Poster Presentation: Lauren Castle

ABSTRACT

In Vivo Cytometry Enables Real Time Quantification of Immune Cell Dynamics

Lauren Castle, Linzheng Shi, Jessica Daher, Andrew Tsao, Bradley E. Hiller, William Polacheck, Joseph P. Mizgerd, Hadi T. Nia

Objective: Immune cells rapidly respond to pathogens or injury, and their circulating concentrations change dynamically based on bone marrow release, proliferation, and recruitment to the site of infection or injury. 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. 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 and neutrophil concentrations during Streptococcus pneumoniae respiratory infection. In ongoing work, we will investigate how aging and immune experience affect circulating immune dynamics during respiratory infections.

Materials/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 using confocal microscopy. Neutrophils and leukocytes were labeled using anti-Ly6G antibody and CD45 nanobody respectively.

Results/Conclusions: We quantified leukocyte (CD45+) and neutrophil (Ly6G) dynamics during S. pneumoniae infection, identifying three peaks in cell concentration 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. Impact: This method enables high temporal resolution immune monitoring that cannot be achieved through standard methods. Ongoing