Journal: Journal of Inflammation Research

Article Title: Cerium Oxide-Loaded Exosomes Derived From Regulatory T Cells Ameliorate Inflammatory Bowel Disease by Scavenging Reactive Oxygen Species and Modulating the Inflammatory Response

doi: 10.2147/JIR.S502388

Figure Lengend Snippet: Synthesis and characterization of Treg-exo@nCeO. ( A ) TEM images of nCeO, scale bars: 10 nm. ( B ) EDS spectrum of nCeO. ( C ) Appearance images of nCeO. ( D – F ) hydrogen peroxide and free radical scavenging assay, n=3; *p < 0.05 vs 0 μg/mL, **p < 0.01 vs 0 μg/mL, ns, no significance vs 0 μg/mL. ( G ) Flow chart of the isolation of CD4+CD25+ T cells. ( H ) Relative proportion of living cells, n=3; **p < 0.01 vs 0 μg/mL, ns, no significance vs 0 μg/mL. ( I ) Uptaken Cy5-labeled nCeO and fluorescence imaging, scale bars: 2 μm. ( J ) NTA of exo and exo@nCeO. ( K ) Western blot assay of the expression of Calnexin, Tsg101 and CD63 in Tregs and exo. ( L ) TEM images and average size of exo and exo@nCeO, n=3, scale bars: 100 nm. ( M ) Uptaken Dio-exo@Cy5-nCeO and fluorescence imaging, scale bars: 5 μm.

Article Snippet: 1×10 6 Tregs were collected and stained with phycoerythrin (PE) monoclonal antibodies against mouse CD25 (E-AB-F1102D, Elabscience, China) and fluorescein isothiocyanate (FITC) conjugated monoclonal antibodies against mouse CD4 (E-AB-F1097C, Elabscience, China) for 30 min, followed by data collection on a flow cytometer (Beckman Coulter, USA).

Techniques: Isolation, Labeling, Fluorescence, Imaging, Western Blot, Expressing

Journal: Frontiers in immunology

Article Title: Targeting Glutamine Metabolism Ameliorates Autoimmune Hepatitis via Inhibiting T Cell Activation and Differentiation.

doi: 10.3389/fimmu.2022.880262

Figure Lengend Snippet: FIGURE 2 | JHU083 inhibited T cells activation in vivo. Mice of Vehicle-AIH and JHU083-AIH group were intravenously injected with 8ml/kg ConA, and Vehicle-WT group was injected with normal saline of equal volume. JHU083-AIH group was gavaged with 0.3mg/kg JHU083 24h and 1h before ConA injection, and mice of Vehicle-WT and Vehicle-AIH group were treated with equal vehicle at the same time points. (A, B) Flow cytometry was applied to analyze the expression of CD25 and CD69 of CD4(+) (A) and CD8(+) (B) T cells. Data were expressed as means ± SEM. *P < 0.05 and **P <0.01.

Article Snippet: The antibodies used in the flow cytometry were as follows: CD4 (FITC, E-AB-F1097C), CD8a (PE, E-AB-F1104D), CD25 (PECy5, E-AB-F1102G), CD69 (PE-Cy7, E-AB-F1187H), IL4 (PE, E-AB-F1204UD), IL17A (APC, E-AB-F1272E) were purchased from Elabscience Biotechnology (Wuhan, China), antibodies IFNg (APC-Cy7, cat# 561479) was purchased from BD Biosciences (UK), and Granzyme B (PE-Cy7, cat# 372213) was purchased from Biolegend (San Diego, CA, USA).

Techniques: Activation Assay, In Vivo, Injection, Saline, Flow Cytometry, Expressing

Journal: Frontiers in immunology

Article Title: Targeting Glutamine Metabolism Ameliorates Autoimmune Hepatitis via Inhibiting T Cell Activation and Differentiation.

doi: 10.3389/fimmu.2022.880262

Figure Lengend Snippet: FIGURE 4 | DON treatment suppressed T cells activation in vitro. Freshly separated spleen cells were stimulated with 1.5mg/ml ConA with or without treating 2.5mM DON, and cells were analyzed 24h after ConA stimulation. (A, B) The expressions of activation markers CD25 and CD69 in CD4(+) (A) and CD8(+) (B) T cells were examined by flow cytometry. Data were expressed as means ± SEM. **P <0.01, and ***P <0.001.

Article Snippet: The antibodies used in the flow cytometry were as follows: CD4 (FITC, E-AB-F1097C), CD8a (PE, E-AB-F1104D), CD25 (PECy5, E-AB-F1102G), CD69 (PE-Cy7, E-AB-F1187H), IL4 (PE, E-AB-F1204UD), IL17A (APC, E-AB-F1272E) were purchased from Elabscience Biotechnology (Wuhan, China), antibodies IFNg (APC-Cy7, cat# 561479) was purchased from BD Biosciences (UK), and Granzyme B (PE-Cy7, cat# 372213) was purchased from Biolegend (San Diego, CA, USA).

Techniques: Activation Assay, In Vitro, Cytometry

Journal: Frontiers in immunology

Article Title: Targeting Glutamine Metabolism Ameliorates Autoimmune Hepatitis via Inhibiting T Cell Activation and Differentiation.

doi: 10.3389/fimmu.2022.880262

Figure Lengend Snippet: FIGURE 7 | The function of DON to mTOR signaling may be mediated by amino transporter amino transporter SLC7A5. (A) qRT-PCR was applied to analyze amino acid transporters SLC7A5 and SLC1A5 mRNA levels. (B, C) Flow cytometry was applied to analyze the degree of activation (expression of CD25 and CD69) of CD4 (+) (B) and CD8(+) (C) T cells with or without treating LAT1-IN1. The levels of phosphorylated mTOR (D) as well as phosphorylated P70S6K (E) were examined by western blotting. Data were expressed as means ± SEM. *P < 0.05, **P <0.01, and ***P <0.001. ns, not significant.

Article Snippet: The antibodies used in the flow cytometry were as follows: CD4 (FITC, E-AB-F1097C), CD8a (PE, E-AB-F1104D), CD25 (PECy5, E-AB-F1102G), CD69 (PE-Cy7, E-AB-F1187H), IL4 (PE, E-AB-F1204UD), IL17A (APC, E-AB-F1272E) were purchased from Elabscience Biotechnology (Wuhan, China), antibodies IFNg (APC-Cy7, cat# 561479) was purchased from BD Biosciences (UK), and Granzyme B (PE-Cy7, cat# 372213) was purchased from Biolegend (San Diego, CA, USA).

Techniques: Quantitative RT-PCR, Flow Cytometry, Activation Assay, Expressing, Western Blot

Journal: eLife

Article Title: SLAMF6​ deficiency augments tumor killing and skews toward an effector phenotype revealing it as a novel T cell checkpoint

doi: 10.7554/eLife.52539

Figure Lengend Snippet: ( A ) SLAMF6 and Vβ13 expression in Pmel-1 or Pmel-1 x SLAMF6 -/- splenocytes measured by flow cytometry. ( B ) Percent CD8+, CD4, and CD19 cells in spleens from Pmel-1 or Pmel-1 x SLAMF6 -/- untreated mice. ( C ) Pmel-1, and Pmel-1 x SLAMF6 -/- CD8+ untreated splenocytes were stained with anti-CD44 and anti-CD62L. One representative experiment is shown. ( D ) Percent CD8+ cells in Pmel-1 or Pmel-1 x SLAMF6 -/- splenocytes after 7 days of in vitro activation with gp100 25-33 peptide and IL-2 (30 IU/ml). ( E ) Flow cytometry for activation markers (CD25, CD69, CD137) in Pmel-1 or Pmel-1 x SLAMF6 -/- splenocytes after 3 days of in vitro activation, as in ( D ). Median fluorescence intensity (MFI) is shown. ( F ) Expression of PD-1 in Pmel-1 or Pmel-1 x SLAMF6 -/- CD8+ T cells after 7 days of in vitro activation, as in ( D ). Median fluorescence intensity (MFI) is shown. ( G, H ) After 7 days of activation, Pmel-1 and Pmel-1 x SLAMF6 -/- CD8+ T cells were stained with anti-CD44 and anti-CD62L. CD8+ subpopulations were defined for each mouse strain. ( G ) One representative experiment and ( H ) summary of subpopulations identified by flow cytometry in five experiments is shown. EM, effector memory, CM, central memory. Student t-test. *, p<0.05, **, p<0.01, ***, p<0.001.

Article Snippet: Antibody , Monoclonal rat anti mouse CD25 (PC61.5) , Biogems, Westlake Village, CA , 07312 , 0.2 μg/100 μl.

Techniques: Expressing, Flow Cytometry, Staining, In Vitro, Activation Assay, Fluorescence

Journal: eLife

Article Title: SLAMF6​ deficiency augments tumor killing and skews toward an effector phenotype revealing it as a novel T cell checkpoint

doi: 10.7554/eLife.52539

Figure Lengend Snippet:

Article Snippet: Antibody , Monoclonal rat anti mouse CD25 (PC61.5) , Biogems, Westlake Village, CA , 07312 , 0.2 μg/100 μl.

Techniques: Generated, Immunohistochemistry, Sequencing, Recombinant, Enzyme-linked Immunosorbent Assay, cDNA Synthesis, Transformation Assay, Clonogenic Cell Survival Assay, Cell Isolation, Isolation, Software

Journal: iScience

Article Title: Hypoxic-immune axis orchestrates metastatic dissemination via HIF isoform imbalance in pancreatic neuroendocrine tumors

doi: 10.1016/j.isci.2025.114340

Figure Lengend Snippet: Immune cell infiltration in KRAS G12C mutant PNETs (A) Quantitative analysis using Image Plus 6.0 software reveals a significant increase in Tregs (CD4 + , CD25 + , and FoxP3 + ) within KRAS- G12C mutant PNETs compared to wild-type KRAS PNET tissues, suggesting KRAS G12C -driven immunosuppressive cell recruitment. (B) Fluorescence intensity analysis demonstrates reduced infiltration of CD8 + cytotoxic T cells and HLA-DR + activated cells in KRAS G12C mutant tumors, indicative of impaired antitumor immunity. (C) Elevated MDSCs counts in KRAS G12C mutant PNETs correlate with enhanced immune evasion.

Article Snippet: PE Anti-Human CD25 Antibody [BC96] , Elabscience , E-AB-F1194D.

Techniques: Mutagenesis, Software, Fluorescence

Journal: iScience

Article Title: Hypoxic-immune axis orchestrates metastatic dissemination via HIF isoform imbalance in pancreatic neuroendocrine tumors

doi: 10.1016/j.isci.2025.114340

Figure Lengend Snippet: Tregs, CD8 + T cells, and HLA-DR + cells in KRAS G12C -mutated PNETs (A) Flow cytometry plots and fluorescence intensity histograms demonstrate elevated CD4 + T cell proportions in KRAS G12C patient blood samples compared to wild-type KRAS tumors and healthy controls. (B) Quantification shows a significant enrichment of CD25 + T cells in KRAS G12C patients, surpassing both wild-type KRAS tumors and normal controls. (C) Quantitative data and histogram overlays confirm a substantial increase in FoxP3 + T cells frequency in KRAS G12C patients, with levels moderately elevated compared to wild-type KRAS and significantly higher than healthy individuals. (D) A slight decrease in CD8 + T cell frequency in KRAS G12C samples relative to wild-type KRAS, with levels significantly lower than those in healthy individuals (E) A moderate reduction in HLA-DR + cell frequency in KRAS G12C patients compared to wild-type KRAS, alongside a marked suppression relative to healthy controls.

Article Snippet: PE Anti-Human CD25 Antibody [BC96] , Elabscience , E-AB-F1194D.

Techniques: Flow Cytometry, Fluorescence

Journal:

Article Title: Mechanism of Reduced T-Cell Effector Functions and Class-Switched Antibody Responses to Herpes Simplex Virus Type 2 in the Absence of B7 Costimulation

doi: 10.1128/JVI.77.4.2426-2435.2003

Figure Lengend Snippet: CD25 expression on CD4+ T cells. At 4 days following infection, (A) spleen and (B) genital lymph node cells from infected BALB/c and B7KO mice were analyzed by flow cytometry with antibodies specific for CD4 and CD25. Numbers within each quadrant represent the percentages of the cells within the lymphocyte gate. Cells from two to five mice were pooled. Data shown are from one of two experiments performed with similar results.

Article Snippet: Single-cell suspensions were prepared as described above, and cells (10 6 ) were stained with anti-CD4-fluorescein isothiocyanate or anti-CD8-fluorescein isothiocyanate (Caltag) and 0.5 μg of anti-CD25-phycoerythrin (Cedarlane, Ontario, Canada) or 1 μg of biotinylated anti-CD40L (PharMingen) followed by streptavidin-CyChrome in phosphate-buffered saline containing 0.2% bovine serum albumin and 0.1% azide.

Techniques: Expressing, Infection, Flow Cytometry

Journal: Frontiers in Oncology

Article Title: CD25 targeting with the afucosylated human IgG1 antibody RG6292 eliminates regulatory T cells and CD25+ blasts in acute myeloid leukemia

doi: 10.3389/fonc.2023.1150149

Figure Lengend Snippet: Regulatory T cells are present in the bone marrow of AML patients and express CD25. (A–C) Data obtained with BM samples in Study #2. (A) Dot plot depicting the expression of 26 markers on annotated T and B cell clusters. Color code indicates the median expression (logicle transformed MFI); point size indicates the fraction of cells positive for each marker. (B) Frequency of CD25+ mature T and B cell subsets. Two-way ANOVA, Sidak’s multiple comparison test. (C) CD25 expression (MFI) on selected T and B cell subsets. Two-way ANOVA, Sidak’s multiple comparison test (for the comparison of Healthy vs AML) or Tukey’s multiple comparison test (for the comparison of expression between cell subsets). (D) Myeloid cell abundance in the bone marrow of HD (n=5), Newly Diagnosed (n=22) and Treated patients (n=11). Data compiled from Study #1 and #2. Kruskal-Wallis with Dunn’s multiple comparison test. (E) Left: Treg abundance in the bone marrow of HD, Newly Diagnosed and Treated patients. Data compiled from Study #1 and #2. Kruskal-Wallis with Dunn’s multiple comparison test. Right: CD25 expression on Treg in the blood vs bone marrow of HD, Newly Diagnosed and Treated patients in Study #2. Multiple t tests, Holm-Sidak correction for multiple comparisons. (F) Frequency of Ki67+ cell populations between HD (n=5), Newly Diagnosed (n=7) and Treated patients (n=10) in Study #2. Multiple t tests, not corrected for multiple comparisons. Significance levels are defined in the methods section. P values > 0.05 are not indicated on the graphs. BM, Bone marrow; HD, Healthy donor; Treg, Regulatory T cell; NK, Natural Killer cell. Significance levels are indicated as follows: ns or not indicated, not significant, P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.

Article Snippet: In the case of monovalent binding of the PE-conjugated anti-human CD25 antibody (clone 24212, R&D Systems), the number of CD25 molecules would be equivalent to the number of PE molecules on the cell surface ( ).

Techniques: Expressing, Transformation Assay, Marker, Comparison

Journal: Frontiers in Oncology

Article Title: CD25 targeting with the afucosylated human IgG1 antibody RG6292 eliminates regulatory T cells and CD25+ blasts in acute myeloid leukemia

doi: 10.3389/fonc.2023.1150149

Figure Lengend Snippet: Peripheral blood offers a window into the bone marrow composition. (A) Correlation between the myeloid cells frequency detected by high dimensional flow cytometry analysis and the blast frequency as per pathology report. (B–D) Correlation between time matched PBMC and BMMC samples from 11 patients of (B) Myeloid cell abundance, (C) Treg abundance and (D) CD25+ AML cells frequency. (E) Correlation of IL2RA gene expression between time matched PBMC and BMMC samples from 9 patients. Pearson correlation r and P values are depicted on the graphs. BMMC: Bone marrow mononuclear cell; PBMC: Peripheral blood mononuclear cell; Treg: Regulatory T cell; CPM: Counts per million.

Article Snippet: In the case of monovalent binding of the PE-conjugated anti-human CD25 antibody (clone 24212, R&D Systems), the number of CD25 molecules would be equivalent to the number of PE molecules on the cell surface ( ).

Techniques: Flow Cytometry, Gene Expression

Journal: Frontiers in Oncology

Article Title: CD25 targeting with the afucosylated human IgG1 antibody RG6292 eliminates regulatory T cells and CD25+ blasts in acute myeloid leukemia

doi: 10.3389/fonc.2023.1150149

Figure Lengend Snippet: Patient-centric approach allows the phenotypic analysis of CD25+ AML clusters. (A) Dot plot depicting the per patient phenotypic analysis of CD25+ AML clusters identified in 14 patients in Study #1 (top) and Study #2 (bottom). See Methods for details. Color code indicates the median expression (logicle transformed MFI); point size indicates the fraction of cells positive for each marker. (B) Bcl-2 expression (logicle transformed MFI) in CD25+ AML clusters identified in Study #2. Unpaired t test. (C) Frequency of CD25+ AML subsets defined according to CD34 and CD38 expression (CD34-, CD34+CD38+, CD34+CD38-) in the 14 pre-selected patients shown in (A) Data compiled from BMMC and PBMC samples analyzed in Study #1 and #2. One-way ANOVA, Tukey’s multiple comparison test. (D) CD25 expression (MFI) on CD25+ AML subsets defined according to CD34 and CD38 expression (CD34-, CD34+CD38+, CD34+CD38-). Data compiled from BMMC and PBMC samples analyzed in Study #2. One-way ANOVA, Tukey’s multiple comparison test. (E) Abundance of CD25+ AML cells in treatment naïve PBMC or BMMC samples according to the FLT3-ITD status. Data compiled from Study #1 and #2. Unpaired t test. The proportion of patients for which CD25 expression was detected on AML cells is shown below the bar graph. Significance levels are defined in the methods section. P values > 0.05 are not indicated on the graphs. BM: Bone marrow; HSC: Hematopoietic stem cell; Chemo: Chemotherapy; HMA: DNA Hypomethylating Agent; VEN: venetoclax; FLT3-ITD: Internal tandem duplications (ITDs) in the fms-like tyrosine kinase 3 (FLT3) gene. Significance levels are indicated as follows: ns or not indicated, not significant, P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.

Article Snippet: In the case of monovalent binding of the PE-conjugated anti-human CD25 antibody (clone 24212, R&D Systems), the number of CD25 molecules would be equivalent to the number of PE molecules on the cell surface ( ).

Techniques: Expressing, Transformation Assay, Marker, Comparison

Journal: Frontiers in Oncology

Article Title: CD25 targeting with the afucosylated human IgG1 antibody RG6292 eliminates regulatory T cells and CD25+ blasts in acute myeloid leukemia

doi: 10.3389/fonc.2023.1150149

Figure Lengend Snippet: CD25 Mab depletes CD25+ AML and Tregs. (A) CD25 expression (MFI) on CD25+ Treg, AML cells or Memory CD4 T cells in Peripheral blood (PB, n = 22) or BM (n = 17) of samples analyzed in Study #2. Two-way ANOVA, Sidak’s multiple comparison test (for the comparison of BM vs PB) or Tukey’s multiple comparison test (for the comparison of expression between cell subsets). (B) Left: NK cell abundance (left) and CD16 expression on NK cells (right panel) in the BM of HD (n=5), Newly Diagnosed (n=22) and Treated patients (n=11). Data compiled from Study #1 and #2. Kruskal- Wallis with Dunn’s multiple comparison test. Right: Frequency of CD16+ NK cells. (C) Density of CD25 molecules on the surface of live target cells (Pfeiffer, EOL-1 and AML-22) in the absence of effector cells and test compounds was determined using BD quantibrite™ Beads, 17h after ADCC assay initiation. The limit of detection (L.O.D) is represented by the dotted line and corresponds to the lowest number of PE molecules present on the BD quantibrite™ Beads. (D) Dose response ADCC activity of the tested compounds (CD25 Mab or isotype control antibodies) as a frequency of CD25+ Target cell killing. Flow cytometric analysis was performed 17h after onset of ADCC assay. Mean +/- SEM represent results obtained with two NK cell donors and technical duplicates. EC50 values were calculated using GraphPad Prism and its inbuilt non-linear regression curve fit (log (agonist) vs response, variable slope, 4 parameter)). (E) Graphical representation of the cytotoxicity assay with allogeneic NK cells and patient material to evaluate CD25 Mab dual mode of action hypothesis. Created with BioRender.com (F) Left: Killing activity of the tested compounds at 10μg/ml (CD25 Mab or isotype control antibodies) as a frequency of CD25+ AML cell killing. See methods section for a detailed description of the experimental setup. Positive control EOL-1 cell line and four AML patient samples from study #2 were used as target cells. Flow cytometric analysis was performed 20h after onset of ADCC assay. Mean +/- SEM represent results obtained with two NK cell donors and technical duplicates. Right: Treg killing activity as described in left panel. Mean +/- SEM represent results obtained with AML Treg (n=3) or healthy BM Treg (n=2). Multiple t tests, Holm-Sidak correction for multiple comparisons. Significance levels are defined in the methods section. P values > 0.05 are not indicated on the graphs. BM, Bone marrow; Treg, Regulatory T cell; NK, Natural Killer cell; ADCC, Antibody dependent cellular cytotoxicity. Significance levels are indicated as follows: ns or not indicated, not significant, P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.

Article Snippet: In the case of monovalent binding of the PE-conjugated anti-human CD25 antibody (clone 24212, R&D Systems), the number of CD25 molecules would be equivalent to the number of PE molecules on the cell surface ( ).

Techniques: Expressing, Comparison, ADCC Assay, Activity Assay, Control, Cytotoxicity Assay, Positive Control

Journal: Frontiers in Oncology

Article Title: CD25 targeting with the afucosylated human IgG1 antibody RG6292 eliminates regulatory T cells and CD25+ blasts in acute myeloid leukemia

doi: 10.3389/fonc.2023.1150149

Figure Lengend Snippet: Depth of NK cell activation does not correlate with their cytotoxic activity. (A) Abundance of CD25+CD69+ NK cells (left) or CD16 expression (MFI) on NK cells (right) after 16-20h incubation in ADCC assay with Target cells (Pfeiffer, EOL-1 or AML22) treated with CD25 Mab or isotype control antibody. (B-D) Data obtained at ADCC assay endpoint (20h). Experimental procedure is described in the Figure 5E . (B) Abundance of CD69+ NK cells represented as count per µl in the absence or presence of target cells and test molecules. Bar graph represents mean +/- SEM for each NK cell donor with technical duplicates. Two-way ANOVA, Tukey’s multiple comparison test. (C) Heatmap of selected cytokine concentrations (mean values in pg/mL) detected in the supernatant by Luminex analysis. Row and column dendrograms represent hierarchical clustering using UPGMA method and Euclidean distance. (D) Log2 fold change of cytokine concentration obtained with CD25 Mab treatment as compared to isotype control antibody for seven differentially expressed cytokines. Each pictograph represents the mean of technical duplicates for each NK cell donor. Significance levels are defined in the methods section. P values > 0.05 are not indicated on the graphs. NK: Natural Killer cell; ADCC: Antibody dependent cellular cytotoxicity. Significance levels are indicated as follows: ns or not indicated, not significant, P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.

Article Snippet: In the case of monovalent binding of the PE-conjugated anti-human CD25 antibody (clone 24212, R&D Systems), the number of CD25 molecules would be equivalent to the number of PE molecules on the cell surface ( ).

Techniques: Activation Assay, Activity Assay, Expressing, Incubation, ADCC Assay, Control, Comparison, Luminex, Concentration Assay

Journal: Frontiers in Oncology

Article Title: CD25 targeting with the afucosylated human IgG1 antibody RG6292 eliminates regulatory T cells and CD25+ blasts in acute myeloid leukemia

doi: 10.3389/fonc.2023.1150149

Figure Lengend Snippet:

Article Snippet: In the case of monovalent binding of the PE-conjugated anti-human CD25 antibody (clone 24212, R&D Systems), the number of CD25 molecules would be equivalent to the number of PE molecules on the cell surface ( ).

Techniques: Transplantation Assay, Fluorescence, Clinical Proteomics, Activation Assay, RNA Sequencing, Wilms Tumor Assay