Murine Lymph Node Research Articles

Approbation of a Homologous Model of the Antitumor Vaccine Based on Mature Mouse Dendritic Cells to Study the Biodistribution of the Cell Product.

Homologous animal cell product was obtained in protocol developed for female BALB/c mice. Dendritic cell (DC) migration from the injection site into the draining lymph nodes was evaluated. The number of DC labeled with carboxyfluorescein succinimidyl ester (CFSE) in draining lymph nodes increased from 5.3% (16 h) to 13.3% (48 h) (p=0.028) with a maximum at 72 h (15.4%, p=0.003). The immunophenotype of CFSE-DC detected in murine lymph nodes corresponded to the immunophenotype of mature vaccine DCs: they expressed differentiation markers CD11c, CD80, CD83, and CD86 (p>0.05 vs initial DC).

A 3D-printed multi-compartment organ-on-chip platform with a tubing-free pump models communication with the lymph node.

Multi-organ-on-chip systems (MOOCs) have the potential to mimic communication between organ systems and reveal mechanisms of health and disease. However, many existing MOOCs are challenging for non-experts to implement, due to complex tubing, electronics, or pump mechanisms. In addition, few MOOCs have incorporated immune organs such as the lymph node (LN), limiting their applicability to critical events such as vaccination. Here we developed a 3D-printed, user-friendly device and companion tubing-free impeller pump with the capacity to co-culture two or more tissue samples, including a LN, under a recirculating common media. Native tissue structure and immune function were incorporated by maintaining slices of murine LN tissue ex vivo in 3D-printed mesh supports for at least 24 hr. In a two-compartment model of a LN and an upstream injection site in mock tissue, vaccination of the multi-compartment chip was similar to in vivo vaccination in terms of locations of antigen accumulation and acute changes in activation markers and gene expression in the LN. We anticipate that in the future, this flexible platform will enable models of multi-organ immune responses throughout the body.

Spatial Patterning Analysis of Cellular Ensembles (SPACE) discovers complex spatial organization at the cell and tissue levels.

Spatial patterns of cells and other biological elements drive both physiologic and pathologic processes within tissues. While many imaging and transcriptomic methods document tissue organization, discerning these patterns is challenging, especially when they involve multiple elements in complex arrangements. To address this challenge, we present Spatial Patterning Analysis of Cellular Ensembles (SPACE), an R package for analysis of high-plex spatial data. SPACE is compatible with any data collection modality that records values (i.e., categorical cell/structure types or quantitative expression levels) at fixed spatial coordinates (i.e., 2d pixels or 3d voxels). SPACE detects not only broad patterns of co-occurrence but also context-dependent associations, quantitative gradients and orientations, and other organizational complexities. Via a robust information theoretic framework, SPACE explores all possible ensembles of tissue elements - single elements, pairs, triplets, and so on - and ranks the most strongly patterned ensembles. For single images, rankings reflect patterns that differ from random assortment. For sets of images, rankings reflect patterns that differ across sample groups (e.g., genotypes, treatments, timepoints, etc.). Further tools then thoroughly characterize the nature of each pattern for intuitive interpretation. We validate SPACE and demonstrate its advantages using murine lymph node images for which ground truth has been defined. We then use SPACE to detect new patterns across varied datasets, including tumors and tuberculosis granulomas.

Enhanced Proteomic Coverage in Tissue Microenvironment by Immune Cell Subtype Library-Assisted DIA-MS

Immune cells that infiltrate the tumor microenvironment (TME) play crucial roles in shaping cancer development and influencing clinical outcomes and therapeutic responses. However, obtaining a comprehensive proteomic snapshot of tumor-infiltrating immunity in clinical specimens is often hindered by small sample amounts and a low proportion of immune infiltrating cells in the TME. To enable in-depth and highly sensitive profiling of microscale tissues, we established an immune cell-enriched library-assisted strategy for data-independent acquisition mass spectrometry (DIA-MS). Firstly, six immune cell subtype-specific spectral libraries were established from sorted cluster of differentiation markers, CD8+, CD4+ T lymphocytes, B lymphocytes, natural killer cells, dendritic cells, and macrophages in murine mesenteric lymph nodes (MLNs), covering 7815 protein groups with surface markers and immune cell-enriched proteins. The feasibility of microscale immune proteomic profiling was demonstrated on 1 μg tissue protein from the tumor of murine colorectal cancer (CRC) models using single-shot DIA; the immune cell-enriched library increased coverage to quantify 7419 proteins compared to directDIA analysis (6978 proteins). The enhancement enabled the mapping of 841 immune function-related proteins and exclusive identification of many low-abundance immune proteins, such as CD1D1, and CD244, demonstrating high sensitivity for immune landscape profiling. This approach was used to characterize the MLNs in CRC models, aiming to elucidate the mechanism underlying their involvement in cancer development within the TME. Even with a low percentage of immune cell infiltration (0.25–3%) in the tumor, our results illuminate downregulation in the adaptive immune signaling pathways (such as C-type lectin receptor signaling, and chemokine signaling), T cell receptor signaling, and Th1/Th2/Th17 cell differentiation, suggesting an immunosuppressive status in MLNs of CRC model. The DIA approach using the immune cell-enriched libraries showcased deep coverage and high sensitivity that can facilitate illumination of the immune proteomic landscape for microscale samples.

Modeling Structural Elements and Functional Responses to Lymphatic-Delivered Cues in a Murine Lymph Node on a Chip.

Lymph nodes (LNs) are organs of the immune system, critical for maintenance of homeostasis and initiation of immune responses, yet there are few models that accurately recapitulate LN functions in vitro. To tackle this issue, an engineered murine LN (eLN) has been developed, replicating key cellular components of the mouse LN; incorporating primary murine lymphocytes, fibroblastic reticular cells, and lymphatic endothelial cells. T and B cell compartments are incorporated within the eLN that mimic LN cortex and paracortex architectures. When challenged, the eLN elicits both robust inflammatory responses and antigen-specific immune activation, showing that the system can differentiate between non specific and antigen-specific stimulation and can be monitored in real time. Beyond immune responses, this model also enables interrogation of changes in stromal cells, thus permitting investigations of all LN cellular components in homeostasis and different disease settings, such as cancer. Here, how LN behavior can be influenced by murine melanoma-derived factors is presented. In conclusion, the eLN model presents a promising platform for in vitro study of LN biology that will enhance understanding of stromal and immune responses in the murine LN, and in doing so will enable development of novel therapeutic strategies to improve LN responses in disease.

Antigen persistence following mRNA vaccination and the immune consequence of lymph node stromal cell delivery

Abstract mRNA vaccines have demonstrated their usefulness on a global scale in recent years. They elicit potent humoral and cell mediated responses, but the specific mechanisms by which these responses are produced still require investigation. Our lab described a process termed antigen archiving, in which antigen is retained within lymph node stromal cell (LNSC) subsets following viral infection and subunit vaccination to bolster/prolong memory CD8 T cell responses. Whether mRNA vaccines utilize this mechanism to promote immunity has yet to be determined. Data suggest mRNA-derived antigen persists within LNSC of C57/BL6 mice for at least 3 wks following subcutaneous or intramuscular delivery. We investigate the immune consequence of this prolonged antigen retention using ionizable lipid nanoparticles (LNP) to deliver mRNA constructs encoding the model antigen ovalbumin as well as a fluorescent siinfekl-GFP to monitor antigen-specific T-cell responses and localization of antigen within murine lymph nodes (LNs). We identified LNP formulations facilitating differential uptake/translation within LNSC which also demonstrated robust antigen-specific CD8 T cell responses and persistence of antigen presentation within LNs for at least 3 wks in vivo. We plan to interrogate the immune consequences of enhanced stromal cell targeting and subsequent antigen retention following mRNA vaccination. A better understanding of mRNA and protein delivery and retention will inform future vaccine design.

Ce3D-IBEX: Achieving Multiplex 3-dimensional Imaging for Deep Phenotyping of Cells in Tissues

Abstract 2D multiplexed antibody-based imaging provides a framework to study cell-cell interactions in thin tissue sections but lacks the ability to interrogate spatial relationships in larger 3D micro-anatomical structures. Several methods for 3D imaging of optically cleared tissue exist, but current approaches do not provide a deep phenotyping capability as afforded by high content 2D imaging. Here,we developed a method to overcome this constraint by combining a fast hydrophilic tissue clearing technique, clearing-enhanced 3D (Ce3D), and the recently developed Iterative Bleaching Extends Multiplexity (IBEX) technique together with a robust image registration algorithm suite to create a highly multiplex 3D imaging method: Ce3D-IBEX. It has been used to analyze murine lung, lymph nodes, small intestine, retina, tumor, and cornea as well as human retina, kidney, liver, tonsil, and jejunum, yielding seamless views of large-scale tissue structures while enabling visualization of high dimensional phenotypic markers at single-cell resolution, permitting the identification of discrete cell subsets, quantification of protein expression level and their mutual spatial association within tissue structures. To date, 25+ markers have been visualized in a single 3D sample. This represents a major advance in the emerging field of highly multiplexed 3D imaging. With aims to open new avenues for characterization of spatial dynamics of immune cells in healthy and diseased tissue.

The temporal expression pattern of classical MHC class I in sleep-restricted mice: Generalizations and broader implications

The intricate relationship between sleep and leukocyte trafficking has garnered intense attention, particularly their homing dynamics to secondary lymphoid organs under normal and restricted sleep (SR). Considering the scarcity of information regarding circadian rhythms in major histocompatibility class I (MHC-I) expression in SR, we designed a study that assessed the temporal expression of MHC-I in murine lymph nodes and spleen and the subsequent effects of sleep recovery. Male C57BL/6, housed in 12:12 light/dark cycle, were grouped into control (C) and SR. SR was carried for one week before lymphoid tissues were sampled at selected time points and assessed for leukocyte number and MHC-I expression.SR resulted in 21% decrease in granulocyte and 24% increase in agranulocyte numbers.In C, MHC-I expression pattern in lymph nodes was bimodal and relatively higher than splenocytes during the animal’s active phase (110.2 ± 1.8 vs 81.9 ± 3.8, respectively; p = 0.002). Splenocytes; however, showed a bimodal pattern upon SR, with higher protein levels during the rest than the activity period (154.6 + 36.2 vs 99.5 + 15.9, respectively; p = 0.002), suggesting preparedness for a potential infection. Furthermore, SR caused a significant drop in MHC-I expression at the onset of rest with 57% and 30% reduction in lymph nodes and splenocytes, respectively. However, the overall protein expression collectively taken from both lymphoid tissues remained stable, emphasizing its indispensable role in immunological homeostasis. This stability coincided with the restoration of protein levels to baseline after a short sleep recovery period, resembling a reset for MHC-I antigen presentation following a week of SR.Understanding the interplay between MHC-I expression and contextual factors could enhance treatment protocols, refining the efficacy and time precision of glucocorticoid-based therapies in immune modulation.

Multiscale elasticity mapping of biological samples in 3D at optical resolution

The mechanical properties of biological tissues have emerged as an integral determinant of tissue function in health and disease. Nonetheless, characterizing the elasticity of biological samples in 3D and at high resolution remains challenging. Here, we present a µElastography platform: a scalable elastography system that maps the elastic properties of tissues from cellular to organ scales. The platform leverages the use of a biocompatible, thermo-responsive hydrogel to deliver compressive stress to a biological sample and track its resulting deformation. By surrounding the specimen with a reference hydrogel of known Young's modulus, we are able to map the absolute values of elastic properties in biological samples. We validate the experimental and computational components of the platform using a hydrogel phantom and verify the system's ability to detect internal mechanical heterogeneities. We then apply the platform to map the elasticity of multicellular spheroids and the murine lymph node. With these applications, we demonstrate the platform's ability to map tissue elasticity at internal planes of interest, as well as capture mechanical heterogeneities neglected by most macroscale characterization techniques. The µElastography platform, designed to be implementable in any biology lab with access to 3D microscopy (e.g., confocal, multiphoton, or optical coherence microscopy), will provide the capability to characterize the mechanical properties of biological samples to labs across the large community of biological sciences by eliminating the need of specialized instruments such as atomic force microscopy. Statement of significanceUnderstanding the elasticity of biological tissues is of great importance, but characterizing these properties typically requires highly specialized equipment. Utilizing stimulus-responsive hydrogels, we present a scalable, hydrogel-based elastography method that uses readily available reagents and imaging modalities to generate resolved maps of internal elasticity within biomaterials and biological samples at optical resolution. This new approach is capable of detecting internal stiffness heterogeneities within the 3D bulk of samples and is highly scalable across both imaging modalities and biological length scales. Thus, it will have significant impact on the measurement capabilities of labs studying engineered biomaterials, mechanobiology, disease progression, and tissue engineering and development.

Spatial Distribution of Non-Immune Cells Expressing Glycoprotein A Repetitions Predominant in Human and Murine Metastatic Lymph Nodes

Several types of cancer spread through the lymphatic system via the sentinel lymph nodes (LNs). Such LN-draining primary tumors, modified by tumor factors, lead to the formation of a metastatic niche associated with an increased number of Foxp3+ regulatory T cells (Tregs). These cells are expected to contribute to the elaboration of an immune-suppressive environment. Activated Tregs express glycoprotein A repetitions predominant (GARP), which binds and presents latent transforming growth factor beta 1 (TGF-β1) at their surface. GARP is also expressed by other non-immune cell types poorly described in LNs. Here, we mapped GARP expression in non-immune cells in human and mouse metastatic LNs. The mining of available (human and murine) scRNA-Seq datasets revealed GARP expression by blood (BEC)/lymphatic (LEC) endothelial, fibroblastic, and perivascular cells. Consistently, through immunostaining and in situ RNA hybridization approaches, GARP was detected in and around blood and lymphatic vessels, in (αSMA+) fibroblasts, and in perivascular cells associated with an abundant matrix. Strikingly, GARP was detected in LECs forming the subcapsular sinus and high endothelial venules (HEVs), two vascular structures localized at the interface between LNs and the afferent lymphatic and blood vessels. Altogether, we here provide the first distribution maps for GARP in human and murine LNs.

A Dissection Method to Harvest Murine Popliteal Draining Lymph Nodes for Ex Vivo Imaging

A Dissection Method to Harvest Murine Popliteal Draining Lymph Nodes for Ex Vivo Imaging

A Dissection Method to Harvest Murine Popliteal Draining Lymph Nodes for Ex Vivo Imaging

A Dissection Method to Harvest Murine Popliteal Draining Lymph Nodes for Ex Vivo Imaging

Lymph node sharing between pancreas, gut, and liver leads to immune crosstalk and regulation of pancreatic autoimmunity

Lymph nodes (LNs) are critical sites for shaping tissue-specific adaptive immunity. However, the impact of LN sharing between multiple organs on such tailoring is less understood. Here, we describe the drainage hierarchy of the pancreas, liver, and the upper small intestine (duodenum) into three murine LNs. Migratory dendritic cells (migDCs), key in instructing adaptive immune outcome, exhibited stronger pro-inflammatory signatures when originating from the pancreas or liver than from the duodenum. Qualitatively different migDC mixing in each shared LN influenced pancreatic β-cell-reactive Tcells to acquire gut-homing and tolerogenic phenotypes proportional to duodenal co-drainage. However, duodenal viral infections rendered non-intestinal migDCs and β-cell-reactive Tcells more pro-inflammatory in all shared LNs, resulting in elevated pancreatic islet lymphocyte infiltration. Our study uncovers immune crosstalk through LN co-drainage as a powerful force regulating pancreatic autoimmunity.

Identifying Regulatory T Cell Subsets from Murine Pancreatic Draining Lymph Nodes

Identifying Regulatory T Cell Subsets from Murine Pancreatic Draining Lymph Nodes

Identifying Regulatory T Cell Subsets from Murine Pancreatic Draining Lymph Nodes

Identifying Regulatory T Cell Subsets from Murine Pancreatic Draining Lymph Nodes

Sirtuin 6 is a negative regulator of the anti-tumor function of natural killer cells in murine inflammatory colorectal cancer

The immune system plays a crucial role in controlling colorectal cancer (CRC) development. Natural killer (NK) cells are tumoricidal but undergo exhaustion in CRC patients. The current research aims to understand the role of sirtuin 6 (SIRT6) in CRC-associated NK cell exhaustion in a murine inflammatory colorectal cancer model. To this end, inflammatory CRC was induced by treating mice with azoxymethane plus dextran sulfate sodium. The expression of SIRT6 in NK cells in murine mesenteric lymph nodes (mLNs) and the CRC tissue was characterized by Immunoblotting. SIRT6 knockdown was achieved by lentiviral transduction of murine splenic NK cells, followed by evaluation of NK cell proliferation and the expression of cytotoxic mediators using flow cytometry. NK cell cytotoxicity was measured by cytotoxicity assays. Adoptive transfer of murine NK cells was applied to analyze the effect of SIRT6 knockdown in vivo. We found that SIRT6 was up-regulated in infiltrating NK cells in the murine CRC tissue, especially NK cells with an exhausted phenotype and impaired cytotoxicity. SIRT6 knockdown significantly boosted murine splenic NK cell functionality, as evidenced by accelerated proliferation, increased production of cytotoxic mediators, and higher tumoricidal activity both in vitro and in vivo. Furthermore, the adoptive transfer of SIRT6-knockdown NK cells into CRC-bearing mice effectively suppressed CRC progression. Therefore, SIRT6 up-regulation is essential for murine NK cell exhaustion in CRC because it impedes the tumoricidal activity of murine NK cells. Artificial SIRT6 down-regulation could boost the function of infiltrating NK cells to oppress CRC progression in mice.

Dictionary of immune responses to 86 cytokines in vivoat single-cell resolution

Abstract Cytokines mediate cell-cell communication in the immune system and represent important therapeutic targets. A myriad of studies have underscored their central role in immune function, yet we lack a global view of the cellular responses of each immune cell type to each cytokine. To address this gap we created the Immune Dictionary – a compendium of single-cell transcriptomic profiles of over 20 cell types in response to each of 86 cytokines in murine lymph nodes in vivo, representing over 1,500 cytokine-cell type combinations. A cytokine-centric view of the dictionary revealed that most cytokines induced highly cell type-specific responses. For example, the inflammatory cytokine IL-1β induced distinct gene programs in almost every cell type. A cell type-centric view of the dictionary identified multiple cytokine-driven cellular polarization states in each immune cell type, including previously uncharacterized states such as an IL-18-induced polyfunctional NK cell state. Based on the dictionary, we developed companion software, Immune Response Enrichment Analysis (IREA), for assessing cytokine activities and immune cell polarization from gene expression data, and applied it to reveal cytokine networks in tumors following immune checkpoint blockade therapy. Our dictionary generates new hypotheses for cytokine functions, illuminates pleiotropic effects of cytokines, expands our knowledge of activation states of each immune cell type, and provides a framework to deduce the roles of specific cytokines and cell-cell communication networks in any immune response. This work was supported by the NIH Grant RM1HG006193 and an Adelson Medical Research Foundation Grant to N.H. This work was also supported by a Natural Sciences and Engineering Research Council of Canada (NSERC) Doctoral Fellowship, Whitaker Health Sciences Fund Fellowship, and Wellington and Irene Loh Fund Fellowship to A.C., and NCI Research Specialist Award (R50CA251956) to S.L.

Age-associated changes to lymph node architecture shape T cell responses

Abstract Aging dramatically impacts T cells, contributing to diminished and delayed responses to infection. While the development and maintenance of lymphocytes certainly undergo intrinsic changes with age, the contribution of cell-extrinsic forces, such as the aging tissue microenvironment, has been difficult to uncouple. Macroscopic changes to the lymph nodes have been well described, but the impact of aging on lymph node stromal subsets is less understood. Here, we visualized naïve T cell migration in the aged murine lymph node using live-cell 2-photon microscopy and determined that T cell motility was impaired in regions of increased fibrotic deposition. Using fluorescent reporters for Ccl19 and Cxcl12, homeostatic chemokines important for stromal:lymphocyte crosstalk, we also found that the distribution of stromal cells expressing these chemokines was altered with age, and that migrating naïve T cells were excluded from regions of chemokine expression. Flow cytometric analyses of fibroblastic reticular cells (FRCs) revealed decreased proportions of FRCs within the mesenteric lymph nodes and decreased putative FRC progenitors in the skin-draining lymph nodes. FRC progenitors at both sites had reduced surface expression of lymphotoxin-beta receptor, an important receptor for FRC differentiation and maintenance. We hypothesize that age-associated changes to FRC maturation contribute to increased lymph node fibrosis and altered localization of homeostatic factors. Current studies have indicated that in vitro cultured lymph node fibroblasts are sensitive to TNF-alpha and respond by increasing expression of alpha-smooth muscle actin, suggesting a mechanism by which aging remodels the lymph node microenvironment.

Suppression of adaptive immunity by Borrelia burgdorferiin the murine host

Abstract Borrelia burgdorferi (Bb), the spirochetal agent of Lyme disease, utilizes a variety of strategies to evade and suppress the host immune response, which enable it to persist chronically. These strategies include complement inhibition, antigenic variation, and suppression of adaptive immunity. Previous studies have shown that lymph node (LN) germinal centers generated in response to Bb collapse one-month post-infection. The resulting immune response is characterized by unusually strong IgM production and a lack of long-lived immunity. Here we aimed to better characterize how Bb manipulates host immunity, ultimately resulting in failure to clear the infection. Mice infected with Bb and concomitantly immunized with recombinant SARS-CoV-2 spike protein had an abrogated antibody response to the immunization. To test how long this humoral immune suppression lasts, mice were immunized at 2-, 4-, and 6-weeks post Bb infection. Suppression of host antibody production against SARS-CoV-2 spike protein peaked at 2 weeks post Bb infection but continued for all timepoints measured. We also examined changes to gene expression in murine LNs 15 days post-infection using RT-qPCR and found that live Bb, but not heat-killed Bb, broadly suppressed many genes related to T cell homing and function. Genes which play a critical role in the establishment and maintenance of T cell zones within the LNs, Ccl19 and Ccr7, were significantly downregulated. In addition, both Il2 and Il2ra were downregulated, which is typically observed during the resolution of infection and indicates a potential lack of antigenic restimulation. These data suggest Bb may disrupt T cell responses, which could play a role in the failure to mount an appropriate humoral immune response. Funding for this work was provided by UNTHSC.

Characterization of murine lymphocyte activation and exhaustion markers by a 14-color flow cytometry panel.

Previously designed flow cytometry panels have provided a framework to analyze T-cell activation; however, few provide an extensive view of lymphocyte populations, and none are optimized for murine models. This article describes a panel designed specifically to assess the expression of activation and exhaustion markers in expanding lymphocyte populations in tumor-bearing mice across two distinct genetic backgrounds: BALB/c and C57BL/6. This comprehensive panel enables the assessment of multiple functional states and immune checkpoint markers across cytotoxic CD8+ T cells, helper and regulatory CD4+ T cells and NK cells in murine whole blood, lymph nodes and tumor.