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: Canadian Journal of Anaesthesia

Article Title: Effects of sevoflurane on natural killer cell-induced apoptosis of malignant tumour cells: an in vitro laboratory study

doi: 10.1007/s12630-026-03129-z

Figure Lengend Snippet: The three natural killer-cell-mediated tumour defense mechanisms. The first mechanism is degranulation with fast kinetics and a directed (cell–cell-contact-mediated) mechanism (A). The second mechanism is the induction of the cytokine-induced death receptor pathway (B). This mechanism is not directed (no cell–cell contact is required) and is promoted by TNF-α and IFN-γ and inhibited by IL-6. The third mechanism is extracellular-vesicle/exosome-mediated tumour defense which is not directed (C). Vesicles containing enzymes such as perforin and granzyme are released by NK cells and may induce distant, not directed target cell apoptosis. The experimental setup of this study is displayed in D and E. The natural killer cell line NK92 was treated with 2.2% sevoflurane for 2 hr. In most experiments, NK92 cells were co-incubated with leukemia cells (K562) as a target cell line for varying durations (between 0 and 48 hr); for the perforin measurements in the NK-cell supernatant and NK-cell cytoplasm, no co-incubation was performed. NK = natural killer; IL-6 = interleukin-6; IFN-γ = interferon-gamma; TNF-α = tumour necrosis factor-alpha; mRNA = messenger RNA

Article Snippet: Magnetic purification of NK cells was performed using the MACSxpress® Whole Blood NK Cell Isolation Kit, human (Miltenyi Biotech, Bergisch Gladbach, Germany).

Techniques: Incubation

Journal: Canadian Journal of Anaesthesia

Article Title: Effects of sevoflurane on natural killer cell-induced apoptosis of malignant tumour cells: an in vitro laboratory study

doi: 10.1007/s12630-026-03129-z

Figure Lengend Snippet: Sevoflurane did not affect natural killer cell viability. Live NK92 cells are defined as Annexin V− and FVS660− cells, determined by flow cytometry analysis. NK92 cells were exposed for 2 hr to sevoflurane (or to air only), followed by a short incubation phase of 1, 2, 4, and 6 hr (A), and a long one of 24 and 48 hr (B). All measurements in Fig. 2 were analyzed using two-way analysis of variance, and multiple comparisons were corrected using the Bonferroni method (four comparisons in A and three comparisons in B). Values represent means (standard deviations). Six independent experiments were included in this analysis. FVS = Fixable Viability Stain; NK = natural killer; Sevo = sevoflurane

Article Snippet: Magnetic purification of NK cells was performed using the MACSxpress® Whole Blood NK Cell Isolation Kit, human (Miltenyi Biotech, Bergisch Gladbach, Germany).

Techniques: Flow Cytometry, Incubation, Staining

Journal: Canadian Journal of Anaesthesia

Article Title: Effects of sevoflurane on natural killer cell-induced apoptosis of malignant tumour cells: an in vitro laboratory study

doi: 10.1007/s12630-026-03129-z

Figure Lengend Snippet: Sevoflurane did not affect natural killer cell degranulation, cytokine messenger ribonucleic acid induction, or the quantity of released extracellular vesicles. NK92 cell degranulation (A) was assessed by the release of CD107a (a marker of degranulation, analyzed by flow cytometry) and induction of IFN-γ (B), TNF-α (C), and IL-6 (D) mRNA (analyzed by real-time polymerase chain reaction) were quantified in NK92 cells with (sevoflurane [sevo]) or without (air) exposure to sevo and incubation with K562 tumour cells. Control cells (ctrl) were not exposed to tumour cells, and the positive degranulation control was NK92 cells stimulated (pos) with PMA and ionomycin. Ribonucleic acid data were normalized. The size (E) and number (F) of EVs released by NK92 cells with (sevo) and without (air) exposure to sevoflurane was quantified by nanotracking analysis. Particles were collected at 48 hr. An unpaired, two-sided Student’s t test was used to compare the air and sevoflurane groups (Fig. 4A–F). Bar graphs show means (standard deviations); box plots illustrate the median [interquartile range], and whiskers indicate min/max values. A minimum of three independent experiments were included in each of the analyses. CD = cluster of differentiation; Ctrl = negative control; IFN-γ = interferon-gamma; IL-6 = interleukin-6; mRNA = messenger ribonucleic acid; NK = natural killer; Pos = positive control; PMA = phorbol 12-myristate 13-acetate; Sevo = sevoflurane; TNF-α = tumour necrosis factor-alpha

Article Snippet: Magnetic purification of NK cells was performed using the MACSxpress® Whole Blood NK Cell Isolation Kit, human (Miltenyi Biotech, Bergisch Gladbach, Germany).

Techniques: Marker, Flow Cytometry, Real-time Polymerase Chain Reaction, Incubation, Control, Negative Control, Positive Control

Journal: Nature Communications

Article Title: Recruitment of plasma cells from IL-21-dependent and IL-21-independent immune reactions to the bone marrow

doi: 10.1038/s41467-024-48570-0

Figure Lengend Snippet: a Number of clonal families of BCRs found within the different BMPC clusters (diversity) and probability of finding different clonal families by random selection of cells (Simpson diversity index). A clonal family was defined by V and J gene composition and a CDR3 region with <20% Hamming distance in both the heavy and light chains originating from one donor. b Mutation rates in the framework regions FR1-3 (left) and in the CDR1-3 regions (right) of the heavy and light chain rearrangements across BMPC clusters. c Bubble plot of the mutation rates in the framework regions FR1-3 (left) and in the CDR1-3 regions (right) of BCRs per isotype and cluster. Colour scale indicates median mutation rates. Values below or above the scale limits are shown in blue or red, respectively. Bubble sizes correspond to the percentage of cells expressing a defined isotype within the indicated cluster. d Violin plot depicting the mutation rates in the V gene of the BCRs of BMPC per cluster. Statistical significance between clusters is shown in Supplementary Data (two-tailed Mann–Whitney U test). Horizontal lines represent the median mutation rate. Violins are coloured by the z score of CD19 gene expression in each cluster. e Identification of SARS-CoV-2 spike-specific and tetanus toxoid-specific public clones (in black) among analysed BMPC. Public clones were defined by exhibiting over 80% CDR3 sequence identity in both heavy and light chains when compared to the BCR of sequenced peripheral blood and bone marrow spike- and tetanus-specific cells from vaccinated individuals (see Supplementary Fig. and Supplementary Data ). f Relative distribution of spike-specific (red) and tetanus-specific (blue) BMPC (depicted in e ) per cluster. g Comparison of mutation rates within the framework regions FR1-3 (left) and the CDR1-3 regions (right) of spike-specific (red) and tetanus-specific (blue) BMPC per isotype. Horizontal lines represent the median mutation rate. Statistics were performed using a two-tailed Mann–Whitney U test. Source data are provided as a Source Data file.

Article Snippet: Plasma cells were enriched from bone marrow using StraightFrom Whole Blood and Bone Marrow CD138 MicroBeads and StraightFrom Whole Blood CD19 MicroBeads (Miltenyi Biotec) according to manufacturer’s instructions.

Techniques: Selection, Mutagenesis, Expressing, Two Tailed Test, MANN-WHITNEY, Gene Expression, Clone Assay, Sequencing, Comparison

Journal: Nature Communications

Article Title: Recruitment of plasma cells from IL-21-dependent and IL-21-independent immune reactions to the bone marrow

doi: 10.1038/s41467-024-48570-0

Figure Lengend Snippet: a , b Gene Set Enrichment Analysis (GSEA) based on gene signatures from ex vivo-differentiated plasmablasts in presence of different cytokine combinations (data sets from Stephenson et al. ). Briefly, naive B cells were cultured in differentiating conditions for 6 days after which different cytokines were added to the culture. After 12 or 24 h, cells were harvested and sequenced. Gene signatures were identified by comparing the different conditions and time points to 0 h. a Density plots of ASC (time points and vaccine protocols combined) with significant enrichment identified by GSEA of gene signatures from ex vivo-differentiated plasmablasts. b Density plots of BMPC with significant enrichment identified by GSEA of gene signatures from ex vivo-differentiated plasmablasts. Violin plots of the normalised enrichment score (NES) per BMPC cluster are depicted in Supplementary Fig. . Statistical significance between NES scores is shown in Supplementary Data (two-tailed Mann–Whitney U test). c , d T follicular helper cells (Tfh, CD19 − CD4 + CD45RA − CXCR5 ++ ) from tonsils or T peripheral helper cells (Tph, CD19 − CD4 + CD45RA − CXCR5 − ) from BAL of sarcoidosis patients were co-cultured 1:1 with tonsillar memory B cells (CD19 + CD4 − IgD − CD38 − ) for 7 days in presence of staphylococcal enterotoxin B (SEB). Where indicated, T-cell help was blocked with an anti-CD40L antibody and/or recombinant soluble IL-21R. c Histograms of CD19 expression by differentiated plasmablasts (see Supplementary Fig. 7). Two Tfh and two Tph donors were analysed. d Frequencies of CD19 hi plasmablasts. Statistics were performed using an RM parametric one-way ANOVA followed by multiple testing according to Fischer’s LSD test. Source data are provided as a Source Data file.

Article Snippet: Plasma cells were enriched from bone marrow using StraightFrom Whole Blood and Bone Marrow CD138 MicroBeads and StraightFrom Whole Blood CD19 MicroBeads (Miltenyi Biotec) according to manufacturer’s instructions.

Techniques: Ex Vivo, Cell Culture, Two Tailed Test, MANN-WHITNEY, Recombinant, Expressing

Journal: Nature Communications

Article Title: Recruitment of plasma cells from IL-21-dependent and IL-21-independent immune reactions to the bone marrow

doi: 10.1038/s41467-024-48570-0

Figure Lengend Snippet: a Representative pseudocolour plots of intracellular double-positive SARS-CoV-2 RBD (left) or tetanus toxoid (TT, right) staining in CD38 high CD138 + CD14 − CD3 − live singlet BMPC (gating strategy in Supplementary Fig. ). b – e Each symbol represents one donor/sample (see Supplementary Table ). Filled symbols represent BMPC samples which were also analysed by single-cell sequencing (Fig. ). b Frequencies of RBD-specific and TT-specific BMPC within total BMPC. Horizontal lines indicate the median. Statistics were performed using the one-tailed Mann–Whitney U test. n = 20 BM samples. c Frequencies of CD19 low cells within RBD-specific BMPC (red), TT-specific BMPC (blue) and total BMPC (black). Horizontal lines indicate the median. Statistics were performed using the Kruskal–Wallis tests with Dunn’s correction for multiple comparisons. n = 20 BM samples. d Frequencies of IgG+ (left) and IgA+ (right) cells within RBD-specific BMPC (red), TT-specific BMPC (blue) and total BMPC (black). Horizontal lines indicate the median. Statistics were performed using the Kruskal–Wallis tests with Dunn’s correction for multiple comparisons. n = 20 BM samples. e Correlation between the frequency of CD19 low RBD-specific BMPC and days after 3rd vaccination against SARS-CoV-2. Statistics were performed using one-tailed Spearman’s correlations. n = 11 BM samples. Source data are provided as a Source Data file.

Article Snippet: Plasma cells were enriched from bone marrow using StraightFrom Whole Blood and Bone Marrow CD138 MicroBeads and StraightFrom Whole Blood CD19 MicroBeads (Miltenyi Biotec) according to manufacturer’s instructions.

Techniques: Staining, Sequencing, One-tailed Test, MANN-WHITNEY