Journal: medRxiv

Article Title: In severe COVID-19, SARS-CoV-2 induces a chronic, TGF-β-dominated adaptive immune response

doi: 10.1101/2020.09.04.20188169

Figure Lengend Snippet: Peripheral blood activated B cells (CD27 high CD38 high ) were isolated and sorted by FACS for single cell sequencing. a) Percentage of CD27 high CD38 high cells among live B cells. Each sample is represented by a circle. The line indicates the median. Unpaired two sided Mann-Whitney U test. b) UMAP representation of 72277 CD27 high CD38 high sorted B cells from 9 COVID-19 ICU patients and 3 healthy controls. Between 2416 and 11229 cells were recovered per sample. Transcriptionally similar clusters were identified using shared nearest neighbor (SNN) modularity optimization (left). The combination of CD19, MS4A1, CD27, CD38, IFIT1, MIKI67, IRF4 and PRDM1 expression were used for annotation of the different activation/differentiation stages (right). c) Heatmap of genes that resemble markers for the six different clusters. Depicted are genes with a p-value < 0.01 (Wilcoxon rank sum test) after bonferroni correction, and an average absolute fold-change > log2(1.3). Shown are z-scores of the average expression. d) UMAP representation of the expression levels of selected signature genes for activated/differentiated B cells. e) UMAP representation of analyzed cells from one healthy control and two ICU patients, representing early (first week, patient #1) and late (>7days, patient #5) phase after ICU admission. f) Percentage of cells belonging to a defined cluster among all sequenced B cells per donor. Each dot represents one time point from one single donor. Donors were grouped as “HC” for healthy controls (n = 3), “1st week” for patients within 7 days after ICU admission (n = 6) and “Late” for patients who have been admitted to the ICU for more than a week at the time of analysis (n = 8). The line indicates the median. Significance was determined by using a two-sided analysis of varience (ANOVA) folowed by Tukey’s multiple comparison test with corrected p values as indicated in the figure.

Article Snippet: Fluorescence-coupled antibodies used: IgA2-PE (Clone REA995, Miltenyi, Cat. No.130-117-763, dilution 1:50); CD27-PE (Clone REA499, Miltenyi, Cat. No. 130-114-166, dilution 1:50); CD38-PE (Clone IB6, Miltenyi, Cat. No. 130-113-427, dilution 1:50) and IgA-PE (Clone IS11–8E10, Miltenyi, Cat. No. 130-114-002, dilution 1:50).

Techniques: Isolation, Sequencing, MANN-WHITNEY, Expressing, Activation Assay, Control, Comparison

Journal: medRxiv

Article Title: In severe COVID-19, SARS-CoV-2 induces a chronic, TGF-β-dominated adaptive immune response

doi: 10.1101/2020.09.04.20188169

Figure Lengend Snippet: a) 5-marker MELC panel of SARS-CoV2-positive and control lungs (SARS-CoV2-negative). Respective patient characteristics are given in supplementary Fig. 4a. Each image of each patient depicts the same field of view of the same section, sequentially stained with the fluorescence-labelled antibodies indicated and the nuclear stain DAPI. Magenta arrows indicate IgA2+IgA+CD27 + CD38 + cells (containing a nucleus). Images contain 2048 × 2048 pixels and are generated using an inverted wide-field fluorescence microscope with a 20x objective, a lateral resolution of 325 nm and an axial resolution above 5 µm. Scale bar: 100 µm. b) Absolute numbers of IgA2+IgA+CD27 + CD38 + cells per field of view in all MELC runs acquired (two runs per patient except for COVID-19_A with four runs and Control_B with one single run; see and supplementary Fig. 4b). Each field of view is represented by a circle. The line indicates the median. Unpaired two sided Mann-Whitney U test. c) Region of interest of one exemplary control and COVID-19 lung (as in a)), showing an overlay of the indicated markers. White arrows point out IgA2+CD27 + CD38 + cells. Scale bar: 20 µm. (d-e) Bronchoalveolar lavage (BAL) cells for single cell sequencing were enriched for CD45 + cells via MACS and live cells were further sorted using FACS (see supplementary Fig. 4c). d) UMAP of 5459 cells representing clusters containing T cells, B cells and CD14 -expressing cells from patient #1 on day 59 following ICU admission. B and T cell cluster identification based on BCR/TCR, CD19, CD3E, CD4 and CD8A expression (see supplementary Fig. 4d). UMAP representation of expression of TGFB1, IL21, CD40LG and IFNG . e) UMAP coordinates and clustering was computed for 433 and 2723 cells from patient #9 on days 31 and 46 following ICU admission, respectively. UMAP representation of expression of TGFB1, IL21, CD40LG and IFNG at the two different time points is shown side by side.

Article Snippet: Fluorescence-coupled antibodies used: IgA2-PE (Clone REA995, Miltenyi, Cat. No.130-117-763, dilution 1:50); CD27-PE (Clone REA499, Miltenyi, Cat. No. 130-114-166, dilution 1:50); CD38-PE (Clone IB6, Miltenyi, Cat. No. 130-113-427, dilution 1:50) and IgA-PE (Clone IS11–8E10, Miltenyi, Cat. No. 130-114-002, dilution 1:50).

Techniques: Marker, Control, Staining, Fluorescence, Generated, Microscopy, MANN-WHITNEY, Sequencing, Expressing

Journal: bioRxiv

Article Title: IL-1R1 Blockade Enhances CD40 Agonist-Mediated Immune Responses but Fails to Increase Efficacy or Mitigate Hepatotoxicity in Pancreatic Cancer

doi: 10.1101/2025.02.23.639774

Figure Lengend Snippet: Mice bearing 9-day-old orthotopic PDAC tumors (confirmed by luminescence imaging) were treated every 3 days with agonistic CD40 antibody or isotype control (200 µg/mouse, intraperitoneal). Bulk transcriptomic analysis of tumors was performed after 2 weeks of treatment initiation (n = 2 mice/group). (A) Volcano plot highlighting differentially expressed genes (DEGs) between CD40-treated and isotype control tumors. (B) Gene Set Enrichment Analysis (GSEA) of bulk RNA-seq data showing hallmark pathways enriched in CD40-treated tumors compared to controls. Upregulated pathways included inflammatory response, IFNγ response, and IL-6/JAK-STAT signaling, while oncogenic pathways such as MYC targets V2, E2F targets, and G2M checkpoint were significantly downregulated (FDR < 0.05). (C) Summary of gene ranks, normalized enrichment scores (NES), p-values, and adjusted p-values (padj) for hallmark pathways. (D) Enrichment plots for select hallmark pathways, illustrating the distribution of pathway-associated genes in the ranked gene list. (E) Heatmap of hallmark gene signatures, depicting relative expression across samples, normalized by row Z-scores. (F) Kaplan-Meier survival curves of orthotopic PDAC-bearing mice treated with CD40 antibody or isotype control (log-rank test, n = 5-6 mice/group).

Article Snippet: Beginning 9 days after tumor implantation, subcutaneous and orthotopic PDAC-bearing mice were treated with anti-IL-1R1 (Clone JAMA-147, BioXCell) or isotype control antibodies (BioXCell) administered intraperitoneally.

Techniques: Imaging, Control, RNA Sequencing, Expressing

Journal: bioRxiv

Article Title: IL-1R1 Blockade Enhances CD40 Agonist-Mediated Immune Responses but Fails to Increase Efficacy or Mitigate Hepatotoxicity in Pancreatic Cancer

doi: 10.1101/2025.02.23.639774

Figure Lengend Snippet: Mice bearing 9-day-old orthotopic PDAC tumors (confirmed by luminescence imaging) were treated every 3 days with the indicated antibodies (200 µg/mouse, intraperitoneal). (a) Bulk transcriptomic analysis of orthotopic PDAC tumors after 2 weeks of treatment initiation (*p < 0.05; unpaired t-test, n = 2 mice/group). (b) Kaplan-Meier survival curves (log-rank test, n = 5-6 mice/group). (c) Tumor growth kinetics in subcutaneous PDAC-bearing mice treated every 3 days with agonistic CD40 antibody, anti-IL-1R1 antibody, their combination, or left untreated (****p < 0.0001; two-way ANOVA, n = 5-8 mice/group). (d) Representative H&E-stained tumor sections showing necrosis after 2 weeks of treatment (n = 3-4 mice/group). (e) Quantification of necrotic areas (**p < 0.01; one-way ANOVA, n = 3-4 mice/group). (f) Flow cytometric analysis of PMN-MDSCs in peripheral blood after 2 weeks of treatment (*p < 0.05; unpaired Student’s t-test, n = 5 mice/group). (g) Volcano plot of differentially expressed genes (DEGs) in subcutaneous PDAC tumors treated with anti-IL-1R1 versus untreated controls (red: upregulated; green: downregulated; 546 up, 530 down; p ≤ 0.05, log2FC ≥ 0). (h–i) Gene Ontology (GO) enrichment analysis of DEGs from (g), showing significantly upregulated (h) and downregulated (i) biological processes. (j) Volcano plot of DEGs comparing combination therapy (CD40 + anti-IL-1R1) versus CD40 monotherapy (524 up, 534 down; p ≤ 0.05, log2FC ≥ 0). (k–l) GO enrichment analysis of DEGs from (j), highlighting upregulated (k) and downregulated (l) pathways. (m) Gene Set Enrichment Analysis (GSEA) plots demonstrating the enrichment of indicated gene sets in the combination therapy group compared to CD40 monotherapy.

Article Snippet: Beginning 9 days after tumor implantation, subcutaneous and orthotopic PDAC-bearing mice were treated with anti-IL-1R1 (Clone JAMA-147, BioXCell) or isotype control antibodies (BioXCell) administered intraperitoneally.

Techniques: Imaging, Staining

Journal: Inflammation and Regeneration

Article Title: Novel artificial nerve transplantation of human iPSC-derived neurite bundles enhanced nerve regeneration after peripheral nerve injury

doi: 10.1186/s41232-024-00319-4

Figure Lengend Snippet: Early-stage tissue evaluation after neurite bundle-derived artificial nerve transplantation. a , b Immunohistochemistry images with anti-NFH and anti-CD31 antibodies. Dotted arrows indicate the proximal side of the intact nerve areas, and solid arrows indicate the NFH- and CD31-positive regeneration tip in the reconstructed tissue. Scale bars = 1 mm. c , d Quantitative comparison of axonal extension distance was indicated by NFH-positive areas and that of vascular elongation distance was demonstrated by CD31-positive areas at 1 and 2 weeks after transplantation. Significant differences between groups elongation distances were observed in 2-week samples only. e , f Iba1 immunohistochemistry image of the proximal part of the regenerated tissue at 2 weeks after transplantation (e). Scale bars = 1 mm. Quantitative analysis of Iba1-positive areas demonstrated that a larger number of macrophages aggregated in the Auto, motor TP and sensory TP (f). ( n = 3). * p < 0.05, ** p < 0.01, N.S. = not significant. Data are represented as the mean ± SEM

Article Snippet: The primary antibodies used in this study were human polyclonal anti-pan-ELAVL (ELAV-like protein 2/3/4) antibody (1:2000, kindly provided from Prof. Robert Darnell, Rockefeller University), rabbit polyclonal anti-TrkA antibody (1:500, kindly provided from Prof. Louis F. Reichardt, UCSF), chicken polyclonal anti-TrkB antibody (1:500, kindly provided from Prof. Louis F. Reichardt, UCSF), goat polyclonal anti-TrkC antibody (AF373, 1:200, R&D systems), rabbit polyclonal anti-parvalbumin antibody (PV-28, 1:1000, Swant), rabbit polyclonal anti-CGRP antibody (BML-CA1134, 1:500, Enzo Life Sciences), Goat polyclonal anti-Choline acetyltransferase antibody (AB144P, 1:200, Chemicon), Mouse monoclonal anti-Islet1 and Islet2 antibody (39.4D5, 1:200, DSHB), rabbit polyclonal anti-Neurofilament heavy polypeptide antibody (ab8135, 1:500, Abcam), goat polyclonal anti-mouse and anti-rat CD31 (AF3628, 1:100, R&D), rabbit polyclonal anti-Iba1 antibody (GtX100042, 1:500, GeneTex), goat anti-GFP antibody (600–101-215, 1:1000, Rockland).

Techniques: Derivative Assay, Transplantation Assay, Immunohistochemistry, Comparison

Journal: bioRxiv

Article Title: Elimination of human papillomavirus 16-induced tumors by a mucosal rAd5 therapeutic vaccination in a pre-clinical study

doi: 10.1101/2024.03.11.584519

Figure Lengend Snippet: T cells in the blood of animals sampled one day after D13 and D20 vaccinations. (A) Total T cells as a percent of lymphocytes (CD45+). (B) CD8+ T cells as a percent of CD3+. (C) CD4+ T cells as a percent of CD4+, were measured by flow cytometry. n=10 mice/group, mean and SEM, 2-way ANOVA.

Article Snippet: Cells were then stained with CD45 BV605 (103155, Biolegend), CD3 PE (130-120-160, Miltenyi), CD4 (130-123-894, Miltenyi), CD8 BV785 (100750, Biolegend), CD62L (BV421 104436, Biolegend), and CD44 VioBright FITC (130-120-213, Miltenyi).

Techniques: Flow Cytometry