Department of Pediatrics, Division of Gastroenterology and Nutrition, Erasmus MC-Sophia Children's Hospital, Rotterdam, The Netherlands.
Am J Gastroenterol. 2011 Jun;106(6):1147-59. doi: 10.1038/ajg.2011.24. Epub 2011 Mar 8.
The aim of this study was to identify new markers of mucosal T cells to monitor ongoing intestinal immune responses in peripheral blood.
Expression of cell-surface markers was studied in mice on ovalbumin (OVA)-specific T cells in the gut-draining mesenteric lymph nodes (MLN) after OVA feed. The effect of the local mucosal mediators retinoic acid (RA) and transforming growth factor-β (TGF-β) on the induction of a mucosal phenotype was determined in in vitro T-cell differentiation assays with murine and human T cells. Tetramer stainings were performed to study gluten-specific T cells in the circulation of patients with celiac disease, a chronic small-intestinal inflammation.
In mice, proliferating T cells in MLN were CD62L(neg)CD38(+) during both tolerance induction and abrogation of intestinal homeostasis. This mucosal CD62L(neg)CD38(+) T-cell phenotype was efficiently induced by RA and TGF-β in mice, whereas for human CD4(+) T cells RA alone was sufficient. The CD4(+)CD62L(neg)CD38(+) T-cell phenotype could be used to identify T cells with mucosal origin in human peripheral blood, as expression of the gut-homing chemokine receptor CCR9 and β(7) integrin were highly enriched in this subset whereas expression of cutaneous leukocyte-associated antigen was almost absent. Tetramer staining revealed that gluten-specific T cells appearing in blood of treated celiac disease patients after oral gluten challenge were predominantly CD4(+)CD62L(neg)CD38(+). The total percentage of circulating CD62L(neg)CD38(+) of CD4 T cells was not an indicator of intestinal inflammation as percentages did not differ between pediatric celiac disease patients, inflammatory bowel disease patients and respective controls. However, the phenotypic selection of mucosal T cells allowed cytokine profiling as upon restimulation of CD62L(neg)CD38(+) cells interleukin-10 (IL-10) and interferon-γ (IFN-γ) transcripts were readily detected in circulating mucosal T cells.
By selecting for CD62L(neg)CD38(+) expression that comprises 5-10% of the cells within the total CD4(+) T-cell pool we are able to highly enrich for effector T cells with specificity for mucosal antigens. This is of pivotal importance for functional studies as this purification enhances the sensitivity of cytokine detection and cellular activation.
本研究旨在鉴定黏膜 T 细胞的新标志物，以监测外周血中持续的肠道免疫反应。
在卵清蛋白（OVA）特异性 T 细胞在肠道引流肠系膜淋巴结（MLN）中的表达，在 OVA 喂养后，研究了小鼠的细胞表面标志物。在体外 T 细胞分化实验中，用鼠和人 T 细胞确定了局部黏膜介质视黄酸（RA）和转化生长因子-β（TGF-β）对诱导黏膜表型的影响。进行四聚体染色以研究乳糜泻患者循环中的麦胶蛋白特异性 T 细胞，乳糜泻是一种慢性小肠炎症。
在小鼠中，MLN 中增殖的 T 细胞在诱导耐受和破坏肠道内稳态时均为 CD62L(neg)CD38(+)。这种黏膜 CD62L(neg)CD38(+)T 细胞表型可被 RA 和 TGF-β在小鼠中有效诱导，而对于人 CD4(+)T 细胞，RA 单独就足够了。CD4(+)CD62L(neg)CD38(+)T 细胞表型可用于鉴定人外周血中具有黏膜起源的 T 细胞，因为肠道归巢趋化因子受体 CCR9 和 β(7)整合素的表达在该亚群中高度富集，而皮肤白细胞相关抗原的表达几乎不存在。四聚体染色显示，在口服麦胶挑战后出现在治疗后乳糜泻患者血液中的麦胶蛋白特异性 T 细胞主要为 CD4(+)CD62L(neg)CD38(+)。循环 CD4 T 细胞中 CD62L(neg)CD38(+)的总百分比不是肠道炎症的指标，因为儿科乳糜泻患者、炎症性肠病患者和各自的对照组之间的百分比没有差异。然而，黏膜 T 细胞的表型选择允许细胞因子谱分析，因为在对 CD62L(neg)CD38(+)细胞进行再刺激时，在循环黏膜 T 细胞中很容易检测到白细胞介素-10（IL-10）和干扰素-γ（IFN-γ）转录本。
通过选择包含总 CD4(+)T 细胞池内 5-10%细胞的 CD62L(neg)CD38(+)表达，我们能够高度富集对黏膜抗原具有特异性的效应 T 细胞。这对于功能研究至关重要，因为这种纯化增强了细胞因子检测和细胞活化的敏感性。
