Intravital three-photon microscopy allows visualization over the entire depth of mouse lymph nodes

Choe, Kibaek; Hontani, Yusaku; Wang, Tianyu; Hebert, Eric; Ouzounov, Dimitre G.; Lai, Kristine; Singh, Ankur; Béguelin, Wendy; Melnick, Ari; Xu, Chris

doi:10.1038/s41590-021-01101-1

Cited by 32 publications

(32 citation statements)

References 53 publications

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“…Studies have suggested that cell speed increases in fluids with higher viscosity [40,41]. Recent studies have found that T cell motility changes throughout the LN, and is potentially dependent on the region within which the T cell is found [42]. Interestingly, we have also observed that diffusion of nanoparticles may change throughout the depth of the LNs (Supplementary Figure 1), suggesting that viscosity may also change throughout the LN.…”

Section: Mpt Reveals Similar Pore Size and Viscosity Within Different...supporting

confidence: 54%

Multiple particle tracking (MPT) using PEGylated nanoparticles reveals heterogeneity within murine lymph nodes and between lymph nodes at different locations

Ramirez¹,

Merwitz²,

Lee³

et al. 2022

Preprint

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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.

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Section: Mpt Reveals Similar Pore Size and Viscosity Within Different...supporting

confidence: 54%

Multiple particle tracking (MPT) using PEGylated nanoparticles reveals heterogeneity within murine lymph nodes and between lymph nodes at different locations

Ramirez¹,

Merwitz²,

Lee³

et al. 2022

Preprint

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“…Humoral immunity against infectious diseases and vaccines is mediated by the B‐cell maturation process in secondary lymphoid organs such as lymph nodes, spleen, and tonsils 1 . Germinal center (GC) reactions are dynamic processes 2–9 that occur in B cell follicles of secondary lymphoid organs that convert a naïve B cell to long‐lived memory B cells (MBCs) and plasma cells 6,7 . MBCs are a unique subset of a heterogeneous population of activated, mature B cells that are multipotent and can rapidly diversify their B‐Cell receptor (BCR) repertoire compared to both terminally differentiated plasma cells and naïve B cells 6,7,10 .…”

Section: Introductionmentioning

confidence: 99%

Engineering early memory B‐cell‐like phenotype in hydrogel‐based immune organoids

Graney

Zhong

Post

et al. 2022

J Biomedical Materials Res

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Memory B cells originate in response to antigenic stimulation in B‐cell follicles of secondary lymphoid organs where naive B cells undergo maturation within a subanatomical microenvironment, the germinal centers. The understanding of memory B‐cell immunology and its regulation is based primarily on sophisticated experiments that involve mouse models. To date, limited evidence exists on whether memory B cells can be successfully engineered ex vivo, specifically using biomaterials‐based platforms that support the growth and differentiation of B cells. Here, we report the characterization of a recently reported maleimide‐functionalized poly(ethylene glycol) (PEG) hydrogels as immune organoids towards the development of early memory B‐cell phenotype and germinal center‐like B cells. We demonstrate that the use of interleukin 9 (IL9), IL21, and bacterial antigen presentation as outer membrane‐bound fragments drives the conversion of naive, primary murine B cells to early memory phenotype in ex vivo immune organoids. These findings describe the induction of early memory B‐cell‐like phenotype in immune organoids and highlight the potential of synthetic organoids as a platform for the future development of antigen‐specific bona fide memory B cells for the study of the immune system and generation of therapeutic antibodies.

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“…Profiling cell motility and other dynamic properties of cells as they move and interact within local microenvironments can provide critical insights into functional cell behaviors in the context of health and disease 3 . Furthermore, quantifying cell motility informs our understanding of many biological processes, including immune locomotion and surveillance 4,5 and cancer metastasis 6,7 . However, conventional cell motility data captured in vitro or in vivo are often performed on small numbers of homogeneous cells and tend to be reported as averaged values for populations of cells, e.g ., average speeds and average displacements.…”

Section: Introductionmentioning

confidence: 99%

Profiling Dynamic Patterns of Single-cell Motility

Maity

Kamat

Zamponi

et al. 2022

Preprint

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Cell motility is essential to many biological processes as cells navigate and interact within their local microenvironments. Currently, most methods to quantify cell motility rely on the ability to follow and track individual cells. However, results from these approaches are typically reported as averaged values across cell populations. While these approaches offer biological simplicity, it limits the ability to assess cellular heterogeneity and infer generalizable patterns of single-cell behaviors at baseline or after perturbations. Here, we present CaMI, a computational framework that takes advantage of the single-cell nature of cell motility data to identify and classify distinct spatio-temporal behaviors of single cells. Using CaMI, we demonstrate the ability to robustly classify single-cell motility patterns in a large dataset (n=74,253 cells), quantify spatio-temporal heterogeneities, determine motility patterns in unclassified cells, and provide a visualization scheme for direct interpretation of dynamic cell behaviors. Furthermore, as a biological proof of concept, we investigate the biphasic spatial and temporal responses of T-cell lymphoma cells moving in Collagen-I gels of varying collagen concentrations and predict the dimensionality (2D vs. 3D) of matched cellular conditions based solely on spatio-temporal heterogeneities. Together, we present a multivariate framework to robustly classify emergent patterns of single-cell behaviors, highlighting cellular heterogeneity as a critical feature that establishes the behaviors of cellular populations.

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Intravital three-photon microscopy allows visualization over the entire depth of mouse lymph nodes

Cited by 32 publications

References 53 publications

Multiple particle tracking (MPT) using PEGylated nanoparticles reveals heterogeneity within murine lymph nodes and between lymph nodes at different locations

Multiple particle tracking (MPT) using PEGylated nanoparticles reveals heterogeneity within murine lymph nodes and between lymph nodes at different locations

Engineering early memory B‐cell‐like phenotype in hydrogel‐based immune organoids

Profiling Dynamic Patterns of Single-cell Motility

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