Journal: Nature Communications

Article Title: Revealing the biophysics of lamina-associated domain formation by integrating theoretical modeling and high-resolution imaging

doi: 10.1038/s41467-025-63244-1

Figure Lengend Snippet: A Representative Voronoi density rendering of STORM images of hMSC nuclei, color-coded to indicate histone H2B density levels (blue for low density and red for high density). B The distribution of heterochromatin domain sizes obtained from STORM images in interior (Top) and at periphery (Bottom). Notably the distributions show a characteristic mean size. C Chromatin-chromatin interactions result in segregation of chromatin into euchromatin and heterochromatin phases. These interactions incorporate chromatin-chromatin interactions, including those mediated by crosslinking molecules such as HP1α, as well as the direct interactions between segments of chromatin. The model also includes the interactions between chromatin and the lamina, mediated by anchoring proteins such as LAP2α/β and LBR. These chromatin-lamina interactions, localized at the nuclear periphery, result in the formation of heterochromatin rich lamina-associated domains (LADs). D Epigenetic factors, such as HDAC and HMT, regulate acetylation and methylation reactions that allow interconversion of heterochromatin and euchromatin phases, captured via first-order reaction kinetics. The diffusion of water and epigenetic markers is included in the continuum model. The anchoring of chromatin to the nuclear lamina is also mediated by HDAC3 , . E The contour plot of free energy density shows the two wells (local minima) corresponding to the two stable phases of chromatin – euchromatin (blue) and heterochromatin (red). We schematically show how an initial homogeneous distribution of chromatin (white circle) will evolve towards the two energy wells.

Article Snippet: The cells were then plated in basal growth medium, which consisted of high-glucose DMEM medium supplemented with 10% penicillin–streptomycin, L-glutamine, and 10% FBS. hMSCs and hTCs were fixed using methanol-ethanol (1:1) at −20 °C for 6 min, followed by blocking with a solution of 10%(w/v) bovine serum albumin (BSA) in phosphate-buffered saline (PBS) for 1 h . Subsequently, the cells were subjected to overnight incubation at 4 °C with a 1:50 dilution of histone H2B anti-rabbit antibody (ProteinTech, #15857-1-AP).

Techniques: Methylation, Diffusion-based Assay

Journal: Nature Communications

Article Title: Revealing the biophysics of lamina-associated domain formation by integrating theoretical modeling and high-resolution imaging

doi: 10.1038/s41467-025-63244-1

Figure Lengend Snippet: A Representative Voronoi density rendering of H2B STORM images, color-coded to indicate density levels (blue for low density and red for high density). B Steady-state chromatin organization predicted by numerical simulations showing phase separation into compacted heterochromatin domains (red) and loosely packed euchromatin domains (blue) and the formation of heterochromatin-rich LADs at the nuclear periphery. Numerical simulations show the synergistic effects of chromatin-lamina affinity and methylation rates on LADs and inner heterochromatin domain morphology. At low levels of chromatin-lamina affinity, as the methylation level increases, the radius of the discrete LADs increases at a fixed value of the contact angle \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$(\theta )$$\end{document} ( θ ) . The morphology of LADs is determined by both the level of methylation and chromatin-lamina affinity. Low affinity results in the formation of isolated LADs, while at greater levels of affinity LAD spread along the lamina. C Schematic illustrating the morphology of LAD at small level of chromatin-lamina affinity. D The scaling relationship between the thickness of LAD and chromatin-lamina affinity at a given methylation level showing three distinct regimes. E The scaling relation between the thickness of LAD and chromatin-lamina affinity at varying methylation levels. F Workflow of the theoretical framework for extracting methylation rates and chromatin-lamina affinity by integrating super resolution STORM images of DNA with theoretical analyses. Within the interior of the nucleus, STORM image analysis gives the distribution of heterochromatin domain radii ( R d ), and at periphery, it provides the distribution of LAD thickness \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{T}}_{{LAD}}$$\end{document} T L A D . The combination of these distributions with the theoretical framework predicts the corresponding distribution of histone methylation rates \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\Gamma }_{{me}}$$\end{document} Γ m e and the chromatin-lamina affinities \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${V}_{{LAD}}$$\end{document} V L A D . Notably the distribution of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${V}_{{LAD}}$$\end{document} V L A D is bimodal. The red curve in all plots shows the smoothed density plot. All source data are provided as a Source Data file.

Article Snippet: The cells were then plated in basal growth medium, which consisted of high-glucose DMEM medium supplemented with 10% penicillin–streptomycin, L-glutamine, and 10% FBS. hMSCs and hTCs were fixed using methanol-ethanol (1:1) at −20 °C for 6 min, followed by blocking with a solution of 10%(w/v) bovine serum albumin (BSA) in phosphate-buffered saline (PBS) for 1 h . Subsequently, the cells were subjected to overnight incubation at 4 °C with a 1:50 dilution of histone H2B anti-rabbit antibody (ProteinTech, #15857-1-AP).

Techniques: Methylation, Isolation

Journal: bioRxiv

Article Title: O-SNAP: A comprehensive pipeline for spatial profiling of chromatin architecture

doi: 10.1101/2025.07.18.665612

Figure Lengend Snippet: (A) Representative Voronoi density map renderings of H2B STORM data for control and TSA-treated fibroblasts. (N=117 nuclei for the control and N=106 for the TSA-treated group). The color code indicates local chromatin compaction from low density (blue) to high density (red). (B) A Volcano plot visualizes fold changes in O-SNAP-generated features of control and TSA-treated fibroblasts. Three O-SNAP features increase in value in TSA-treated cells. The statistical significance was calculated using a two-sided t-test followed by Benjamini-Hochberg adjustment for multiple comparisons. (C) Visualizations of the chromatin packing domains, (i.e. the compact chromatin compartment segmented using DBSCAN), from the representative nuclei from panel A. Top: Packing domains are color-coded based on their respective localization density for control (left) or TSA-treated cells (right). Bottom: Each point represents the centroid position of a single chromatin packing domain. (D) FSEA indicates which overall nucleus characteristic TSA-treated samples trend towards, here being features related to a larger nucleus and those that indicate more domains at the nucleus interior. The color code corresponds to the statistical significance of the normalized enrichment score, measured by the False Discovery Rate (FDR) q-value, and the size of the icon corresponds to the number of features contained in each feature set. (E) Aggregated MRMR score across the five folds. Three features (I.5, P.12 and V4.2) were selected. (F) Classification results across five training/test folds for a gaussian SVM model trained to discriminate control from TSA-treated fibroblasts using the features selected in (E) and labels with the ground-truth cell state, which has an overall accuracy of 91.76 ± 5.22%. Top: ROC curve where the solid line indicates the average value across the folds, and the shaded region indicates the ±1 standard deviation interval. Bottom: A confusion matrix shows the model’s average accuracy for the control and TSA-treated conditions across the five folds.

Article Snippet: Primary antibodies were diluted in blocking buffer and used as follows: H2B (Proteintech, #15857-1-AP) 1:25; H3K27me3 (Thermo Fisher Scientific; #MA5-11198) 1:100; and H3K9ac (Thermo Fisher Scientific, #MA5-11195) 1:50; and incubated 12h–15 h at 4°C in a humidified chamber.

Techniques: Control, Generated, Standard Deviation

Journal: bioRxiv

Article Title: O-SNAP: A comprehensive pipeline for spatial profiling of chromatin architecture

doi: 10.1101/2025.07.18.665612

Figure Lengend Snippet: (A) Representative Voronoi density map renderings of H2B STORM data of tenocyte cells derived from healthy donors or those with tendinosis (N=19 for healthy cells and N=21 for tendinosis cells). The color code indicates local chromatin compaction from low density (blue) to high density (red). (B) A Volcano plot visualizes fold changes in O-SNAP-generated features of nuclei from healthy or tendinosis donors. One feature increased in value in tendinosis cells while 22 features increased in value in healthy cells. The statistical significance was calculated using a two-sided t-test followed by Benjamini-Hochberg adjustment for multiple comparisons. (C) (i) Violin plots showing the distribution of Feature I.2, the number of packing domains at the nucleus interior, for the healthy and tendinosis nuclei imaged, showing a decrease in tendinosis nuclei (ii) Representative nuclei, where each point represents the centroid position of a single packing domain for healthy (left) and tendinosis (right) nuclei. (D) FSEA indicates which overall nucleus characteristics tendinosis tenocytes trend towards. Features related to more heterogeneous domains at the periphery and rounder nuclei trend towards enrichment but are not statistically significant for α < 0.05. The color code corresponds to the statistical significance of the normalized enrichment score, measured by the False Discovery Rate (FDR) q-value, and the size of the icon corresponds to the number of features contained in each feature set.

Article Snippet: Primary antibodies were diluted in blocking buffer and used as follows: H2B (Proteintech, #15857-1-AP) 1:25; H3K27me3 (Thermo Fisher Scientific; #MA5-11198) 1:100; and H3K9ac (Thermo Fisher Scientific, #MA5-11195) 1:50; and incubated 12h–15 h at 4°C in a humidified chamber.

Techniques: Derivative Assay, Generated

Journal: bioRxiv

Article Title: O-SNAP: A comprehensive pipeline for spatial profiling of chromatin architecture

doi: 10.1101/2025.07.18.665612

Figure Lengend Snippet: (A) Representative Voronoi density map renderings of H2B STORM images of control human fibroblasts (hFbs), mouse ESCs, and hFb nuclei 48-hr post cell fusion (N=151 nuclei for control hFbs, N=164 for control mESCs, and N=99 for 48-hr hFb nuclei post cell fusion). The color code indicates local chromatin compaction from low density in blue to high density in red. (B) A logistic regression classification model trained on the heterokaryon H2B data across five training/test folds using ground truth labels. The results of the classifier are shown with a ROC curve (top) and confusion matrix (bottom) across the five folds, where the overall classification accuracy is 80.94 ± 5.23%. (C) An ensemble boosted trees classification model across five training/test folds, shuffling the cell state labels of the heterokaryon H2B system. The average results of the ROC curve (top) and confusion matrix (bottom), where the overall classification accuracy across the five folds is 41.08 ± 6.71%. (D) Representative Voronoi density map renderings of H3K27me3 STORM data of somatic heterokaryon nucleus at different timepoints post-fusion (N=92 nuclei for control hFbs and N=61 for 48-hr hFb nuclei post cell fusion). (E) Representative Voronoi density map renderings of H3K9ac STORM data of somatic heterokaryon nucleus at different timepoints post-fusion (N=93 nuclei for control hFbs and N=62 for 48-hr hFb nuclei post cell fusion). (D,E) The color code indicates local chromatin compaction from low density in blue to high density in red. (F) Volcano plot visualizing fold changes in H3K27me3 O-SNAP-generated features of control fibroblast nuclei or fibroblast nuclei within hFb nuclei 48-hr post cell fusion. 36 features increased in value in control hFbs and one increased for 48-hr hFb nuclei post cell fusion. (G) Volcano plot visualizing fold changes in H3K9ac O-SNAP-generated features of control fibroblast nuclei or fibroblast nuclei within hFb nuclei 48-hr post cell fusion. Three features increase in value in the 48-hr hFb nuclei post cell fusion and two features increase in value in control hFbs. (F, G) The statistical significance was calculated using a two-sided t-test followed by Benjamini-Hochberg adjustment for multiple comparisons. (H) Top: Histogram of assigned pseudotimes calculated using the Slingshot trajectory inference method on O-SNAP features generated from the H3K27me3 heterokaryon SMLM data. Bottom: A heatmap of H3K27me3 O-SNAP features changing with pseudotime. The features displayed are a subset of those determined to be most predictive to pseudotime.

Article Snippet: Primary antibodies were diluted in blocking buffer and used as follows: H2B (Proteintech, #15857-1-AP) 1:25; H3K27me3 (Thermo Fisher Scientific; #MA5-11198) 1:100; and H3K9ac (Thermo Fisher Scientific, #MA5-11195) 1:50; and incubated 12h–15 h at 4°C in a humidified chamber.

Techniques: Control, Generated

Journal: bioRxiv

Article Title: O-SNAP: A comprehensive pipeline for spatial profiling of chromatin architecture

doi: 10.1101/2025.07.18.665612

Figure Lengend Snippet: (A) Representative Voronoi density map renderings of H2B STORM images of the MCF10A/NLS-DAO system with H3.1 overexpression in control cells and those treated with 10 nM D-Ala for 4 hours (N=39 nuclei for control and N=32 for D-Ala treated cells). (B) Representative Voronoi density map renderings of H2B STORM images of the MCF10A/NLS-DAO system with H3.2 overexpression in control cells and those treated with 10 nM D-Ala for 4 hours. (A, B) The color code indicates local chromatin compaction from low density in blue to high density in red. (C) Volcano plot visualizing fold changes in O-SNAP-generated features of H2B STORM data of MCF10A/NLS-DAO cells in control versus D-Ala treated cells. 5 features increase in value in the treatment group compared to the control, and 3 features increase in value in the control group (N=41 nuclei for control and N=29 for D-Ala treated cells). (D) Volcano plot visualizing fold changes in O-SNAP-generated features of H2B STORM data of MCF10A/NLS-DAO cells in control versus D-Ala treated cells. (C, D) The statistical significance was calculated using a two-sided t-test followed by Benjamini-Hochberg adjustment for multiple comparisons. (E) Classification results of a quadratic discriminant classification model trained to discriminate between control and D-Ala treated cells in the MCF10A/NLS-DAO, H3.1 overexpression system based on H2B STORM data with an overall validation accuracy of 90.19 ± 3.67%. (F) Classification results of a neural network model trained to discriminate between control and D-Ala treated cells in the MCF10A/NLS-DAO, H3.2 overexpression system based on H2B STORM data with an overall validation accuracy of 71.43 ± 7.95%. (E, F) The ROC curve (left) and a confusion matrix (right) indicate the performance for the control and D-Ala treatment conditions across the five folds. (G, H) The distribution of packing domain localization density of the MCF10A/NLS-DAO H3.1 (G) and H3.2 (H) overexpression system, where the solid line shows the percentage of packing domains that have a given localization density value for a given bin, averaged from over all nuclei of either the control or D-Ala treatment condition. The shaded region represents ±1 standard deviation interval from the mean for each bin.

Article Snippet: Primary antibodies were diluted in blocking buffer and used as follows: H2B (Proteintech, #15857-1-AP) 1:25; H3K27me3 (Thermo Fisher Scientific; #MA5-11198) 1:100; and H3K9ac (Thermo Fisher Scientific, #MA5-11195) 1:50; and incubated 12h–15 h at 4°C in a humidified chamber.

Techniques: Over Expression, Control, Generated, Biomarker Discovery, Standard Deviation

Journal: bioRxiv

Article Title: O-SNAP: A comprehensive pipeline for spatial profiling of chromatin architecture

doi: 10.1101/2025.07.18.665612

Figure Lengend Snippet: (A) Representative Voronoi density map renderings of H2B STORM images of human chondrocytes at Passage 0 (P0), Passage 3 (P3), and Passage 6 (P6) from Donor A (N=14 nuclei for P0, N=11 for P3, and N=14 for P6). The color code indicates local chromatin compaction from low density in blue to high density in red. (B) Classification results for a KNN model discriminating P0, P3, and P6 chondrocytes based on H2B STORM data across five training/test folds on Donor A. Top: The ROC curves across the five folds. Bottom: A confusion matrix demonstrating the classifier performance for each passage. The overall classification accuracy across the five folds is 82.14 ± 6.56%. (C) Volcano plot visualizing fold changes in O-SNAP-generated features of H2B STORM data of human chondrocytes at P0 compared to P6. Top: Donor A demonstrated 2 features significantly increase in value at P0 whereas 4 features increase in value at P6. Top: Donor A demonstrated 2 features significantly increased in value at P0 whereas 4 features increased in value at P6. Bottom: For Donor B, 4 features decrease in value for P0 and 3 increase at P6. The color-coded features (red, blue, yellow, green and purple) correspond to those that are the same between the two donors. The statistical significance was calculated using a two-sided t-test followed by Benjamini-Hochberg adjustment for multiple comparisons. (D) Violin plots of common features identified in (C) and Supplementary Figure 9C for P0 and P6 chondrocytes for each donor. Top row, left: The asymmetry of the domain size distribution at the nucleus periphery decreases in both donors. Top row, right: Model LAD thickness increases with passage. The number of localizations assigned to packing domains at both the nucleus interior (middle row, left) and periphery (middle row, right) increase by P6. Bottom row, left: The number of Voronoi clusters per unit area generated using a threshold of Voronoi area < 17.78 nm 2 increases with passage.

Article Snippet: Primary antibodies were diluted in blocking buffer and used as follows: H2B (Proteintech, #15857-1-AP) 1:25; H3K27me3 (Thermo Fisher Scientific; #MA5-11198) 1:100; and H3K9ac (Thermo Fisher Scientific, #MA5-11195) 1:50; and incubated 12h–15 h at 4°C in a humidified chamber.

Techniques: Generated

Journal: bioRxiv

Article Title: Self-Reactive B Cells in Artery Tertiary Lymphoid Organs Encode Pathogenic High Affinity Autoantibody in Atherosclerosis

doi: 10.1101/2025.07.10.664251

Figure Lengend Snippet: (A) Identification of the cognate autoantigen of A6. Schematic view shows the workflow to identify atherosclerosis autoantigens using diseased artery-detecting antibodies. A6-loaded protein A beads were incubated with tissue lysates prepared from diseased mouse arteries followed by elution of proteins from the beads to undergo autoantigen screening of candidates by mass spectrometry (MS) analyses. Several autoantigen-antibody interaction assays were used to confirm protein-protein interaction in vitro including western blots, ELISA, biolayer interferometry (BLI) and surface plasmon resonance (SPR) analyses using various expression-cloned autoantibodies derived from RLNs and ATLO GCs. (B) A6 reacts with mouse aorta-associated atherosclerotic plaques; co-staining of A6, macrophages/DCs and nuclei in Apoe -/- atherosclerosis plaque sections. A6 (red), CD11c (green) and nuclei (DAPI, blue). Bars 100 μm. (C) A6 reacts with human aorta or carotid artery atherosclerotic plaques; staining of A6 in human carotid atherosclerosis plaques by IHC. Bars 50 µm. (D) A6 identifies a 10-15 kDa protein in mouse diseased artery lysates; western blot (WB) shows A6 staining of aorta tissues obtained from 3 individual aged Apoe -/- mice. (E) Immunoprecipitation (IP) combined with MS analyses to identify autoantigen candidates. The ATLO GC-derived A6 antibody was used as a bait to capture autoantigens from protein lysates of diseased aorta of 78-weeks aged Apoe -/- mice. Beads without primary antibody was used as control. Label-free quantitation (LFQ) values of A6 were compared to controls. A6 antibody-enriched candidates are shown as a Venn diagram from three aorta samples from three individual aged Apoe -/- mice. The molecular weight (MW) of each candidate protein is shown. (F) A6 antibody captured histone 2b (H2B) protein in the diseased aorta. The ATLO GC-derived A6 antibody was used as a bait to capture autoantigens from protein lysates of diseased aorta of 32-weeks Apoe -/- mice. An isotype control antibody and the ATLO-GC-derived A11 antibody were used as controls. The peptide-spectrum matches (PSM) values of H2B are shown. Each dot represents one individual biological repeat. Three 32-weeks old Apoe -/- aortas were combined as one sample. (G) A6 binding to recombinant H2B by WB; recombinant H2a, H2b, H4 were used for comparison. (H) A6 reacts with human recombinant H2B at high-affinity; binding affinity of A6 to human recombinant H2B by SPR and BLI analyses. (I) A6 acquires high-affinity binding to the H2B autoantigens via SHM from its unmutated ancestor; binding of ATLO GC-derived A6 antibody to H2B determined by ELISA: Mutated A6 (red line), the unmutated parent of A6 (unmutated-A6, blue line), and the isotype control antibody (black line). (J) Summary figure: A6 acquires high-affinity binding to H2B via ATLO GC responses indicating that negative selection of GC autoreactive BCRs to prevent high-affinity autoantibody generation is compromised in ATLOs.

Article Snippet: Equal amounts of TiterMax@Gold Adjuvant (Sigma, T2684-1ML) and recombinant Histone H2B protein (Active motif company, https://www.activemotif.com/ ) was used to prepare the adjuvant – H2B emulsion.

Techniques: Immunopeptidomics, Incubation, Mass Spectrometry, In Vitro, Western Blot, Enzyme-linked Immunosorbent Assay, SPR Assay, Expressing, Clone Assay, Derivative Assay, Staining, Immunoprecipitation, Control, Quantitation Assay, Molecular Weight, Binding Assay, Recombinant, Comparison, Selection

Journal: bioRxiv

Article Title: Self-Reactive B Cells in Artery Tertiary Lymphoid Organs Encode Pathogenic High Affinity Autoantibody in Atherosclerosis

doi: 10.1101/2025.07.10.664251

Figure Lengend Snippet: (A) Anti-H2B autoantibody titers increase in Apoe -/- mice during aging. Anti-H2B IgG1 serum antibody titers of WT and Apoe -/- mice during aging were examined by ELISA; each dot represents one mouse. n= 8-weeks WT (n = 11), Apoe -/- (n = 12); 32-weeks WT (n = 12), Apoe -/- (n = 11); 78-weeks WT (n = 13), Apoe -/- (n = 16). Two-tailed student T test. (B) H2B vaccination promotes atherosclerosis. Vaccination using recombinant H2B in young Apoe -/- mice. Apoe -/- mice were vaccinated with recombinant H2B with adjuvant (termed as H2B vaccine), adjuvant alone, or PBS alone at the age of 8 weeks with a boost at the age of 12 weeks. The experiment was completed at 32 weeks. Blood was collected at different time points before and after the injection; en face staining for whole aorta. Atherosclerotic plaques were quantified, as described in Methods. Scale bar 5 mm. for PBS control (n = 7 mice), adjuvant control (n = 7), H2B + adjuvant (n = 8). (C-D) H2B vaccination induced long-lasting T cell dependent autoantibody responses in Apoe -/- mice. Serum anti-H2B total antibody and anti-H2B IgG1 antibody titers were determined by ELISA. n = 7 PBS, 7 adjuvant, and 8 H2B + adjuvant. (E) A6 antibody transfer accelerated atherosclerosis in chow diet-fed Apoe -/- mice. 100 µg A6 antibody was transferred via intraperitoneal (i.p.) injection at 10 weeks, and injections were repeated every 10 days. The experiment was completed at 32 weeks. Blood was collected at multiple time points before and after the injection; en face staining for whole aorta. Atherosclerotic plaques were quantified, as described in Methods. Scale bar 5 mm for PBS control (n = 17 mice), isotype antibody control (n = 12), A6 antibody (n = 18). (F) A6 antibody transfer accelerated atherosclerosis in high fat diet-fed Apoe -/- mice. 100 µg A6 antibody was transferred via i.p. injection at 10 weeks, and injections were repeated every 7 days. The experiment was completed at 14 weeks. Blood was collected at several time points before and after the injection; en face staining for whole aorta. Atherosclerotic plaques were quantified, as described in Methods. Scale bar 5 mm for PBS control (n = 8 mice), irrelevant isotype antibody control (n = 7), A6 antibody (n = 9).

Article Snippet: Equal amounts of TiterMax@Gold Adjuvant (Sigma, T2684-1ML) and recombinant Histone H2B protein (Active motif company, https://www.activemotif.com/ ) was used to prepare the adjuvant – H2B emulsion.

Techniques: Enzyme-linked Immunosorbent Assay, Two Tailed Test, Recombinant, Adjuvant, Injection, Staining, Control

Journal: bioRxiv

Article Title: Self-Reactive B Cells in Artery Tertiary Lymphoid Organs Encode Pathogenic High Affinity Autoantibody in Atherosclerosis

doi: 10.1101/2025.07.10.664251

Figure Lengend Snippet: (A) study design of the clinical cohort. A cross-sectional cohort of 495 individuals of the general population aged between 30 - 70 years who underwent routine medical screening of preventive health care was studied. Exclusion criteria included patients with previously diagnosed cardiovascular diseases, strokes, autoimmune diseases requiring systemic treatment, active malignancies, and severe kidney or liver dysfunctions. 84.4% (418 out of 495) cases voluntarily underwent chest 3-dimentional computed tomography (CT) scans as part of the preventive health screening program. Serum anti-H2B antibody titers were examined. (B) Serum anti-H2B antibody titers is an independent risk factor for aorta and coronary artery calcification. Anti-H2B antibody titers were logarithmically transformed before entering the model to approximate Gaussian distribution. Odds ratios and 95% confidence intervals (CI) are calculated by binary logistic regression with simultaneous entry of all variables (Cox & Snell R 2 0.296, P <0.001). eGFR, estimated glomerular filtration rate; HbA1c, glycated hemoglobin A1c; DBP, diastolic blood pressure; SBP, systolic blood pressure; BMI, body mass index; (C) Serum anti-H2B antibody is an independent risk factor for atherosclerosis.

Article Snippet: Equal amounts of TiterMax@Gold Adjuvant (Sigma, T2684-1ML) and recombinant Histone H2B protein (Active motif company, https://www.activemotif.com/ ) was used to prepare the adjuvant – H2B emulsion.

Techniques: Computed Tomography, Transformation Assay, Filtration

Journal: bioRxiv

Article Title: Self-Reactive B Cells in Artery Tertiary Lymphoid Organs Encode Pathogenic High Affinity Autoantibody in Atherosclerosis

doi: 10.1101/2025.07.10.664251

Figure Lengend Snippet: (A) Identification of the cognate autoantigen of A6. Schematic view shows the workflow to identify atherosclerosis autoantigens using diseased artery-detecting antibodies. A6-loaded protein A beads were incubated with tissue lysates prepared from diseased mouse arteries followed by elution of proteins from the beads to undergo autoantigen screening of candidates by mass spectrometry (MS) analyses. Several autoantigen-antibody interaction assays were used to confirm protein-protein interaction in vitro including western blots, ELISA, biolayer interferometry (BLI) and surface plasmon resonance (SPR) analyses using various expression-cloned autoantibodies derived from RLNs and ATLO GCs. (B) A6 reacts with mouse aorta-associated atherosclerotic plaques; co-staining of A6, macrophages/DCs and nuclei in Apoe -/- atherosclerosis plaque sections. A6 (red), CD11c (green) and nuclei (DAPI, blue). Bars 100 μm. (C) A6 reacts with human aorta or carotid artery atherosclerotic plaques; staining of A6 in human carotid atherosclerosis plaques by IHC. Bars 50 µm. (D) A6 identifies a 10-15 kDa protein in mouse diseased artery lysates; western blot (WB) shows A6 staining of aorta tissues obtained from 3 individual aged Apoe -/- mice. (E) Immunoprecipitation (IP) combined with MS analyses to identify autoantigen candidates. The ATLO GC-derived A6 antibody was used as a bait to capture autoantigens from protein lysates of diseased aorta of 78-weeks aged Apoe -/- mice. Beads without primary antibody was used as control. Label-free quantitation (LFQ) values of A6 were compared to controls. A6 antibody-enriched candidates are shown as a Venn diagram from three aorta samples from three individual aged Apoe -/- mice. The molecular weight (MW) of each candidate protein is shown. (F) A6 antibody captured histone 2b (H2B) protein in the diseased aorta. The ATLO GC-derived A6 antibody was used as a bait to capture autoantigens from protein lysates of diseased aorta of 32-weeks Apoe -/- mice. An isotype control antibody and the ATLO-GC-derived A11 antibody were used as controls. The peptide-spectrum matches (PSM) values of H2B are shown. Each dot represents one individual biological repeat. Three 32-weeks old Apoe -/- aortas were combined as one sample. (G) A6 binding to recombinant H2B by WB; recombinant H2a, H2b, H4 were used for comparison. (H) A6 reacts with human recombinant H2B at high-affinity; binding affinity of A6 to human recombinant H2B by SPR and BLI analyses. (I) A6 acquires high-affinity binding to the H2B autoantigens via SHM from its unmutated ancestor; binding of ATLO GC-derived A6 antibody to H2B determined by ELISA: Mutated A6 (red line), the unmutated parent of A6 (unmutated-A6, blue line), and the isotype control antibody (black line). (J) Summary figure: A6 acquires high-affinity binding to H2B via ATLO GC responses indicating that negative selection of GC autoreactive BCRs to prevent high-affinity autoantibody generation is compromised in ATLOs.

Article Snippet: In order to determine the titer of anti-histone H2b antibodies in the circulation of humans and mice, 100 μl purified recombinant histone H2b proteins (Active motif company, Cat: 31892) at 2 μg/ml concentration in the carbonate-bicarbonate buffer (Sigma, C3041-50CAP) were immobilized on high binding capacity microliter plates (Costar, 9018) overnight at 4 °C.

Techniques: Immunopeptidomics, Incubation, Mass Spectrometry, In Vitro, Western Blot, Enzyme-linked Immunosorbent Assay, SPR Assay, Expressing, Clone Assay, Derivative Assay, Staining, Immunoprecipitation, Control, Quantitation Assay, Molecular Weight, Binding Assay, Recombinant, Comparison, Selection

Journal: bioRxiv

Article Title: Self-Reactive B Cells in Artery Tertiary Lymphoid Organs Encode Pathogenic High Affinity Autoantibody in Atherosclerosis

doi: 10.1101/2025.07.10.664251

Figure Lengend Snippet: (A) Anti-H2B autoantibody titers increase in Apoe -/- mice during aging. Anti-H2B IgG1 serum antibody titers of WT and Apoe -/- mice during aging were examined by ELISA; each dot represents one mouse. n= 8-weeks WT (n = 11), Apoe -/- (n = 12); 32-weeks WT (n = 12), Apoe -/- (n = 11); 78-weeks WT (n = 13), Apoe -/- (n = 16). Two-tailed student T test. (B) H2B vaccination promotes atherosclerosis. Vaccination using recombinant H2B in young Apoe -/- mice. Apoe -/- mice were vaccinated with recombinant H2B with adjuvant (termed as H2B vaccine), adjuvant alone, or PBS alone at the age of 8 weeks with a boost at the age of 12 weeks. The experiment was completed at 32 weeks. Blood was collected at different time points before and after the injection; en face staining for whole aorta. Atherosclerotic plaques were quantified, as described in Methods. Scale bar 5 mm. for PBS control (n = 7 mice), adjuvant control (n = 7), H2B + adjuvant (n = 8). (C-D) H2B vaccination induced long-lasting T cell dependent autoantibody responses in Apoe -/- mice. Serum anti-H2B total antibody and anti-H2B IgG1 antibody titers were determined by ELISA. n = 7 PBS, 7 adjuvant, and 8 H2B + adjuvant. (E) A6 antibody transfer accelerated atherosclerosis in chow diet-fed Apoe -/- mice. 100 µg A6 antibody was transferred via intraperitoneal (i.p.) injection at 10 weeks, and injections were repeated every 10 days. The experiment was completed at 32 weeks. Blood was collected at multiple time points before and after the injection; en face staining for whole aorta. Atherosclerotic plaques were quantified, as described in Methods. Scale bar 5 mm for PBS control (n = 17 mice), isotype antibody control (n = 12), A6 antibody (n = 18). (F) A6 antibody transfer accelerated atherosclerosis in high fat diet-fed Apoe -/- mice. 100 µg A6 antibody was transferred via i.p. injection at 10 weeks, and injections were repeated every 7 days. The experiment was completed at 14 weeks. Blood was collected at several time points before and after the injection; en face staining for whole aorta. Atherosclerotic plaques were quantified, as described in Methods. Scale bar 5 mm for PBS control (n = 8 mice), irrelevant isotype antibody control (n = 7), A6 antibody (n = 9).

Article Snippet: In order to determine the titer of anti-histone H2b antibodies in the circulation of humans and mice, 100 μl purified recombinant histone H2b proteins (Active motif company, Cat: 31892) at 2 μg/ml concentration in the carbonate-bicarbonate buffer (Sigma, C3041-50CAP) were immobilized on high binding capacity microliter plates (Costar, 9018) overnight at 4 °C.

Techniques: Enzyme-linked Immunosorbent Assay, Two Tailed Test, Recombinant, Adjuvant, Injection, Staining, Control

Journal: bioRxiv

Article Title: Self-Reactive B Cells in Artery Tertiary Lymphoid Organs Encode Pathogenic High Affinity Autoantibody in Atherosclerosis

doi: 10.1101/2025.07.10.664251

Figure Lengend Snippet: (A) study design of the clinical cohort. A cross-sectional cohort of 495 individuals of the general population aged between 30 - 70 years who underwent routine medical screening of preventive health care was studied. Exclusion criteria included patients with previously diagnosed cardiovascular diseases, strokes, autoimmune diseases requiring systemic treatment, active malignancies, and severe kidney or liver dysfunctions. 84.4% (418 out of 495) cases voluntarily underwent chest 3-dimentional computed tomography (CT) scans as part of the preventive health screening program. Serum anti-H2B antibody titers were examined. (B) Serum anti-H2B antibody titers is an independent risk factor for aorta and coronary artery calcification. Anti-H2B antibody titers were logarithmically transformed before entering the model to approximate Gaussian distribution. Odds ratios and 95% confidence intervals (CI) are calculated by binary logistic regression with simultaneous entry of all variables (Cox & Snell R 2 0.296, P <0.001). eGFR, estimated glomerular filtration rate; HbA1c, glycated hemoglobin A1c; DBP, diastolic blood pressure; SBP, systolic blood pressure; BMI, body mass index; (C) Serum anti-H2B antibody is an independent risk factor for atherosclerosis.

Article Snippet: In order to determine the titer of anti-histone H2b antibodies in the circulation of humans and mice, 100 μl purified recombinant histone H2b proteins (Active motif company, Cat: 31892) at 2 μg/ml concentration in the carbonate-bicarbonate buffer (Sigma, C3041-50CAP) were immobilized on high binding capacity microliter plates (Costar, 9018) overnight at 4 °C.

Techniques: Computed Tomography, Transformation Assay, Filtration