Mazzarella Giuseppe, Stefanile Rosita, Camarca Alessandra, Giliberti Paolo, Cosentini Elena, Marano Caterina, Iaquinto Gaetano, Giardullo Nicola, Auricchio Salvatore, Sette Alessandro, Troncone Riccardo, Gianfrani Carmen
Institute of Food Sciences, CNR, Avellino, Italy.
Gastroenterology. 2008 Apr;134(4):1017-27. doi: 10.1053/j.gastro.2008.01.008. Epub 2008 Jan 11.
BACKGROUND & AIMS: The extensive infiltration of CD8(+) T cells in the intestinal mucosa of celiac disease (CD) patients is a hallmark of the disease. We identified a gliadin peptide (pA2) that is selectively recognized by CD8(+) T cells infiltrating intestinal mucosa of HLA-A2(+) CD patients. Herein, we investigated the phenotype, the tissue localization, and the effector mechanism of cells responsive to pA2 by using the organ culture of CD intestinal mucosa. The target of pA2-mediated cytotoxicity was also investigated by using the intestinal epithelial cell lines Caco2 and HT29, A2(+) and A2(-), respectively, as target cells.
Jejunal biopsy specimens from CD patients were cultured in vitro with pA2, and cellular activation was evaluated by immunohistochemistry and cytofluorimetric analysis. Cytotoxicity of pA2-specific, intestinal CD8(+) T cells was assayed by granzyme-B and interferon-gamma release and by apoptosis of target cells.
pA2 challenge of A2(+) CD mucosa increased the percentage of CD8(+)CD25(+) and of CD80(+) cells in the lamina propria, the former mainly localized beneath the epithelium, as well as the number of terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling-positive cells (TUNEL(+)) in the epithelium. Intraepithelial CD3(+) cells and enterocyte expression of Fas were also increased. CD8(+)CD25(+) and CD8(+)FASL(+) T cells were significantly increased in cell preparations from biopsy specimens cultured with pA2. CD8(+) T-cell lines released both granzyme-B and interferon-gamma following recognition of pA2 when presented by Caco2 and not by HT29.
These data indicate that gliadins contain peptides able to activate, through a TCR/HLA class I interaction, CD8-mediated response in intestinal CD mucosa and to induce the enterocyte apoptosis.
乳糜泻（CD）患者肠黏膜中CD8(+) T细胞的广泛浸润是该疾病的一个标志。我们鉴定出一种麦醇溶蛋白肽（pA2），其被浸润HLA - A2(+) CD患者肠黏膜的CD8(+) T细胞选择性识别。在此，我们通过使用CD肠黏膜的器官培养来研究对pA2有反应的细胞的表型、组织定位及效应机制。还分别使用肠上皮细胞系Caco2（A2(+)）和HT29（A2(-)）作为靶细胞来研究pA2介导的细胞毒性的靶标。
将CD患者的空肠活检标本与pA2进行体外培养，通过免疫组织化学和细胞荧光分析评估细胞活化情况。通过颗粒酶B和干扰素 - γ释放以及靶细胞凋亡来检测pA2特异性肠CD8(+) T细胞的细胞毒性。
用pA2刺激A2(+) CD黏膜可增加固有层中CD8(+)CD25(+)和CD80(+)细胞的百分比，前者主要位于上皮下方，同时也增加上皮中末端脱氧核苷酸转移酶介导的dUTP缺口末端标记阳性细胞（TUNEL(+)）的数量。上皮内CD3(+)细胞和肠上皮细胞Fas的表达也增加。在用pA2培养的活检标本的细胞制剂中，CD8(+)CD25(+)和CD8(+)FASL(+) T细胞显著增加。当由Caco2而非HT29呈递pA2时，CD8(+) T细胞系在识别pA2后释放颗粒酶B和干扰素 - γ。
这些数据表明麦醇溶蛋白包含能够通过TCR/HLA I类相互作用激活肠CD黏膜中CD8介导的反应并诱导肠上皮细胞凋亡的肽。
