Single cell in vivo analysis of type I IFN and NK cell-mediated control of B cell infection densities during acute gammaherpesvirus infection

Immunodeficient patients are at risk of severe complications following infection with the human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposis sarcoma-associated herpesvirus (KSHV). Due to the strict host specificity of human gammaherpesviruses, murine gammaherpesvirus 68 (MHV68) is widely used as an in vivo model to study gammaherpesvirus pathogenesis. While type I interferons (IFNs) and natural killer (NK) cells are known to contribute to antiviral defense during MHV68 infection, how these innate immune mechanisms control infection within lymphoid tissues at the level of individual infected cells remains incompletely understood. Here, we used an MHV68-DsRed reporter virus to visualize infected cells in the lymph nodes of wildtype and type I IFN receptor-deficient (Ifnar1-/-) mice by flow cytometry, immunohistochemistry, and two-photon microscopy. We found that type I IFN signaling plays a major role in limiting both local viral infection in draining lymph nodes and systemic dissemination to the spleen during acute infection. In the absence of IFNAR signaling, infected B cells accumulated to higher numbers, including an increased proportion of infected germinal center phenotype B cells, and this was accompanied by enhanced activation of T and B lymphocytes. Using two-photon microscopy, we further examined NK cell behavior within infected lymph nodes. NK cells were rapidly recruited to sites of infection but did not form stable clusters or prolonged contacts with infected cells. Nevertheless, NK cell depletion resulted in increased numbers of infected cells, indicating that NK cells contribute to the control of acute MHV68 infection despite the absence of detectable long-lasting interactions with infected cells. Together, our findings provide a single-cell view of acute gammaherpesvirus infection and innate immune control within lymph nodes in vivo. These data refine our understanding of how type I IFN responses and NK cells restrict early gammaherpesvirus spread and shape infection dynamics within lymphoid tissues. Author SummaryCertain viruses can remain in the body for life and cause little harm in healthy individuals. However, when the immune system is weakened, these infections can lead to cancer or other life-threatening diseases. To study human gammaherpesviruses within the living body, scientists often use a closely related mouse virus to understand how the immune system controls these infections. In this study, we tracked virus-infected cells within lymph nodes, important sites of the immune responses. Using a fluorescent virus together with advanced imaging techniques, we visualized infected cells directly in intact tissues. We found that type I interferons, a key component of the early antiviral defense, strongly limited the number of infected cells. Mice that could not respond to type I interferons developed substantially larger infections. We also investigated the role of natural killer cells, immune cells that provide rapid protection against viral infections. Although these cells quickly accumulated at sites of infection, they rarely formed prolonged contacts with infected cells. Together, our findings provide a detailed view of how innate immune defenses restrict gammaherpesvirus infection within lymph nodes. By revealing how early immune responses limit viral spread, this work improves our understanding of mechanisms that protect against severe herpesvirus-associated disease.