Journal: Genome Medicine

Article Title: Blocking CXCR4 + CD4 + T cells reprograms T reg -mediated immunosuppression via modulating the Rho-GTPase/NF-κB signaling axis

doi: 10.1186/s13073-025-01515-8

Figure Lengend Snippet: Clinical trial data and human organoids confirm that blocking CXCR4 enhances antitumor immunotherapy efficacy. A Schematic overview of clinical trial NCT02826486 , including treatment timeline and peripheral blood sampling schedule. B Longitudinal analysis of lymphocyte counts, CXCR4 + CD4 + T cells, CD4 + CD25 + FOXP3 + regulatory T cells, and CD69 + CD4 + T cells following CXCR4 antagonist monotherapy and combination therapy. Gray lines denote geometric means; blue and red lines indicate the lower and upper bounds of the 95% confidence intervals, respectively. M, monotherapy; Pre, pre-treatment; Post, post-treatment; D, day. Statistical comparisons were performed using two-sided t -tests based on geometric means and derived standard deviations. Significance thresholds were adjusted using Bonferroni correction. C Schematic of RNA-seq workflow from trial NCT04516616 . Cervical cancer patients receiving neoadjuvant chemotherapy plus anti-PD-1 therapy were stratified into pathological complete response (pCR) and non-pCR groups, and tumor samples were subjected to transcriptomic profiling. D Heatmap showing immune cell-type enrichment scores, as assessed by xCell algorithm analysis, comparing pCR and non-pCR groups. E Paired comparisons of gene expression and immune cell scores before and after immune checkpoint blockade (ICB) in pCR and non-pCR groups. p values calculated by paired t -test in GraphPad Prism. F Correlation analysis of gene expression profiles of pre- and post-ICB in non-pCR patients. G Schematic of experimental setup in which PBMCs from non-pCR patients were co-cultured with cervical cancer-derived organoids. H Representative images of cervical cancer organoid and PBMC co-culture system under different treatments. Tumor cell apoptosis was evaluated by caspase-3 staining. ***, p < 0.001

Article Snippet: Briefly, human PBMCs from healthy donors were isolated using a CD4 + CD25 + Regulatory T Cell Isolation Kit (Miltenyi Biotec, Cat. No. 130091301), followed by stimulation with varying concentrations of human recombinant IFN-α (0 mg/mL, 0.25 mg/mL, 0.5 mg/mL, and 1 mg/mL) (Abcam, Cat. No. ab48750).

Techniques: Blocking Assay, Sampling, Derivative Assay, RNA Sequencing, Gene Expression, Cell Culture, Co-Culture Assay, Staining

Journal: Genome Medicine

Article Title: Blocking CXCR4 + CD4 + T cells reprograms T reg -mediated immunosuppression via modulating the Rho-GTPase/NF-κB signaling axis

doi: 10.1186/s13073-025-01515-8

Figure Lengend Snippet: Single-cell transcriptomic analysis identified that CXCR4 expression was associated with T reg cell developmental trajectories. A UMAP plot of CD4 + T cells ( n = 45,363) in lung adenocarcinoma. B Correlation analysis of gene expression ( CXCR4 and CTLA4 ) and the proportion of CD4-CTLA4-T reg cells. C UMAP plot of CD4 + T cells ( n = 11,166) in non-small cell lung cancer. D Correlation analysis of gene expression ( CXCR4 and FOXP3 ) and the proportion of CD4-CTLA4-Treg cells. E UMAP plot of CD4 + T cells ( n = 63,965) in multi-cancer. F Correlation analysis of CXCR4 expression and the proportion of CD4-LEF1-T reg and CD4-CTLA4-T reg cells. G Developmental trajectories of T reg cells in breast cancer from GSE156728 via diffusion map. Expression levels of marker genes and CXCR4 were shown. H Developmental trajectories of T reg cells in pancreatic cancer from GSE156728 via diffusion map analysis. Expression levels of marker genes and CXCR4 were shown

Article Snippet: Briefly, human PBMCs from healthy donors were isolated using a CD4 + CD25 + Regulatory T Cell Isolation Kit (Miltenyi Biotec, Cat. No. 130091301), followed by stimulation with varying concentrations of human recombinant IFN-α (0 mg/mL, 0.25 mg/mL, 0.5 mg/mL, and 1 mg/mL) (Abcam, Cat. No. ab48750).

Techniques: Expressing, Gene Expression, Diffusion-based Assay, Marker

Journal: Genome Medicine

Article Title: Blocking CXCR4 + CD4 + T cells reprograms T reg -mediated immunosuppression via modulating the Rho-GTPase/NF-κB signaling axis

doi: 10.1186/s13073-025-01515-8

Figure Lengend Snippet: Blocking CXCR4 in CD4 + T cells reduces the activated T reg phenotypes in vivo. A Gross anatomy of subcutaneous tumors (U14) in mice treated with CXCR4 antagonists. B Flow cytometry and statistical analysis of tumor models (4T1 and ID8) under different treatments; p values were calculated using the unpaired, parametric t -test. C UMAP plot of CD4 + T meta-cell clusters of scRNA-seq data from pan-cancer analysis. Exhausted clusters are indicated. T n , naïve T cells; T m , memory T cells; T em , effector memory T cells; T emra , terminally differentiated effector memory or effector; Tfh, follicular helper T cell; Th1, T helper 1; ISG, interferon-stimulated genes. D Violin plots showing the expression levels of canonical marker genes across CD4 + T cell clusters. E Schematic diagram illustrating the experimental design for CXCR4 antagonist treatment in U14 tumor-bearing immunocompetent syngeneic mice. Flow cytometry analysis ( F ) and corresponding quantification ( G ) of CD4 + T cell subsets in tumors across different treatment groups. H Schematic diagram and experimental design for tumor-bearing (U14) Cxcr4 -cKO and control mice. Flow cytometry analysis ( I ) and quantification ( J ) of CD4. + T cell subsets in spleens from tumor-bearing control and cKO mice. p values were determined by unpaired, parametric t -test. ** p < 0.01; *** p < 0.001

Article Snippet: Briefly, human PBMCs from healthy donors were isolated using a CD4 + CD25 + Regulatory T Cell Isolation Kit (Miltenyi Biotec, Cat. No. 130091301), followed by stimulation with varying concentrations of human recombinant IFN-α (0 mg/mL, 0.25 mg/mL, 0.5 mg/mL, and 1 mg/mL) (Abcam, Cat. No. ab48750).

Techniques: Blocking Assay, In Vivo, Flow Cytometry, Expressing, Marker, Control

Journal: Genome Medicine

Article Title: Blocking CXCR4 + CD4 + T cells reprograms T reg -mediated immunosuppression via modulating the Rho-GTPase/NF-κB signaling axis

doi: 10.1186/s13073-025-01515-8

Figure Lengend Snippet: Blocking CXCR4 + CD4 + T cells enhances anti-PD-1 immunotherapy efficacy by reprogramming T reg -mediated immunosuppressive TME. A Mean tumor volumes and gross anatomy of U14 tumors in primary recipients with various treatments (saline and IgG, CXCR4 antagonist, anti-PD-1, combination). p values were assessed using two-way ANOVA. B Tumor volume analysis of 4T1-tumor-bearing animals treated with indicated drugs; each line represents an individual mouse. After 40 days of combination treatment, mice showing complete response to combined treatment were rechallenged with 4T1 tumors and treated with CXCR4 agonist and control vehicle. p values were assessed using two-way ANOVA. C RNA heatmaps of marker genes in U14 tumors from different treatments. D UMAP plots of 11 cell clusters and treatment samples based on scRNA-seq data. E UMAP plots of five CD4 + T cell subtypes and their treatment samples based on scRNA-seq data. F Venny plot of DGEs in CD4 + T cells compared to controls. The bottom panel shows pathway enrichment analysis of the overlapped DEGs. G Pseudotime analysis of differentially expressed heatmaps for CD4 + T cells. H Violin plot of gene expression in DC and T reg cells from different treatments based on scRNA-seq data. I Schematic illustrating the mechanism by which blocking CXCR4 + CD4 + T cells regulates T reg -mediated immunosuppression. CXCR4 inhibition reduces the recruitment and suppressive function of T reg cells, characterized by downregulation of key immunosuppressive cytokines (e.g., IL-10, TGF-β) and immune checkpoint molecules (e.g., CTLA-4, TIM-3). This leads to diminished T reg –APC interactions and decreased expression of co-inhibitory ligands (e.g., PD-L1, CD80, CD86) on APCs. *** p < 0.001

Article Snippet: Briefly, human PBMCs from healthy donors were isolated using a CD4 + CD25 + Regulatory T Cell Isolation Kit (Miltenyi Biotec, Cat. No. 130091301), followed by stimulation with varying concentrations of human recombinant IFN-α (0 mg/mL, 0.25 mg/mL, 0.5 mg/mL, and 1 mg/mL) (Abcam, Cat. No. ab48750).

Techniques: Blocking Assay, Saline, Control, Marker, Gene Expression, Inhibition, Expressing

Journal: Genome Medicine

Article Title: Blocking CXCR4 + CD4 + T cells reprograms T reg -mediated immunosuppression via modulating the Rho-GTPase/NF-κB signaling axis

doi: 10.1186/s13073-025-01515-8

Figure Lengend Snippet: Phosphoproteome and ChIP-seq analyses reveal that targeting CXCR4 + CD4 + T cells reduces T reg -associated suppressive genes via modulation of the Rho-GTPase/NF-κB signaling axis. A Experimental design for phosphoproteome analysis in human T reg cells treated with CXCR4 antagonist. Human T reg cells were purified by FACS and stimulated in vitro. B Heatmap displaying the differentially regulated phosphorylation sites in T reg cells before and after CXCR4 antagonist treatment. Each row represents a phosphorylation site, and each column represents a biological replicate. C Pathway enrichment analysis based on proteins with different phosphorylation sites in T reg cells pre- and post-CXCR4 antagonist treatment. D Overview of phosphorylation level changes associated with the Rho-GTPase/NF-κB signaling axis induction by CXCR4 antagonist treatment. Differentially regulated phosphorylation sites related Rho-GTPase/NF-κB signaling axis were indicated. Protein–protein interaction networks represent differentially regulated phosphorylation sites. E The ex vivo co-culture system using human cervical cancer organoids and PBMCs derived from two patients at different treatment timepoints. Patient-derived PBMCs were co-cultured with autologous organoids under various treatment conditions. F Violin plots showing quantification of apoptosis rates in cervical cancer organoids under different treatment conditions, as assessed by caspase-3 staining. Each dot represents an independent measurement from different patient-derived samples. p values were calculated by the Mann–Whitney test in GraphPad Prism. G ChIP-seq analysis of chromatin occupancy in T reg -associated genes based on NFKB2 and RelB ChIP-seq data. H ChIP-PCR validation of NFKB2 and RelB binding to the promoters of selected T reg signature genes in T reg cells before and after CXCR4 antagonist exposure. Data are presented as mean ± SD. * p < 0.05; ** p < 0.01; ns, no significance

Article Snippet: Briefly, human PBMCs from healthy donors were isolated using a CD4 + CD25 + Regulatory T Cell Isolation Kit (Miltenyi Biotec, Cat. No. 130091301), followed by stimulation with varying concentrations of human recombinant IFN-α (0 mg/mL, 0.25 mg/mL, 0.5 mg/mL, and 1 mg/mL) (Abcam, Cat. No. ab48750).

Techniques: ChIP-sequencing, Purification, In Vitro, Phospho-proteomics, Ex Vivo, Co-Culture Assay, Derivative Assay, Cell Culture, Staining, MANN-WHITNEY, Biomarker Discovery, Binding Assay

Journal: Genome Medicine

Article Title: Blocking CXCR4 + CD4 + T cells reprograms T reg -mediated immunosuppression via modulating the Rho-GTPase/NF-κB signaling axis

doi: 10.1186/s13073-025-01515-8

Figure Lengend Snippet: Single-cell multi-omic analyses reveal that blocking CXCR4 + CD4 + T cells epigenetically reprogram T reg -associated suppressive genes. A tSNE plots of 14 cell clusters based on scRNA-seq and scATAC-seq data from U14 tumor-bearing Cxcr4 flox/flox Lck Cre and Cxcr4 flox/flox mice. B Chromatin accessibility analysis of marker genes in cell clusters. C Representative mIHC staining of spleens from tumor-bearing Cxcr4 flox/flox Lck Cre and Cxcr4 flox/flox mice. D tSNE plots of four subtypes of CD4 + T cells based on scRNA-seq and scATAC-seq data from tumor-bearing Cxcr4 flox/flox Lck Cre and Cxcr4 flox/flox mice. E tSNE and violin plots displaying CXCR4 expression levels in T reg cells based on scRNA-seq analysis. p values were determined by the Wilcoxon rank-sum test. F Motif scores in T reg cells from scATAC-seq analysis. G Chromatin accessibility analysis of marker genes in T reg cells. H Pathway enrichment analysis based on the top 50 different peaks in T reg cells between Cxcr4 flox/flox Lck Cre and Cxcr4 flox/flox mice. I Chromatin accessibility analysis of marker genes in B cells, macrophages, and DCs. J Representative mIHC staining of tumors from tumor-bearing Cxcr4 flox/flox Lck Cre and Cxcr4 flox/flox mice. ***, p < 0.001

Article Snippet: Briefly, human PBMCs from healthy donors were isolated using a CD4 + CD25 + Regulatory T Cell Isolation Kit (Miltenyi Biotec, Cat. No. 130091301), followed by stimulation with varying concentrations of human recombinant IFN-α (0 mg/mL, 0.25 mg/mL, 0.5 mg/mL, and 1 mg/mL) (Abcam, Cat. No. ab48750).

Techniques: Blocking Assay, Marker, Staining, Expressing

Journal: Genome Medicine

Article Title: Blocking CXCR4 + CD4 + T cells reprograms T reg -mediated immunosuppression via modulating the Rho-GTPase/NF-κB signaling axis

doi: 10.1186/s13073-025-01515-8

Figure Lengend Snippet: Main content analysis and findings of this study. This study integrates multi-omic profiling and functional validation experiments to investigate how CXCR4 + CD4 + T cells contribute to tumor immunosuppression and how CXCR4 blockade can reprogram this process. Approaches included single-cell RNA-seq, single-cell multi-omics, bulk RNA-seq, phosphoproteomics, mouse tumor models, patient-derived organoid PBMC co-culture systems, and clinical cohort analyses. Mechanistically, CXCR4 antagonism disrupted the Rho GTPase/NF-κB signaling pathways, leading to reduced transcription of T reg -associated suppressive molecules (e.g., CTLA-4, PD-1, TIM-3, and TNFRSF members). This downregulation impaired T reg -mediated suppression, ultimately rebalancing the tumor immune microenvironment in favor of antitumor immunity and enhancing the efficacy of anti-PD-1 immunotherapy

Article Snippet: Briefly, human PBMCs from healthy donors were isolated using a CD4 + CD25 + Regulatory T Cell Isolation Kit (Miltenyi Biotec, Cat. No. 130091301), followed by stimulation with varying concentrations of human recombinant IFN-α (0 mg/mL, 0.25 mg/mL, 0.5 mg/mL, and 1 mg/mL) (Abcam, Cat. No. ab48750).

Techniques: Functional Assay, Biomarker Discovery, RNA Sequencing, Phospho-proteomics, Derivative Assay, Co-Culture Assay, Protein-Protein interactions

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