The role of pulmonary endothelial cell antigen presentation in sequential influenza infection

Summary: Background and Significance: The increasing prominence of highly pathogenic H5N1 avian influenza virus is a potential public threat given its high mortality. In conjunction with continued widespread H1N1 infection, it is imperative we understand any protection afforded from previous infection and the underlying mechanisms of sequential influenza infection. Our laboratory has demonstrated that endothelial cells (ECs), uniquely positioned between circulating immune cells and tissues, present vial antigen to T cells contributing to the pulmonary viral response. What remains a pervasive gap, is what role ECs play in immunological protection from sequential infections and the formation of immunological memory. Methods: We hypothesize that EC antigen presentation of H1N1 (A/Puerto Rico/8/1934(Cambridge)) antigens confers resistance to subsequent H5N1 (A/Texas/37/2024 rH5N1) infections by immune regulation of the adaptive immune system. Using reporter viruses and EC-specific MHC knockout mice (tdTomatofl/fl:B2mfl/fl:Cdh5-CreERT2 (tdT-B2mEC mice)), we will specifically evaluate the effects of sequential infections on ECs and immune cells through in vitro and in vivo assays. Results: Preliminary body weight and survival results show sequential infection of both H1N1-H1N1 and H1N1-H5N1 is physiologically protective compared to acute infection controls. Mice infected with H5N1 alone perished, while mice H1N1-H5N1 infected survived in a H5N1-dose dependent manner. Flow cytometry of ECs and CD8+ T cells from H1N1-H1N1 infected C57BL/6J indicates memory generation and decreased inflammatory EC response compared to acute infection. H1N1-H1N1 resulted in reduced EC VCAM-1 and ICAM-1 expression, and increased tetramer-specific CD8+ T resident memory (RM) and effector memory (EM) populations. Results from B2mEC-KO mice indicate ECs are important for survival and T cell memory formation. B2mEC-KO mice infected with H5N1 had decreased survival compared to controls. Flow cytometry results from H1N1 infected tdT-B2mEC mice showed an aberrant CD8+ inflammatory phenotype and reduced response specificity. At an acute infection timepoint, tetramer specific CD8+ populations and CD8+ cytotoxic (IFNy+ and Granzyme B+) markers decreased compared to B2m-WT controls. Conclusions: Preliminary results indicate ECs, important in generating a specific CD8+ T cell response during the acute infections, play a critical role in sequential H1N1-H5N1 infection by establishing or promoting CD8+ T memory populations during the first infection that generate immunity to subsequent infection. Future studies may better leverage the role of EC antigen presentation in generating immunological memory. Kamal Bagale, Yuanyun Ao, Jiayue Lu, Dejuanna Chan, Lianghui Zhang