# Isolation and preliminary characterization of bovine Th17 lymphocytes

Patricia Cunha, Christophe Gitton, Pierre Germon, Gilles Foucras, Pascal Rainard

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Patricia Cunha, Christophe Gitton, Pierre Germon, Gilles Foucras, Pascal Rainard. Isolation and preliminary characterization of bovine Th17 lymphocytes. 12. International Veterinary Immunology Symposium (IVIS), Aug 2019, Seattle, United States. , 176 p., 2019, P023. hal-02735890

HAL Id: hal-02735890

https://hal.inrae.fr/hal-02735890

Submitted on 2 Jun 2020

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ABSTRACT BOOK

International Veterinary
Immunology Symposium

AUG 13 – 16, 2019 | RENAISSANCE SEATTLE, SEATTLE, USA
P022

Predicted microRNA biomarkers of
*Mycobacterium avium* subspecies
*paratuberculosis* using an *in-silico*
*bioinformatics approach*

Wright K.E., Plain K.M., Purdie A.C., de Silva K.

University of Sydney, Sydney School of
Veterinary Science, Camden, Australia

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne's Disease (JD) or paratuberculosis, a chronic granulomatous enteritis affecting ruminants. Typified by long and silent subclinical disease phases, there is a defined need for the early detection and diagnosis of MAP infection. Biomarkers are defined as objectively measurable indicators of normal or disease processes and may provide further insight into the interplay between host immune systems and pathogenic bacteria. microRNA (miRNA), a small non-coding subset of RNA, have been proposed as potential biomarker targets as they are highly stable and exert control over protein translation and gene expression. Previous studies have shown modulation of miRNA responses to mycobacterial infection, making them ideal candidates for biomarkers for the diagnosis and differentiation between disease states of paratuberculosis. This study aimed to generate a list of candidate biomarkers for the diagnosis of paratuberculosis in cattle and sheep using bioinformatic predictions and Ingenuity Pathway Analysis software (IPA).

A full literature search was carried out for
the terms "Mycobacteria + paratuberculosis
+ miRNA + microRNA + mycobacterium"
using the Scopus database and Abcam's
Firefly Discovery Engine. A list of potential

miRNAs was compiled and uploaded to IPA.
A core analysis was run, and new miRNAs
added based on suggested function and
commonalities between networks. Key
networks were generated based on function
and relevance to MAP pathogenesis.

Eight key networks were obtained from the
original core analysis and a list of candidate
biomarkers for identifying MAP infection was
generated.

This list of candidate biomarkers for
MAP infection provides potential miRNA
biomarkers that may aid in the diagnosis of
paratuberculosis in sheep and cattle. These
candidates will not only provide the basis for
future investigations, but may also further
elucidate the molecular mechanisms of
MAP pathogenesis through their associated
modulation of target molecules and
immunological pathways.

P023

Isolation and preliminary characterization of
bovine Th17 lymphocytes

Cunha P.¹, Gitton C.¹, Germon P.¹, Foucras G.², Rainard P.¹

¹ISP, INRA, UMR 1282, Université de Tours, Nouzilly, France, ²IHAP, Université de Toulouse, ENVT, INRA, Toulouse, France

Interleukin 17A-producing T helper cells (Th17)
are effector memory CD4+ T cells that are
crucial to adaptive immunity to extracellular
bacteria. The activities of these cells in the
bovine species are not yet defined for want
of straightforward cultivation and isolation procedures. We have developed a method to cultivate, expand, sort and maintain in culture bovine Th17 cells from circulating CD4+ T cells of adult cows. Using polyclonal stimulation (antibodies to CD3 and CD28), we expanded positive CD4+ IL-17A+ T (Th17) cells in a cell culture medium without serum supplemented with TGF-β1 and IL-6. We used IL-2 to expand the cells, which were characterized by intracellular labeling for IL-17A and IFN-γ. Then, we isolated populations of CD4+ T cells producing IL-17A, IFN-γ or both by labeling surface IL-17A with either a complex of biotinylated anti-CD45 antibodies-streptavidin-biotinylated anti-IL-17A antibodies or by direct surface labeling with antibodies to IL-17A, followed by flow cytometry cell sorting. The percentages of surface-labeled IL-17A-secreting cells were quite similar to the percentages of intracellular-labeled IL-17A-producing cells of the same cultures, and the two labeling procedures of live cells yielded similar results. The sorted IL-17A+ cells were restimulated and expanded. After expansion, 80% of the isolated Th17 cells were positive for IL-17A intracellular labeling. The sorted IL-17A+ cells can be frozen, stored and expanded again. The sorted Th17 cells secreted much more IL-17A and IL-17F than did CD4+ IL-17- cells. Notably, most of Th17 cells secreted IFN-γ, although in lower amounts than did CD4+ IL-17- cells. Sorted cells were characterized by transcriptomic profiling. Genes coding for Th17 signature cytokines (IL-17A, IL-17F, IL-26) and transcription factors (RORγt, RORα) were overexpressed in Th17 cells. The techniques developed will make it possible to investigate the phenotypic and functional profiles of bovine Th17 cells along with their stability or plasticity.

P024

Measuring CMI responses using the PrimeFlow RNA assay; a potential new method of evaluating BVDV vaccination response

Falkenberg S.¹, Dassanayake R.¹, Walz P.², Neill J.¹, Ridpath J.¹, Roth J.³

¹USDA-ARS-National Animal Disease Center, Ruminant Diseases and Immunology Unit, Ames, United States, ²Auburn University, College of Veterinary Medicine, Department of Pathobiology, Auburn, United States, ³Iowa State University, College of Veterinary Medicine, Department of Veterinary Microbiology and Preventive Medicine, Ames, United States

Current methods for evaluating bovine viral diarrhea virus (BVDV) vaccination response typically rely on measurement of humoral responses as determined by neutralizing antibody titers against BVDV. While virus neutralization titers (VNT) are correlated with increased protection, research has shown that T-cell mediated immunity (CMI) is an important component of a protective response against BVDV. For example, improved protection to BVDV by modified-live viral (MLV) vaccines as compared to killed vaccines for BVDV is thought to be due to better CMI induced by MLV. The goal of this work was to evaluate the immune response in vaccinated calves using methods that quantitated both humoral and CMI responses. Classic VNT was used to measure the humoral response while a new method based on the PrimeFlow RNA assay was used for measuring CMI. PBMC from both vaccinated (MLV) and non-vaccinated calves were isolated and stimulated with BVDV. The frequency of IFN-γ, IL-2, and BVDV positive