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 C. Hope , Elizabeth J. Glass , Gary Entrican

AbstractThe 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 (defined as CD4+ve, CD8+ve or WC-1+ve) and sheep (defined as CD4+ve or WC-1+ve) 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.

Domaines: Sciences du Vivant [q-bio]

Sean R. Wattegedera, Yolanda Corripio-Miyar, Yvonne Pang, David Frew, Tom N. Mcneilly, et al.. Enhancing the toolbox to study IL-17A in cattle and sheep. Veterinary Research, 2017, 48 (1), pp.20. ⟨10.1186/s13567-017-0426-5⟩. ⟨hal-01504132⟩