Enhancing the toolbox to study IL-17A in cattle and sheep

Sean R Wattegedera, Yolanda Corripio-Miyar, Yvonne Pang, David Frew, Tom N McNeilly, Javier Palarea-Albaladejo, Colin J McInnes, Jayne Hope, Elizabeth Glass, Gary Entrican

Research output: Contribution to journalArticlepeer-review

Abstract

The development of methods to detect cytokine expression by T cell subsets in ruminants is fundamental to strategic development of new livestock vaccines for prevention of infectious diseases. It has been possible to detect T cell expression of IFN-γ, IL-4 and IL-10 in ruminants for many years but methods to detect expression of IL-17A are relatively limited. To address this gap in capability we have cloned bovine and ovine IL-17A cDNAs and expressed biologically-active recombinant proteins in Chinese Hamster Ovary (CHO) cells. We used the transfected CHO cells to screen commercially-available antibodies for their ability to detect IL-17A expression intracellularly and in culture supernates. We demonstrate that an ELISA for bovine IL-17A detects native ovine IL-17A. Moreover, the constituent polyclonal antibodies (pabs) in the ELISA were used to enumerate peripheral blood mononuclear cells (PBMC) expressing IL-17A from cattle and sheep by ELISpot. We identified two monoclonal antibodies (mabs) that detect recombinant intracellular IL-17A in CHO cells by flow cytometry. One of these mabs was used to detect native intracellular IL-17A expression in PBMC in conjunction with cell surface phenotyping mabs (CD4+ve, CD8+ve and Workshop Cluster 1 [WC-1]+ve gamma-delta [γδ]) we show that distinct T cell subsets in cattle and sheep can express IL-17A following activation. These novel techniques provide a solid basis to investigate IL-17A expression and define specific CD4+ve T cell subset activation in ruminants.
Original languageEnglish
Article number20
JournalVeterinary Research
Volume48
Issue number1
Early online date8 Apr 2017
DOIs
Publication statusE-pub ahead of print - 8 Apr 2017

Keywords

  • ovine
  • bovine
  • IL-17A
  • T cell subsets
  • intracellular cytokine staining
  • ELISA
  • ELISpot

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