title: Flow Cytometric and Cytokine ELISpot Approaches To Characterize the Cell-Mediated Immune Response in Ferrets following Influenza Virus Infection.
abstract: Influenza virus infections represent a significant socioeconomic and public health burden worldwide. Although ferrets are considered by many to be ideal for modeling human responses to influenza infection and vaccination, efforts to understand the cellular immune response have been severely hampered by a paucity of standardized procedures and reagents. In this study, we developed flow cytometric and T cell enzyme-linked immunosorbent spot (ELISpot) approaches to characterize the leukocyte composition and antigen-specific T cell response within key lymphoid tissues following influenza virus infection in ferrets. Through a newly designed and implemented set of serological reagents, we used multiparameter flow cytometry to directly quantify the frequency of CD4(+) and CD8(+) T cells, Ig(+) B cells, CD11b(+) myeloid-derived cells, and major histocompatibility complex (MHC) class II-positive antigen-presenting cells (APCs) both prior to and after intranasal infection with A/California/04/09 (H1N1). We found that the leukocyte composition was altered at 10 days postinfection, with notable gains in the frequency of T cells and myeloid cells within the draining lymph node. Furthermore, these studies revealed that the antigen specificity of influenza virus-reactive CD4 and CD8 T cells was very broad, with recognition of the viral HA, NA, M1, NS1, and NP proteins, and that total reactivity to influenza virus postinfection represented approximately 0.1% of the circulating peripheral blood mononuclear cells (PBMC). Finally, we observed distinct patterns of reactivity between individual animals, suggesting heterogeneity at the MHC locus in ferrets within commercial populations, a finding of considerable interest in efforts to move the ferret model forward for influenza vaccine and challenge studies.
  
  
  
  Ferrets are an ideal animal model to study transmission, diseases, and vaccine efficacies of respiratory viruses because of their close anatomical and physiological resemblances to humans. However, a lack of reagents has limited our understanding of the cell-mediated immune response following infection and vaccination. In this study, we used cross-reactive and ferret-specific antibodies to study the leukocyte composition and antigen-specific CD4 and CD8 T cell responses following influenza A/California/04/09 (H1N1) virus infection. These studies revealed strikingly distinct patterns of reactivity between CD4 and CD8 T cells, which were overlaid with differences in protein-specific responses between individual animals. Our results provide a first, in-depth look at the T cell repertoire in response to influenza infection and suggest that there is considerable heterogeneity at the MHC locus, which is akin to that in humans and an area of intense research interest.
authors.affiliation: Department of Microbiology and Immunology, David H. Smith Center for Vaccine Biology and Immunology, University of Rochester Medical Center, Rochester, New York, USA.
authors.forename: Anthony
authors.initials: A
authors.lastname: DiPiazza
authors.affiliation: Department of Microbiology and Immunology, David H. Smith Center for Vaccine Biology and Immunology, University of Rochester Medical Center, Rochester, New York, USA.
authors.forename: Katherine
authors.initials: K
authors.lastname: Richards
authors.affiliation: Department of Microbiology and Immunology, David H. Smith Center for Vaccine Biology and Immunology, University of Rochester Medical Center, Rochester, New York, USA.
authors.forename: Frances
authors.initials: F
authors.lastname: Batarse
authors.affiliation: Department of Microbiology and Immunology, David H. Smith Center for Vaccine Biology and Immunology, University of Rochester Medical Center, Rochester, New York, USA.
authors.forename: Laura
authors.initials: L
authors.lastname: Lockard
authors.affiliation: Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
authors.forename: Hui
authors.initials: H
authors.lastname: Zeng
authors.affiliation: Department of Microbiology, Icahn School of Medicine at Mt. Sinai, New York, New York, USA Global Health and Emerging Pathogens Institute at Icahn School of Medicine, New York, New York, USA Department of Medicine, Icahn School of Medicine at Mt. Sinai, New York, New York, USA.
authors.forename: Adolfo
authors.initials: A
authors.lastname: García-Sastre
authors.affiliation: Department of Microbiology, Icahn School of Medicine at Mt. Sinai, New York, New York, USA Global Health and Emerging Pathogens Institute at Icahn School of Medicine, New York, New York, USA.
authors.forename: Randy A
authors.initials: RA
authors.lastname: Albrecht
authors.affiliation: Department of Microbiology and Immunology, David H. Smith Center for Vaccine Biology and Immunology, University of Rochester Medical Center, Rochester, New York, USA andrea_sant@urmc.rochester.edu.
authors.forename: Andrea J
authors.initials: AJ
authors.lastname: Sant
mesh_terms: D000818:Animals; D000938:Antigen-Presenting Cells / Q000276:immunology; D001402:B-Lymphocytes / Q000276:immunology; D016207:Cytokines / Q000032:analysis*; D058501:Enzyme-Linked Immunospot Assay; D005260:Female; D005289:Ferrets; D005434:Flow Cytometry; D007111:Immunity, Cellular; D053118:Influenza A Virus, H1N1 Subtype / Q000276:immunology*; D008198:Lymph Nodes / Q000276:immunology; D022423:Myeloid Cells / Q000276:immunology; D009976:Orthomyxoviridae Infections / Q000276:immunology*; D013601:T-Lymphocytes / Q000276:immunology
chemical_list: D016207:Cytokines