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ARS Home » Midwest Area » Ames, Iowa » National Animal Disease Center » Food Safety and Enteric Pathogens Research » Research » Publications at this Location » Publication #406919
Research Project: Intestinal Microbial Ecology and Non-Antibiotic Strategies to Limit Shiga Toxin-Producing Escherichia coli (STEC) and Antimicrobial Resistance Transmission in Food Animals

Location: Food Safety and Enteric Pathogens Research

Title: Detection B cell and antibody-secreting cell populations in pigs at RNA and protein levels

Author
item WIARDA, JAYNE - Oak Ridge Institute For Science And Education (ORISE)
item Shircliff, Adrienne
item Stasko, Judith
item SIVASANKARAN, SATHESH - Iowa State University
item Ackermann, Mark
item TUGGLE, CHRISTOPHER - Iowa State University
item Loving, Crystal

Submitted to: International Veterinary Immunology Symposium
Publication Type: Abstract Only
Publication Acceptance Date: 9/11/2023
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: The study of B cells and antibody-secreting cells in pigs is limited by the minimal toolbox of B cell-specific reagents and lack of comprehensive analysis to simultaneously evaluate distinct B cell subsets. Query of porcine intestinal B cell subsets defined via single-cell RNA sequencing (scRNA-seq) revealed PAX5, CD79A, BCL6, MKI67, IRF4, and PRDM1 gene expression defined functionally-distinct B cell subsets. Antibodies reactive to proteins encoded by the six genes (Pax5, CD79a, Bcl-6, Ki-67, IRF4, Blimp-1) were used in flow cytometry for identification of B cell subsets in porcine intestinal cells and peripheral blood. Resting B cells, activated/cycling B cells, transitioning B cells, and antibody-secreting cells were readily identified, with distinct B cell subsets noted between intestine and circulation. B cell subset proportions identified via flow cytometry were similar to proportions of corresponding populations identified via scRNA-seq in pig intestine, indicating protein expression as a suitable proxy to the defined gene expression for B cell subsets. To understand locational context of intestinal B cell subsets, we developed immunohistochemistry and RNA in situ hybridization protocols for staining of proteins and RNA for the six biomarkers. In situ staining patterns between RNA and encoded proteins were highly similar and coincided with patterns observed through spatial transcriptomic (STomics) analysis of pig intestinal tissue. Integrating scRNA-seq and STomics data further predicted distinct localization of B cell subsets in the intestine, similar to in situ labeling. Results indicate a six-marker panel at both RNA and protein levels can successfully identify functionally distinct B cell subsets in pigs.