Comprehensive and Automated Mouse Immune Profiling with CyTOF Technology Reveals Aging-Associated Alterations in Lymphoid and Pulmonary Tissues 2259224

Abstract Introduction Immunosenescence disrupts surveillance and contributes to age-related diseases, yet its mechanisms remain incompletely understood. Deep immune profiling in mouse models is essential to elucidate age-related modulators of disease. High-parameter cytometry with CyTOF™ technology enables identification of 50-plus markers at the single-cell level, providing exceptional resolution without spectral unmixing or compensation. This study leverages CyTOF technology to characterize aging-associated immune alterations in spleen and lung tissues of aged and young adult mice. Methods Aged (75—76 weeks) and young adult (6—8 weeks) C57BL/6J mice were evaluated. Spleen and lung tissues were processed and stained using the 33-antibody Maxpar™ OnDemand Mouse Immune Profiling Panel Kit for spleen and an expanded customized 50-parameter panel for lung. Live-cell CD45 barcoding enabled pooled sample analysis. Data was acquired on a Helios™ CyTOF instrument and analyzed using Maxpar Pathsetter software for automated identification of 38 splenic and 55 pulmonary immune subsets. Results In spleen, aged mice showed reduced frequencies of CD4 and CD8 T cells, NK cells and γδ T cells, with increased PD-1+ T cells and B cells. NK cell maturation was impaired, with altered expression of PD-L1, CD11c and CD49b. In lungs, aged mice exhibited higher frequencies of exhausted CD8 T cells (PD-1+TIM-3+), cytotoxic T cells (granzyme B+) and monocyte-derived dendritic cells, alongside a pro-inflammatory alveolar macrophage phenotype. Overall, 26 splenic and 28 pulmonary subsets showed significant age-related differences. Conclusion This study demonstrates the power of CyTOF immune profiling with automated analysis to uncover aging-associated immune alterations. The Mouse Immune Profiling Panel and Maxpar Pathsetter software provide a scalable and reproducible solution for deep phenotyping in mouse models, facilitating mechanistic insights into immunosenescence and supporting translational research in aging and immune modulation. Funding Source n/a Topic Categories Immune Response Regulation: Cellular Mechanisms (IRC)