Human Lymph Nodes Research Articles

TCR-CD3 signal strength regulates plastic coexpression of IL-4 and IFN-γ in Tfh-like cells

The development of T follicular helper (Tfh) cells is an ongoing process resulting in the formation of various Tfh subsets. Despite advancements, the precise impact of T cell receptor (TCR) stimulation on this process remains incompletely understood. This study explores how TCR-CD3 signaling strength influences naive CD4+ T cell differentiation into Tfh-like cells and the concurrent expression of interleukin-21 (IL-21), interleukin-4 (IL-4), and interferon-gamma (IFN-γ). Strong TCR-CD3 stimulation induces proliferation and increased IL-21 expression in Tfh-like cells, which exhibit a characteristic phenotype expressing CXCR5 and PD1. The coexpression of IL-4 and IFN-γ in IL-21-producing Tfh-like cells is controlled by the strength TCR-CD3 stimulation; low stimulation favors IL-4, while strong stimulation enhances IFN-γ secretion. Exogenous addition of the effector cytokines IL-21 and IL-4 further modulate cytokine coexpression. These findings highlight the intricate regulatory mechanisms governing cytokine production and plasticity in Tfh-like cells, providing insights into B cell response modulation. In vivo, antigen availability may regulate Tfh cell plasticity, impacting subsequent B cell differentiation, emphasizing the need for further exploration through animal models or antigen-specific Tfh cell analyses in human lymph node biopsies

Consensus tissue domain detection in spatial omics data using multiplex image labeling with regional morphology (MILWRM).

Spatially resolved molecular assays provide high dimensional genetic, transcriptomic, proteomic, and epigenetic information in situ and at various resolutions. Pairing these data across modalities with histological features enables powerful studies of tissue pathology in the context of an intact microenvironment and tissue structure. Increasing dimensions across molecular analytes and samples require new data science approaches to functionally annotate spatially resolved molecular data. A specific challenge is data-driven cross-sample domain detection that allows for analysis within and between consensus tissue compartments across high volumes of multiplex datasets stemming from tissue atlasing efforts. Here, we present MILWRM (multiplex image labeling with regional morphology)-a Python package for rapid, multi-scale tissue domain detection and annotation at the image- or spot-level. We demonstrate MILWRM's utility in identifying histologically distinct compartments in human colonic polyps, lymph nodes, mouse kidney, and mouse brain slices through spatially-informed clustering in two different spatial data modalities from different platforms. We used tissue domains detected in human colonic polyps to elucidate the molecular distinction between polyp subtypes, and explored the ability of MILWRM to identify anatomical regions of the brain tissue and their respective distinct molecular profiles.

Aberrant BCAT1 expression augments MTOR activity and accelerates disease progression in chronic lymphocytic leukemia.

We performed gene expression profiling of mRNA/cDNA isolated from N = 117 flow sorted CLL. We detected aberrant expression of the metabolic enzyme branched chain amino acid transferase (BCAT1) in CLL with del17p/TP53mut. Through extensive validation, we confirmed the highly preferential expression of BCAT1 in CLL with del17p/TP53mut (66%) or trisomy 12 (77%). BCAT1 was not expressed in B cells isolated from normal human lymph nodes. The products of the bidirectional BCAT1 reaction, including leucine, acetyl-CoA, and alpha-ketoglutarate are known activators of MTOR. We measured an ~two-fold higher MTOR activity via normalized p-S6K levels in primary CLL with BCAT1 high versus absent expression before and after sIgM crosslinking. Through steady state metabolomics and heavy isotope metabolic tracing in primary CLL cells, we demonstrate that CLL cells are avid consumers of branched chain amino acids (BCAAs) and that BCAT1 in CLL engages in bidirectional substrate reactions. Of additional interest, CLL with aberrant BCAT1 expression were less sensitive to Venetoclax-induced apoptosis. Biologically, three CLL-derived cell lines with disruption of BCAT1 had substantially reduced growth ex vivo. Clinically, the expression of any detectable BCAT1 protein in CLL independently associated with shorter median survival (125 months versus 296 months; p < 0.0001), even after exclusion of del17p/TP53mut cases.

On-chip human lymph node stromal network for evaluating dendritic cell and T-cell trafficking

The lymph node paracortex, also known as the T-cell zone, consists of a network of fibroblastic reticular cells (FRCs) that secrete chemokines to induce T-cell and dendritic cell (DC) trafficking into the paracortex. To model the lymph node paracortex, we utilize multi-channel microfluidic devices to engineer a 3D lymph node stromal network from human cultured FRCs embedded in a collagen I-fibrin hydrogel. In the hydrogel, the FRCs self-assemble into an interconnected network, secrete the extracellular matrix proteins entactin, collagen IV, and fibronectin, as well as express an array of immune cell trafficking chemokines. Although the engineered FRC network did not secrete characteristic CCR7-ligand chemokines (i.e. CCL19 and CCL21), human primary TNF-αmatured monocyte-derived DCs, CD45RA+T-cells, and CD45RA-T-cells migrate toward the lymph node stromal network to a greater extent than toward a blank hydrogel. Furthermore, the FRCs co-recruit DCs and antigen-specific T-cells into the lymph node stromal network. This engineered lymph node stromal network may help evaluate how human DCs and T-cells migrate into the lymph node paracortex via CCR7-independent mechanisms.

Neurodegenerative fluid biomarkers are enriched in human cervical lymph nodes.

In animal models, brain neurodegeneration biomarkers drain into cervical lymph nodes (CLNs), and this drainage function is reduced with ageing. If this occurred in humans, CLNs may provide a readily accessible measure of this aspect of protein clearance. We tested this hypothesis in people using ultrasound-guided fine needle aspiration (FNA). We measured amyloid-beta 40 and 42, phospho-Tau-181, glial-fibrillary-acidic-protein, and neurofilament-light using single molecule array in CLN aspirates and plasma from: i) a discovery cohort of 25 autoimmune patients, and from ii) plasma, CLNs and capillary blood in four healthy volunteers, an optimisation cohort. FNA was well-tolerated by all participants. In both cohorts, all biomarkers were detected in all plasma and CLN samples, other than neurofilament-light (8/17 of discovery cohort). CLN biomarker concentrations were significantly greater than plasma concentrations for all except neurofilament-light, most markedly for phospho-Tau-181 (266-fold; P<0.02), whose CLN concentrations decreased with age (Spearman r=-0.66, P=0.001). This study presents the first evidence that neurodegenerative biomarkers are detectable in human CLNs. Raised CLN:plasma biomarker ratios suggest their concentration in CLNs may offer a distinct compartment for minimally-invasive measurement of brain clearance and lymphatic drainage, with potential applicability to study of ageing and future clinical trials.

Innate immune cell activation by adjuvant AS01 in human lymph node explants is age-independent.

Vaccine adjuvants are thought to work by stimulating innate immunity in the draining lymph node (LN), although this has not been proven in humans. To bridge data obtained in animals to humans, we have developed an in situ human LN explant model to investigate how adjuvants initiate immunity. Slices of explanted LNs were exposed to vaccine adjuvants and revealed responses that were not detectable in LN cell suspensions. We used this model to compare the liposome-based AS01 with its components MPL and QS-21, and TLR ligands. Liposomes were predominantly taken up by subcapsular sinus-lining macrophages, monocytes and dendritic cells. AS01 induced dendritic cell maturation and a strong pro-inflammatory cytokine response in intact LN slices but not in dissociated cell cultures, in contrast to R848. This suggests the onset of the immune response to AS01 requires a coordinated activation of LN cells in time and space. Consistent with the robust immune response observed in older adults with AS01-adjuvanted vaccines, the AS01 response in human LNs was independent of age, unlike R848. This human LN explant model is a valuable tool for studying the mechanism of action of adjuvants in humans and for screening new formulations to streamline vaccine development.

Graph Fourier transform for spatial omics representation and analyses of complex organs

Spatial omics technologies decipher functional components of complex organs at cellular and subcellular resolutions. We introduce Spatial Graph Fourier Transform (SpaGFT) and apply graph signal processing to a wide range of spatial omics profiling platforms to generate their interpretable representations. This representation supports spatially variable gene identification and improves gene expression imputation, outperforming existing tools in analyzing human and mouse spatial transcriptomics data. SpaGFT can identify immunological regions for B cell maturation in human lymph nodes Visium data and characterize variations in secondary follicles using in-house human tonsil CODEX data. Furthermore, it can be integrated seamlessly into other machine learning frameworks, enhancing accuracy in spatial domain identification, cell type annotation, and subcellular feature inference by up to 40%. Notably, SpaGFT detects rare subcellular organelles, such as Cajal bodies and Set1/COMPASS complexes, in high-resolution spatial proteomics data. This approach provides an explainable graph representation method for exploring tissue biology and function.

TFH cell responses endure in human lymph nodes after vaccination.

TFH cell responses endure in human lymph nodes after vaccination.

ULTRA-SR Challenge: Assessment of Ultrasound Localization and TRacking Algorithms for Super-Resolution Imaging.

With the widespread interest and uptake of super-resolution ultrasound (SRUS) through localization and tracking of microbubbles, also known as ultrasound localization microscopy (ULM), many localization and tracking algorithms have been developed. ULM can image many centimeters into tissue in-vivo and track microvascular flow non-invasively with sub-diffraction resolution. In a significant community effort, we organized a challenge, Ultrasound Localization and TRacking Algorithms for Super-Resolution (ULTRA-SR). The aims of this paper are threefold: to describe the challenge organization, data generation, and winning algorithms; to present the metrics and methods for evaluating challenge entrants; and to report results and findings of the evaluation. Realistic ultrasound datasets containing microvascular flow for different clinical ultrasound frequencies were simulated, using vascular flow physics, acoustic field simulation and nonlinear bubble dynamics simulation. Based on these datasets, 38 submissions from 24 research groups were evaluated against ground truth using an evaluation framework with six metrics, three for localization and three for tracking. In-vivo mouse brain and human lymph node data were also provided, and performance assessed by an expert panel. Winning algorithms are described and discussed. The publicly available data with ground truth and the defined metrics for both localization and tracking present a valuable resource for researchers to benchmark algorithms and software, identify optimized methods/software for their data, and provide insight into the current limits of the field. In conclusion, Ultra-SR challenge has provided benchmarking data and tools as well as direct comparison and insights for a number of the state-of-the art localization and tracking algorithms.

Transcriptomic diversity of innate lymphoid cells in human lymph nodes compared to BM and spleen

Innate lymphoid cells (ILCs) are largely tissue-resident, mostly described within the mucosal tissues. However, their presence and functions in the human draining lymph nodes (LNs) are unknown. Our study unravels the tissue-specific transcriptional profiles of 47,287 CD127+ ILCs within the human abdominal and thoracic LNs. LNs contain a higher frequency of CD127+ ILCs than in BM or spleen. We define independent stages of ILC development, including EILP and pILC in the BM. These progenitors exist in LNs in addition to naïve ILCs (nILCs) that can differentiate into mature ILCs. We define three ILC1 and four ILC3 sub-clusters in the LNs. ILC1 and ILC3 subsets have clusters with high heat shock protein-encoding genes. We identify previously unrecognized regulons, including the BACH2 family for ILC1 and the ATF family for ILC3. Our study is the comprehensive characterization of ILCs in LNs, providing an in-depth understanding of ILC-mediated immunity in humans.

Spatial Characterization of the Immune Microenvironment within HIV+ Lymph Nodes

Abstract Background: Persistent HIV replication is hypothesized to induce chronic inflammation in ART-suppressed people living with HIV (PLWH), however defining this chronic inflammation within tissues is limited. Molecular and spatial characterization of chronic inflammation within human lymph nodes from PLWH provide valuable information on the immunological microenvironment that limits viral eradication. Methods: We spatially characterized the transcriptomic and proteomic profiles of the lymph node microenvironment from 6 human lymph nodes: three from ART-suppressed PLWH, two from chronically infected PLWH, and one from a male HIV- control donor. For spatial transcriptomics analysis, lymph nodes were either from fresh-frozen or FFPE tissue. Lymph node sections (10μm) were placed onto the 10X Visium slides, stained with H&amp;E, and imaged prior to RNA-Seq library prep. Following sequencing cluster identification was done with single cell RNA-seq datasets and downstream bioinformatics analyses were performed. Results: Following the bioinformatic integration of spatial transcriptomic datasets, cluster analysis identified pro-inflammatory T cells within the HIV+ tissues compared to the HIV- tissue within the T cell zone. Additionally, two sets of proinflammatory myeloid cells were identified that were spatially located within the afferent and efferent lymphatics in the HIV+ tissues. These proinflammatory clusters further characterize chronic inflammation that occurs in PLWH.

Fine needle aspiration of human lymph nodes reveals cell populations and soluble interactors pivotal to immunological priming.

Lymph node (LN) fine needle aspiration (LN FNA) represents a powerful technique for minimally invasive sampling of human LNs in vivo and has been used effectively to directly study aspects of the human germinal center response. However, systematic deep phenotyping of the cellular populations and cell-free proteins recovered by LN FNA has not been performed. Thus, we studied human cervical LN FNAs as a proof-of-concept and used single-cell RNA-sequencing and proteomic analysis to benchmark this compartment, define the purity of LN FNA material, and facilitate future studies in this immunologically pivotal environment. Our data provide evidence that LN FNAs contain bone-fide LN-resident innate immune populations, with minimal contamination of blood material. Examination of these populations reveals unique biology not predictable from equivalent blood-derived populations. LN FNA supernatants represent a specific source of lymph- and lymph node-derived proteins, and can, aided by transcriptomics, identify likely receptor-ligand interactions. This represents the first description of the types and abundance of immune cell populations and cell-free proteins that can be efficiently studied by LN FNA. These findings are of broad utility for understanding LN physiology in health and disease, including infectious or autoimmune perturbations, and in the case of cervical nodes, neuroscience.

Rapid Generation of hPSC-Derived High Endothelial Venule Organoids with In Vivo Ectopic Lymphoid Tissue Capabilities.

Bioengineering strategies for the fabrication of implantable lymphoid structures mimicking lymph nodes (LNs) and tertiary lymphoid structures (TLS) could amplify the adaptive immune response for therapeutic applications such as cancer immunotherapy. No method to date has resulted in the consistent formation of high endothelial venules (HEVs), which is the specialized vasculature responsible for naïve T cell recruitment and education in both LNs and TLS. Here orthogonal induced differentiation of human pluripotent stem cells carrying a regulatable ETV2 allele is used to rapidly and efficiently induce endothelial differentiation. Assembly of embryoid bodies combining primitive inducible endothelial cells and primary human LN fibroblastic reticular cells results in the formation of HEV-like structures that can aggregate into 3D organoids (HEVOs). Upon transplantation into immunodeficient mice, HEVOs successfully engraft and form lymphatic structures that recruit both antigen-presenting cells and adoptively-transferred lymphocytes, therefore displaying basic TLS capabilities. The results further show that functionally, HEVOs can organize an immune response and promote anti-tumor activity by adoptively-transferred T lymphocytes. Collectively, the experimental approaches represent an innovative and scalable proof-of-concept strategy for the fabrication of bioengineered TLS that can be deployed in vivo to enhance adaptive immune responses.

Schwann cell-derived exosomes promote lung cancer progression via miRNA-21-5p.

Schwann cells (SCs), the primary glial cells of the peripheral nervous system, which have been identified in many solid tumors, play an important role in cancer development and progression by shaping the tumor immunoenvironment and supporting the development of metastases. Using different cellular, molecular, and genetic approaches with integrated bioinformatics analysis and functional assays, we revealed the role of human SC-derived exosomal miRNAs in lung cancer progression in vitro and in vivo. We found that exosomal miRNA-21 from SCs up-regulated the proliferation, motility, and invasiveness of human lung cancer cells in vitro, which requires functional Rab small GTPases Rab27A and Rab27B in SCs for exosome release. We also revealed that SC exosomal miRNA-21-5p regulated the functional activation of tumor cells by targeting metalloprotease inhibitor RECK in tumor cells. Integrated bioinformatic analyses showed that hsa-miRNA-21-5p is associated with poor prognosis in patients with lung adenocarcinoma and can promote lung cancer progression through multiple signaling pathways including the MAPK, PI3K/Akt, and TNF signaling. Furthermore, in mouse xenograft models, SC exosomes and SC exosomal hsa-miRNA-21-5p augmented human lung cancer cell growth and lymph node metastasis in vivo. Together our data revealed, for the first time, that SC-secreted exosomes and exosomal miRNA-21-5p promoted the proliferation, motility, and spreading of human lung cancer cells in vitro and in vivo. Thus, exosomal miRNA-21 may play an oncogenic role in SC-accelerated progression of lung cancer and this pathway may serve as a new therapeutic target for further evaluation.

MarsGT: Multi-omics analysis for rare population inference using single-cell graph transformer

Rare cell populations are key in neoplastic progression and therapeutic response, offering potential intervention targets. However, their computational identification and analysis often lag behind major cell types. To fill this gap, we introduce MarsGT: Multi-omics Analysis for Rare population inference using a Single-cell Graph Transformer. It identifies rare cell populations using a probability-based heterogeneous graph transformer on single-cell multi-omics data. MarsGT outperforms existing tools in identifying rare cells across 550 simulated and four real human datasets. In mouse retina data, it reveals unique subpopulations of rare bipolar cells and a Müller glia cell subpopulation. In human lymph node data, MarsGT detects an intermediate B cell population potentially acting as lymphoma precursors. In human melanoma data, it identifies a rare MAIT-like population impacted by a high IFN-I response and reveals the mechanism of immunotherapy. Hence, MarsGT offers biological insights and suggests potential strategies for early detection and therapeutic intervention of disease.

Ultrasound-Guided Fine Needle Biopsy of Human Axillary Lymph Nodes to Assess B Cell Responses to Vaccination.

Ultrasound-guided fine needle biopsy, also known as fine needle aspiration, of human axillary lymph nodes is a safe and effective procedure to assess the immune response within the lymph nodes following vaccination. Once acquired, lymph node cells can be characterized via flow cytometric immunophenotyping and/or single-cell RNA sequencing for gene expression and T and B cell receptors. Analysis of the immune cells from the lymph nodes enables the investigation of T and B cells that may interact at this site. These interactions may lead to germinal center formation and expansion, critical for the generation of effective immunity to vaccination. Directly studying the dynamic processes and interaction of the key cells has been challenging in humans due to the anatomically protected location of these cells. Here, we describe the methods involved in ultrasound-guided fine needle biopsy of human axillary lymph nodes in response to vaccination and subsequent analyses of the B cell populations.

Age-associated reduction of sinus macrophages in human mesenteric lymph nodes.

There are numerous macrophages and dendritic cells in lymph nodes (LNs). Recent studies have highlighted that sinus macrophages (SMs) in LNs possess antigen-presenting capabilities and are related to anti-cancer immune responses. In this study, we assessed the distribution of SMs in mesenteric LNs removed during surgery for colorectal cancer. A marked reduction of SMs was noted in elderly patients, particularly those over 80 years old. We observed a disappearance of CD169-positive cells in LNs where SMs were reduced. In silico analysis of publicly available single-cell RNA sequencing data from LNs revealed that CD169-positive macrophages express numerous genes associated with antigen presentation and lymphocyte proliferation, similar to dendritic cells' functions. In conclusion, our study demonstrates that SMs, potentially crucial for immune activation, diminish in the LNs of elderly patients. This reduction of SMs may contribute to the immune dysfunction observed in the elderly.

Assessment of CXCL13 plasma level in chronic lymphocytic leukemia and its relation to other prognostic markers

Abstract: BACKGROUND: Chronic lymphocytic leukemia is a mature B-cell malignancy where there is a progressive accumulation of leukemic cells with a distinctive immunophenotype as consequence to defective apoptosis and survival signals derived from the microenvironment. CXC chemokine ligand 13 (CXCL13) chemokines have recently emerged as crucial orchestrators for lymphocyte trafficking and activation. These secreted polypeptides exert their function by binding to specific cell surface receptors and can be divided into two categories: homeostatic and inflammatory. The CXCL13 is an efficacious attractant of naive B-cells in vitro and has been shown to be produced constitutively by stromal cells in lymphoid follicles of human lymph nodes. The CXCL13-CXCR5 axis has been previously shown to contribute to the progression of several malignancies and possibly CLL relapse. OBJECTIVE: The aim of this study to compare the plasma level of CXCL13 in a patient with CLL with healthy normal control and to correlate the plasma level of CXCL13 to beta-2 microglobulin (β2 M) and other hematological parameters in CLL. PATIENTS, MATERIALS AND METHODS: This cross-sectional study was conducted on 50 CLL patients who were newly diagnosed. A total of 30 healthy individuals were included in this study as a control group. Measurement of plasma CXCL13 and beta-2 microglobulin levels was done by the enzyme-linked immunosorbent assay. RESULTS: Fifty CLL patients were studied and compared with thirty control group of healthy individuals. The mean level of CXCL13 was 67.48 pg/ml in CLL patients while it was 69.2 pg/ml in control, so it is statistically not significant (P = 0.363). The mean level of β2 M was 50.89 ug/ml in CLL patients while it was 50.59 ug/ml in control, so it is statistically not significant (P = 0.702). The percentage of stage A of CLL patients was 22.44%, stage B was18.36%, and stage C was 10.20%. The percentages of lymphadenopathy, splenomegaly, and hepatomegaly were 66%, 32%, and 16%, respectively. The mean of malignant cell percentage in stage A was 55.18%; in stage B, it was 69.28%; and in stage C, it was 81%, so it is statistically significant (P &lt; 0.001). CXCL13 shows no statistically significant between Cd38+ and CD38− and P value was 0.950. CONCLUSIONS: There was no correlation in level of CXCL13 between the CLL group and the control group. There was no correlation in level of CXCL13 and β2 M in the CLL group.

Seeing or believing in hyperplexed spatial proteomics via antibodies: New and old biases for an image-based technology.

Hyperplexed in-situ targeted proteomics via antibody immunodetection (i.e., >15 markers) is changing how we classify cells and tissues. Differently from other high-dimensional single-cell assays (flow cytometry, single-cell RNA sequencing), the human eye is a necessary component in multiple procedural steps: image segmentation, signal thresholding, antibody validation, and iconographic rendering. Established methods complement the human image evaluation, but may carry undisclosed biases in such a new context, therefore we re-evaluate all the steps in hyperplexed proteomics. We found that the human eye can discriminate less than 64 out of 256 gray levels and has limitations in discriminating luminance levels in conventional histology images. Furthermore, only images containing visible signals are selected and eye-guided digital thresholding separates signal from noise. BRAQUE, a hyperplexed proteomic tool, can extract, in a marker-agnostic fashion, granular information from markers which have a very low signal-to-noise ratio and therefore are not visualized by traditional visual rendering. By analyzing a public human lymph node dataset, we also found unpredicted staining results by validated antibodies, which highlight the need to upgrade the definition of antibody specificity in hyperplexed immunostaining. Spatially hyperplexed methods upgrade and supplant traditional image-based analysis of tissue immunostaining, beyond the human eye contribution.

Serological, Morphological and Molecular Diagnosis of Mycobacterium bovis in Cattle at Sulaimani Province of Iraq

Background:Mycobacterium bovis (M. bovis) causes bovine tuberculosis (BTB), is an endemic disease in cattle and poses a high risk of spreading to humans.Objective:This study aimed to determine M. bovis in cattle and assess the similarities between cattle and humans through molecular methods and histopathological examinations.Methodology:Randomly, blood samples from 411 healthy appearance cows (1% of the target population) in five districts in Sulaimani province, Iraq, were collected from January to March 2022. Sera were obtained immediately and used for ELISA test to determine M. bovis. Additionally, the disease prevalence was confirmed by gross lesions at the slaughterhouse and histopathological examination of collected lymph nodes. Moreover, a PCR assay was used to detect M. bovis in suspected cow samples and previously diagnosed human samples. Gene sequencing and phylogenetic tree analysis were also done to determine the molecular differences between animal and human M. bovis.Results:Using an ELISA test, 46 (11.11%) of 414 samples were positive, while 368 (88.89%) were negative without significant differences between the districts (p&gt;0.05). According to postmortem lesions at the slaughterhouse, only three cows were infected with TB, and typical gross lesions were calcified necrotic and multiple well-demarcated granulomas. The molecular test using two primers (CSB2 and oxyR gene) revealed that M. bovis was found in animal and human extra-pulmonary lymph nodes with no molecular change.Conclusion:Healthy cows harbored M. bovis, the causative agent of a contagious disease that spreads and causes a persistent health problem in humans.