magnetic beads  (Miltenyi Biotec)

Journal: Science Advances

Article Title: CD318 expression defines a novel subset of human CD8 + regulatory T cells

doi: 10.1126/sciadv.adz4203

Figure Lengend Snippet: ( A ) Purified human CD4 T cells were incubated with rhCD318 (10402-CU-050, R&D System) for 30 min on ice, followed by staining with anti–CD6-BV421 and anti–CD318-APC to measure the binding of rhCD318 on CD4 T cells. Data shown are representative of individual donors from multiple experiments. n = 5, n indicates biological replicates. ( B ) Je6-NF-κB::eGFP Jurkat cells were incubated for 24 hours with superantigen pulsed BSM and Priess, and the expression of CD69 and CD25 was measured. n = 3 to 6, n indicates technical replicates. ( C ) Je6-NF-κB::eGFP Jurkat cells were CD6 KD by nucleofection. CD6 expression was measured by after 24 hours. ( D ) CD318 KD Je6-NF-κB::eGFP Jurkat cells were incubated for 24 hours with superantigen pulsed Priess, and the expression of CD69 and NF-κB::eGFP was measured. n = 3, n indicates technical replicates. Data represent mean ± SEM. Statistical significance was assessed using a one-way ANOVA followed by multiple-comparison analysis. * P < 0.05, *** P < 0.001, and **** P < 0.0001.

Article Snippet: For suppression assay, we isolated CD4 + CD25 + CD127dim/ − T regs by magnetic beads (130-094-775, Miltenyi Biotech).

Techniques: Purification, Incubation, Staining, Binding Assay, Expressing, Comparison

Journal: Science Advances

Article Title: CD318 expression defines a novel subset of human CD8 + regulatory T cells

doi: 10.1126/sciadv.adz4203

Figure Lengend Snippet: ( A ) Human CD4 and CD8 T cells were tested for the expression of early T cell activation markers after 24 hours of stimulation with anti-CD3 (0.1 μg/ml), without (white dots) or with (black dots) rhCD318 (5 μg/ml, 4°C O/N coat). n = 3. ( B ) Purified CD4 and CD8 T cells were stimulated with plate-bound anti-CD3 (0.5 μg/ml), without (white dots) or with (black dots) rhCD318 (5 μg/ml, 4°C O/N coat). Seven days poststimulation, T cells were restimulated with PMA and ionomycin to measure cytokine expression. The expansion fold was normalized by control anti-CD3 alone (dotted line). Data shown are representative of individual donors from multiple experiments. n = 6 to 10. ( C ) CD127 − CD25 + FOXP3 + T reg proliferation analysis. Microbead-sorted CFSE-labeled T regs were stimulated with CD3 (0.5 μg/ml), without (white dots) or with (black dots) rhCD318 (5 μg/ml, plate coated) at 1 × 10 6 cells/ml with IL-2 (10 IU/ml) for 7 days. T reg expansion is shown on the y axis as the fold change relative to anti-CD3 alone (dotted line). n = 4, n indicates biological replicates. CFSE profiling and cytokine expression were evaluated by flow cytometry. n indicates biological replicates. Data are presented as the mean ± SEM. Statistical significance was assessed using a two-tailed Student’s t test. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. n = 6 to 10.

Article Snippet: For suppression assay, we isolated CD4 + CD25 + CD127dim/ − T regs by magnetic beads (130-094-775, Miltenyi Biotech).

Techniques: Expressing, Activation Assay, Purification, Control, Labeling, Flow Cytometry, Two Tailed Test

Journal: Science Advances

Article Title: CD318 expression defines a novel subset of human CD8 + regulatory T cells

doi: 10.1126/sciadv.adz4203

Figure Lengend Snippet: ( A ) ND PBMCs were stimulated with anti-CD3 (1 μg/ml) for 7 days ± rhIL-2 (50 IU/ml). Levels of CD318 were measured from total live cells. n = 4. ( B ) Expression of CD318 on CD4 (white dots) and CD8 (black dots) at days 7, 14, and 21 after stimulation and expansion in vitro. n = 7 to 10. Activated PBMCs were split at day 7 and expanded. For expansion, half of the media was replaced with fresh media containing rhIL-2 (final concentration, 50 IU/ml). ( C ) Cytokine expression in CD318 − (white dots) and CD318 + (black dots) CD8 T cells was measured at day 7 after stimulation. Flow cytometry data shown are representative of three individual donors. n = 3. ( D ) Cytokine secretion was measured from CD318 − (white dots) and CD318 + (black dots) CD8 T cells expanded for 21 days. Supernatants were collected from CD318 − and CD318 + CD8 T cells isolated from 21-day culture-expanded PBMCs and then restimulated with anti-CD3 (1 μg/ml) for 24 hours. n = 3. Cytokine levels were measured by ELISA. ( E ) IFN-γ and TNFα expression on CD318 + CD8 T cells. CD8 T cells expanded for 21 days were enriched with CD318 + CD8 T cells. CD318 + CD8 T cells were restimulated with anti-CD3 (1 μg/ml) alone, or with anti-CD28 (2 μg/ml), anti-CD318 (5 μg/ml), or both anti-CD28 and anti-CD318 together for 24 hours. Cytokine expression was determined by flow cytometry. n = 7, n indicates biological replicates. Data are presented as the mean ± SEM. Statistical significance was assessed using a two-tailed Student’s t test (A) or two-way to [(B) to (D)] or one-way ANOVA followed by multiple-comparison analysis (E). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: For suppression assay, we isolated CD4 + CD25 + CD127dim/ − T regs by magnetic beads (130-094-775, Miltenyi Biotech).

Techniques: Expressing, In Vitro, Concentration Assay, Flow Cytometry, Isolation, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Comparison

Journal: Science Advances

Article Title: CD318 expression defines a novel subset of human CD8 + regulatory T cells

doi: 10.1126/sciadv.adz4203

Figure Lengend Snippet: ( A ) Phenotypic comparison between CD318 − (red dots) and CD318 + (blue dots) CD8 T cells at day 7 after stimulation. Data are representative of three individual donors. n = 3. ( B ) FlowSOM and hierarchical clustering to identify unique CD318 + CD8 T cell clusters. ( C ) Suppressive function of CD318 + CD8 T cells. Expanded CD318 + CD8 T cells were isolated at day 21. CFSE-labeled autologous PBMCs were stimulated with T cell activator anti-CD2/3/28–coated microbeads in the presence of varying numbers of CD318 + (white dots) CD8 T cells and purified natural T regs for positive control (black dots). CFSE-labeled autologous CD4 and CD8 T cell proliferation was evaluated by flow cytometry at day 5. Data shown are representative of multiple experiments. n = 3 to 8, n indicates biological replicates. Data are presented as the mean ± SEM. Statistical significance was assessed using a two-tailed Student’s t test or two-way ANOVA followed by multiple-comparison analysis. *** P < 0.001 and **** P < 0.0001.

Article Snippet: For suppression assay, we isolated CD4 + CD25 + CD127dim/ − T regs by magnetic beads (130-094-775, Miltenyi Biotech).

Techniques: Comparison, Isolation, Labeling, Purification, Positive Control, Flow Cytometry, Two Tailed Test

Journal: Science Advances

Article Title: CD318 expression defines a novel subset of human CD8 + regulatory T cells

doi: 10.1126/sciadv.adz4203

Figure Lengend Snippet: ( A ) CD318 costimulation induce phosphorylation of SHP2 in T cell. Human PBMCs were immobilized on plates coated with anti-CD3 and stimulated in the presence or absence of CD318 (5 μg/ml, 4°C O/N coat). n = 4. ( B ) CD318 suppresses T cell proliferation independently of SHP2 inhibition. PBMCs stimulated with plate-bound anti-CD3, in the presence or absence of rhCD318 (5 μg/ml, 4°C O/N coat) and of a SHP2 inhibitor (2 μM) were assessed for T cell proliferation using CFSE profiles at 4 days after stimulation. n = 4. ( C ) Purified CD4 T cells were incubated with plate-bound anti-CD3 ± rhCD318 for 10 min at 37°C. Cells were harvested and immediately analyzed for phosphorylation of ERK1/2, Zap70, and MAPK-p38 by flow cytometry. n = 3, n indicates biological replicates. Data are presented as the mean ± SEM. Statistical significance was assessed using a one-way ANOVA or two-tailed Student’s t test followed by multiple-comparison analysis. * P < 0.05, ** P < 0.01, and **** P < 0.0001.

Article Snippet: For suppression assay, we isolated CD4 + CD25 + CD127dim/ − T regs by magnetic beads (130-094-775, Miltenyi Biotech).

Techniques: Phospho-proteomics, Inhibition, Purification, Incubation, Flow Cytometry, Two Tailed Test, Comparison

Journal: Science Advances

Article Title: CD318 expression defines a novel subset of human CD8 + regulatory T cells

doi: 10.1126/sciadv.adz4203

Figure Lengend Snippet: ( A ) Baseline expression of CD318 in CD4 and CD8 T cells. CD4 and CD8 T cells from ND and T1D PBMCs were measured for CD318 expression by flow cytometry. n = 2 to 9. ( B ) CD318 expression in CD4 and CD8 T cells from ND (white dots) and T1D (black dots) after 7-day stimulation with anti-CD3 + rhIL-2 (50 IU/ml). n = 3 to 10. ( C ) Phosphorylation of mTOR and S6 in CD8 and CD4 T cells. PBMCs from ND and T1D were stimulated with anti-CD3 + rhIL-2 (50 IU/ml) for 16 hours. Phosphorylation was measured in CD8 + and CD4 + T cells by flow cytometry. n = 2 to 4, n indicates biological replicates. Flow cytometry data are representative of multiple experiments. ( D ) Phosphorylation of mTOR and S6 in CD8 T cells at 48 hours. PBMCs from ND and T1D were stimulated with anti-CD3 + rhIL-2 for 48 hours. Phosphorylation was measured in CD8 + T cells by flow cytometry. n = 2 to 4. n indicates biological replicates. Flow cytometry data are representative of two experiments. Data are presented as the mean ± SEM. Statistical significance was assessed using a two-tailed Student’s t test [(B) and (D)] or one-way ANOVA followed by multiple-comparison analysis (C). * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: For suppression assay, we isolated CD4 + CD25 + CD127dim/ − T regs by magnetic beads (130-094-775, Miltenyi Biotech).

Techniques: Expressing, Flow Cytometry, Phospho-proteomics, Two Tailed Test, Comparison

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