Establishing a Multicolor Flow Cytometry to Characterize Cellular Immune Response in Chickens Following H7N9 Avian Influenza Virus Infection

Abstract

Avian influenza virus (AIV) emerged and has continued to re-emerge, continuously posing great threats to animal and human health. The detection of hemagglutination inhibition (HI) or virus neutralization antibodies (NA) is essential for assessing immune protection against AIV. However, the HI/NA-independent immune protection is constantly observed in vaccines’ development against H7N9 subtype AIV and other subtypes in chickens and mammals, necessitating the analysis of the cellular immune response. Here, we established a multi-parameter flow cytometry to examine the innate and adaptive cellular immune responses in chickens after intranasal infection with low pathogenicity H7N9 AIV. This assay allowed us to comprehensively define chicken macrophages, dendritic cells, and their MHC-II expression, NK cells, γδ T cells, B cells, and distinct T cell subsets in steady state and during infection. We found that NK cells and KUL01+ cells significantly increased after H7N9 infection, especially in the lung, and the KUL01+ cells upregulated MHC-II and CD11c expression. Additionally, the percentages and numbers of γδ T cells and CD8 T cells significantly increased and exhibited an activated phenotype with significant upregulation of CD25 expression in the lung but not in the spleen and blood. Furthermore, B cells showed increased in the lung but decreased in the blood and spleen in terms of the percentages or/and numbers, suggesting these cells may be recruited from the periphery after H7N9 infection. Our study firstly disclosed that H7N9 infection induced local and systemic cellular immune responses in chickens, the natural host of AIV, and that the flow cytometric assay developed in this study is useful for analyzing the cellular immune responses to AIVs and other avian infectious diseases and defining the correlates of immune protection.

An Anti-Tumor Vaccine Against Marek's Disease Virus Induces Differential Activation and Memory Response of γδ T Cells and CD8 T Cells in Chickens

“…Single cell suspension from spleens and lungs were prepared as previously described ( 34 ) and peripheral blood mononuclear cells (PBMC) were isolated from blood as per previous report ( 35 ). Briefly, spleens were mechanically disrupted and pushed through a 70 μm nylon cell strainer and the cells were resuspended in 10 ml with phosphate buffered saline (PBS) containing 2% fetal bovine serum (FBS).…”

The Evaluation of Cellular Immunity to Avian Viral Diseases: Methods, Applications, and Challenges

“…While a basic flow cytometric assay has been used in the abovementioned studies for numerating immune cells, to develop more advanced multi-parameter flow cytometry for analyzing both frequency and function/activation markers of immune cells in chicken is still challenging ( Yang et al, 2020 ). Recently, Hao et al (2020) integrated most of the antibodies against chicken markers available commercially and developed two panels of 6- and 7-color flow cytometry for comprehensively analyzing chicken myeloid lineage cells, partial NK cells, and T cell subsets as well as their activation. Employing this assay, they found that H7N9 AIV intranasal infection-induced distinct local γδ T and CD8 T cell responses in the lung and systemic NK and monocyte/macrophage responses in chickens ( Hao et al, 2020 ).…”

“…Recently, Hao et al (2020) integrated most of the antibodies against chicken markers available commercially and developed two panels of 6- and 7-color flow cytometry for comprehensively analyzing chicken myeloid lineage cells, partial NK cells, and T cell subsets as well as their activation. Employing this assay, they found that H7N9 AIV intranasal infection-induced distinct local γδ T and CD8 T cell responses in the lung and systemic NK and monocyte/macrophage responses in chickens ( Hao et al, 2020 ). Using same method, Hao et al (2021) performed a comprehensive analysis T cell responses to a MD vaccine CVI988/Rispens and revealed the dynamic changes and early activation state of chicken γδ T and CD8 T cells as well as a potential role of γδ T cells in vaccine-induced protection against MD.…”

“…Thus, the clone 5C.123.02 may not be an optimal antibody for intracellular staining of chicken IFN-γ. We also tested other 10 clones of anti-chicken IFN-γ antibodies that are commercially available ( Table 3 ), none of them worked for ICS ( Hao et al, 2020 ). A recent study showed that newly-developed six clones of anti-chicken IFN-γ antibodies have strong reactivity with chicken IFN-γ in ICS, which might be a better antibody for intracellular staining of chicken IFN-γ ( Lagler et al, 2019 ).…”

Special Issue—Immunity to Influenza Viruses

“…Wang, Wei, and Liu [11] provide an overview of mucosal immunity to influenza viruses, focusing on infection of avian hosts and strategies available to mitigate influenza infections in birds. Hao et al [12] developed a multicolor flow cytometry panel to assess cellular immunity in chickens infected with an H7N9 virus.…”