Enhancing the toolbox to study IL-17A in cattle and sheep
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 ye...
| Main Authors: | , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
BMC
2017-04-01
|
| Series: | Veterinary Research |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s13567-017-0426-5 |
| _version_ | 1828451789603602432 |
|---|---|
| author | 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 |
| author_facet | 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 |
| author_sort | Sean R. Wattegedera |
| collection | DOAJ |
| description | 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 (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. |
| first_indexed | 2024-12-10T23:41:34Z |
| format | Article |
| id | doaj.art-effc8b9d0e004693a9f25bf7a939d628 |
| institution | Directory Open Access Journal |
| issn | 1297-9716 |
| language | English |
| last_indexed | 2024-12-10T23:41:34Z |
| publishDate | 2017-04-01 |
| publisher | BMC |
| record_format | Article |
| series | Veterinary Research |
| spelling | doaj.art-effc8b9d0e004693a9f25bf7a939d6282022-12-22T01:29:01ZengBMCVeterinary Research1297-97162017-04-0148112010.1186/s13567-017-0426-5Enhancing the toolbox to study IL-17A in cattle and sheepSean R. Wattegedera0Yolanda Corripio-Miyar1Yvonne Pang2David Frew3Tom N. McNeilly4Javier Palarea-Albaladejo5Colin J. McInnes6Jayne C. Hope7Elizabeth J. Glass8Gary Entrican9Moredun Research Institute, International Research CentreMoredun Research Institute, International Research CentreMoredun Research Institute, International Research CentreMoredun Research Institute, International Research CentreMoredun Research Institute, International Research CentreBiomathematics and Statistics Scotland, JCMBMoredun Research Institute, International Research CentreThe Roslin Institute and Royal (Dick) School of Veterinary Studies, The University of EdinburghThe Roslin Institute and Royal (Dick) School of Veterinary Studies, The University of EdinburghMoredun Research Institute, International Research CentreAbstract 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 (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.http://link.springer.com/article/10.1186/s13567-017-0426-5Peripheral Blood Mononuclear CellChinese Hamster Ovary CellChinese Hamster OvaryChinese Hamster Ovary Cell LinePermeabilisation Buffer |
| spellingShingle | 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 Enhancing the toolbox to study IL-17A in cattle and sheep Veterinary Research Peripheral Blood Mononuclear Cell Chinese Hamster Ovary Cell Chinese Hamster Ovary Chinese Hamster Ovary Cell Line Permeabilisation Buffer |
| title | Enhancing the toolbox to study IL-17A in cattle and sheep |
| title_full | Enhancing the toolbox to study IL-17A in cattle and sheep |
| title_fullStr | Enhancing the toolbox to study IL-17A in cattle and sheep |
| title_full_unstemmed | Enhancing the toolbox to study IL-17A in cattle and sheep |
| title_short | Enhancing the toolbox to study IL-17A in cattle and sheep |
| title_sort | enhancing the toolbox to study il 17a in cattle and sheep |
| topic | Peripheral Blood Mononuclear Cell Chinese Hamster Ovary Cell Chinese Hamster Ovary Chinese Hamster Ovary Cell Line Permeabilisation Buffer |
| url | http://link.springer.com/article/10.1186/s13567-017-0426-5 |
| work_keys_str_mv | AT seanrwattegedera enhancingthetoolboxtostudyil17aincattleandsheep AT yolandacorripiomiyar enhancingthetoolboxtostudyil17aincattleandsheep AT yvonnepang enhancingthetoolboxtostudyil17aincattleandsheep AT davidfrew enhancingthetoolboxtostudyil17aincattleandsheep AT tomnmcneilly enhancingthetoolboxtostudyil17aincattleandsheep AT javierpalareaalbaladejo enhancingthetoolboxtostudyil17aincattleandsheep AT colinjmcinnes enhancingthetoolboxtostudyil17aincattleandsheep AT jaynechope enhancingthetoolboxtostudyil17aincattleandsheep AT elizabethjglass enhancingthetoolboxtostudyil17aincattleandsheep AT garyentrican enhancingthetoolboxtostudyil17aincattleandsheep |