Journal:

Article Title: Suppression of HIV-specific T cell activity by lymph node CD25 + regulatory T cells from HIV-infected individuals

doi: 10.1073/pnas.0611423104

Figure Lengend Snippet: Representative phenotypic analysis of CD25 and FoxP3 expression in CD4+ T cells of PBMC compared with LNMC. (A) Freshly isolated PBMC and LNMC were stained for CD3, CD4, CD25 and intracellular FoxP3. CD25 and FoxP3 expression was analyzed in the CD3+CD4+ population. (A) CD25+ Treg cells in PBMC and LNMC were quantified as FoxP3+ and CD25hi (MFI ≥70) as indicated in the box in the upper right quadrant. (B) Representative example of CD25+hi expression on purified CD4+ T cell subsets used in functional assays. (C) Representative example of FoxP3 expression intensity (MFI) on CD25+ CD4+ T cells in LN and PB.

Article Snippet: CD25 hi cells were obtained by using anti-human CD25 PE conjugated Mab (3 μl/10 6 cells) followed by a short incubation (3 min) with anti-PE immunomagnetic beads (Miltenyi, Auburn, CA).

Techniques: Expressing, Isolation, Staining, Purification, Functional Assay

Journal:

Article Title: Suppression of HIV-specific T cell activity by lymph node CD25 + regulatory T cells from HIV-infected individuals

doi: 10.1073/pnas.0611423104

Figure Lengend Snippet: The frequency and MFI of FoxP3 expression in CD4+ T cell subsets within LNMC is significantly greater than within PBMC. CD25hi (Top) and CD25− (Middle) FoxP3+ CD4+ T cell frequencies and FoxP3 MFI in CD25+ CD4+ T cells (Bottom) in parallel LNMC and PBMC isolated from 11 chronically HIV-infected individuals.

Article Snippet: CD25 hi cells were obtained by using anti-human CD25 PE conjugated Mab (3 μl/10 6 cells) followed by a short incubation (3 min) with anti-PE immunomagnetic beads (Miltenyi, Auburn, CA).

Techniques: Expressing, Isolation, Infection

Journal:

Article Title: Suppression of HIV-specific T cell activity by lymph node CD25 + regulatory T cells from HIV-infected individuals

doi: 10.1073/pnas.0611423104

Figure Lengend Snippet: Comparison of PB and LN for the expression of surface markers used to distinguish CD25+ Treg cells and normal CD4+ T cells. LNMC and PBMC were stained for CD4, CD25, intracellular FoxP3, and either sCTLA-4, GITR, or CD127. CD25−FoxP3− and CD25hiFoxP3+ CD4+ LNMC and PBMC subsets were analyzed for expression of CD127, sCTLA-4, and GITR. P values compare LNMC and PBMC subsets. Data are of mean percent ± SD of data obtained from LNMC and PBMC isolated from 11 chronically HIV-infected individuals.

Article Snippet: CD25 hi cells were obtained by using anti-human CD25 PE conjugated Mab (3 μl/10 6 cells) followed by a short incubation (3 min) with anti-PE immunomagnetic beads (Miltenyi, Auburn, CA).

Techniques: Comparison, Expressing, Staining, Isolation, Infection

Journal:

Article Title: Suppression of HIV-specific T cell activity by lymph node CD25 + regulatory T cells from HIV-infected individuals

doi: 10.1073/pnas.0611423104

Figure Lengend Snippet: CD25+ Treg suppressive capacity assessed in HIV p24-specific proliferation assays. Total and CD25+ cell-depleted CD4+ T cells isolated from LNMC and PBMC of subjects 1–5 were stimulated with HIV p24 protein for 6 days, then pulsed with 3H thymidine 16 h. Percent suppression of proliferation by CD25+ cells was determined by comparing net cpm obtained in total versus CD25+ cell-depleted CD4+ T cell cultures (≥40% was arbitrarily considered to be a significant level of suppression). N.R., no response.

Article Snippet: CD25 hi cells were obtained by using anti-human CD25 PE conjugated Mab (3 μl/10 6 cells) followed by a short incubation (3 min) with anti-PE immunomagnetic beads (Miltenyi, Auburn, CA).

Techniques: Isolation

Journal:

Article Title: Suppression of HIV-specific T cell activity by lymph node CD25 + regulatory T cells from HIV-infected individuals

doi: 10.1073/pnas.0611423104

Figure Lengend Snippet: Representative example of CD25+ Treg suppressive capacity assessed in flow cytometry-based CTL assays by using Granzyme-B substrate cleavage as a read out. HIV Gag pre-stimulated effector unfractionated (Top plots) and CD25+ cell-depleted LNMC, alone or plus 30% CD25+CD8− MC, (Middle and Bottom Right plots, respectively) were cultured for 1 h with FL-4-labeled un-pulsed or HIV Gag peptide-pulsed autologous CD25−CD8− target cells in the presence of a Granzyme-B substrate that fluoresces when cleaved. Percent target cell killing (indicated within each plot) was defined by the frequency of substrate cleavage high events within the FL-4+ target cell gate; analyses are of FL-4+ target cells only. Substrate cleavage in target cells cultured in the absence of effector cells is shown in the Bottom Left plot.

Article Snippet: CD25 hi cells were obtained by using anti-human CD25 PE conjugated Mab (3 μl/10 6 cells) followed by a short incubation (3 min) with anti-PE immunomagnetic beads (Miltenyi, Auburn, CA).

Techniques: Flow Cytometry, Cell Culture, Labeling

Journal:

Article Title: Suppression of HIV-specific T cell activity by lymph node CD25 + regulatory T cells from HIV-infected individuals

doi: 10.1073/pnas.0611423104

Figure Lengend Snippet: Comparison of PB and LN CD25+ Treg cell-mediated suppression of HIV-specific CTL activity. (A) Mean (± SD) HIV-specific CTL activity in unfractionated and CD25+ cell-depleted PBMC and LNMC. (B) Mean (± SD) percent inhibition of HIV-specific CTL activity mediated by CD25+ cells in LNMC and PBMC of patients stratified by VL (HIV RNA copies per ml). (C) Comparison of the suppressive effects of LN and PB-derived CD25hi+ CD4+ T cells on HIV-specific CTL activity. Purified CD25− or CD25hi+ CD4+ T cells were isolated from the PB and LN (subjects 3–5) and added to autologous CD25+ cell-depleted MC (at 10%) at the time of primary stimulation with HIV Gag peptides. CTL assays were performed 6 days later after exposure to HIV Gag peptide-pulsed target cells. Data are of mean (±SD) percent suppression of HIV-specific CTL activity present in control CD25+ cell-depleted MC cultured alone.

Article Snippet: CD25 hi cells were obtained by using anti-human CD25 PE conjugated Mab (3 μl/10 6 cells) followed by a short incubation (3 min) with anti-PE immunomagnetic beads (Miltenyi, Auburn, CA).

Techniques: Comparison, Activity Assay, Inhibition, Derivative Assay, Purification, Isolation, Control, Cell Culture

Journal: Scientific Reports

Article Title: Tuning IL-2 signaling by ADP-ribosylation of CD25

doi: 10.1038/srep08959

Figure Lengend Snippet: Splenocytes from DEREG mice were incubated in the absence or presence of the indicated concentrations of NAD + before staining with fluorochrome-conjugated mAbs directed against CD4 and CD25 (mAb PC61 or mAb 7D4). Gating was performed on CD4 + cells. (a) Representative dot plots of cells incubated in the absence or presence of 12 μM NAD + . (b) Percentages of CD4 + GFP + cells staining with anti-CD25 mAbs plotted as a function of the concentration of added NAD + . Results are representative of two independent experiments.

Article Snippet: Alternatively, after erythrocyte lysis with Ack lysis buffer (Bio Whittaker) and depletion of B-cells with Dynabead-conjugated sheep anti-mouse IgG (Invitrogen), Tregs were positively selected by magnetic cell sorting using PE conjugated anti-CD25 (7D4) and magnetic bead-conjugated anti-PE antibodies according to the manufacturer's instructions (Miltenyi Biotec).

Techniques: Incubation, Staining, Concentration Assay

Journal: Cell Reports Medicine

Article Title: Ablation of ERO1A induces lethal endoplasmic reticulum stress responses and immunogenic cell death to activate anti-tumor immunity

doi: 10.1016/j.xcrm.2023.101206

Figure Lengend Snippet: ERO1A induces T cell dysfunction in response to aPD-1 treatment (A) t-distributed stochastic neighbor embedding (t-SNE) visualization of major cell clusters, colored by cell subtype. Tumors were collected after two rounds of aPD-1 treatment and processed for scRNA-seq (n = 5 mice/group). CAFs, cancer-associated fibroblasts. DCs, dendritic cells. NK, natural killer. (B) t-SNE map of major cell types in MC-38 Ero1a WT and Ero1a KO tumors. Colored by cell subtype. (C) t-SNE map indicating the macrophage clusters based on the scRNA-seq data. Colored by cell subtype. (D) t-SNE map showing the sample origins of macrophages in MC-38 Ero1a WT and Ero1a KO tumors. Colored by cell subtype. (E) t-SNE map depicting the M1 and M2 macrophage signatures based on the scRNA-seq data. (F) Uniform manifold approximation and projection (UMAP) plot showing the RNA velocity of CD8 + T cell subsets. RNA velocities were visualized on the UMAP of Mki67 + Tex, Il2ra + Texp, Tnfrsf9 + Texp, Gzmf + Tem, Ifng + Tm, Ifit1 + Tcm, and CD28 + Tm using Gaussian smoothing on a regular grid. (G and H) Diffusion map of CD8 + T cell clusters shows a resting-to-activated trajectory. The pseudotime expression changes in Lag3 , Pdcd1 , Havcr2 , and Gzmb in CD8 + T cells (G). Pseudotime trajectory of CD8 + T cell subsets in MC-38 Ero1a WT and Ero1a KO tumors (H). Colored by cell subtype. (I and J) Projection of effective CD8 + T cells (I) and proliferative CD8 + T cells (J) based on cell activation, degranulation, and proliferation levels in MC-38 Ero1a WT and Ero1a KO tumors. (K) Bar plot showing the inflammatory cytokines of CD8 + T cells in MC-38 Ero1a WT and Ero1a KO tumors, assessed by scRNA-seq. Data presented as means ± SDs. ∗∗∗p < 0.001. Two-sided Student’s t test.

Article Snippet: Anti-mouse CD25 PE , Tonbo , Cat# 50-0251; RRID: AB_2621757.

Techniques: Diffusion-based Assay, Expressing, Activation Assay

Journal: Cell Reports Medicine

Article Title: Ablation of ERO1A induces lethal endoplasmic reticulum stress responses and immunogenic cell death to activate anti-tumor immunity

doi: 10.1016/j.xcrm.2023.101206

Figure Lengend Snippet:

Article Snippet: Anti-mouse CD25 PE , Tonbo , Cat# 50-0251; RRID: AB_2621757.

Techniques: Control, Purification, Virus, Recombinant, CCK-8 Assay, Transfection, Protease Inhibitor, SYBR Green Assay, Saline, Lysis, Multiplex Assay, Cytotoxicity Assay, Cell Isolation, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Software

Journal: iScience

Article Title: Akkermansia muciniphila ameliorates fatty liver through microbiota-derived α-ketoisovaleric acid metabolism and hepatic PI3K/Akt signaling

doi: 10.1016/j.isci.2025.112458

Figure Lengend Snippet: Akk improves gut immune responses in obese mice (A) PHATE of T cells clustering in mice intestinal lymph nodes. (B–E) Percentages of CD3 + CD4 + Tregs, CD3 + CD8 + Tregs, CD4 + CD25 + Tregs, and CD4 + CD25 + Foxp3 + Tregs. Data are expressed as mean ± SD ( n = 6), ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, compared with the Mod group, and p value was calculated using Dunnet’s post hoc test in one-way ANOVA.

Article Snippet: Anti-Mouse CD25 + PE , BioGems , Cat# 07312-60.

Techniques:

Journal: iScience

Article Title: Akkermansia muciniphila ameliorates fatty liver through microbiota-derived α-ketoisovaleric acid metabolism and hepatic PI3K/Akt signaling

doi: 10.1016/j.isci.2025.112458

Figure Lengend Snippet: Gut fungi is the key in Akk improving gut immune responses in organoids (A) Co-culture of colonic organoids, fecal microbiota and Akk. (B) Fluorescent images of colonic organoids with DAPI-labeled cells, and number of organoid crypts. Scale bars: 100 μm. (C) Relative abundance of fungal in co-culture system. (D) Percentages of CD3 + CD4 + , CD3 + CD8 + , CD4 + CD25 + , and CD4 + CD25 + Foxp3 + Tregs in organoids. (E) Relative content of metabolites in co-culture system. (F) Co-culture of colonic organoids, fluconazole, fecal microbiota and Akk. (G) Fluorescent images of colonic organoids with DAPI-labeled cells, and number of organoid crypts. Scale bars: 50 μm. (H) Percentages of CD3 + CD4 + , CD3 + CD8 + , CD4 + CD25 + , and CD4 + CD25 + Foxp3 + Tregs in organoids. (I and J) Relative content of metabolites (α-ketoisovaleric acid and glycylleucine) in co-culture system. Figures 7 A–7E and Figures 7 F–7J represent experiments from different batches, respectively. Data are expressed as mean ± SD (n = 6–8), ns p >0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, compared with the Mod group, and p value was calculated using Dunnet’s post hoc test in one-way ANOVA.

Article Snippet: Anti-Mouse CD25 + PE , BioGems , Cat# 07312-60.

Techniques: Co-Culture Assay, Labeling

Journal: Cancers

Article Title: The Immunocytokine FAP-IL-2v Enhances Anti-Neuroblastoma Efficacy of the Anti-GD 2 Antibody Dinutuximab Beta

doi: 10.3390/cancers14194842

Figure Lengend Snippet: Flow cytometry analysis of tumor-infiltrating lymphocytes. To investigate effects of the combinatorial DB + FAP-IL-2v treatment on tumor-infiltrating lymphocytes, primary tumor tissue was analyzed using flow cytometry. After resection, primary tumor tissue samples were enzymatically digested to obtain a single-cell solution. To assess NK ( A ) and cytotoxic T cells ( B ) as well as Treg ( C ), the effector-cell-population-specific antigens CD335 and CD8 as well as CD25 and FocP3 were marked, respectively. Additionally, the ratio of cytotoxic T cells to Treg ( D ) was calculated. Results are presented as a percentage of the respective effector cell population cells relative to all viable CD45- or CD3-positive leukocytes for NK and cytotoxic T cells or Treg, respectively. ANOVA followed by appropriate post-hoc comparison test and t -test. ( A ) * p < 0.05 vs. FAP-IL-2v, ( B ) * p < 0.05 vs. DB + FAP-IL-2v, ( C ) * p < 0.05 vs. DB + FAP-IL-2v, # p < 0.05 vs. FAP-IL-2v, ( D ) * p < 0.05 vs. IL-2.

Article Snippet: For detection of different tumor-infiltrating leukocyte populations described below, the following Ab were utilized: APC-Vio ® 770-labeled anti-mouse CD3 REAfinityTM Ab, VioGreenTM-labeled anti-mouse CD4 Antibody REAfinityTM, PerCP-labeled anti-mouse CD8a REAfinityTM Ab, Vio ® Bright FITC-labeled anti-mouse CD11b REAfinityTM Ab, PE-Vio ® 770-labeled anti-mouse CD25 REAfinityTM Ab, PE-labeled anti-mouse CD335 REAfinityTM Ab, APC-labeled anti-mouse FoxP3 REAfinityTM Ab (all Miltenyi Biotec, Teterow, Germany), APC/Cy-7-labeled anti-mouse Ly6C Ab and PerCP-labeled anti-mouse Ly6G Ab (BioLegend, San Diego, CA, USA).

Techniques: Flow Cytometry, Comparison

Journal: Gut Microbes

Article Title: Targeting the gut microbiota with dietary fibers: a novel approach to prevent the development cardiovascular complications linked to systemic lupus erythematosus in a preclinical study

doi: 10.1080/19490976.2023.2247053

Figure Lengend Snippet: Key antibodies for flow cytometry.

Article Snippet: anti-CD25 (RRID:AB_2784091, PE-VIO770, clone 7D4) , Miltenyi.

Techniques: Cytometry, Staining