Spatially Validated Human Lymphoid Follicles-on-Chip for Modeling Inflammaging and Age-Related Immune Dysfunction - PROJECT SUMMARY/ABSTRACT Aging is accompanied by a chronic, low-grade inflammatory state known as inflammaging, which drives immune dysfunction and contributes to systemic diseases such as vascular and metabolic disorders. Within lymphoid tissues, aging leads to spatial disorganization, loss of germinal center integrity, and altered immune composition. Recurrent pathogen challenges further amplify these processes, sustaining chronic inflammation. However, the mechanisms linking lymphoid tissue remodeling, chronic immune activation, and inflammation remain poorly understood, largely due to the absence of physiologically relevant human models. Preliminary data suggest that the proposed studies are built upon a robust and well-validated experimental foundation. We have established and functionally validated a lymphoid follicle organ-on-chip (LF-Chip) platform that recapitulates key hallmarks of human lymphoid function, including germinal center formation, AID expression, and antiviral antibody production. Controlled viral infection experiments demonstrate that the LF- Chip supports viral replication and immune activation, resulting in increased follicle number and size, confirming its ability to model physiologic immune responses. In addition, we have optimized workflows to integrate LF- Chip models with high-content spatial profiling platforms, ensuring compatibility with multiplex imaging and transcriptomic assays. In parallel, we developed IN-DEPTH, a same-slide spatial multi-omics workflow that enables integrated RNA and protein detection alongside spatial assays. These preliminary findings collectively establish the feasibility, technical maturity, and readiness of the proposed approach. In alignment with the RFA, this project will build upon the validated LF-Chip platform to model inflammaging by incorporating spatially profiled young and aged human lymphoid tissues, introducing pathogen-mimetic inflammatory stimulation, and functionally assessing its downstream impact on vascular dysfunction as a proof- of-concept. Together, these efforts will mechanistically link age, viral reactivation, and immune remodeling within a human- relevant model of inflammaging. Successful completion will yield the first validated lymphoid organ-on-chip model of immune aging, providing a transformative platform for dissecting pathways that drive immunosenescence and for testing strategies to mitigate chronic inflammation underlying age-related diseases.
