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Agulla B, García-Sancho M, Sainz Á, et al. Isolation and immunophenotyping by flow cytometry of canine peripheral blood and intraepithelial and lamina propria duodenal T lymphocytes. Vet Immunol Immunopathol. 2021;239:110305doi: 10.1016/j.vetimm.2021.110305.
Agulla, B., García-Sancho, M., Sainz, �. �., Rodríguez-Franco, F., Díaz-Regañón, D., Rodríguez-Bertos, A., & Villaescusa, A. (2021). Isolation and immunophenotyping by flow cytometry of canine peripheral blood and intraepithelial and lamina propria duodenal T lymphocytes. Veterinary immunology and immunopathology, 239110305. https://doi.org/10.1016/j.vetimm.2021.110305
Agulla, Beatriz, et al. "Isolation and immunophenotyping by flow cytometry of canine peripheral blood and intraepithelial and lamina propria duodenal T lymphocytes." Veterinary immunology and immunopathology vol. 239 (2021): 110305. doi: https://doi.org/10.1016/j.vetimm.2021.110305
Agulla B, García-Sancho M, Sainz Á, Rodríguez-Franco F, Díaz-Regañón D, Rodríguez-Bertos A, Villaescusa A. Isolation and immunophenotyping by flow cytometry of canine peripheral blood and intraepithelial and lamina propria duodenal T lymphocytes. Vet Immunol Immunopathol. 2021 Sep;239:110305. doi: 10.1016/j.vetimm.2021.110305. Epub 2021 Jul 28. PMID: 34352607.
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Vet Immunol Immunopathol. 2021 Sep;239:110305. doi: 10.1016/j.vetimm.2021.110305. Epub 2021 Jul 28.

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

Veterinary immunology and immunopathology

Beatriz Agulla, Mercedes García-Sancho, Ángel Sainz, Fernando Rodríguez-Franco, David Díaz-Regañón, Antonio Rodríguez-Bertos, Alejandra Villaescusa

Affiliations

  1. Department of Animal Medicine and Surgery, College of Veterinary Medicine, Complutense University of Madrid, Avda. Puerta de Hierro s/n, 28040, Madrid, Spain. Electronic address: [email protected].
  2. Department of Animal Medicine and Surgery, College of Veterinary Medicine, Complutense University of Madrid, Avda. Puerta de Hierro s/n, 28040, Madrid, Spain.
  3. Department of Animal Medicine and Surgery, College of Veterinary Medicine, Complutense University of Madrid, Avda. Puerta de Hierro s/n, 28040, Madrid, Spain; VISAVET Health Surveillance Centre, Complutense University of Madrid, Avda. Puerta de Hierro s/n, 28040, Madrid, Spain.

PMID: 34352607 DOI: 10.1016/j.vetimm.2021.110305

Abstract

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.

Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.

Keywords: Dog; Flow cytometry; Intraepithelial lymphocytes; Lamina propria lymphocytes; Peripheral blood lymphocytes; T lymphocytes

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