abs against cd25 antibody  (Cell Signaling Technology Inc)

Journal: Frontiers in Immunology

Article Title: Cysteine-specific 89 Zr-labeled anti-CD25 IgG allows immuno-PET imaging of interleukin-2 receptor-α on T cell lymphomas

doi: 10.3389/fimmu.2022.1017132

Figure Lengend Snippet: 89 Zr-CD25 IgG characterization. (A) Schema of 89 Zr-CD25 IgG (left). SDS PAGE of intact, TCEP reduced, and DFO-maleimide conjugated anti-human and anti-mouse CD25 IgG (middle). Autoradiography of column-eluted 89 Zr-CD25 IgG fractions on native PAGE (right). (B) PD10 column chromatography of anti-human and anti-mouse 89 Zr-CD25 IgG (left), and in vitro radiochemical stability in PBS and FBS over days (right; mean of duplicate samples per group).

Article Snippet: Mouse monoclonal IgG2a anti- CD25 Ab (7G7/B6) that is specific against human CD25 and does not react with mouse CD25, rat monoclonal IgG1 Ab against mouse CD25 (PC-61.5.3), and IgG2a isotype control Ab were from BioXcell (West Lebanon, NH).

Techniques: SDS Page, Autoradiography, Clear Native PAGE, Column Chromatography, In Vitro

Journal: Frontiers in Immunology

Article Title: Cysteine-specific 89 Zr-labeled anti-CD25 IgG allows immuno-PET imaging of interleukin-2 receptor-α on T cell lymphomas

doi: 10.3389/fimmu.2022.1017132

Figure Lengend Snippet: CD25 expression and 89 Zr-CD25 IgG binding to human lymphoma cells. (A) Western blots of CD25 protein and anti-human 89 Zr-CD25 IgG uptakes in H9, Jurkat, and SUDHL1 lymphoma cells (left). Complete blocking of SUDHL1 cell binding by 0.5 μM of unlabeled anti-CD25 IgG (right). (B) Lindmo binding assays for determining the immunoreactive fraction. Five concentrations of SUDHL1 cells were used, from 0.5 to 16 million cells/ml. The final concentration of 89 Zr-CD25 IgG was 50 ng/ml, and nonspecific binding was assessed with 5 μg of unlabeled anti-CD25 IgG. A conventional plot of specific and nonspecific binding over total applied radioactivity, as a function of increasing cell concentration is shown (left). A double inverse plot was then drawn using the same data as total applied radioactivity over specific binding, as a function of the inverse cell concentration (right). The immunoreactive fraction was determined through linear extrapolation to the ordinate. Data represent the mean ± S.E of values from two independent experiments [total n = 5 per group; uptake in (A) ], or the mean ± S.D of data from a single representative experiment [n = 3 per group; (B) ].

Article Snippet: Mouse monoclonal IgG2a anti- CD25 Ab (7G7/B6) that is specific against human CD25 and does not react with mouse CD25, rat monoclonal IgG1 Ab against mouse CD25 (PC-61.5.3), and IgG2a isotype control Ab were from BioXcell (West Lebanon, NH).

Techniques: Expressing, Binding Assay, Western Blot, Blocking Assay, Concentration Assay, Radioactivity

Journal: Frontiers in Immunology

Article Title: Cysteine-specific 89 Zr-labeled anti-CD25 IgG allows immuno-PET imaging of interleukin-2 receptor-α on T cell lymphomas

doi: 10.3389/fimmu.2022.1017132

Figure Lengend Snippet: Circulating free CD25 level and effect on blood 89 Zr-CD25 IgG. (A) Western blots of CD25 protein in blood obtained from the heart of 16 SUDHL1 lymphoma-bearing mice (top). Relations of tumor weight with blood 89 Zr-CD25 IgG (bottom left) and blood 89 Zr-CD25 IgG with blood human CD25 level (bottom right). (B) Relations of tumor weight with 89 Zr-CD25 IgG tumor uptake (left) and blood activity (middle) in another group of mice. Western blots and quantified band intensities of tumor CD25 protein according to tumor weight group is shown in the right. Lines were fitted by linear regression and correlation was based on nonparametric Spearman’s analysis. Bars represent the mean ± S.D of four samples per group. N.S., not significant.

Article Snippet: Mouse monoclonal IgG2a anti- CD25 Ab (7G7/B6) that is specific against human CD25 and does not react with mouse CD25, rat monoclonal IgG1 Ab against mouse CD25 (PC-61.5.3), and IgG2a isotype control Ab were from BioXcell (West Lebanon, NH).

Techniques: Western Blot, Activity Assay

Journal: Frontiers in Immunology

Article Title: Cysteine-specific 89 Zr-labeled anti-CD25 IgG allows immuno-PET imaging of interleukin-2 receptor-α on T cell lymphomas

doi: 10.3389/fimmu.2022.1017132

Figure Lengend Snippet: Pharmacokinetics, PET imaging, and biodistribution in mice. (A) Anti-human 89 Zr-CD25 IgG activity in whole blood, plasma, and blood cells of SUDHL1 lymphoma mice following intravenous injection (left). Data are the mean ± SD of five mice for each time group. Representative maximum intensity projection PET images at 5 days (right). (B) Representative coronal (top left) and transaxial (top right) PET images. Biodistribution at 5 days in mice at baseline or after three intraperitoneal PMA injections. Data are the mean ± S.E from two independent experiments (n = 5 per group).

Article Snippet: Mouse monoclonal IgG2a anti- CD25 Ab (7G7/B6) that is specific against human CD25 and does not react with mouse CD25, rat monoclonal IgG1 Ab against mouse CD25 (PC-61.5.3), and IgG2a isotype control Ab were from BioXcell (West Lebanon, NH).

Techniques: Drug discovery, Imaging, Activity Assay, Clinical Proteomics, Injection

Journal: Frontiers in Immunology

Article Title: Cysteine-specific 89 Zr-labeled anti-CD25 IgG allows immuno-PET imaging of interleukin-2 receptor-α on T cell lymphomas

doi: 10.3389/fimmu.2022.1017132

Figure Lengend Snippet: Target Specificity of tumor uptake in vivo . Representative coronal (left) and transaxial (right) PET images at 5 days of SUDHL1 lymphoma mice co-injected with anti-human 89 Zr-CD25 IgG and 0.8 mg of unlabeled anti-CD25 IgG for blocking (top), or with 89 Zr-labeled IgG2a isotype control Ab (middle). Biodistribution data are shown (bottom) as the mean ± S.E of values obtained from two independent experiments (n = 5 per group).

Article Snippet: Mouse monoclonal IgG2a anti- CD25 Ab (7G7/B6) that is specific against human CD25 and does not react with mouse CD25, rat monoclonal IgG1 Ab against mouse CD25 (PC-61.5.3), and IgG2a isotype control Ab were from BioXcell (West Lebanon, NH).

Techniques: In Vivo, Injection, Blocking Assay, Labeling, Control

Journal: Frontiers in Immunology

Article Title: Cysteine-specific 89 Zr-labeled anti-CD25 IgG allows immuno-PET imaging of interleukin-2 receptor-α on T cell lymphomas

doi: 10.3389/fimmu.2022.1017132

Figure Lengend Snippet: 89 Zr-CD25 IgG uptake in mouse cells in vitro and in mouse lymphoma in vivo . (A) CD25 expression (left) and anti-mouse 89 Zr-CD25 IgG binding (right) in mouse thymus Treg lymphocytes and EL4 mouse lymphoma cells. Cell uptake was compared by using equivalent amounts of cells. Blocking was done by adding 0.5 μM of unlabeled anti-CD25 IgG. (B) Representative coronal (left) and transaxial (right) PET images at 5 days of EL4 tumor-bearing immunocompetent C57/Bl6 mice administered with anti-mouse 89 Zr-CD25 IgG alone (top) or co-injected and 0.8 mg of unlabeled anti-CD25 IgG for blocking (middle). Biodistribution data are shown (bottom) as the mean ± S.E of values obtained from two independent experiments (n = 6 per group) N.S., not significant.

Article Snippet: Mouse monoclonal IgG2a anti- CD25 Ab (7G7/B6) that is specific against human CD25 and does not react with mouse CD25, rat monoclonal IgG1 Ab against mouse CD25 (PC-61.5.3), and IgG2a isotype control Ab were from BioXcell (West Lebanon, NH).

Techniques: In Vitro, In Vivo, Expressing, Binding Assay, Blocking Assay, Injection

Journal: Frontiers in Immunology

Article Title: Cysteine-specific 89 Zr-labeled anti-CD25 IgG allows immuno-PET imaging of interleukin-2 receptor-α on T cell lymphomas

doi: 10.3389/fimmu.2022.1017132

Figure Lengend Snippet: Autoradiography and histologic staining of microsections of SUDHL1 tumors from 89 Zr-CD25 IgG- injected mice. (Top) Photograph of a film exposed to a microsection of a small tumor showing radioactivity efficiently delivered into the inner tumor regions (left). A microscopic image of the tumor region in the boxed area demonstrates a focus of greater radioactivity (middle) that contained microvessels shown in red by alkaline phosphatase staining in an immediately adjacent microsection (right). (Bottom) Photograph of a film exposed to a microsection of a larger tumor showing greater radiation accumulated in the tumor periphery (left). A microscopic image of the tumor region in the boxed area demonstrates radioactivity distribution (middle) that correlated with immunohistochemistry staining for human CD25 (right).

Article Snippet: Mouse monoclonal IgG2a anti- CD25 Ab (7G7/B6) that is specific against human CD25 and does not react with mouse CD25, rat monoclonal IgG1 Ab against mouse CD25 (PC-61.5.3), and IgG2a isotype control Ab were from BioXcell (West Lebanon, NH).

Techniques: Autoradiography, Staining, Injection, Radioactivity, Immunohistochemistry

Journal: PLoS ONE

Article Title: Differential regulation of C5a receptor 1 in innate immune cells during the allergic asthma effector phase

doi: 10.1371/journal.pone.0172446

Figure Lengend Snippet: (A) Gating strategy to identify CD4 + T cells. T cells were subdivided into naive (CD44 - CD62L + ) and effector (CD44 - CD62L + ) T cells. (B) Recruitment of different T cell populations into the lungs of PBS-treated or OVA-immunized WT or GFP-C5aR1 flox/flox animals. Values shown are the mean ± SEM; n = 9–15 per group. (C) Expression of FoxP3 (Tregs), GATA3 (Th2), RORγT (Th17), and TBX21 (Th1) transcripts in sorted CD44 + CD62L - T cells. The abundance of transcripts was evaluated after reverse transcription by real-time PCR. Values shown are the mean abundance of target mRNA as compared to actin analyzed by Mann-Whitney test (n = 4–11). (D) Comparison of IL-13 and IL-17A mRNA expression levels in WT and GFP-C5aR1 flox/flox mice in sorted CD44 + CD62L - T cells as determined by real-time PCR. Values shown are the mean abundance of target mRNA as compared to actin analyzed by Mann-Whitney test (n = 4–11). (E) Gating strategy used to identify ILC2 in lung tissue (Lin - CD25 + CD90.2 + CD127 + ). (F) ILC2 cell numbers in lung tissue of PBS-treated or OVA-immunized WT or GFP-C5aR1 flox/flox animals. Values shown are the mean ± SEM; n = 4–11 per group. * indicates significant differences between PBS or OVA-treated groups; § indicates significant differences between WT and GFP-C5aR1 flox/flox OVA-treated groups. § p < 0.05, ** p < 0.01, *** p <0.001.

Article Snippet: Per-CP–Cy5.5–labeled Ab against CD3 (17A2), CD5 (53–73), CD27 (LG.7F9), NK1.1 (PK136), TCRβ (H57-597), CD11b (MI/70); eF780–labeled Ab against CD11c (N418), B220 (RA3-6B2), CD49b (DX5); eF450–labeled Ab against CD25 (PC61.5); PE-labeled Ab against CD90.2 (30-H12); PE-cy5-labeled Ab against CD127 (A7R34) were purchased from eBioscience (Affimetrix) except anti-CD90.2 (BD Pharmingen)

Techniques: Expressing, Real-time Polymerase Chain Reaction, MANN-WHITNEY

Journal: International Journal of Cell Biology

Article Title: Regulatory T Cells Resist Cyclosporine-Induced Cell Death via CD44-Mediated Signaling Pathways

doi: 10.1155/2015/614297

Figure Lengend Snippet: CD44 cross-linking and IL-2 both promote Foxp3 expression and Treg persistence despite CSA treatment. (a) Representative flow cytometric analysis of CD25 labeled and GFP/Foxp3+ cells after 3 days in culture in the presence of anti-CD3 and anti-CD28 alone or with the addition of anti-CD44, and with or without CSA (50 ng/mL) alone or together with IL-2 (20 IU/mL). (b) Fold increase (FI) in GFP/Foxp3 MFI after 3 days of culture in the presence of anti-CD3 and anti-CD28 alone or in conjunction with anti-CD44 Ab, with or without CSA (50 ng/mL) alone or together with IL-2 (20 IU/mL). N = 4 independent experiments, among these are included . (c) Fold Increase in GFP/FoxP3 MFI in the presence of anti-CD3 and anti-CD28 alone, or in conjunction with anti-CD44 and increasing concentrations of CSA. Data are representative of two experiments. (d) Fold increase in the fraction of viable GFP/FoxP3+ cells (Annexin V-, 7AAD-) upon culture with aCD3/28 or aCD3/28/44 with or without CSA (50 ng/mL) alone or together with IL-2 (20 IU/mL). N = 4 experiments among these are included in and the other experiments are in .

Article Snippet: Where indicated, the following reagents were incubated at 37°C in a CO 2 incubator with the cells 30 minutes prior to cross-linking CD44: CSA (50 ng/mL or at concentrations indicated) and neutralizing Ab against CD25 (100 μ g/mL; R&D Systems, Cat number AB-223-NA).

Techniques: Expressing, Labeling

Journal: International Journal of Cell Biology

Article Title: Regulatory T Cells Resist Cyclosporine-Induced Cell Death via CD44-Mediated Signaling Pathways

doi: 10.1155/2015/614297

Figure Lengend Snippet: CD44 cross-linking promotes Foxp3 expression in an IL-2-independent manner . (a) Fold Increase (FI) in GFP/FoxP3 MFI for Treg activated with anti-CD3 and anti-CD28 Ab alone or in conjunction with plate-bound anti-CD44 Ab with or without anti-IL-2 Ab (Anti-IL-2), recombinant CD25 (rCD25), or IL-2 ( n = 7). (b) Representative histograms demonstrating GFP/Foxp3 expression by Treg isolated from GFP/Foxp3 knock-in mice on a conventional B6 background mice or on a CD25 −/− background (B6 GFP/Foxp3.CD25 −/− mice) following 3 days of culture with anti-CD3 and anti-28 alone or in conjunction with plate-bound anti-CD44. (c) Representative histograms illustrating GFP/FoxP3 expression of Treg following 3 days of culture with anti-CD3 and anti-CD28 alone, or in conjunction with plate-bound CD44 Ab, and with or without varying doses of IL-2. Data are representative of two experiments.

Article Snippet: Where indicated, the following reagents were incubated at 37°C in a CO 2 incubator with the cells 30 minutes prior to cross-linking CD44: CSA (50 ng/mL or at concentrations indicated) and neutralizing Ab against CD25 (100 μ g/mL; R&D Systems, Cat number AB-223-NA).

Techniques: Expressing, Recombinant, Isolation, Knock-In

Journal: International Journal of Cell Biology

Article Title: Regulatory T Cells Resist Cyclosporine-Induced Cell Death via CD44-Mediated Signaling Pathways

doi: 10.1155/2015/614297

Figure Lengend Snippet: Inhibition of HA synthesis impairs Treg homeostasis which can be overcome with exogenous IL-2 or CD44-cross-linking . (a) Representative histograms of GFP/FoxP3 expression of Treg following 3 days of culture in the presence of anti-CD3 and anti-CD28 alone or with IL-2, CD44 cross-linking, or exogenous plate-bound HA. N = 3 independent experiments. (b) Representative FACS plots illustrating GFP/Foxp3 and CD25 expression on Day 0 immediately following isolation of CD4+GFP/Foxp3+ Treg from murine splenocytes and following 3 days of culture with anti-CD3 and anti-CD28 Ab alone or in conjunction with plate-bound anti-CD44 Ab, the HA synthesis inhibitor 4-MU, and/or IL-2. (c) Fold change in GFP/Foxp3 MFI for the same conditions as in (b), here for N = 3 independent experiments. (d) Viability (the percentage of GFP/Foxp3+ cells negative for 7AAD and Annexin V) for Treg cultured in the setting of either DMSO or 4MU.

Article Snippet: Where indicated, the following reagents were incubated at 37°C in a CO 2 incubator with the cells 30 minutes prior to cross-linking CD44: CSA (50 ng/mL or at concentrations indicated) and neutralizing Ab against CD25 (100 μ g/mL; R&D Systems, Cat number AB-223-NA).

Techniques: Inhibition, Expressing, Isolation, Cell Culture