Type I interferons (IFN-I) are central to antiviral immunity but can drive immunopathology when dysregulated. Plasmacytoid dendritic cells (pDCs) are the major source of IFN-I during viral infection; however, the mechanisms by which they recognize viruses and initiate IFN-I responses in vivo remain unresolved. Previous in vitro studies suggest that pDCs detect virus-infected cells, but physiological relevance of this recognition mechanism is unknown.

Here, we demonstrate that pDCs initiate IFN-I responses through contact-dependent recognition of infected cells rather than through direct infection of themselves. Utilizing a subcutaneous viral infection model, we studied the early IFN-I responses in skin-draining lymph nodes (dLNs). We found that subcapsular sinus (SCS) macrophages became infected but did not produce IFN-I, whereas the pDCs remained uninfected yet were the primary sources of early IFN-I. Notably, this pDC-mediated IFN-I response was specific, directional and spatially restricted within infected dLNs, suggesting tightly controlled local activation. Integrating confocal and intravital multiphoton microscopy with scRNA-seq, we identified contact-dependent interactions between pDCs and infected macrophages that drive IFN-I production by pDC. These findings provide direct in vivo evidence that pDCs detect infection through intercellular contacts, revealing that pDC activation is driven by intercellular sensing of infected cells rather than direct pathogen recognition. This unique mechanism may enable amplification of local antiviral responses while limiting systemic interferon exposure.

Collectively, our study uncovers a previously unrecognized contact-dependent immune surveillance by pDCs as a key regulator of the IFN-I responses, suggesting new strategies for selectively modulating interferon-driven pathology in viral infection and autoimmunity.