Tissue resident host responses to microbial infections in the respiratory tract are highly dynamic in space and time and rely on the interaction of a multitude of cell types. To model these multicellular responses reliably in cell culture, we compare here the global transcriptional antimicrobial response to infection with influenza A virus (IAV) in precision cut lung slices (PCLS), volume-defined organ discs largely maintaining the cellular composition and 3D architecture of the donor lung. To permit a fair comparison of host responses in an isogenic background, we first challenged mice <italic toggle="yes">in vivo</i> and murine PCLS (mPCLS) and assessed host transcriptomic changes by unbiased RNA-sequencing. While core antiviral responses overlapped substantially, mPCLS lacked certain features—such as type II interferon expression—likely due to the absence of infiltrating immune cell responses. Importantly, when expanding our findings to immune-experienced human precision cut lung slices (hPCLS), we find a much broader antiviral response after IAV challenge, including types I, II, and III interferons, suggesting the presence of responsive tissue resident lymphocytes. To prove the specificity of this response, we infected hPCLS with <italic toggle="yes">Streptococcus pneumoniae. Ex vivo</i> tissues responded with a distinct proinflammatory gene profile including IL1A, IL1B, and IL17 expression. Blocking of IL-1β signaling partially inhibited the proinflammatory response, suggesting cellular cross talk and a complex and specific antimicrobial reaction in this <italic toggle="yes">ex vivo</i> model. In conclusion, a diversified tissue resident immune cell compartment distinguishes the human <italic toggle="yes">ex vivo</i> model, making it an ideal system for microbiological and immunological research.

IMPORTANCE : Pathogen interactions with the lungs are very dynamic processes. In biomedical research, it is paramount to model these processes in the laboratory as accurately as possible. Influenza A virus has been extensively studied in epithelial cell culture models, including advanced organoids and organ-on-a-chip systems. Here, we use ex vivo cultured precision cut lung slices (PCLS) and generate transcriptomic data to assess the global tissue resident host response to viral and bacterial challenges. Our data show (i) that murine PCLS faithfully reflect core responses to viral infection, while missing proinflammatory responses linked to infiltrating immune cells and (ii) that human PCLS show a highly diversified tissue resident immune response to viral infection due to previous exposures of the host to this pathogen. These responses are clearly distinct from antibacterial gene profiles. Our data advertise PCLS as a complex and realistic model to study tissue resident immune responses to microbes in a human system.