Journal: Nature Communications

Article Title: Autoantibody landscape and functional role of anti-C-C motif chemokine receptor 8 autoantibodies in systemic sclerosis: post-hoc analysis of a B-cell depletion trial

doi: 10.1038/s41467-025-66974-4

Figure Lengend Snippet: A Representative histograms from flow cytometry showing binding of anti-C-C motif chemokine receptor 8 (CCR8)-positive IgG from systemic sclerosis (SSc) patients or healthy controls (HC) to CCR8-overexpressing HEK293 cells. A fluorophore-conjugated anti-human IgG Fc antibody was used for detection. B Quantification of mean fluorescence intensity (MFI) in CCR8-overexpressing cells incubated with anti-CCR8-positive or control IgG, with or without preincubation with blocking anti-CCR8 monoclonal antibody ( n = 4 technical replicates per group). C ERK phosphorylation levels in CCR8-overexpressing HEK293 cells after stimulation with CCL1 in the presence of anti-CCR8-positive or control IgG, as measured by ELISA ( n = 8 biological replicates per group). D Treg migration assay in Transwell culture systems. The number of migrated Tregs in response to CCL1 with anti-CCR8-positive IgG or control IgG ( n = 6 biological replicates per group) are presented. Statistical significance was assessed using Welch’s ANOVA with Dunnett’s T3 multiple comparison adjustment. Error bars are defined as the standard error of the mean. Source data are provided as a Source Data file.

Article Snippet: Tregs were isolated from human peripheral blood using the MACSxpress Whole Blood Treg Isolation Kit (#130-109-557, Miltenyi Biotec, Bergisch Gladbach, North Rhine-Westphalia, Germany), following the manufacturer’s instructions with minor modifications.

Techniques: Flow Cytometry, Binding Assay, Fluorescence, Incubation, Control, Blocking Assay, Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Migration, Comparison

Journal: Nature Communications

Article Title: Autoantibody landscape and functional role of anti-C-C motif chemokine receptor 8 autoantibodies in systemic sclerosis: post-hoc analysis of a B-cell depletion trial

doi: 10.1038/s41467-025-66974-4

Figure Lengend Snippet: A Experimental protocol of the skin sclerosis mouse model. Six-week-old female C57BL/6NcrSlc mice (MGI ID: MGI:5295404) received daily subcutaneous injections of bleomycin (BLM, 200 µg) or PBS as a control for two weeks. Mice were also treated intraperitoneally with either control (Ctrl) IgG or anti-C-C motif chemokine receptor 8 (CCR8) antibody (Ab) once on Day 8. Skin biopsies were collected at Week 2 for histological analysis and next-generation sequencing (NGS). This figure was partially created with BioRender ( https://BioRender.com/slxff50 ). Representative histological images and quantitative analyses of dermal fibrosis and Treg infiltration in mouse skin. H&E-stained B and Masson’s trichrome–stained C skin sections show increased dermal thickness and collagen deposition following BLM treatment. Images were captured at ×200 magnification, and scale bars represent 50 μm. D Quantification of dermal thickness and E Foxp3⁺ Treg counts are presented in the accompanying bar graphs ( n = 6 biological replicates for PBS group; n = 5 biological replicates for BLM-treated groups). Statistical significance was assessed using Welch’s ANOVA with Dunnett’s T3 multiple comparison adjustment. Error bars are defined as the standard error of the mean. Source data are provided as a Source Data file.

Article Snippet: Tregs were isolated from human peripheral blood using the MACSxpress Whole Blood Treg Isolation Kit (#130-109-557, Miltenyi Biotec, Bergisch Gladbach, North Rhine-Westphalia, Germany), following the manufacturer’s instructions with minor modifications.

Techniques: Control, Next-Generation Sequencing, Staining, Comparison

Journal: bioRxiv

Article Title: UBA1 Mutations Drive RIPK1-Mediated Cell Death and Monocyte Dysfunction in VEXAS Syndrome

doi: 10.1101/2025.10.06.680650

Figure Lengend Snippet: Regulated cell death pathways are activated in myeloid cells from VEXAS patients. ( A ) Leucocytes, neutrophils, monocytes, lymphocytes count from patients with VEXAS and elderly gender-matched healthy controls (HC). Panels show individual data (dots) and means ± SEM (histograms). P values were determined by the Mann-Whitney test. ( B ) Hematoxylin-eosin staining of UBA1-mutated Sweet-like lesion revealing karyorrhectic nuclei and apoptotic debris in VEXAS (arrows). Illustrative picture is shown (Magnification x10 and x40, scale bar 100 μm and 50µm). ( C ) Representative immunofluorescence images of pMLKL (green) staining on CD14+ sorted cells form 3 active VEXAS patients and 3 elderly gender-matched HC. Nuclei are stained in blue with Hoechst. Percentage of pMLKL cell surface area per CD14+ cells, data are shown and means (Histograms) ± SEM. Forty cells were quantified for each patient. ( D ) Multiplex Immunofluorescence of skin biopsy samples from VEXAS skin lesion showing the co-expression of cleaved GSDMD, MLKL, cleaved caspase-3 and phosphorylated RIPK1 within CD68+ infiltrates in VEXAS. ( E ) Gene set enrichment analysis of apoptosis (KEGG), necroptosis (GOBP) and pyroptosis (GOBP) pathways enriched in VEXAS lesional skin (n=6) versus non lesional skin from HC (n=5) adapted form from dataset GSE245639. *P□<□0.05; **P□<□0.01; ***P□<□0.001, ****P□<□0.0001. SEM, HC, Healthy Control; Standard Error of the Mean; VEXAS, Vacuoles, E1 enzyme, X-linked, Autoinflammatory, Somatic; WT, Wild-Type.

Article Snippet: For CD14+ cells form patients, cells were sorted from 2mL fresh Whole Blood collected in EDTA tube and sorted using StraightFrom® Whole Blood CD14 MicroBeads and Whole Blood column kit (Milteny Biotec, #130-090-879) according to the manufacturer’s instructions.

Techniques: MANN-WHITNEY, Staining, Immunofluorescence, Multiplex Assay, Expressing, Control

Journal: eBioMedicine

Article Title: The impact of oral cannabis consumption during pregnancy on maternal spiral artery remodelling, fetal growth and offspring behaviour in mice

doi: 10.1016/j.ebiom.2025.105572

Figure Lengend Snippet: Cannabinoids decrease IFN- γ expression in human NK cells . a–c: PBMCs were isolated from healthy donor blood and incubated in the presence of absence of THC or CBD for 24 h and then stained for IFN- γ . a: Schematic illustrating experimental design. b: IFN- γ expression in NK cells relative to control following incubation in THC (n = 5 per group). c: IFN- γ expression in NK cells relative to control following incubation in CBD (n = 3–5 per group). d–f: Isolated pbNK cells were converted to NKreg cells and then incubated with THC or CBD. d: Schematic illustrating experimental design. e: VEGF amount relative to control following THC incubation (n = 3–4 per group). f: VEGF amount relative to control following CBD incubation (n = 5 per group). Data are means ± SEM, ∗P < 0.05, ∗∗P < 0.01 (b Kruskal–Wallis test, c, e and f one-way ANOVA).

Article Snippet: NK cells were isolated from female human whole blood using MACSxpress Whole Blood NK Cell Isolation Kit (Miltenyi Biotec #130-127-695).

Techniques: Expressing, Isolation, Incubation, Staining, Control

Journal: Translational Lung Cancer Research

Article Title: Enumeration and molecular characterization of circulating tumor cells enriched by microcavity array from stage III non-small cell lung cancer patients

doi: 10.21037/tlcr-20-841

Figure Lengend Snippet: Contrived samples in HDB. (A) Representative on-chip immunofluorescence image of cells captured with the cartridge of the Hitachi MCA system. Red: CD45; blue: DAPI; green: cytokeratin, white: brightfield image of 8 µm × 100 µm microcavity, imaged at 100×. CTCs are defined as cells that lack CD45 but have a defined DAPI nuclear stain and cytokeratin. (B) Recovery of tumor cell lines spiked into healthy donor blood. The recovery rates of spiked cell lines were 91.5% for H358 and 84.3% for A549. HDB, healthy donor blood; MCA, Micro Cavity Array.

Article Snippet: To generate this leukocyte-poor fraction, we incubated peripheral blood samples with StraightFrom Whole Blood CD45 MicroBeads at 50 μL per mL of blood passed through a magnetic column (Miltenyi Biotec) before enrichment by the MCA system.

Techniques: Immunofluorescence, Staining

Journal: Translational Lung Cancer Research

Article Title: Enumeration and molecular characterization of circulating tumor cells enriched by microcavity array from stage III non-small cell lung cancer patients

doi: 10.21037/tlcr-20-841

Figure Lengend Snippet: Image of CD45 depletion. (A) Shows an MCA cartridge imaged by immunofluorescence where the leukocytes were depleted using CD45 Microbeads prior to enrichment. (B) Shows high levels of co-eluting leukocytes in an MCA cartridge without pre-treatment with CD45 microbeads. MCA, Micro Cavity Array.

Article Snippet: To generate this leukocyte-poor fraction, we incubated peripheral blood samples with StraightFrom Whole Blood CD45 MicroBeads at 50 μL per mL of blood passed through a magnetic column (Miltenyi Biotec) before enrichment by the MCA system.

Techniques: Immunofluorescence

Journal: Translational Lung Cancer Research

Article Title: Enumeration and molecular characterization of circulating tumor cells enriched by microcavity array from stage III non-small cell lung cancer patients

doi: 10.21037/tlcr-20-841

Figure Lengend Snippet: Multivariate analyses

Article Snippet: To generate this leukocyte-poor fraction, we incubated peripheral blood samples with StraightFrom Whole Blood CD45 MicroBeads at 50 μL per mL of blood passed through a magnetic column (Miltenyi Biotec) before enrichment by the MCA system.

Techniques:

Journal: Translational Lung Cancer Research

Article Title: Enumeration and molecular characterization of circulating tumor cells enriched by microcavity array from stage III non-small cell lung cancer patients

doi: 10.21037/tlcr-20-841

Figure Lengend Snippet: Multivariate analyses with CTC count

Article Snippet: To generate this leukocyte-poor fraction, we incubated peripheral blood samples with StraightFrom Whole Blood CD45 MicroBeads at 50 μL per mL of blood passed through a magnetic column (Miltenyi Biotec) before enrichment by the MCA system.

Techniques:

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: B cells were enriched from peripheral blood using StraightFrom® Whole Blood CD19 MicroBeads (Miltenyi Biotec) according to manufacturer’s instructions.

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: B cells were enriched from peripheral blood using StraightFrom® Whole Blood CD19 MicroBeads (Miltenyi Biotec) according to manufacturer’s instructions.

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

Journal: Cancer Research

Article Title: Leptin Receptor–Related Immune Response in Colorectal Tumors: The Role of Colonocytes and Interleukin-8

doi: 10.1158/0008-5472.can-08-1017

Figure Lengend Snippet: Figure 5. Colonocyte-immune cell effects. The human colon cancer cell line HT-29 was cultured and used for leptin-induced stimulatory effect on CD8+ cells. Fresh media without or with recombinant human leptin at three concentrations (10, 50, and 100 nmol/L) were added and the incubation was continued for another 24 h. After this period, the supernatants were gently collected and transferred directly to CD8+ T-cell culture flasks (v:v), which were obtained from PBMCs of six healthy persons by Ficoll-Hypaque. A, perforin concentrations were measured in the supernatants of the human CD8+ enriched T-cell culture flasks by ELISA test. B, Western blotting of perforin from human CD8+ enriched T cells by using a mouse monoclonal IgG2a perforin 1 and a h-actin mouse monoclonal IgG1. Immunoreactivity was visualized with goat anti-mouse IgG-HRP and revealed by enhanced chemiluminescence. C, isolated colonocytes from male adult BALB/c mice were incubated for 24 h, the media were discarded, and cell layers were washed. New fresh media with or without recombinant mouse leptin (498-OB-01M) were added (10, 50, and 100 nmol/L) and the incubation was continued for another 24 h. The supernatants were then collected and transferred (v:v) directly to mouse splenocytes or mouse CD8+ enriched T-cell culture flasks. Culture flasks containing nonseparated splenocytes and those with CD8+ enriched cells were incubated for 24 h, and then supernatants of mouse control- or leptin-treated colonocytes were added and incubation was continued for another 48 h. The remaining monolayer splenocytes in culture flasks were harvested and tested for CD8 expression by using semiquantitative RT-PCR (top); RNA sequences were reverse transcribed to complementary DNAs and PCR was done. Sense and antisense primer sequences were CD8 sense, 5¶-TCACAGGCGAAGTCCAATCC-3¶; CD8 antisense, 5¶-AGGATGCTCTTGGCTCTTCC-3¶; h-actin sense, 5¶-GGGTCAGAAGGATTCCTATG-3¶; and h-actin antisense, 5¶-GGTCTCAAACATGATCTGGG-3¶. Results were normalized by using the housekeeping gene b-actin in the same sample. Lysates of the splenocyte culture flasks were prepared for Western blotting by using a mouse monoclonal IgG1 against granzyme B and a h-actin mouse monoclonal IgG1 (bottom). D, cytotoxic activity of CD8+ enriched splenocytes was determined in a standard 51Cr release assay. These effector cells were harvested after incubation with supernatant collected from control- or leptin-treated colonocytes and incubated for 4 h with 51Cr-labeled (Amersham) P815 cells used as syngenic target cells and EL4 as control target cells for specificity. Varying numbers of effector cells were added to 10,000 51Cr-labeled target cells in appropriate effector/target cell ratios. Supernatants were removed, and radioactivity was counted, to determine the percentage of specific lysis, as 100 [(cpm released from target cells in the presence of effector cells cpm released from target cells alone) / (cpm released from target cells lysed with 10% Triton X-100 cpm released from target cells alone)], where cpm is counts per minute. Total cell lysis was determined in control target cell samples (n = 3) after Triton X-100 was added and radioactivity quantified.

Article Snippet: One part of these cells was labeled with human whole blood CD8 MicroBeads (130-090-878 Miltenyi Biotec; 50 AL/mL of sample) according to the manufacturer’s protocol.

Techniques: Cell Culture, Recombinant, Incubation, Enzyme-linked Immunosorbent Assay, Western Blot, Isolation, Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Reverse Transcription, Activity Assay, Release Assay, Labeling, Radioactivity, Lysis

Journal: Cancer Research

Article Title: Leptin Receptor–Related Immune Response in Colorectal Tumors: The Role of Colonocytes and Interleukin-8

doi: 10.1158/0008-5472.can-08-1017

Figure Lengend Snippet: Figure 6. Interaction between colon cells and immune cells. Leptin exerts its effects via ObRb, the large-size leptin receptor that is located on both colon cells and various immune cells in the immune system (see review in ref. 6). Cytokines are released from normal colon and tumor cells in proportions depending on the density of ObRb. The products of colon cells act to recruit lymphocytes. Regulatory T lymphocyte activity may be suppressed and CD8+ T cells may be activated. In the present study, we established production of cytotoxic markers such as perforin and granzyme B produced from normal PBMCs and normal splenocytes in human and in mice, respectively.

Article Snippet: One part of these cells was labeled with human whole blood CD8 MicroBeads (130-090-878 Miltenyi Biotec; 50 AL/mL of sample) according to the manufacturer’s protocol.

Techniques: Activity Assay, Produced