Isolation and immunophenotyping by flow cytometry of canine peripheral blood and intraepithelial and lamina propria duodenal T lymphocytes



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Agulla Pérez, Beatriz and García-Sancho Téllez, Mercedes Guadalupe and Sainz Rodríguez, Ángel and Rodríguez Franco, Fernando and Díaz-Regañón Fernández, David Rafael and Rodríguez Bertos, Antonio Manuel and Villaescusa Fernández, Alejandra (2021) Isolation and immunophenotyping by flow cytometry of canine peripheral blood and intraepithelial and lamina propria duodenal T lymphocytes. Veterinary Immunology and Immunopathology, 239 . p. 110305. ISSN 01652427

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The gut associated lymphoid tissue (GALT) effector sites play a crucial role on the pathogenesis of many immunemediated 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.

Item Type:Article
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CRUE-CSIC (Acuerdos Transformativos 2021)

Uncontrolled Keywords:Flow cytometry, Intraepithelial lymphocytes, Lamina propria lymphocytes, T lymphocytes, Dog, Peripheral blood lymphocytes
Subjects:Medical sciences > Veterinary
Medical sciences > Biology > Molecular biology
ID Code:69609
Deposited On:17 Jan 2022 16:12
Last Modified:18 Feb 2022 11:03

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