cd4 cd25 cd127 dim  (Miltenyi Biotec)

Journal: Frontiers in Immunology

Article Title: IL-33 and IL-3 synergistically induce CD25 expression on human basophils without functional IL-2 signaling: a potential marker of severe COVID-19

doi: 10.3389/fimmu.2025.1718240

Figure Lengend Snippet: IL-33 upregulates IL-2 receptor alpha chain (CD25) expression in IL-3 primed human basophils. Basophils isolated from PBMCs of healthy donors were cultured alone or in the presence of various stimuli with or without IL-3 (10 ng/ml) priming. (A) Each of the indicated stimulatory agents was added either alone or after 1 hr of IL-3 priming, and CD25 expression was evaluated by FACS after 24 hrs. Summarized data (% positive cells and MFI) for n = 6 independent donors. (B) Dose response of IL-33 cytokine on the expression of CD25 (MFI, n = 3). (C) Time kinetics of IL-3 and IL-33 on the expression of CD25 up to 96 hrs compared to time 0. IL-33 was added at 5 ng/ml after 1 hr of priming with IL-3 (10 ng/ml). The culture was supplemented every 24 hrs with additional IL-33 (5 ng/ml) (MFI, n = 3). (D) Representative FACS histogram for CD25 expression on basophils compared to isotype control. (E) Levels of soluble CD25 in the culture supernatants of basophils either cultured alone or stimulated with IL-3 and IL-33 at different time points (n = 6). Data are represented as mean ± SEM. * P < 0.05 , ** P < 0.01 , *** P < 0.001 , **** P < 0.0001 ; ns, not significant by one-way ANOVA (A, B) or 2-way ANOVA (C, E) followed by Tukey’s multiple comparison test. Abbreviation: MFI, median fluorescence intensity.

Article Snippet: CD4 + CD25 + CD127 dim/– regulatory T Cell Isolation Kit II (Miltenyi Biotec) was used for the isolation of CD4 + CD25 + CD127 dim/– Treg cells from human PBMCs.

Techniques: Expressing, Isolation, Cell Culture, Control, Comparison, Fluorescence

Journal: Frontiers in Immunology

Article Title: IL-33 and IL-3 synergistically induce CD25 expression on human basophils without functional IL-2 signaling: a potential marker of severe COVID-19

doi: 10.3389/fimmu.2025.1718240

Figure Lengend Snippet: Expression of IL-2 receptor alpha (CD25), beta (CD122) and gamma (CD132) chains on IL-3 + IL-33 stimulated basophils. CD25 (IL-2Rα), CD122 (IL-2Rβ), and CD132 (IL-2Rγ) expression was analyzed on basophils after 24 hrs of stimulation with or without IL-3 and IL-33 cytokines as indicated. (A-C) Representative FACS histogram and summarized data for the expression level of CD25, CD122, and CD132 (% positive cells and MFI). (D) Percentage of cells expressing the trimeric IL-2 receptor was identified by successive gating on each of the receptor chains. Data are represented as mean ± SEM (n = 8). * P < 0.05 , ** P < 0.01, ***P < 0.001, and ****P < 0.0001 ; ns, not significant by one-way ANOVA followed by Tukey’s multiple comparison test. Abbreviation: CA, cells alone; MFI, median fluorescence intensity.

Article Snippet: CD4 + CD25 + CD127 dim/– regulatory T Cell Isolation Kit II (Miltenyi Biotec) was used for the isolation of CD4 + CD25 + CD127 dim/– Treg cells from human PBMCs.

Techniques: Expressing, Comparison, Fluorescence

Journal: Frontiers in Immunology

Article Title: IL-33 and IL-3 synergistically induce CD25 expression on human basophils without functional IL-2 signaling: a potential marker of severe COVID-19

doi: 10.3389/fimmu.2025.1718240

Figure Lengend Snippet: Binding of IL-2 to CD25-expressing basophils neither induces downstream signaling nor limits IL-2 availability to Treg cells. Isolated basophils were cultured alone or in the presence of IL-3 and IL-33 for 24 hrs to induce the expression of CD25. (A) Cultured basophils were treated with 5 μg/mL biotinylated-IL-2 for 30 min and stained with PE-streptavidin to detect extracellular IL-2 binding to CD25. Representative FACS histograms and summarized data for binding of IL-2 to basophils (% positive cells and MFI, n = 4). (B) Cultured basophils were treated with IL-2 (3000 U/ml) for 30 min, followed by intracellular staining for phosphorylated STAT5 (pSTAT5). IL-3 (10 ng/ml) was added for 30 min on untreated basophils as a positive control for STAT5 activation on basophils. Representative FACS histograms and summarized data showing pSTAT5 expression (% positive cells, n = 3). (C) Representative histogram showing the p-STAT5 expression in isolated regulatory T cells following stimulation with IL-2 (3000 U/ml). (D) Basophils were treated with IL-3 and IL-33 for 72 hrs, and IL-2 (20 U/ml) was added during last 24 hrs. Subsequently, Treg cells were cocultured with these basophils at a 1:1 ratio, and the viability of Treg cells was analyzed by Annexin-V and PI staining after 48 hrs. Representative dot plots and summarized data from different donors are presented (n = 6). Data are represented as mean ± SEM. * P < 0.05 , ** P < 0.01 , **** P < 0.0001 ; ns, not significant by One-way ANOVA followed by Tukey’s multiple comparison test. Abbreviation: CA, cells alone; MFI, median fluorescence intensity; PI, propidium iodide.

Article Snippet: CD4 + CD25 + CD127 dim/– regulatory T Cell Isolation Kit II (Miltenyi Biotec) was used for the isolation of CD4 + CD25 + CD127 dim/– Treg cells from human PBMCs.

Techniques: Binding Assay, Expressing, Isolation, Cell Culture, Staining, Positive Control, Activation Assay, Comparison, Fluorescence

Journal: Frontiers in Immunology

Article Title: IL-33 and IL-3 synergistically induce CD25 expression on human basophils without functional IL-2 signaling: a potential marker of severe COVID-19

doi: 10.3389/fimmu.2025.1718240

Figure Lengend Snippet: Basophils from severe COVID-19 patients display enhanced expression of IL2RA and IL2RG . (A) Expression of IL2 receptor subunit transcripts in BALF basophils of healthy controls, mild, and severe COVID-19 patients. Violin plots showing the normalized expression levels of IL2RA , IL2RB , and IL2RG in BALF basophils from healthy controls (Cnt, blue), mild (mild, green), and severe (Sev, red) COVID-19 patients. Individual dots indicate single-cell expression values (B) Global Expression of IL3 and IL33 in cells from BALF of healthy controls, mild, and severe COVID-19 patients. Violin plot showing the normalized expression of IL3 and IL33 in BALF cells from healthy controls (Cnt, blue), mild (mild, green), and severe (Sev, red) COVID-19 patients. Individual dots indicate single-cell expression values. Statistical significance was assessed using Wilcoxon rank-sum test; **** P < 0.0001 , * P < 0.05 . (C) Graphical abstract. IL-3 and IL-33 cytokines synergistically induce CD25 expression in basophils. Transient IL-2 sequestration by CD25 + basophils or basophil-derived soluble CD25 could increase the bioavailability of low-dose IL-2 for Treg cells and thereby have a positive impact on their survival. On the other hand, the expression of CD25 and CD132 on basophils might serve as potential biomarkers of severe inflammation like COVID-19. Figure created at BioRender.com .

Article Snippet: CD4 + CD25 + CD127 dim/– regulatory T Cell Isolation Kit II (Miltenyi Biotec) was used for the isolation of CD4 + CD25 + CD127 dim/– Treg cells from human PBMCs.

Techniques: Expressing, Derivative Assay

Journal: Nature Cardiovascular Research

Article Title: Low-dose interleukin-2 induces clonal expansion of BACH2-repressed effector regulatory T cells following acute coronary syndrome

doi: 10.1038/s44161-025-00652-y

Figure Lengend Snippet: a , LILACS study sample collection and workflow schematic. Three treatment groups from Part B of LILACS are evaluated in this study: placebo ( n = 4), 1.5 MIU d −1 IL-2 ( n = 6) and 2.5 MIU d −1 IL-2 ( n = 6). b , Uniform manifold approximation and projection (UMAP) visualization of unsupervised clustering revealed 8 distinct T cell populations ( n = 41,050 cells). c , Heat map with the average expression of canonical T cell function-associated genes. The histogram on the right of the heat map shows the number of cells within each cluster. d , Single-cell TCR workflow schematic. e , Gene segment usage and gene–gene pairing landscapes are illustrated graphically using four vertical stacks (one for each V and J segment) connected by curved segments with thickness proportional to the number of TCRs with the respective gene pairing. Each stack has genes with the highest frequencies stacked on top with subsequent genes in descending order. f , Plot showing the frequency of clonotype sizes across all T cells, CD4 + T cells or CD8 + T cells. g , Network analysis showing larger ( n ≥ 15) clonotypes. Each number is a clonotype identification number, each individual color represents a single patient and the size of the dots represents the number of cells. Linked dots are clonotypes that are closely related. Panels a and d created with BioRender.com .

Article Snippet: Primary human T reg cells were isolated from fresh (collected within 12 h) apheresis cones using a Lymphoprep gradient and a subsequent EasySep Human CD4 + CD127 low CD25 + Regulatory T Cell Isolation Kit (Stemcell) or Human CD4 + CD25 + CD127 dim/− Regulatory T Cell Isolation Kit II (Miltenyi).

Techniques: Expressing, Cell Function Assay

Journal: Nature Cardiovascular Research

Article Title: Low-dose interleukin-2 induces clonal expansion of BACH2-repressed effector regulatory T cells following acute coronary syndrome

doi: 10.1038/s44161-025-00652-y

Figure Lengend Snippet: a , scRNA-seq data showing the effect of IL-2 versus placebo on T cell subsets. There was a significant increase in T reg cell numbers after 1.5 MIU d −1 and 2.5 MIU d −1 of IL-2 treatment compared with pooled pretreatment counts: * P = 0.034; **P = 0.0072 by two-tailed t -test. b , Histogram showing the distribution of clonotype size across 5 bins: 1, 2–5, 6–10, 11–20 and 21+. The bars represent the proportion of that clonotype size in each treatment allocation. *** P < 0.001 (by chi-squared (one-tailed) test), indicating significant difference in clonotype size proportions between 1.5 MIU d − 1 or 2.5 MIU d −1 doses and placebo. c , Histograms showing the effect of the treatment allocations on TCR diversity using Shannon entropy and D50. d , The left panel shows a UMAP with the CD4 + T cell subsets overlaid. The other panels show the effect of either placebo or IL-2 treatment on CD4 + T cell clonotype size. The shades of red or blue represent the size of clonotypes on a scale from 0 to >5. Clonotype size is defined as the number of CD4 + T cells that share highly similar TCR sequences .

Article Snippet: Primary human T reg cells were isolated from fresh (collected within 12 h) apheresis cones using a Lymphoprep gradient and a subsequent EasySep Human CD4 + CD127 low CD25 + Regulatory T Cell Isolation Kit (Stemcell) or Human CD4 + CD25 + CD127 dim/− Regulatory T Cell Isolation Kit II (Miltenyi).

Techniques: Two Tailed Test, One-tailed Test

Journal: Nature Cardiovascular Research

Article Title: Low-dose interleukin-2 induces clonal expansion of BACH2-repressed effector regulatory T cells following acute coronary syndrome

doi: 10.1038/s44161-025-00652-y

Figure Lengend Snippet: a , Venn diagrams showing the overlap of CD4 + TCR clonotypes (and associated T cells) that match between the pre- and post-dose timepoints in the various treatment allocations. *** P < 0.001 (by chi-squared (one-tailed) test), indicating a significant difference. b , Schematic showing the concept of TCR tracking. We use the TCR as a barcode to track cells over two timepoints before and after dosing. By then pairing scTCR-seq and scRNA-seq, we can phenotype these tracked cells. c , Using TCR tracking, we show the effect of acute MI (treated with placebo) on matched TCRs and their cell phenotype over two timepoints. Connected lines from the pre- and post-dose timepoints represent CD4 + T cells that have the same TCR clonotype. The labels on the sides describe the scRNA-seq phenotype of the cell. Only those clonotypes tracked onto T reg cells at either the pre- or post-dose timepoints are shown. The numbers of cells underlying this figure are provided in Supplementary Table . d , Results of transition matrix calculation within each treatment group followed by outgoing flow visualization using CellRank 2. Panel b created with BioRender.com .

Article Snippet: Primary human T reg cells were isolated from fresh (collected within 12 h) apheresis cones using a Lymphoprep gradient and a subsequent EasySep Human CD4 + CD127 low CD25 + Regulatory T Cell Isolation Kit (Stemcell) or Human CD4 + CD25 + CD127 dim/− Regulatory T Cell Isolation Kit II (Miltenyi).

Techniques: One-tailed Test

Journal: Nature Cardiovascular Research

Article Title: Low-dose interleukin-2 induces clonal expansion of BACH2-repressed effector regulatory T cells following acute coronary syndrome

doi: 10.1038/s44161-025-00652-y

Figure Lengend Snippet: A , Weblogo plot showing differences in aligned CDR3 composition in clonally expanded CD4+ non-Tregs. The height of the letters represent the relative frequency with decreasing frequency stacked on top. B , Volcano plot showing differentially expressed genes in expanded CD4+ non-Tregs vs. nonexpanded CD4+ non-Tregs (enriched in the expanded (red) or non-expanded (blue).

Article Snippet: Primary human T reg cells were isolated from fresh (collected within 12 h) apheresis cones using a Lymphoprep gradient and a subsequent EasySep Human CD4 + CD127 low CD25 + Regulatory T Cell Isolation Kit (Stemcell) or Human CD4 + CD25 + CD127 dim/− Regulatory T Cell Isolation Kit II (Miltenyi).

Techniques:

Journal: Nature Cardiovascular Research

Article Title: Low-dose interleukin-2 induces clonal expansion of BACH2-repressed effector regulatory T cells following acute coronary syndrome

doi: 10.1038/s44161-025-00652-y

Figure Lengend Snippet: a , Network plot from regulon analysis of expanded versus non-expanded T reg cells using SCENIC. b , Expression of BACH2 and encompassing regulons in expanded versus non-expanded T reg cells from the LILACS dataset. c , Representative data from flow cytometry analysis ( n = 3) of pSTAT5 levels in primary human T reg cells after 48 h of stimulation and an 8-h rest in serum-free media ± BACH2 inhibitor (inh.) (RGFP966), followed by 15 min of IL-2 treatment. Numeric values indicate percentages. SSC, side scatter; MFI, mean fluorescence intensity; FC norm, normalized fold change. d , pSTAT5 and CD25 levels assessed via flow cytometry ( n = 3). e , RT-qPCR analysis of IL-32 ( n = 5) and IL-10 ( n = 5) after 24 h in primary human T reg cells treated with BACH2 inhibitor and/or IL-2 (error bars represent s.e.m.; P values were determined by two-tailed independent t -test; n = number of independent experiments).

Article Snippet: Primary human T reg cells were isolated from fresh (collected within 12 h) apheresis cones using a Lymphoprep gradient and a subsequent EasySep Human CD4 + CD127 low CD25 + Regulatory T Cell Isolation Kit (Stemcell) or Human CD4 + CD25 + CD127 dim/− Regulatory T Cell Isolation Kit II (Miltenyi).

Techniques: Expressing, Flow Cytometry, Fluorescence, Quantitative RT-PCR, Two Tailed Test