The gut associated lymphoid tissue (GALT) effector sites play a crucial role on the pathogenesis of many immune-mediated gastrointestinal diseases. The lymphocytes at these effector sites are principally T cells which present important morphological, phenotypical and functional differences. Flow cytometry (FC) is one of the most commonly used techniques to characterize intestinal lymphocytes in human and animal models. Published studies with a focus on dogs for intraepithelial lymphocytes (IEL) immunophenotyping exist in very limited numbers. Moreover, no lamina propria lymphocytes (LPL) isolation protocols in the canine species have been described for FC evaluation. In addition to immune intestinal dysregulation, imbalances in the peripheral blood immune system have been described in both human and animal gastrointestinal disorders. The aim of this study was to provide a protocol for canine IEL and LPL isolation for FC immunophenotyping of T cells subsets. Specifically, T helper, T cytotoxic, activated Th and Tc lymphocytes, regulatory, double negative, double positive, IFN-γ and IL-4 producing T cells, and to compare their respective populations between these effector sites and with the blood stream compartment in healthy dogs. The potential relationship of these cells distributions with age, sex and breed was also evaluated. This study included sixteen healthy dogs of different sexes and breeds with a mean age of 4.55 ± 2.93 years old. The selected protocols for the three immune compartments showed proper cell yield, purity, viability, and the absence of phenotypic and functional disturbances. Histologically, an adequate separation of the duodenal epithelium from the lamina propria was also observed. All the proposed T cells subsets were identified in the three immune compartments studied, showing some statistically significant differences in their distributions at these locations that highlight the importance of their individual evaluation. This study provides an adequate method for canine small intestine IEL and LPL isolation for FC immunophenotyping and is key for future studies on the gastrointestinal immune system associated with different canine diseases.

肠道相关淋巴组织 (GALT) 效应位点在许多免疫介导的胃肠道疾病的发病机制中起着至关重要的作用。这些效应部位的淋巴细胞主要是 T 细胞，它们呈现出重要的形态、表型和功能差异。流式细胞术 (FC) 是在人类和动物模型中表征肠道淋巴细胞的最常用技术之一。以狗为重点的上皮内淋巴细胞 (IEL) 免疫表型研究已发表的研究数量非常有限。此外，没有描述犬科动物固有层淋巴细胞 (LPL) 分离方案用于 FC 评估。除了免疫肠道失调外，外周血免疫系统的失衡在人类和动物胃肠道疾病中都有描述。本研究的目的是为犬 IEL 和 LPL 分离提供一种方案，用于 T 细胞亚群的 FC 免疫表型分析。具体来说，T 辅助细胞、T 细胞毒性细胞、活化的 Th 和 Tc 淋巴细胞、调节性、双阴性、双阳性、产生 IFN-γ 和 IL-4 的 T 细胞，并比较它们在这些效应位点之间以及与血流区室中各自的群体健康的狗。还评估了这些细胞分布与年龄、性别和品种的潜在关系。这项研究包括 16 只不同性别和品种的健康犬，平均年龄为 4.55 ± 2.93 岁。三个免疫隔间的选定方案显示出适当的细胞产量、纯度、活力，并且没有表型和功能障碍。组织学上，还观察到十二指肠上皮与固有层充分分离。所有提议的 T 细胞亚群都在所研究的三个免疫区室中被识别出来，显示出它们在这些位置的分布存在一些统计学上的显着差异，这突出了它们个体评估的重要性。该研究为犬小肠 IEL 和 LPL 分离提供了一种充分的方法，用于 FC 免疫表型分析，并且是未来研究与不同犬类疾病相关的胃肠道免疫系统的关键。