化学（Immunohistochemistry，标记物为FoxP3，检测移植物样本）分析人体样本中的Treg频率。构建大鼠同种异体异位小肠移植模型，将实验动物分为3组：无免疫抑制治疗组、雷帕霉素（2 mg/kg）给药组及他克莫司（0.6 mg/kg）给药组。急性细胞排斥反应（Acute cellular rejection, ACR）的诊断依据为临床与组织病理学表现，同时通过实时定量聚合酶链反应（RT-qPCR）检测移植物内促炎与抗炎介质的基因表达水平，通过Luminex平台检测血清IL-6、IL-10水平，并采用流式细胞术（标记方案为CD4+CD25highFoxP3+）分析Treg频率。
结果：发生ACR的患者血液样本中Treg数量较移植物功能正常的患者显著降低；同样，发生ACR的黏膜样本中FoxP3+细胞数量亦明显减少。在大鼠实验模型中，雷帕霉素给药组动物的临床与组织病理学表现与未接受免疫抑制治疗的动物相似。值得注意的是，ACR与高CD8/CD4比值、T细胞嵌合状态丧失、促炎基因mRNA上调及移植物内Treg频率降低显著相关。与之相反，他克莫司治疗可预防ACR发生，并促进血液及移植物内Treg扩增。尤为关键的是，移植物内出现Treg扩增的受体在停用免疫抑制治疗后仍未发生ACR，这一现象与血清IL-10水平升高显著相关。
结论：本研究的临床与实验结果证实了肠移植术后Treg频率与移植物排斥反应的关联，提示可通过促进肠黏膜内Treg扩增的策略改善患者长期预后。