Background Influenza vaccination and bacterial colonization both shape immunity in the respiratory tract, yet their combined impact on the human lung mucosa remains poorly understood. Secondary bacterial pneumonia following influenza infection is a leading cause of mortality, underscoring the need to define how vaccines and microbes intersect at the airway interface.

Methods Using the Experimental Human Pneumococcal Challenge (EHPC) model, we examined how intramuscular inactivated (TIV) and nasal live attenuated (LAIV) influenza vaccines, with or without Streptococcus pneumoniae colonization, modulate lower airway immunity. Bronchoalveolar lavage samples from 22 adults were profiled by single-cell RNA-seq (>40,000 cells), flow cytometry, cytokine multiplexing, and macrophage functional assays.

Findings LAIV recipients who became colonized with S. pneumoniae displayed heightened influenza-specific CD4□ T cell responses and enhanced alveolar macrophage (AM) opsonophagocytic activity, showing that nasal bacterial colonization can act as natural mucosal adjuvant. Single-cell transcriptomics revealed four AM gene modules; among them, an interferon-driven “anti-microbial” program correlated with enhanced phagocytosis, whereas a complement- and antigen-presentation module associated with IFNγ-iNOS/ROS signaling was attenuated in colonized vaccinees. Given that AMs are poor antigen-presenting cells, this shift likely reflects reprogramming toward cytokine-mediated immune modulation rather than direct T cell activation. The elevated influenza-specific CD4□ T cell responses may instead represent feedback from enhanced local activation. Together, these data indicate that vaccination and colonization synergize to rewire AM-T cell communication, fine-tuning antiviral and antibacterial defenses. Similar transcriptional perturbations in public COVID-19 and lung cancer datasets underscore the broader relevance of these macrophage modules across lung disease contexts.

Conclusions Our findings define how influenza vaccination and pneumococcal colonization converge in the human lung to reprogram AM-T cell crosstalk, enhancing local immune responses and protective immunity. By uncovering conserved macrophage modules and mechanisms that shape mucosal defense, this study provides a framework for designing next-generation respiratory vaccines and strategies to mitigate post-viral bacterial pneumonia.