Abstract
Ferrets are an ideal animal model in which to study the transmission of respiratory viruses as well as disease progression and vaccine efficacy because of their close anatomical and physiological resemblances to humans. However, a paucity of reagents and standardized procedures has hampered research progress, especially for studying cell-mediated immunity. The approaches described here—leukocyte isolation from whole blood and secondary lymphoid tissues—are generalizable, highly reproducible, and deliver single cell suspensions with excellent cell viability. Importantly, we have now developed assays to quantify key cellular components and antigen-specific T cell responses at the single cell level from multiple tissue compartments following influenza infection in ferrets. Collectively, these methods were instrumental in flow cytometry studies that revealed alterations in immune cell composition and distribution across lymphoid tissues following viral infection. Furthermore, sorting of T cell populations and peptide restimulation ex vivo in cytokine ELISpot assays has provided novel insight into the influenza-specific CD4 and CD8 T cell repertoire. The detailed procedures for these techniques are described in this chapter and can likely be adapted for the analyses of responses to many respiratory pathogens.
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Acknowledgments
This work was funded by the following grants from HHS, NIH, National Institute of Allergy and Infectious Diseases (NIAID)—T32AI007285, HHSN272201300005C, HHSN272201400008C, and P01AI097092. WCL is a recipient of a training fellowship from the Taiwan Ministry of Science and Technology (MOST 105-2917-I-564-006-A1). We thank Hui Zeng and Taronna Maines (Centers for Disease Control and Prevention, Atlanta, GA) for providing ferret tissue samples that were instrumental in preliminary experiments that led to the development of the immune reagents and procedures described in this article. We also thank BEI Resources (NIAID) for the influenza-derived peptide arrays used for characterizing T cell responses following infection.
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DiPiazza, A.T., Richards, K.A., Liu, WC., Albrecht, R.A., Sant, A.J. (2018). Analyses of Cellular Immune Responses in Ferrets Following Influenza Virus Infection. In: Yamauchi, Y. (eds) Influenza Virus. Methods in Molecular Biology, vol 1836. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8678-1_24
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DOI: https://doi.org/10.1007/978-1-4939-8678-1_24
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