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Lymph nodes (LNs) are specialized secondary lymphoid tissues essential to the priming and maintenance of adaptive immune responses, including the B cell germinal center response; thus, they are central to immunity. However, the anatomically restricted and time‐resolved nature of immune priming means that sampling disease‐relevant human LNs requires specialized techniques. This article describes the application of ultrasound‐guided fine‐needle aspiration (FNA) to sample LNs, using cervical LNs of the head and neck as an exemplar. This minimally invasive technique allows collection of both immune cells and cell‐free material that are relevant to both neuroimmune diseases and basic lymphatic functions. Downstream use of cellular material can include multiplexed flow cytometry, single‐cell transcriptome sequencing (RNA‐seq), and B cell cultures. The cell‐free supernatant can be used for proteomics or other similar ‘omics approaches. This unit describes collection of samples by FNA as well as processing and storage of samples for downstream assays. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Sampling of human cervical lymph nodes by ultrasound‐guided fine‐needle aspirationAlternate Protocol: Sampling of human lymph nodes by ultrasound‐guided fine‐needle aspiration with negative pressureBasic Protocol 2: Processing and storage of human lymph node samples
Lymph nodes (LNs) are specialized secondary lymphoid tissues essential to the priming and maintenance of adaptive immune responses, including the B cell germinal center response; thus, they are central to immunity. However, the anatomically restricted and time‐resolved nature of immune priming means that sampling disease‐relevant human LNs requires specialized techniques. This article describes the application of ultrasound‐guided fine‐needle aspiration (FNA) to sample LNs, using cervical LNs of the head and neck as an exemplar. This minimally invasive technique allows collection of both immune cells and cell‐free material that are relevant to both neuroimmune diseases and basic lymphatic functions. Downstream use of cellular material can include multiplexed flow cytometry, single‐cell transcriptome sequencing (RNA‐seq), and B cell cultures. The cell‐free supernatant can be used for proteomics or other similar ‘omics approaches. This unit describes collection of samples by FNA as well as processing and storage of samples for downstream assays. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Sampling of human cervical lymph nodes by ultrasound‐guided fine‐needle aspirationAlternate Protocol: Sampling of human lymph nodes by ultrasound‐guided fine‐needle aspiration with negative pressureBasic Protocol 2: Processing and storage of human lymph node samples
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.
Type 1 diabetes (T1D) affects a genetically susceptible population that develops autoreactive T cells attacking insulin-producing pancreatic β cells. Increasingly, neoantigens are recognized as critical drivers of this autoimmune response. Here, we report a novel insulin neoepitope generated via post-translational cysteine-to-serine conversion (C>S) in human patients, which is also seen in the autoimmune-prone non-obese diabetic (NOD) mice. This modification is driven by oxidative stress within the microenvironment of pancreatic β cells and is further amplified by T1D-relevant inflammatory cytokines, enhancing neoantigen formation in both pancreatic β cells and dendritic cells. We discover that C>S-modified insulin is specifically recognized by CD4+ T cells in human T1D patients and NOD mice. In humans with established T1D, HLA-DQ8-restricted, C>S-specific CD4+ T cells exhibit an activated memory phenotype and lack regulatory signatures. In NOD mice, these neoepitope-specific T cells can orchestrate islet infiltration and promote diabetes progression. Collectively, these data advance a concept that microenvironment-driven and context-dependent post-translational modifications (PTMs) can generate neoantigens that contribute to organ-specific autoimmunity.
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