Once regarded as passive bystander cells of the tissue stroma, fibroblasts have emerged as active orchestrators of tissue homeostasis and disease. From regulating immunity and controlling tissue remodelling to governing cell growth and differentiation, fibroblasts assume myriad roles in guiding normal tissue development, maintenance and repair. By comparison, in chronic inflammatory diseases such as rheumatoid arthritis, fibroblasts recruit and sustain inflammatory leukocytes, become dominant producers of pro-inflammatory factors and catalyse tissue destruction. In other disease contexts, fibroblasts promote fibrosis and impair host control of cancer. Single-cell studies have uncovered striking transcriptional and functional heterogeneity exhibited by fibroblasts in both normal tissues and diseased tissues. In particular, advances in the understanding of fibroblast pathology in rheumatoid arthritis have shed light on pathogenic fibroblast states in other chronic diseases. The differentiation and activation of these fibroblast states is driven by diverse physical and chemical cues within the tissue microenvironment and by cell-intrinsic signalling and epigenetic mechanisms. These insights into fibroblast behaviour and regulation have illuminated therapeutic opportunities for the targeted deletion or modulation of pathogenic fibroblasts across many diseases.

Fibroblastic reticular cells (FRCs) are the master regulators of the lymph node (LN) microenvironment. However, the role of specific FRC subsets in controlling alloimmune responses remains to be studied. Single-cell RNA sequencing (scRNA-Seq) of naive and draining LNs (DLNs) of heart-transplanted mice and human LNs revealed a specific subset of CXCL12hi FRCs that expressed high levels of lymphotoxin-β receptor (LTβR) and are enriched in the expression of immunoregulatory genes. CXCL12hi FRCs had high expression of CCL19, CCL21, indoleamine 2,3-dioxygenase (IDO), IL-10, and TGF-β1. Adoptive transfer of ex vivo-expanded FRCs resulted in their homing to LNs and induced immunosuppressive environments in DLNs to promote heart allograft acceptance. Genetic deletion of LTβR and Cxcl12 in FRCs increased alloreactivity, abrogating the effect of costimulatory blockade in prolonging heart allograft survival. As compared with WT recipients, CXCL12+ FRC-deficient recipients exhibited increased differentiation of CD4+ T cells into Th1 cells. Nano delivery of CXCL12 to DLNs improved allograft survival in heart-transplanted mice. Our study highlights the importance of DLN CXCL12hi FRCs in promoting transplant tolerance.

Fibroblasts are cells of mesenchymal origin with a range of phenotypic diversity and heterogeneity. One of the major functions of fibroblasts is the formation and turnover of the extracellular matrix and establishing a tissue structure by forming a matrisome from embryonic development to the adult stage. It plays an indispensable role in extracellular matrix remodeling during injury, repair, and infection, providing a scaffold for cell-to-cell interaction. Despite their important pathophysiological roles, molecular markers for tissue-resident fibroblasts are only now being identified. Fibroblasts acquire molecular signatures based on anatomical locations, thus impacting their phenotypic heterogeneity despite their overlapping morphology. Fibroblasts are now recognized as key immune sentinel cells, capable of regulating the inflammatory milieu through their distinct functional subsets that are designed to respond differently with unique immune signatures. Fibroblasts can detect pathogenic and danger signals through their diverse pattern recognition receptors (PRRs) and release soluble mediators that can modulate the immune infiltrates at the site of tissue injury and repair. This review discusses the diversity and heterogeneity of fibroblasts in secondary lymphoid organs such as lymph nodes, spleen, and Peyer's patches, and their contributions to a range of pathological and physiological processes. The role of trans-differentiated effector fibroblast phenotypes that modulate the expression and function of various innate immune components (PRRs, cytokines, chemokines, and complement) in maintaining homeostasis has also been discussed.

Lymph node (LN) fibroblastic reticular cells (FRCs) are key regulators of mammalian adaptive immune responses. Together with their deposited extracellular matrix (ECM), FRCs form a reticular network that provides mechanical strength to LNs. Furthermore, the ECM regulates various cell functions including proliferation and differentiation. The ECM is dynamically remodeled in activated LNs, thereby affecting immune cell survival and function. Although both the LN ECM and FRCs can affect immune reactivity, a link between altered LN ECM during an immune response and ECM-producing FRCs is lacking. We explore recent work on the complex interplay between FRCs, ECM, and immune cells in health and disease, and provide guidance for future research to understand the complex regulation of the adaptive immune system within LNs.

Ageing is an inevitable biological process that impacts the immune system, leading to immunosenescence and inflammaging, which contribute to increased susceptibility to infections, autoimmune diseases and cancers in individuals over the age of 65. This review focuses on the ageing of lymph node stromal cells (LNSCs), which are crucial for maintaining lymph node (LN) structure and function. Age-related changes in LNs, such as fibrosis and lipomatosis, disrupt the LN architecture and reduce immune cell recruitment and function, impairing immune responses to infections and vaccinations. The review discusses the structural and functional decline of various LNSC subsets, including fibroblastic reticular cells (FRCs), lymphatic endothelial cells (LECs) and blood endothelial cells (BECs), highlighting their roles in immune cell activation and homeostasis. Potential strategies to restore aged LNSC function, such as enhancing LNSC activation during vaccination and using senotherapeutics, are explored. Outstanding questions regarding the mechanisms of LNSC ageing and how ageing of the LN stroma might impact autoimmune disorders are also addressed. This review aims to stimulate further research into the characterisation of aged LNSCs and the development of therapeutic interventions to improve immune function in the older adults.

Many viruses induce viremia (virus in the blood) and disseminate throughout the body via the bloodstream from the initial infection site. However, viruses must often pass through the lymphatic system to reach the blood. The lymphatic system comprises a network of vessels distinct from blood vessels, along with interconnected lymph nodes (LNs). The complex network has become increasingly appreciated as a crucial host factor that contributes to both the spread and control of viral infections. Viruses can enter the lymphatics as free virions or along with migratory cells. Once virions arrive in the LN, sinus-resident macrophages remove infectious virus from the lymph. Depending on the virus, macrophages can eliminate infection or propagate the virus. A virus released from an LN is eventually deposited into the blood. This unique pathway highlights LNs as targets for viral infection control and for modulation of antiviral response development. Here, we review the lymphatic system and viruses that disseminate through this network. We discuss infection of the LN, the generation of adaptive antiviral immunity, and current knowledge of protection within the infected node. We conclude by sharing insights from ongoing efforts to optimize lymphatic targeting by vaccines and pharmaceuticals. Understanding the lymphatic system's role during viral infection enhances our knowledge of antiviral immunity and virus-host interactions and reveals potential targets for next-generation therapies.

Cancer progression is significantly influenced by the complex interactions within the tumor microenvironment (TME). Immune cells, in particular, play a critical role by infiltrating tumors from the circulation and surrounding lymphoid tissues in an attempt to control their spread. However, they often fail in this task. Current in vivo and in vitro preclinical models struggle to fully capture these intricate interactions affecting our ability to understand immune evasion and predict drugs behaviour in the clinic. To address this challenge, biofabrication and particularly 3D bioprinting has emerged as a promising tool for modeling both tumors and the immune system. Its ability to incorporate multiple cell types into 3D matrices, enable tissue compartmentalization with high spatial accuracy, and integrate vasculature makes it a valuable approach. Nevertheless, limited research has focused on capturing the complex tumor-immune interplay in vitro. This review highlights the composition and significance of the TME, the architecture and function of lymphoid tissues, and innovative approaches to modeling their interactions in vitro, while proposing the concept of an extended TME.

The imbalance between estrogen and androgen may be an important mechanism of BPH, but the specific mechanism remains unclear. We used mixed sustained-release pellets made of testosterone and estradiol (T + E2) to stimulate the establishment of a BPH rat model. Compared to the prostate hyperplasia rat model using only androgens, the new prostate hyperplasia rat model can be observed to have better macroscopic and pathological characteristics of prostate hyperplasia. We used RNA-seq and bioinformatics to detect differentially expressed genes (DEGs) between the prostate tissue of the novel benign prostatic hyperplasia rat group and the control group, including 458 DEGs, of which 336 were upregulated and 122 were downregulated. Then, RT-qPCR confirmed the authenticity of sequencing results. The analysis results showed that Kif4a and Mki67 were the top core genes in the PPI network. Moreover, we found that these two genes have a positive correlation with each other in multiple cancer tissues, normal tissues, and cancer cells. The DEGs were mainly involved in mitotic nuclear division, nuclear chromosome segregation, and cytokine cell receptor interactions. DEGs were also regulated by 250 miRNAs. In conclusion, we built a novel T + E2-induced rat BPH model, and discovered potentially important genes, pathways, and miRNA-mRNA regulatory networks.

Inflammation is crucial in neurodegenerative and chronic diseases, including Alzheimer disease (AD) and liver fibrosis. To gain a deeper understanding of lysosomal functions in cellular physiology and disease mechanisms, we developed a carbazole-based BODIPY lysosomal probe, designated LysoI. This probe specifically targets lysosomes within 15 min and exhibits a Stokes shift of approximately 180 nm, enabling continuous incubation for up to 5 h without the need for washing steps. Interestingly, LysoI remained effective for long-term imaging, even up to 24 h poststaining. Despite varying pH values and conditions, such as autophagy, apoptosis, and inflammation, it consistently provides excellent lysosomal imaging. Notably, inflammation disrupts lysosomal morphology and motility, as evidenced by an increased size, a decrease in number, and a reduction in movement speed, as observed with LysoI. Furthermore, lysosomal rupture and impaired clearance may exacerbate inflammation and contribute to cellular apoptosis. These findings suggest that lysosomal dysfunction is closely associated with disease progression; therefore, protection and repair targeting lysosomes may offer promising strategies for treating inflammation-related diseases.

The study explores the development and characterization of lymph node stromal cell cultures (LNSCs) from patients with oral squamous cell carcinoma (OSCC), highlighting the importance of understanding tumor-node cross-talk for effective prognostic and therapeutic interventions. Herein, we describe the development and characterization of primary lymph node stromal cells (LNSCs, N = 14) from nodes of metastatic and non-metastatic OSCC patients. Primary cultures were established by the explant method from positive (N + ; N = 2), and negative nodes (N0m; N = 4) of the metastatic patients (N = 3) as well as negative (N0nm; N = 8) nodes from non-metastatic (N = 4) patients. STR profiling confirmed the purity and novelty, while characterization by immunocytochemistry/flow cytometry revealed heterogeneous cell populations consisting of fibroblastic reticular cells (CD31-Gp38 +) and double negative cells (CD31-Gp38-). Transcriptomic profiling indicated molecular alterations in the cells based on the non-metastatic, the pre-metastatic or metastatic status of the nodes, pro-inflammatory, matrix remodeling, and immune evasion being the primary pathways. Assessment of the protein levels for five selected markers (MX1, ISG15, CPM, ITGB4 and FOS) in the cell lines revealed that CPM levels were significantly reduced in the N + and N0m nodes whereas ISG15 levels reduced in N0m. Significantly, the profiling also provided insights into possible glycosylation of CPM (N0nm) and ISGylation of ISG15 (N0m). Cytokine profiling indicated release of chemokines/anti-proliferative cytokines from the negative nodes, while angiogenic/pro-metastatic cytokines were released from the nodes of metastatic patients. The lymph node stromal cell models established in the study with distinctive transcriptomic/cytokine characteristics will be invaluable in delineating the processes underlying nodal metastasis.

Advances in genetic, pharmacologic, and sequencing technology have led to new insight into the role of lymphatics in health and disease. This includes fundamental aspects of the crosstalk between immune cells with cardiac lymphatics. At the interface between leukocytes and lymphatic endothelial cells, myeloid populations are sources of lymphatic growth factors during inflammation. Lymphatic endothelial cells also secrete signals that activate leukocytes, including to antigen presenting cells. Taken together, a view of the lymphatic vasculature as a supplemental cardiac immune hub is emerging. Herein, we discuss reciprocal cell and molecular crosstalk between leukocytes and lymphatics in the myocardium, with implications for health and cardiac inflammation.

Lymph node (LN) lymphatic endothelial cells (LEC) actively acquire and archive foreign antigens. Here, we address questions of how LECs achieve durable antigen archiving and whether LECs with high levels of antigen express unique transcriptional programs. We used single cell sequencing in dissociated LN tissue and spatial transcriptomics to quantify antigen levels in LEC subsets and dendritic cell populations at multiple time points after immunization and determined that ceiling and floor LECs archive antigen for the longest duration. We identify, using spatial transcriptomics, antigen positive LEC-dendritic cell interactions. Using a prime-boost strategy we find increased antigen levels within LECs after a second immunization demonstrating that LEC antigen acquisition and archiving capacity can be improved over multiple exposures. Using machine learning we defined a unique transcriptional program within archiving LECs that predicted LEC archiving capacity in mouse and human independent data sets. We validated this modeling, showing we could predict lower levels of LEC antigen archiving in chikungunya virus-infected mice and demonstrated in vivo the accuracy of our prediction. Collectively, our findings establish unique properties of LECs and a defining transcriptional program for antigen archiving that can predict antigen archiving capacity in different disease states and organisms.

The lymphatic system is a multifaceted regulator of tissue homeostasis and an integral part of immune responses. Previous studies had shown that subsets of lymphatic endothelial cells (LEC) express PTX3, an essential component of humoral innate immunity and tissue homeostasis.

In the present study using whole-mount imaging and image-based morphometric quantifications, Ptx3-targeted mice and in vivo functional analysis, we investigated the involvement of PTX3 in shaping and function of the lymphatic vasculature.

We found that PTX3 is localized in the extracellular matrix (ECM) surrounding human and murine lymphatic vessels (LV). In murine tissues, PTX3 was localized in the ECM close to LV terminals and sprouting. Ptx3-deficient mice showed LV abnormalities in the colon submucosa and diaphragm, including a disorganized pattern and hyperplasia of initial LV capillaries associated with altered distribution of tight junction-associated molecules. Mice with LEC-restricted PTX3 gene inactivation showed morphological and organization abnormalities similar to those observed in Ptx3-deficient animals. Ptx3-deficient mice showed defective fluid drainage from footpads and defective dendritic cell (DC) trafficking.

Thus, PTX3 is strategically localized in the ECM of specialized LV, playing an essential role in their structural organization and immunological function.

Recent breakthroughs in research have sparked a paradigm shift in our understanding of cancer biology, uncovering the critical role of the crosstalk between tumor cells and the immune cells of the tumor microenvironment (TME) in malignant transformation. Fibroblasts have long been viewed as ancillary participants in cancer progression, often eclipsed by the prominence given to malignant cells. Novel investigations, however, have increasingly acknowledged the essential part played by the fibroblasts and their phenotypic doppelganger cancer-associated fibroblasts (CAFs) in fostering immunosuppression and promoting tumor progression. Here we review the cell-of-origin from which CAFs derive and their altered programs compared to their normal counterpart. We will also discuss the complex interplay between CAFs and the surrounding immune cells of the TME in the context of solid tumors and B cell lymphomas, with a focus on the "reprogrammable" role of CAFs in immunosuppression, immuno-activation and immuno-avoidance, and their implications on drug resistance. Finally, we will examine the existing and plausible therapeutic approaches targeting CAFs as a strategy to enhance treatment response.

Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 is a clinical and research standard for evaluating malignant tumors and lymph node metastasis. However, quantitative analysis of nodal status is limited to measurement of short axis diameter (SAD), and metastatic lymph nodes below 10 mm in SAD are often not detected. The purpose of this study was to evaluate the value of multifrequency magnetic resonance elastography (MRE) when added to RECIST 1.1 for detection of lymph node metastasis.

Twenty-five benign and 82 metastatic lymph nodes were prospectively examined by multifrequency MRE at 1.5 T using tomoelastography postprocessing at 30, 40, 50, and 60 Hz (total scan time of 4 minutes). Shear wave speed as a surrogate of soft tissue stiffness was provided in m/s. Positron emission tomography-computed tomography was used as reference standard for identification of abdominal lymph node metastasis from histologically confirmed primary tumors. The diagnostic performance of MRE was compared with that of SAD according to RECIST 1.1 and evaluated by receiver operating characteristic curve analysis using generalized linear mixed models and binary logistic mixed models. Sensitivity, specificity, and predictive values were calculated for different cutoffs.

Metastatic lymph nodes (1.90 ± 0.57 m/s) were stiffer than benign lymph nodes (0.98 ± 0.20 m/s, P < 0.001). An area under the curve of 0.95 for a cutoff of 1.32 m/s was calculated. Using a conservative approach with 1.0 specificity, we found sensitivity (SAD/MRE/MRE + SAD, 0.56/0.84/0.88), negative predictive values (0.41/0.66/0.71), and overall accuracy (0.66/0.88/0.91) to be improved using MRE and even higher for combined MRE and SAD.

Multifrequency MRE improves metastatic abdominal lymph node detection by 25% based on higher tissue stiffness-even for lymph nodes with an SAD ≤10 mm. Stiffness information is quick to obtain and would be a promising supplement to RECIST.

Profiling studies using reverse transcription quantitative PCR (RT-qPCR) require reliable normalization to reference genes to accurately interpret the results. A stable reference gene panel was established to profile metastatic and non-metastatic lymph nodes in patients with oral squamous cell carcinoma. The stability of 18S ribosomal RNA (18SrRNA), ribosomal Protein Lateral Stalk Subunit P0 (RPLP0), ribosomal Protein L27 (RPL27), TATA-box binding protein (TBP), hypoxanthine phosphoribosyl-transferase 1 (HPRT1), beta-actin (ACTB), glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) and vimentin (VIM) was evaluated, as reference genes for profiling patient-derived lymph node stromal cells (LNSCs; N=8; N0:6, N+:2) and lymph node tissues (Patients:14, Nodes=20; N0:7; N+:13). The genes were initially assessed based on their expression levels, specificity, and stability rankings to identify the best combination of reference genes. VIM was excluded from the final analysis because of its low expression (high quantification cycle >32) and multiple peaks in the melting curve. The stability analysis was performed using Reffinder, which utilizes four tools; geNorm, NormFinder, BestKeeper and Comparative ∆Ct methods, thereby enabling the computing of a comprehensive ranking. Evaluation of the gene profiles indicated that while RPLP0 and 18SrRNA were stable in both lymph node tissues and LNSCs, HPRT1, RPL27 were uniquely stable in these tissues whereas ACTB and TBP were most stable in LNSCs. The present study identified the most stable reference gene panel for the RT-qPCR profiling of lymph node tissues and patient-derived LNSCs. The observation that the gene panel differed between the two model systems further emphasized the need to evaluate the reference gene subset based on the disease and cellular context.

The extracellular matrix (ECM) is known to regulate innate immune cells but its role in T cell functions is poorly understood. Here, we show a protective role for ECM proteoglycans, lumican and biglycan in hapten-induced contact dermatitis that is achieved through limiting proinflammatory CD4 + T cells. Lumican and biglycan-null mice develop significant inflammation with greater numbers of CD4 + T cells in hapten-challenged ear pinnae, while their draining lymph nodes show increased T-bet-STAT1 signaling, Th1 commitment, and IFN-γ secreting CD4 + T cell proliferation. Wild type mouse lymph node fibroblastic reticular cells secrete lumican, biglycan and decorin, a related proteoglycan, while none are expressed by naive or activated T cells. In vitro , lumican and biglycan co-localize with LFA-1 on T cell surfaces, and all three proteoglycans suppress LFA-1 mediated T cell activation. Overall, this study elucidates a novel paracrine regulation of Th1 cells by ECM proteoglycans to limit inflammation and tissue damage.

Integrative analysis of heterogeneous expression data remains challenging due to variations in platform, RNA quality, sample processing, and other unknown technical effects. Selecting the approach for removing unwanted batch effects can be a time-consuming and tedious process, especially for more biologically focused investigators.

Here, we present BatchFLEX, a Shiny app that can facilitate visualization and correction of batch effects using several established methods. BatchFLEX can visualize the variance contribution of a factor before and after correction. As an example, we have analyzed ImmGen microarray data and enhanced its expression signals that distinguishes each immune cell type. Moreover, our analysis revealed the impact of the batch correction in altering the gene expression rank and single-sample GSEA pathway scores in immune cell types, highlighting the importance of real-time assessment of the batch correction for optimal downstream analysis.

Our tool is available through Github https://github.com/shawlab-moffitt/BATCH-FLEX-ShinyApp with an online example on Shiny.io https://shawlab-moffitt.shinyapps.io/batch_flex/.

Desmoplasia in fibroblasts within metastatic lymph nodes (MLNs) serves as an indicator of extranodal extension (ENE), which led mortality in oral squamous cell carcinoma (OSCC). However, systematic studies on fibroblasts in MLNs are lacking. Therefore, this study characterized the differences in phenotype, function, and origin of fibroblasts between primary tumors (PTs) and lymph nodes (LNs) in OSCC. We generated single-cell maps of PTs and paired MLNs and draining LNs from three OSCC patients. The transcriptomic atlas, pseudotime analysis, intercellular communication networks and enrichment analysis of the single cells were characterized. Phenotype and function heterogeneity of fibroblast cells between PTs and MLNs were further verified in vitro. Among 44,052 fibroblasts, we identified two distinct subpopulations of cancer-associated myofibroblastic cells (mCAFs): RGS4+ mCAF1 and COMP + mCAF2. Notably, they exhibited distinct distributions, with mCAF1 predominantly localized in the PTs and mCAF2 in the MLNs. Moreover, pseudotime analysis revealed their distinct origins: mCAF1 originated from inherent normal myofibroblastic cells in the PT, whereas mCAF2 originated from fibroblastic reticular cells in the LNs. Further functional experiments using primary fibroblasts revealed that, compared to mCAF1, mCAF2 in MLNs exhibited weaker crosstalk with immune cells but enhanced extracellular matrix activity, which is closely linked to ENE formation in OSCC. Additionally, we identified two fibroblast subgroups in a transforming state, indicating a potential epithelial-mesenchymal transition. Our research offers profound insights into the heterogeneity of fibroblasts between the PT and MLN in OSCC, serving as an essential resource for future drug discovery endeavors.

Macrophages play a pivotal role in tissue homeostasis, pathogen defense, and inflammation resolution. M1 and M2 macrophage phenotypes represent two faces in a spectrum of responses to microenvironmental changes, crucial in both physiological and pathological conditions. Neuraminidase 1 (Neu1), a lysosomal and cell surface sialidase responsible for removing terminal sialic acid residues from glycoconjugates, modulates several macrophage functions, including phagocytosis and Toll-like receptor (TLR) signaling. Current evidence suggests that Neu1 expression influences M1/M2 macrophage phenotype alterations in the context of cardiovascular diseases, indicating a potential role for Neu1 in macrophage polarization. For this reason, we investigated the impact of Neu1 deficiency on macrophage polarization in vitro and in vivo. Using bone marrow-derived macrophages (BMDMs) and peritoneal macrophages from Neu1 knockout (Neu1-/- ) mice and wild-type (WT) littermate controls, we demonstrated that Neu1-deficient macrophages exhibit an aberrant M2-like phenotype, characterized by elevated macrophage mannose receptor 1 (MMR/CD206) expression and reduced responsiveness to M1 stimuli. This M2-like phenotype was also observed in vivo in peritoneal and splenic macrophages. However, lymph node (LN) macrophages from Neu1-/- mice exhibited phenotypic alterations with reduced CD206 expression. Further analysis revealed that peripheral LNs from Neu1-/- mice were highly fibrotic, with overexpression of transforming growth factor-beta 1 (TGF-β1) and hyperactivated TGF-β signaling in LN macrophages. Consistently, TGF-β1 was found to alter M1/M2 macrophage polarization in vitro. Our findings showed that Neu1 deficiency prompts macrophages towards an M2 phenotype and that microenvironmental changes, particularly increased TGF-β1 in fibrotic tissues such as peripheral LNs in Neu1-/- mice, further influence M1/M2 macrophage polarization, highlighting its sensitivity to the local microenvironment. Therapeutic interventions targeting Neu1 or TGF-β signaling pathways may offer the potential to regulate macrophage behavior across different diseases.

Cancer dissemination to lymph nodes (LN) is associated with a worse prognosis, increased incidence of distant metastases and reduced response to therapy. The LN microenvironment puts selective pressure on cancer cells, creating cells that can survive in LN as well as providing survival advantages for distant metastatic spread. Additionally, the presence of cancer cells leads to an immunosuppressive LN microenvironment, favoring the evasion of anti-cancer immune surveillance. However, recent studies have also characterized previously unrecognized roles for tumor-draining lymph nodes (TDLNs) in cancer immunotherapy response, including acting as a reservoir for pre-exhausted CD8+ T cells and stem-like CD8+ T cells. In this review, we will discuss the spread of cancer cells through the lymphatic system, the roles of TDLNs in metastasis and anti-cancer immune responses, and the therapeutic opportunities and challenges in targeting LN metastasis.

Vaccination is one of the most effective medical interventions, saving millions of lives and reducing the morbidity of infections across the lifespan, from infancy to older age. The generation of plasma cells and memory B cells that produce high affinity class switched antibodies is central to this protection, and these cells are the ultimate output of the germinal centre response. Optimal germinal centre responses require different immune cells to interact with one another in the right place and at the right time and this delicate cellular ballet is coordinated by a network of interconnected stromal cells. In this review we will discuss the various types of lymphoid stromal cells and how they coordinate immune cell homeostasis, the induction and maintenance of the germinal centre response, and how this is disorganised in older bodies.

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.

Lymph node stromal cells (LNSCs) are an often overlooked component of the immune system but play a crucial role in maintaining tissue homeostasis and orchestrating immune responses. Our understanding of the functions these cells serve in the context of bacterial infections remains limited. We previously showed that Listeria monocytogenes, a facultative intracellular foodborne bacterial pathogen, must replicate within an as-yet-unidentified cell type in the mesenteric lymph node (MLN) to spread systemically. Here, we show that L. monocytogenes could invade, escape from the vacuole, replicate exponentially, and induce a type I interferon response in the cytosol of 2 LNSC populations infected in vitro, fibroblastic reticular cells (FRCs) and blood endothelial cells (BECs). Infected FRCs and BECs also produced a significant chemokine and proinflammatory cytokine response after in vitro infection. Flow cytometric analysis confirmed that GFP+  L. monocytogenes were associated with a small percentage of MLN stromal cells in vivo following foodborne infection of mice. Using fluorescent microscopy, we showed that these cell-associated bacteria were intracellular L. monocytogenes and that the number of infected FRCs and BECs changed over the course of a 3-day infection in mice. Ex vivo culturing of these infected LNSC populations revealed viable, replicating bacteria that grew on agar plates. These results highlight the unexplored potential of FRCs and BECs to serve as suitable growth niches for L. monocytogenes during foodborne infection and to contribute to the proinflammatory environment within the MLN that promotes clearance of listeriosis.

Antigens from protein subunit vaccination traffic from the tissue to the draining lymph node, either passively via the lymph or carried by dendritic cells at the local injection site. Lymph node (LN) lymphatic endothelial cells (LEC) actively acquire and archive foreign antigens, and archived antigen can be released during subsequent inflammatory stimulus to improve immune responses. Here, we answer questions about how LECs achieve durable antigen archiving and whether there are transcriptional signatures associated with LECs containing high levels of antigen. We used single cell sequencing in dissociated LN tissue to quantify antigen levels in LEC and dendritic cell populations at multiple timepoints after immunization, and used machine learning to define a unique transcriptional program within archiving LECs that can predict LEC archiving capacity in independent data sets. Finally, we validated this modeling, showing we could predict antigen archiving from a transcriptional dataset of CHIKV infected mice and demonstrated in vivo the accuracy of our prediction. Collectively, our findings establish a unique transcriptional program in LECs that promotes antigen archiving that can be translated to other systems.

Fibroblastic reticular cells (FRCs) construct microanatomical niches that support lymph node (LN) homeostasis and coordination of immune responses. Transcription factors regulating the functionality of FRCs remain poorly understood. Here, we investigated the role of the transcription factor SpiB that is expressed in LN FRCs. Conditional ablation of SpiB in FRCs impaired the FRC network in the T-cell zone of LNs, leading to reduced numbers of FRCs and altered homeostatic functions including reduced CCL21 and interleukin-7 expression. The size and cellularity of LNs remained intact in the absence of SpiB but the space between the reticular network increased, indicating that although FRCs were reduced in number they stretched to maintain network integrity. Following virus infection, antiviral CD8+ T-cell responses were impaired, suggesting a role for SpiB expression in FRCs in orchestrating immune responses. Together, our findings reveal a new role for SpiB as an important regulator of FRC functions and immunity in LNs.

Antigens from viruses or immunizations can persist or are archived in lymph node stromal cells such as lymphatic endothelial cells (LEC) and fibroblastic reticular cells (FRC). Here, we find that, during the time frame of antigen archiving, LEC apoptosis caused by a second, but unrelated, innate immune stimulus such as vaccina viral infection or CpG DNA administration resulted in cross-presentation of archived antigens and boosted memory CD8 + T cells specific to the archived antigen. In contrast to "bystander" activation associated with unrelated infections, the memory CD8 + T cells specific to the archived antigen from the immunization were significantly higher than memory CD8 + T cells of a different antigen specificity. Finally, the boosted memory CD8 + T cells resulted in increased protection against Listeria monocytogenes expressing the antigen from the immunization, but only for the duration that the antigen was archived. These findings outline an important mechanism by which lymph node stromal cell archived antigens, in addition to bystander activation, can augment memory CD8 + T cell responses during repeated inflammatory insults.

Fibroblasts are ubiquitous cells that can adopt many functional states. As tissue-resident sentinels, they respond to acute damage signals and shape the earliest events in fibrotic and immune-mediated inflammatory diseases. Upon sensing an insult, fibroblasts produce chemokines and growth factors to organize and support the response. Depending on the size and composition of the resulting infiltrate, these activated fibroblasts may also begin to contract or relax thus changing local stiffness within the tissue. These early events likely contribute to the divergent clinical manifestations of fibrotic and immune-mediated inflammatory diseases. Further, distinct changes to the cellular composition and signaling dialogue in these diseases drive progressive fibroblasts specialization. In fibrotic diseases, fibroblasts support the survival, activation and differentiation of myeloid cells, granulocytes and innate lymphocytes, and produce most of the pathogenic extracellular matrix proteins. Whereas, in immune-mediated inflammatory diseases, sequential accumulation of dendritic cells, T cells and B cells programs fibroblasts to support local, destructive adaptive immune responses. Fibroblast specialization has clear implications for the development of effective induction and maintenance therapies for patients with these clinically distinct diseases.

Infection with chikungunya virus (CHIKV) causes disruption of draining lymph node (dLN) organization, including paracortical relocalization of B cells, loss of the B cell-T cell border, and lymphocyte depletion that is associated with infiltration of the LN with inflammatory myeloid cells. Here, we found that, during the first 24 hours of infection, CHIKV RNA accumulated in MARCO-expressing lymphatic endothelial cells (LECs) in both the floor and medullary LN sinuses. The accumulation of viral RNA in the LN was associated with a switch to an antiviral and inflammatory gene expression program across LN stromal cells, and this inflammatory response - including recruitment of myeloid cells to the LN - was accelerated by CHIKV-MARCO interactions. As CHIKV infection progressed, both floor and medullary LECs diminished in number, suggesting further functional impairment of the LN by infection. Consistent with this idea, antigen acquisition by LECs, a key function of LN LECs during infection and immunization, was reduced during pathogenic CHIKV infection.

The constant exposure of the fish branchial cavity to aquatic pathogens causes local mucosal immune responses to be extremely important for their survival. Here, we used a marker for T lymphocytes/natural killer (NK) cells (ZAP70) and advanced imaging techniques to investigate the lymphoid architecture of the zebrafish branchial cavity. We identified a sub-pharyngeal lymphoid organ, which we tentatively named "Nemausean lymphoid organ" (NELO). NELO is enriched in T/NK cells, plasma/B cells, and antigen-presenting cells embedded in a network of reticulated epithelial cells. The presence of activated T cells and lymphocyte proliferation, but not V(D)J recombination or hematopoiesis, suggests that NELO is a secondary lymphoid organ. In response to infection, NELO displays structural changes including the formation of T/NK cell clusters. NELO and gill lymphoid tissues form a cohesive unit within a large mucosal lymphoid network. Collectively, we reveal an unreported mucosal lymphoid organ reminiscent of mammalian tonsils that evolved in multiple teleost fish families.

The secondary lymphoid organs (SLOs) undergo structural changes with age, which correlates with diminishing immune responses against infectious disease. A growing body of research suggests that the aged tissue microenvironment can contribute to decreased immune function, independent of intrinsic changes to hematopoietic cells with age. Stromal cells impart structural integrity, facilitate fluid transport, and provide chemokine and cytokine signals that are essential for immune homeostasis. Mechanisms that drive SLO development have been described, but their roles in SLO maintenance with advanced age are unknown. Disorganization of the fibroblasts of the T cell and B cell zones may reduce the maintenance of naïve lymphocytes and delay immune activation. Reduced lymphatic transport efficiency with age can also delay the onset of the adaptive immune response. This review focuses on recent studies that describe age-associated changes to the stroma of the lymph nodes and spleen. We also review recent investigations into stromal cell biology, which include high-dimensional analysis of the stromal cell transcriptome and viscoelastic testing of lymph node mechanical properties, as they constitute an important framework for understanding aging of the lymphoid tissues.

In this review we highlight emerging immune regulatory functions of lumican, keratocan, fibromodulin, biglycan and decorin, which are members of the small leucine-rich proteoglycans (SLRP) of the extracellular matrix (ECM). These SLRPs have been studied extensively as collagen-fibril regulatory structural components of the skin, cornea, bone and cartilage in homeostasis. However, SLRPs released from a remodeling ECM, or synthesized by activated fibroblasts and immune cells contribute to an ECM-free pool in tissues and circulation, that may have a significant, but poorly understood foot print in inflammation and disease. Their molecular interactions and the signaling networks they influence also require investigations. Here we present studies on the leucine-rich repeat (LRR) motifs of SLRP core proteins, their evolutionary and functional relationships with other LRR pathogen recognition receptors, such as the toll-like receptors (TLRs) to bring some molecular clarity in the immune regulatory functions of SLRPs. We discuss molecular interactions of fragments and intact SLRPs, and how some of these interactions are likely modulated by glycosaminoglycan side chains. We integrate findings on molecular interactions of these SLRPs together with what is known about their presence in circulation and lymph nodes (LN), which are important sites of immune cell regulation. Recent bulk and single cell RNA sequencing studies have identified subsets of stromal reticular cells that express these SLRPs within LNs. An understanding of the cellular source, molecular interactions and signaling consequences will lead to a fundamental understanding of how SLRPs modulate immune responses, and to therapeutic tools based on these SLRPs in the future.

Infection with chikungunya virus (CHIKV) causes disruption of draining lymph node (dLN) organization, including paracortical relocalization of B cells, loss of the B cell-T cell border, and lymphocyte depletion that is associated with infiltration of the LN with inflammatory myeloid cells. Here, we find that during the first 24 h of infection, CHIKV RNA accumulates in MARCO-expressing lymphatic endothelial cells (LECs) in both the floor and medullary LN sinuses. The accumulation of viral RNA in the LN was associated with a switch to an antiviral and inflammatory gene expression program across LN stromal cells, and this inflammatory response, including recruitment of myeloid cells to the LN, was accelerated by CHIKV-MARCO interactions. As CHIKV infection progressed, both floor and medullary LECs diminished in number, suggesting further functional impairment of the LN by infection. Consistent with this idea, we find that antigen acquisition by LECs, a key function of LN LECs during infection and immunization, was reduced during pathogenic CHIKV infection.

Viral and vaccine antigens persist or are archived in lymph node stromal cells (LNSC) such as lymphatic endothelial cells (LEC) and fibroblastic reticular cells (FRC). Here, we find that, during the time frame of antigen archiving, LEC apoptosis caused by a second, but unrelated, innate immune stimulus such as vaccina viral infection or CpG DNA administration boosted memory CD8+ T cells specific to the archived antigen. In contrast to "bystander" activation associated with unrelated infections, the memory CD8+ T cells specific to the vaccine archived antigen were significantly higher than memory CD8+ T cells of a different antigen specificity. Finally, the boosted memory CD8+ T cells resulted in increased protection against Listeria monocytogenes expressing the vaccine antigen, but only for the duration that the vaccine antigen was archived. These findings outline a novel mechanism by which LNSC archived antigens, in addition to bystander activation, can augment memory CD8+ T cell responses during repeated inflammatory insults.

Nonhematopoietic lymph node stromal cells (LNSCs) regulate lymphocyte trafficking, survival, and function for key roles in host defense, autoimmunity, alloimmunity, and lymphoproliferative disorders. However, the study of LNSCs in human diseases is complicated by a dependence on viable lymphoid tissues, which are most often excised prior to establishment of a specific diagnosis. Here, we demonstrate that cryopreservation can be used to bank lymphoid tissue for the study of LNSCs in human disease. Using human tonsils and lymph nodes (LN), lymphoid tissue fragments were cryopreserved for subsequent enzymatic digestion and recovery of viable nonhematopoietic cells. Flow cytometry and single-cell transcriptomics identified comparable proportions of LN stromal cell types in fresh and cryopreserved tissue. Moreover, cryopreservation had little effect on transcriptional profiles, which showed significant overlap between tonsils and LN. The presence and spatial distribution of transcriptionally defined cell types were confirmed by in situ analyses. Our broadly applicable approach promises to greatly enable research into the roles of LNSCs in human disease.

In recent years, there has been an explosion of interest in how fibroblasts initiate, sustain, and resolve inflammation across disease states. Fibroblasts contain heterogeneous subsets with diverse functionality. The phenotypes of these populations vary depending on their spatial distribution within the tissue and the immunopathologic cues contributing to disease progression. In addition to their roles in structurally supporting organs and remodeling tissue, fibroblasts mediate critical interactions with diverse immune cells. These interactions have important implications for defining mechanisms of disease and identifying potential therapeutic targets. Fibroblasts in the respiratory tract, in particular, determine the severity and outcome of numerous acute and chronic lung diseases, including asthma, chronic obstructive pulmonary disease, acute respiratory distress syndrome, and idiopathic pulmonary fibrosis. Here, we review recent studies defining the spatiotemporal identity of the lung-derived fibroblasts and the mechanisms by which these subsets regulate immune responses to insult exposures and highlight past, current, and future therapeutic targets with relevance to fibroblast biology in the context of acute and chronic human respiratory diseases. This perspective highlights the importance of tissue context in defining fibroblast-immune crosstalk and paves the way for identifying therapeutic approaches to benefit patients with acute and chronic pulmonary disorders.

The lymph node (LN) is home to resident macrophage populations that are essential for immune function and homeostasis, but key factors controlling this niche are undefined. Here, we show that fibroblastic reticular cells (FRCs) are an essential component of the LN macrophage niche. Genetic ablation of FRCs caused rapid loss of macrophages and monocytes from LNs across two in vivo models. Macrophages co-localized with FRCs in human LNs, and murine single-cell RNA-sequencing revealed that FRC subsets broadly expressed master macrophage regulator CSF1. Functional assays containing purified FRCs and monocytes showed that CSF1R signaling was sufficient to support macrophage development. These effects were conserved between mouse and human systems. These data indicate an important role for FRCs in maintaining the LN parenchymal macrophage niche.

Recent transcriptomic-based analysis of diffuse large B cell lymphoma (DLBCL) has highlighted the clinical relevance of LN fibroblast and tumor-infiltrating lymphocyte (TIL) signatures within the tumor microenvironment (TME). However, the immunomodulatory role of fibroblasts in lymphoma remains unclear. Here, by studying human and mouse DLBCL-LNs, we identified the presence of an aberrantly remodeled fibroblastic reticular cell (FRC) network expressing elevated fibroblast-activated protein (FAP). RNA-Seq analyses revealed that exposure to DLBCL reprogrammed key immunoregulatory pathways in FRCs, including a switch from homeostatic to inflammatory chemokine expression and elevated antigen-presentation molecules. Functional assays showed that DLBCL-activated FRCs (DLBCL-FRCs) hindered optimal TIL and chimeric antigen receptor (CAR) T cell migration. Moreover, DLBCL-FRCs inhibited CD8+ TIL cytotoxicity in an antigen-specific manner. Notably, the interrogation of patient LNs with imaging mass cytometry identified distinct environments differing in their CD8+ TIL-FRC composition and spatial organization that associated with survival outcomes. We further demonstrated the potential to target inhibitory FRCs to rejuvenate interacting TILs. Cotreating organotypic cultures with FAP-targeted immunostimulatory drugs and a bispecific antibody (glofitamab) augmented antilymphoma TIL cytotoxicity. Our study reveals an immunosuppressive role of FRCs in DLBCL, with implications for immune evasion, disease pathogenesis, and optimizing immunotherapy for patients.

The cellular dynamics in the tumour microenvironment (TME) along with non-small cell lung cancer (NSCLC) progression remain unclear.

Multiplex immunofluorescence test detecting 10 immune-related markers on 553 primary tumour (PT) samples of NSCLC was conducted and spatial information in TME was assessed by the StarDist depth learning model. The single-cell transcriptomic atlas of PT (n = 4) and paired tumour-draining lymph nodes (TDLNs) (n = 5 for tumour-invaded, n = 3 for tumour-free) microenvironment was profiled. Various bioinformatics analyses based on Gene Expression Omnibus, TCGA and Array-Express databases were also used to validate the discoveries.

Spatial distances of CD4+ T cells-CD38+ T cells, CD4+ T cells-neutrophils and CD38+ T cells-neutrophils prolonged and they were replaced by CD163+ macrophages in PT along with tumour progression. Neutrophils showed unique stage and location-dependent prognostic effects. A high abundance of stromal neutrophils improved disease-free survival in the early-stage, whereas high intratumoural neutrophil infiltrates predicted poor prognosis in the mid-to-late-stage. Significant molecular and functional reprogramming in PT and TDLN microenvironments was observed. Diverse interaction networks mediated by neutrophils were found between positive and negative TDLNs. Five phenotypically and functionally heterogeneous subtypes of tumour-associated neutrophil (TAN) were further identified by pseudotime analysis, including TAN-0 with antigen-presenting function, TAN-1 with strong expression of interferon (IFN)-stimulated genes, the pro-tumour TAN-2 subcluster, the classical subset (TAN-3) and the pro-inflammatory subtype (TAN-4). Loss of IFN-stimulated signature and growing angiogenesis activity were discovered along the transitional trajectory. Eventually, a robust six neutrophil differentiation relevant genes-based model was established, showing that low-risk patients had longer overall survival time and may respond better to immunotherapy.

The cellular composition, spatial location, molecular and functional changes in PT and TDLN microenvironments along with NSCLC progression were deciphered, highlighting the immunoregulatory roles and evolutionary heterogeneity of TANs.