Journal: Frontiers in Immunology

Article Title: Phosphoproteomics Reveals Regulatory T Cell-Mediated DEF6 Dephosphorylation That Affects Cytokine Expression in Human Conventional T Cells

doi: 10.3389/fimmu.2017.01163

Figure Lengend Snippet: Experimental setup and quality controls for phosphoproteomics in primary human T cells. (A) Conventional CD4 + CD25 – T cells (Tcons) were cocultured either with allogeneic Tcons or regulatory T cells (Tregs), and cocultures were stimulated for 5 min with cross-linked anti-CD3/anti-CD28 antibodies. Stimulation was stopped on ice. Tstim (blue) and Tsup (red) were obtained after separation of T cell receptor (TCR)-stimulated Tcon:Tcon or Tcon:Treg cocultures, respectively. Unstimulated Tcons (Trest; gray) from the same donor were processed in parallel. Proteins were digested, peptides dimethyl-labeled and mixed, before phosphopeptides were enriched and measured by mass spectrometry (MS). Relative abundance of phosphopeptides was quantified by calculating the intensity ratios between the different samples as indicated. (B) An aliquot of cells used for phosphoproteomics was stimulated for 3 h before coculture separation, and suppression of cytokine mRNA was measured in re-isolated responder Tcons [Trest, Tstim, and Tsup as in panel (A) ]. As additional control, responder Tcons were stimulated without allogeneic Tcons (control Tstim). IL2 and IFNG mRNA were measured by quantitative RT-PCR, normalized to GAPDH mRNA. Results are presented as fold change compared to Trest (set to 1). The upper panel shows a representative donor (mean ± SD of technical PCR duplicates). Percentage suppression of respective cytokines in Tsup as compared to Tstim was calculated and is summarized for the three phosphoproteomics donors (lower panel). T cells were processed in three independent experiments (one experiment/donor) and phosphopeptide enrichment was performed in two independent experiments. (C) The number of unique phosphopeptides detected in each donor was determined, and the overlap is depicted as Venn diagram.

Article Snippet: We first isolated CD25 high cells with CD25-specific MACS beads (2 μl/10 7 cells, Miltenyi Biotec; cat. no. 130-092-983) as described previously ( ).

Techniques: Phospho-proteomics, Labeling, Mass Spectrometry, Isolation, Control, Quantitative RT-PCR

Journal: Pharmaceutics

Article Title: CD25-Targeted Aptamer–Drug Conjugate for the Treatment of CD25-Expressing Hematological Malignancies

doi: 10.3390/pharmaceutics18020217

Figure Lengend Snippet: The structure and characterization of CD25 aptamer. ( A ) The secondary structure of the CD25 aptamer was estimated by RNAstructure software v6.4 of Mathews Lab. The sequence of CD25 aptamer is shown with modifications indicated (Z, 5-[ N -(1-naphthylmethyl)carboxamide]-2′-deoxyuridine; N me , 2′- O -methyl nucleosides). ( B ) The binding affinity of CD25 aptamer to the CD25 recombinant protein was determined by the BLI method. Ni-NTA probes were immobilized with His-tag CD25 protein, followed by incubation with the aptamer 125 (green), 250 (yellow), or 500 nM (red). The binding signal over time is shown. Kd is expressed as mean ± SD. ( C ) The cells were stained with biotin-aptamer combined with NeutrAvidin DyLight 650 or APC-conjugated anti-CD25 monoclonal antibody (mAb), and then the specificity of the antibody and the aptamer to the cells was examined by flow cytometry (control for aptamer: DyLight 650 only; control for antibody: not stained).

Article Snippet: A 5 μg/mL recombinant human CD25 His-tagged protein solution (R&D Systems, Minneapolis, MN, USA) was immobilized onto Ni-NTA probes (Gator Bio).

Techniques: Software, Sequencing, Binding Assay, Recombinant, Incubation, Staining, Flow Cytometry, Control

Journal: Pharmaceutics

Article Title: CD25-Targeted Aptamer–Drug Conjugate for the Treatment of CD25-Expressing Hematological Malignancies

doi: 10.3390/pharmaceutics18020217

Figure Lengend Snippet: The CD25 aptamer specifically binds and internalizes into CD25-positive cells. ( A ) The cell internalization of Cy-5-labeled CD25 aptamer (red) was visualized for 0, 1, and 4 h using confocal fluorescence microscopy using CD25-positive Karpas299 and CD25-negative Daudi cell lines. The nuclei were stained with DAPI (blue). ( B ) The rate of internalized CD25 aptamer was determined using the MFI value of flow cytometry analysis at 0 to 240 min. ( C ) Cellular trafficking of the CD25 aptamer. Fluorescence microscopy visualized the lysosomal delivery of pHrodo-labeled CD25 aptamer (red) for up to 4 h.

Article Snippet: A 5 μg/mL recombinant human CD25 His-tagged protein solution (R&D Systems, Minneapolis, MN, USA) was immobilized onto Ni-NTA probes (Gator Bio).

Techniques: Labeling, Fluorescence, Microscopy, Staining, Flow Cytometry

Journal: Pharmaceutics

Article Title: CD25-Targeted Aptamer–Drug Conjugate for the Treatment of CD25-Expressing Hematological Malignancies

doi: 10.3390/pharmaceutics18020217

Figure Lengend Snippet: Effects of the CD25 aptamer on CD25/IL-2 signaling. ( A ) A competitive binding assay was performed by adding biotinylated IL-2 proteins to 96-well plates coated with CD25 proteins, in the presence or absence of the CD25 aptamer. ( B , C ) Karpas299 cells were pre-treated with the CD25 aptamer for 30 min, followed by stimulation with IL-2 for 15 min. The levels of pSTAT5 protein and TGF-β mRNA were analyzed by Western blotting and quantitative RT-PCR, respectively. ( D , E ) HuT78 cells were treated with IL-2 in the presence of either the CD25 aptamer or the anti-CD25 antibody Daclizumab. The expression of pSTAT5 was then assessed by Western blot analysis. ( F ) HuT78 cells were pre-treated with the indicated concentrations of the CD25 aptamer, stimulated with IL-2, and the secretion of IL-4 was measured as described in the Materials and Methods. Results are expressed as mean ± SD. ** p < 0.01, *** p < 0.001.

Article Snippet: A 5 μg/mL recombinant human CD25 His-tagged protein solution (R&D Systems, Minneapolis, MN, USA) was immobilized onto Ni-NTA probes (Gator Bio).

Techniques: Competitive Binding Assay, Western Blot, Quantitative RT-PCR, Expressing

Journal: Pharmaceutics

Article Title: CD25-Targeted Aptamer–Drug Conjugate for the Treatment of CD25-Expressing Hematological Malignancies

doi: 10.3390/pharmaceutics18020217

Figure Lengend Snippet: In vitro cytotoxicity of CD25 aptamer–MMAE conjugates. Karpas299 and Daudi Cells were treated with CD25-ApDC MMAE1 ( A ) or CD25-ApDC MMAE3 ( B ) for 3 days, after which cell viability was assessed, as described in the Materials and Methods. ( C ) Karpas299 and HuT78 cells were co-cultured at a 1:1 ratio for 24 h, stained with anti-CD4 and anti-CD25 antibodies, and analyzed by flow cytometry. The co-cultured cells were subsequently incubated with 45 nM CD25-ApDC MMAE3 for 24, 48, or 72 h, and analyzed again using flow cytometry. ( D ) Cells were treated with increasing concentrations of MMAE or CD25-ApDC MMAE3 for 24 h. Western blot analysis of total PRAP, cleaved PARP, total caspase-3, and cleaved caspase-3 was performed. ( E ) The cell cycle was analyzed using flow cytometry after staining with PI. Results are expressed as mean ±SD. * p < 0.05, *** p < 0.001.

Article Snippet: A 5 μg/mL recombinant human CD25 His-tagged protein solution (R&D Systems, Minneapolis, MN, USA) was immobilized onto Ni-NTA probes (Gator Bio).

Techniques: In Vitro, Cell Culture, Staining, Flow Cytometry, Incubation, Western Blot

Journal: Pharmaceutics

Article Title: CD25-Targeted Aptamer–Drug Conjugate for the Treatment of CD25-Expressing Hematological Malignancies

doi: 10.3390/pharmaceutics18020217

Figure Lengend Snippet: In vivo antitumor efficacy of CD25 aptamer–MMAE conjugates in xenograft models. Tumor growth curves were generated by measuring tumor volumes in Karpas299 tumor-bearing mice following intravenous administration of CD25 aptamer–MMAE conjugates when tumors reached an average volume of 150 mm 3 . ( A ) Red arrows indicate the time points of injection with CD25-ApDC MMAE1 at doses of 1, 2, or 4 mg/kg. ( B ) Mice were treated either four times with 4 mg/kg (red arrows) or twice with 12 mg/kg (green arrows). ( C ) Tumor-bearing mice received a single dose of 0.4, 0.8, or 1.6 mg/kg, or were administered doses three times (once per week) with 0.8 or 1.6 mg/kg CD25-ApDC MMAE3 . Data are the mean tumor volume ±SE of eight animals per group. ( D ) NOD/SCID mice were systemically inoculated with Karpas299 cells and treated intravenously with the indicated dose of CD25-ApDC MMAE1 or CD25-ApDC MMAE3 twice per week for 3 weeks. Kaplan–Meier survival curves show the percentage of survival for each group, with statistical comparison performed using log-rank tests.

Article Snippet: A 5 μg/mL recombinant human CD25 His-tagged protein solution (R&D Systems, Minneapolis, MN, USA) was immobilized onto Ni-NTA probes (Gator Bio).

Techniques: In Vivo, Generated, Injection, Comparison

Journal: Pharmaceutics

Article Title: CD25-Targeted Aptamer–Drug Conjugate for the Treatment of CD25-Expressing Hematological Malignancies

doi: 10.3390/pharmaceutics18020217

Figure Lengend Snippet: The structure and characterization of CD25 aptamer. ( A ) The secondary structure of the CD25 aptamer was estimated by RNAstructure software v6.4 of Mathews Lab. The sequence of CD25 aptamer is shown with modifications indicated (Z, 5-[ N -(1-naphthylmethyl)carboxamide]-2′-deoxyuridine; N me , 2′- O -methyl nucleosides). ( B ) The binding affinity of CD25 aptamer to the CD25 recombinant protein was determined by the BLI method. Ni-NTA probes were immobilized with His-tag CD25 protein, followed by incubation with the aptamer 125 (green), 250 (yellow), or 500 nM (red). The binding signal over time is shown. Kd is expressed as mean ± SD. ( C ) The cells were stained with biotin-aptamer combined with NeutrAvidin DyLight 650 or APC-conjugated anti-CD25 monoclonal antibody (mAb), and then the specificity of the antibody and the aptamer to the cells was examined by flow cytometry (control for aptamer: DyLight 650 only; control for antibody: not stained).

Article Snippet: Recombinant human CD25 protein (R&D Systems) was immobilized on high-binding 96-well ELISA plates (Corning) at a concentration of 0.2 μg/mL in PBS overnight at 4 °C.

Techniques: Software, Sequencing, Binding Assay, Recombinant, Incubation, Staining, Flow Cytometry, Control

Journal: Pharmaceutics

Article Title: CD25-Targeted Aptamer–Drug Conjugate for the Treatment of CD25-Expressing Hematological Malignancies

doi: 10.3390/pharmaceutics18020217

Figure Lengend Snippet: The CD25 aptamer specifically binds and internalizes into CD25-positive cells. ( A ) The cell internalization of Cy-5-labeled CD25 aptamer (red) was visualized for 0, 1, and 4 h using confocal fluorescence microscopy using CD25-positive Karpas299 and CD25-negative Daudi cell lines. The nuclei were stained with DAPI (blue). ( B ) The rate of internalized CD25 aptamer was determined using the MFI value of flow cytometry analysis at 0 to 240 min. ( C ) Cellular trafficking of the CD25 aptamer. Fluorescence microscopy visualized the lysosomal delivery of pHrodo-labeled CD25 aptamer (red) for up to 4 h.

Article Snippet: Recombinant human CD25 protein (R&D Systems) was immobilized on high-binding 96-well ELISA plates (Corning) at a concentration of 0.2 μg/mL in PBS overnight at 4 °C.

Techniques: Labeling, Fluorescence, Microscopy, Staining, Flow Cytometry

Journal: Pharmaceutics

Article Title: CD25-Targeted Aptamer–Drug Conjugate for the Treatment of CD25-Expressing Hematological Malignancies

doi: 10.3390/pharmaceutics18020217

Figure Lengend Snippet: Effects of the CD25 aptamer on CD25/IL-2 signaling. ( A ) A competitive binding assay was performed by adding biotinylated IL-2 proteins to 96-well plates coated with CD25 proteins, in the presence or absence of the CD25 aptamer. ( B , C ) Karpas299 cells were pre-treated with the CD25 aptamer for 30 min, followed by stimulation with IL-2 for 15 min. The levels of pSTAT5 protein and TGF-β mRNA were analyzed by Western blotting and quantitative RT-PCR, respectively. ( D , E ) HuT78 cells were treated with IL-2 in the presence of either the CD25 aptamer or the anti-CD25 antibody Daclizumab. The expression of pSTAT5 was then assessed by Western blot analysis. ( F ) HuT78 cells were pre-treated with the indicated concentrations of the CD25 aptamer, stimulated with IL-2, and the secretion of IL-4 was measured as described in the Materials and Methods. Results are expressed as mean ± SD. ** p < 0.01, *** p < 0.001.

Article Snippet: Recombinant human CD25 protein (R&D Systems) was immobilized on high-binding 96-well ELISA plates (Corning) at a concentration of 0.2 μg/mL in PBS overnight at 4 °C.

Techniques: Competitive Binding Assay, Western Blot, Quantitative RT-PCR, Expressing

Journal: Pharmaceutics

Article Title: CD25-Targeted Aptamer–Drug Conjugate for the Treatment of CD25-Expressing Hematological Malignancies

doi: 10.3390/pharmaceutics18020217

Figure Lengend Snippet: In vitro cytotoxicity of CD25 aptamer–MMAE conjugates. Karpas299 and Daudi Cells were treated with CD25-ApDC MMAE1 ( A ) or CD25-ApDC MMAE3 ( B ) for 3 days, after which cell viability was assessed, as described in the Materials and Methods. ( C ) Karpas299 and HuT78 cells were co-cultured at a 1:1 ratio for 24 h, stained with anti-CD4 and anti-CD25 antibodies, and analyzed by flow cytometry. The co-cultured cells were subsequently incubated with 45 nM CD25-ApDC MMAE3 for 24, 48, or 72 h, and analyzed again using flow cytometry. ( D ) Cells were treated with increasing concentrations of MMAE or CD25-ApDC MMAE3 for 24 h. Western blot analysis of total PRAP, cleaved PARP, total caspase-3, and cleaved caspase-3 was performed. ( E ) The cell cycle was analyzed using flow cytometry after staining with PI. Results are expressed as mean ±SD. * p < 0.05, *** p < 0.001.

Article Snippet: Recombinant human CD25 protein (R&D Systems) was immobilized on high-binding 96-well ELISA plates (Corning) at a concentration of 0.2 μg/mL in PBS overnight at 4 °C.

Techniques: In Vitro, Cell Culture, Staining, Flow Cytometry, Incubation, Western Blot

Journal: Pharmaceutics

Article Title: CD25-Targeted Aptamer–Drug Conjugate for the Treatment of CD25-Expressing Hematological Malignancies

doi: 10.3390/pharmaceutics18020217

Figure Lengend Snippet: In vivo antitumor efficacy of CD25 aptamer–MMAE conjugates in xenograft models. Tumor growth curves were generated by measuring tumor volumes in Karpas299 tumor-bearing mice following intravenous administration of CD25 aptamer–MMAE conjugates when tumors reached an average volume of 150 mm 3 . ( A ) Red arrows indicate the time points of injection with CD25-ApDC MMAE1 at doses of 1, 2, or 4 mg/kg. ( B ) Mice were treated either four times with 4 mg/kg (red arrows) or twice with 12 mg/kg (green arrows). ( C ) Tumor-bearing mice received a single dose of 0.4, 0.8, or 1.6 mg/kg, or were administered doses three times (once per week) with 0.8 or 1.6 mg/kg CD25-ApDC MMAE3 . Data are the mean tumor volume ±SE of eight animals per group. ( D ) NOD/SCID mice were systemically inoculated with Karpas299 cells and treated intravenously with the indicated dose of CD25-ApDC MMAE1 or CD25-ApDC MMAE3 twice per week for 3 weeks. Kaplan–Meier survival curves show the percentage of survival for each group, with statistical comparison performed using log-rank tests.

Article Snippet: Recombinant human CD25 protein (R&D Systems) was immobilized on high-binding 96-well ELISA plates (Corning) at a concentration of 0.2 μg/mL in PBS overnight at 4 °C.

Techniques: In Vivo, Generated, Injection, Comparison

Journal: iScience

Article Title: DUSP6 deletion protects mice and reduces disease severity in autoimmune arthritis

doi: 10.1016/j.isci.2024.110158

Figure Lengend Snippet:

Article Snippet: anti-CD25-APC (clone: 4E3) , Miltenyi Biotec, Germany , Cat# 130-113-846.

Techniques: Transgenic Assay, Knock-Out

Journal: Stem Cell Research & Therapy

Article Title: Multiple intravenous injections of allogeneic equine mesenchymal stem cells do not induce a systemic inflammatory response but do alter lymphocyte subsets in healthy horses

doi: 10.1186/s13287-015-0050-0

Figure Lengend Snippet: Multiple allogeneic mesenchymal stem cell (MSC) injections result in changes in splenic regulatory T cell percentages. (A-D) There were no significant changes in splenic CD21 + B-cell (A) , CD4 + T-cell (B) , or CD8 + T-cell percentages (C) or CD4/CD8 ratios (D) following multiple MSC injections. (E) There were no significant changes in activated (CD25 + ) lymphocyte proportions. (F) There were significantly higher percentages of splenic FoxP3 + regulatory T cells in the horses injected with bone marrow (BM)-derived MSCs compared with horses injected with adipose tissue (AT)-derived MSCs. Data are presented as mean ± standard error of the mean. * P <0.05.

Article Snippet: The following antibodies were used: mouse-anti-equine CD3 (clone UC F6G 1:250; Jeffery Stott, University of California, Davis, CA, USA) [ ], mouse-anti-human CD21 (clone B-ly4 1:20; BD Pharmingen, San Jose, CA, USA) [ , ], polyclonal goat-anti-human CD25 (clone AF-223; R&D Systems) [ ], rat-anti-mouse/human FoxP3 (clone PCH101; ebioscience, San Diego, CA, USA) [ ], mouse-anti-CD4 (clone HB61A 1:133; VMRD, Pullman, WA, USA) [ ], mouse-anti-equine CD8 (clone F18P 1:500; J. Stott) [ ], and a donkey-anti-mouse secondary when necessary (1:50; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA).

Techniques: Injection, Derivative Assay