Todd D, Singh A J, Greiner D L, Mordes J P, Rossini A A, Bortell R
Department of Medicine, University of Massachusetts Medical School, Worcester 01655, USA.
J Immunol Methods. 1999 Apr 22;224(1-2):111-27. doi: 10.1016/s0022-1759(99)00015-0.
Intraepithelial lymphocytes (IELs) play critical roles in gut immunity. In mice, gammadelta T cells are a large component of the IEL population. In the rat, gammadelta IELs are reportedly much less common, but technical issues suggest that previous analyses should be interpreted cautiously. The study of IELs in rats has been impeded by isolation procedures that are lengthy and complex, leading to small cell yields. For this reason, it is possible that rat IELs analyzed in previous studies have not been representative of the entire IEL compartment. We report a new method for the isolation of rat IELs that is based on the selective removal of intestinal epithelial cells under conditions that leave the basement membrane undisturbed. The method is rapid and requires neither enzymatic digestion, nor surgical removal of Peyer's patches, nor vigorous mechanical manipulation of the intestine. The yield of rat IELs using this method is 5- to 10-fold greater than that reported for other methods. Morphological and phenotypic analyses demonstrated that the purified cell population is comprised of IELs and is not contaminated with lamina propria or Peyer's patch lymphocytes. Phenotypic analysis revealed five major subsets of IELs based on differential cell surface expression of CD4, CD8, and alphabeta T cell receptor (TcR). Among the alphabetaTcR- cells was a population of gammadelta T cells present at levels not previously detected. The isolation of IEL sub-populations using this methodology should facilitate studies of the function of these cells in gut immunity.
上皮内淋巴细胞（IELs）在肠道免疫中发挥着关键作用。在小鼠中，γδ T细胞是IEL群体的重要组成部分。据报道，在大鼠中，γδ IELs要少见得多，但技术问题表明，此前的分析结果应谨慎解读。大鼠IELs的研究一直受到分离程序冗长复杂的阻碍，导致细胞产量较低。因此，以往研究中分析的大鼠IELs可能并不代表整个IEL区室。我们报告了一种分离大鼠IELs的新方法，该方法基于在不破坏基底膜的条件下选择性去除肠道上皮细胞。该方法快速，既不需要酶消化，也不需要手术切除派尔集合淋巴结，也不需要对肠道进行剧烈的机械操作。使用该方法获得的大鼠IELs产量比其他方法报道的产量高5至10倍。形态学和表型分析表明，纯化的细胞群体由IELs组成，未被固有层或派尔集合淋巴结淋巴细胞污染。表型分析基于CD4、CD8和αβ T细胞受体（TcR）的细胞表面差异表达，揭示了IELs的五个主要亚群。在αβTcR-细胞中，存在一群γδ T细胞，其水平是此前未检测到的。使用该方法分离IEL亚群应有助于研究这些细胞在肠道免疫中的功能。
