The lymph node (LN) is the main site where adaptive immunity is shaped, through education of B and T cells. LNs are highly structured lymphoid organs that compartmentalize B and...

The lymph node (LN) is the main site where adaptive immunity is shaped, through education of B and T cells. LNs are highly structured lymphoid organs that compartmentalize B and T cells in the outer cortex and inner paracortex, respectively, and are supported by a collagen-rich reticular network. Tissue material properties like viscoelasticity and diffusion of materials within extracellular spaces and their implications on cellular behavior have been a recent hot topic of investigation. Researchers have shown that mechanical properties of LNs are dependent on reticular network and extracellular matrix, and the LN mesh spacing has been estimated at 10 - 20 um. Here, we developed a nanoparticle system to investigate the rheological properties, including pore size and viscoelasticity, through multiple particle tracking (MPT) combined with LN slice cultures. We first determined that dense coatings with polyethylene glycol (PEG) are necessary to allow nanoparticles to diffuse within the extracellular spaces of the LNs. We demonstrate that despite differences in functionality, extracellular tissue properties and mesh spacing do not change significantly in the cortex and paracortex, where B and T cells are educated, respectively, though nanoparticle diffusion was slightly reduced in B cell zones, indicating a higher viscosity. Our studies also confirm that LNs exhibit viscoelastic properties, with an initial solid-like response followed by stress-relaxation at higher frequencies. Finally, we found that nanoparticle diffusion is dependent on LN location, with nanoparticles in skin draining LNs (sdLNs) exhibiting higher diffusion compared to mesenteric LNs (mLNs). This phenomenon is also reflected in the slightly reduced nanoparticle diffusion coefficient and pore size in mLNs. Our data shed new light onto LN interstitial tissue properties, pore size, and define surface chemistry parameters required for nanoparticles to diffuse within LN interstitium. These studies provide both a tool for studying LNs interstitium and design criteria for nanoparticles targeting LN interstitial spaces, which has recently received increasing attention.