C96-04 In Vivo Cytometry Enables Real Time Quantification of Immune Cell Dynamics During Respiratory Infections

Abstract Rationale Immune cells rapidly respond to respiratory pathogens, and their circulating concentrations change dynamically based on bone marrow release, proliferation, and recruitment to the lung and other impacted organs. Understanding these temporal dynamics is critical for therapeutic intervention and immunology research. However, conventional techniques rely on terminal or sparse sampling, preventing continuous immune monitoring. Other in vivo cytometry platforms have been developed, but mice must be restrained and anesthetized, limiting measurements to short time periods. Additionally, anesthesia has immunomodulatory effects. To overcome these challenges, we developed an in vivo cytometry platform for continuous immune cell imaging in awake, freely moving mice for up to 5 days. Here, we demonstrate the application of this technology by quantifying leukocyte (CD45+) and neutrophil (Ly6G+) concentrations during a terminal Streptococcus pneumoniae respiratory infection. In ongoing work, we will investigate how aging and immune experience affect circulating immune dynamics during respiratory infections. Methods Catheters were placed into the carotid artery and jugular vein, enabling continuous blood access. After recovery, mice were connected to the in vivo cytometry platform (Fig. 1A-1B) where fluorescently labeled circulating immune cells were imaged continuously using confocal microscopy. Neutrophils and leukocytes were labeled using anti-Ly6G antibody and CD45 nanobody respectively. Results We quantified leukocyte (CD45+) and neutrophil (Ly6G) dynamics during S. pneumoniae infection for 24 hours. In our preliminary study, we found both leukocyte and neutrophil concentrations fluctuated and exhibited distinct peaks and troughs. Three clear peaks in cell concentrations emerged for both populations at ∼0-, 6- and 15-hours post-infection (Fig. 1C), likely reflecting heightened bone marrow and splenic production and mobilization, with the largest peak occurring immediately after infection. For leukocytes, the second and third peaks were 1.36 and 3.30-fold lower than the initial peak. For neutrophils, both later peaks were 1.18-fold lower than the initial response. Conclusions This method enables real time immune monitoring throughout the entire course of respiratory infections that cannot be achieved through existing methods. Here, we demonstrate how leukocyte and neutrophil circulating populations increase and decrease throughout a terminal S. pneumoniae infection. In ongoing work, we examine how immune experience, and aging impact the responses of these cells and others, as well as comparing the immune response of different respiratory infections, such as influenza. This abstract is funded by: DP2HL168562, Beckman Young Investigator Award, NSF CAREER Award, NSFGRFP