College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China.
Institute of Comparative Medicine, Yangzhou University, Yangzhou 225009, China.
Viruses. 2020 Dec 6;12(12):1396. doi: 10.3390/v12121396.
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.
禽流感病毒（AIV）不断出现并持续重现，对动物和人类健康构成了巨大威胁。血凝抑制（HI）或病毒中和抗体（NA）的检测对于评估针对 AIV 的免疫保护至关重要。然而，在针对 H7N9 亚型 AIV 和鸡和哺乳动物中其他亚型的疫苗开发中，不断观察到 HI/NA 非依赖性免疫保护，这需要分析细胞免疫反应。在这里，我们建立了一种多参数流式细胞术，以检测低致病性 H7N9 AIV 鼻内感染后鸡的固有和适应性细胞免疫反应。该测定法使我们能够全面定义鸡的巨噬细胞、树突状细胞及其 MHC-II 表达、NK 细胞、γδ T 细胞、B 细胞和稳定状态和感染期间的不同 T 细胞亚群。我们发现，NK 细胞和 KUL01 细胞在 H7N9 感染后显着增加，特别是在肺部，并且 KUL01 细胞上调 MHC-II 和 CD11c 表达。此外，γδ T 细胞和 CD8 T 细胞的百分比和数量显着增加，并在肺部表现出激活表型，CD25 表达显着上调，但在脾脏和血液中则没有。此外，B 细胞在肺部的百分比或/和数量增加，而在血液和脾脏中的百分比或/和数量减少，表明这些细胞可能在 H7N9 感染后从外围募集。我们的研究首次揭示，H7N9 感染诱导了鸡（AIV 的天然宿主）的局部和全身细胞免疫反应，并且本研究中开发的流式细胞术测定法可用于分析针对 AIV 和其他禽传染性疾病的细胞免疫反应，并确定免疫保护的相关性。
