cd45ro microbeads  (Miltenyi Biotec)

Journal: Molecular Medicine

Article Title: Dysfunctional glycolysis-UCP2-fatty acid oxidation promotes CTLA4 int FOXP3 int regulatory T-cell production in rheumatoid arthritis

doi: 10.1186/s10020-025-01372-6

Figure Lengend Snippet: Increased CTLA4 int FOXP3 int Treg production in patients with RA. A - D CTLA4 high Treg subsets among PBMCs and SFs from patients with RA and OA. A Representative plots for PBMCs. B Percentages of CD25 high CTLA4 high FOXP3 high CD4 high Tregs among PBMCs examining 15 RA and 15 OA patients. C Representative plots for SFs. D Percentages of CD25 high CTLA4 high FOXP3 high CD4 high Tregs among SFs examining 4 RA and 4 OA patients. E–F CD4 + T cells from RA patients and healthy controls (HCs) were stimulated with anti-CD3/CD28 beads for 5 days, and E Representative plots. F Percentages of CD25 high CTLA4 high FOXP3 high CD4 high T cells examining 11 RA patients and 11 HCs were presented. G – R T cell differentiation: CD4 + CD45RO − T cells were cultured under Treg-polarizing conditions. G - H Percentages of CD25 high CTLA4 high FOXP3 high CD4 high and CD25 inter CTLA4 inter FOXP3 inter CD4 high Tregs under Treg-polarizing conditions examining 6 RA patients and 6 HCs were presented. I Expression of IL-10 was measured by FACS. J - L Tregs isolated from RA patients and HCs co-cultured with CFSE-labeled CD4 + CD25 − T cells and stimulated with anti-CD3/CD28 beads for 5 days. J Proliferation of the T cells was analyzed by quantifying CFSE dilution at ( K ). L Cytokine Secretion was measured by ELISA. M Tregs isolated from RA patients and HCs cocultured with conventional CD4 + T cells. CD40L and ICOS levels in CD4 + T cells were analyzed by flow cytometry. N B cells isolated from same donors were divided into two parts and cocultured with either RA- or HCs-derived Tregs. Cultures were examined for the frequencies of CD19 + CD38 high IgD low B cells. O - R CD25 high CTLA4 high FOXP3 high CD4 high and CD25 inter CTLA4 inter FOXP3 inter CD4 high Tregs were sorted from same RA patients and cocultured with CFSE-labeled CD4 + CD25 − T cells. O - P Proliferation of the CD4 + T cells were analyzed by quantifying CFSE dilution. Q CD25 high CTLA4 high FOXP3 high CD4 high and CD25 inter CTLA4 inter FOXP3 inter CD4 high Tregs were cocultured with B cells. Frequencies of CD19 + CD38 high IgD. low B cells were examined. R The production levels of cytokines were measured by ELISA. All data were presented as the mean ± SEM. Paired Student t-test, ** p < 0.01; *** p < 0.001

Article Snippet: To sort CD4 + CD45RO − cells, Peripheral blood mononuclear cells (PBMCs) were negatively selected with CD45RO microbeads (130–046-001, Miltenyi Biotec Inc., Auburn, USA), followed by positive selection with CD4 micro-beads (130–097-048, Miltenyi Biotec Inc.) using autoMACS (130–097-048, Miltenyi Biotec Inc.).

Techniques: Cell Differentiation, Cell Culture, Expressing, Isolation, Labeling, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Derivative Assay

Journal: Molecular Medicine

Article Title: Dysfunctional glycolysis-UCP2-fatty acid oxidation promotes CTLA4 int FOXP3 int regulatory T-cell production in rheumatoid arthritis

doi: 10.1186/s10020-025-01372-6

Figure Lengend Snippet: An increase in caveolar expression promotes CTLA4 endocytosis in RA Tregs. CD4 + CD45RO − T cells from RA patients and HCs were stimulated with anti-CD3/CD28 beads. A Expression of CTLA4 in the T cells from RA patients and HCs on day 2 after CD3/CD28 stimulation were measured by qPCR. B Surface and cytoplasm distribution of CTLA4 was detected by western blotting. A representative blot from three experiments is shown and quantified at ( C ). D - E Cell surface distribution of CTLA4 was detected by CTLA4 staining (red) and T cell membrane was shown by lipid raft staining by cholera toxin Subunit B. Bar, 20 μm. D Representative images from 3 experiments were qualified at ( E ). F - Q T cell differentiation: CD4 + CD45RO − T cells were cultured under Treg-polarizing condition. F - G The CTLA4 Surface expression levels in Treg were analyzed by FACS on day 5. H CTLA4 endocytosis. CTLA4 Surface and cytoplasm distribution at 30 min after CTLA4 antibody stimulation were detected by FACS, representative plots were from 6 RA and 6 HCs. I Intracellular caveolar levels were detected by FACS and data from 6 RA patients and 5 HCs were quantified. J Intracellular clathrin level were detected by FACS. K Proteins from RA and HCs Tregs were subjected to immunoprecipitation with CTLA4 antibody followed by blotting with and caveolin-1 or clathrin antibodies. L - O CTLA4 (red) endocytosis patterns were determined with co-staining with caveolin-1 (green) and clathrin (green) antibodies. Fluorescence intensities of colocalization signal of CTLA4-clathrin and CTLA4-caveolae examining 6 HCs and 6 RA were presented. Bar, 20 μm. P - Q CD4 + CD45RO − T cells cultured under Treg-polarizing condition were treated with caveolar inhibitor Methyl-β-cyclodextrin (MT-β-CD, 10 mM) or clathrin inhibitor Pitstop2 (10 µM) for 24 h on day 3. The CTLA4 surface expression levels of Tregs were analyzed by FACS. All data were presented as the mean ± SEM. * p < 0.05; *** p < 0.001; n.s., non-significance

Article Snippet: To sort CD4 + CD45RO − cells, Peripheral blood mononuclear cells (PBMCs) were negatively selected with CD45RO microbeads (130–046-001, Miltenyi Biotec Inc., Auburn, USA), followed by positive selection with CD4 micro-beads (130–097-048, Miltenyi Biotec Inc.) using autoMACS (130–097-048, Miltenyi Biotec Inc.).

Techniques: Expressing, Western Blot, Staining, Membrane, Cell Differentiation, Cell Culture, Immunoprecipitation, Fluorescence

Journal: Molecular Medicine

Article Title: Dysfunctional glycolysis-UCP2-fatty acid oxidation promotes CTLA4 int FOXP3 int regulatory T-cell production in rheumatoid arthritis

doi: 10.1186/s10020-025-01372-6

Figure Lengend Snippet: Inadequate FAO pathway induces caveolae-mediated CTLA4 endocytosis . CD4 + CD45RO − T cells from RA patients and HCs were stimulated with anti-CD3/CD28 beads and cultured under Treg-polarizing condition. A - B Neutral Lipid droplets were stained with Nile red and data examining 5 RA patients and 5 HCs were quantified at ( B ). C - D Time course and rate of increase in oleate-driven Fatty acid β-oxidation (FAO) assay. The data was recorded every 5 min. T cells treated with FCCP were used as positive control, and cells treated with Etomoxir were used as negative control. Relative FAO rate analyzing 6 RA patients and 6 HCs were quantified at ( D ). E – F PFKPB3 expression was detected by FACS and results analyzing 10 RA patients and 10 HCs were quantified. G CPT1A and CPT2 gene expression in Tregs from RA patients and HCs. H - I Representative blotting for CPT1A and CPT2 protein expression. Results analyzing 6 RA patients and 6 HCs were quantified at ( I ). J - M CPT1A and CPT2 expression were detected by FACS and result examining 6 RA patients and 6 HCs were quantified. N - R CTLA4 cell surface distribution regulated by CPT2. CD4 + T cells isolated from HCs were cultured under Treg condition and transfected with control and CPT2 siRNA on day 3. N CPT2 protein level was examined by western blotting. Representative blotting from 3 experiments. O - P Caveolar expression was detected by FACS and data from 3 experiments were quantified at ( P ). Q - R CTLA4 cell Surface expression were detected by FACS and data from 3 experiments were quantified at ( R ). All data were presented as the mean ± SEM. * p < 0.05; *** p < 0.001; n.s., non-significance

Article Snippet: To sort CD4 + CD45RO − cells, Peripheral blood mononuclear cells (PBMCs) were negatively selected with CD45RO microbeads (130–046-001, Miltenyi Biotec Inc., Auburn, USA), followed by positive selection with CD4 micro-beads (130–097-048, Miltenyi Biotec Inc.) using autoMACS (130–097-048, Miltenyi Biotec Inc.).

Techniques: Cell Culture, Staining, Positive Control, Negative Control, Expressing, Gene Expression, Isolation, Transfection, Control, Western Blot

Journal: Molecular Medicine

Article Title: Dysfunctional glycolysis-UCP2-fatty acid oxidation promotes CTLA4 int FOXP3 int regulatory T-cell production in rheumatoid arthritis

doi: 10.1186/s10020-025-01372-6

Figure Lengend Snippet: CTLA4 endocytosis increasing is associated with impaired glycolysis-UCP2-FAO system. CD4 + CD45RO − T cells from RA and HCs were stimulated with anti-CD3/CD28 beads. A UCP1 , UCP2 and UCP3 gene expression was quantified by qPCR in CD4 + T cells from RA patients and HCs. B UCP2 gene expression on day 0, day 2, and day 4 after anti-CD3/CD28 stimulation. C Representative blotting for UCP1, UCP2 and UCP3 expression. D UCP2 insufficiency is independent of drug treatment. E UCP2 protein expression correlation with disease activity. F - N CD4 + CD45RO − T cells from RA patients and HCs were cultured under Treg-polarizing condition. F - G UCP2 expression in Treg from RA patients and HCs were detected by FACS. The results analyzing 6 RA and 6 HCs were quantified at ( G ). H - I Representative blotting for UCP2 expression in Tregs. Data analyzing 6 RA and 6 HCs were quantified at ( I ). J UCP2 expression in 11 RA patients was assessed for correlation with the caveolar expression. K RA-derived Tregs were transfected with GFP-labeled control vectors or GFP-labeled CPT2-expressing plasmids on day 3. The UCP2 and caveolar levels in Tregs were analyzed by FACS. L HC-derived Tregs were treated with a FAO inhibitor Etomoxir 20 μM on day 3. The UCP2 and caveolar levels in Tregs were analyzed by FACS. M – N UCP2 protein expression in Tregs from 11 RA patients was assessed for correlation with the CPT1A and CPT2 expression. All data were presented as the mean ± SEM. *** p < 0.001; n.s., non-significance

Article Snippet: To sort CD4 + CD45RO − cells, Peripheral blood mononuclear cells (PBMCs) were negatively selected with CD45RO microbeads (130–046-001, Miltenyi Biotec Inc., Auburn, USA), followed by positive selection with CD4 micro-beads (130–097-048, Miltenyi Biotec Inc.) using autoMACS (130–097-048, Miltenyi Biotec Inc.).

Techniques: Gene Expression, Expressing, Activity Assay, Cell Culture, Derivative Assay, Transfection, Labeling, Control

Journal: Molecular Medicine

Article Title: Dysfunctional glycolysis-UCP2-fatty acid oxidation promotes CTLA4 int FOXP3 int regulatory T-cell production in rheumatoid arthritis

doi: 10.1186/s10020-025-01372-6

Figure Lengend Snippet: UCP2 signaling regulates caveolae-mediated CTLA4 surface distribution through CPT2. CD4+CD45RO− T cells from RA patients and HCs were cultured under Treg-polarizing condition. A-K UCP2 inhibitor treatment. CD4+ T from RA patients were treated with vehicle or UCP2 inhibitor Genipin (25 μM) on day 3 under Treg polarization conditions A CPT2 expression was analyzed by FACS. Representative histograms examining 8 RA patients were quantified. B PFKPB3 expression was detected by FACS. C Time course and rate of increase in oleate-driven FAO assay. Relative FAO rate analyzing 3 RA were quantified. D The caveolar levels were analyzed by FACS. E Co-immunoprecipitation with CTLA4 antibody followed by blotting with caveolin-1 antibody. F The CTLA4 surface expression levels were analyzed by FACS. G Proliferation of the CD4+ T cells was analyzed by CFSE dilution. H CD40L and ICOS protein levels in CD4+ T cells were analyzed by flow cytometry. I A representative density plot of the expression of CD38 and IgD on CD19+ B cells. J The production levels of cytokines were measured by ELISA. K CTLA4 endocytosis patterns were determined with co-immunostaining CTLA4 with caveolin-1. Fluorescence intensities of caveolae and CTLA4 in the RA Tregs from 3 experiments were quantified. L-W UCP2 overexpression. HC-derived CD4+ T cells were transfected with control or mcherry-UCP2 on day 3 under Treg polarization conditions. L Transfection efficiency. M CPT2 expression was analyzed by FACS. N PFKPB3 expression was detected by FACS. O Time course and rate of increase in oleate-driven FAO assay. P Caveolar levels were analyzed by FACS. Q Co-immunoprecipitation for CTLA4 and caveolin-1. R CTLA4 surface expression levels were analyzed by FACS. S Proliferation of the CD4+ T cells was analyzed by CFSE dilution. T CD40L and ICOS levels were analyzed by FACS. U The frequencies of CD19+CD38highIgDlow B cells. V Expression of cytokines. W CTLA4 endocytosis was determined with co-immunostaining CTLA4 with caveolin-1. Bar, 20 μm. All data were presented as the mean ± SEM. ** p < 0.01; *** p < 0.001.

Article Snippet: To sort CD4 + CD45RO − cells, Peripheral blood mononuclear cells (PBMCs) were negatively selected with CD45RO microbeads (130–046-001, Miltenyi Biotec Inc., Auburn, USA), followed by positive selection with CD4 micro-beads (130–097-048, Miltenyi Biotec Inc.) using autoMACS (130–097-048, Miltenyi Biotec Inc.).

Techniques: Cell Culture, Expressing, Immunoprecipitation, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Immunostaining, Fluorescence, Over Expression, Derivative Assay, Transfection, Control

Journal: Molecular Medicine

Article Title: Dysfunctional glycolysis-UCP2-fatty acid oxidation promotes CTLA4 int FOXP3 int regulatory T-cell production in rheumatoid arthritis

doi: 10.1186/s10020-025-01372-6

Figure Lengend Snippet: UCP2 suppresses CPT2 expression by impairing oxidative signaling and protein acetylation. CD4 + CD45RO − T cells from HCs were treated with vehicle or Genipin at indicated dose on day 3 after anti-CD3/CD28 bead stimulation. A Correlation of mitochondrial membrane potential (MΦ) with UCP2 activity. MΦ was assessed by FACS. B Correlation of CPT2 expression with MΦ. CPT2 expression and MΦ were assessed by FACS. C-E CPT2 expression regulated by ROS. C ROS levels from RA patients and HCs were measured with H2DCFDA probe on day 3. D RA-derived CD4 + T cells were treated with Genipin on day 3 and ROS levels were measured after 24 h. E HC-derived CD4 + T cells were transfected with control and mCherry-UCP2 plasmids on day 3 and ROS levels were measured after 24 h. F HC-derived CD4 + T cells were treated with vehicle or TEMPO (20 μM) on day 3 for 24 h. CPT2 levels were measured by FACS. G HC-derived CD4 + T cells were treated with vehicle or NAC 20 μM on day 3 for 24 h. CPT2 levels were measured by FACS. H - I CPT2 expression after TEMPO and NAC treatment was measured by Western blotting and quantified at ( I ). J - K HC-derived CD4 + T cells were treated with vehicle, NAC 20 mM, or NAC and Genipin 25 μM on day 3 for 24 h. J CPT2 levels were measured by FACS and quantified at ( K ). L - M CPT2 protein expression after NAC 20 mM, or NAC and Genipin treatment were measured by Western blotting. N CPT2 acetylation was determined by a Co-immunoprecipitation assay with anti-CPT2 followed by immunoblotting with anti-acetylated-lysine antibody. O RA-derived CD4 + T cells were treated with Genipin and CPT2 acetylation were measured by Co-immunoprecipitation. All data were presented as the mean ± SEM. ** p < 0.01; *** p < 0.001; n.s., non-significance

Article Snippet: To sort CD4 + CD45RO − cells, Peripheral blood mononuclear cells (PBMCs) were negatively selected with CD45RO microbeads (130–046-001, Miltenyi Biotec Inc., Auburn, USA), followed by positive selection with CD4 micro-beads (130–097-048, Miltenyi Biotec Inc.) using autoMACS (130–097-048, Miltenyi Biotec Inc.).

Techniques: Expressing, Membrane, Activity Assay, Derivative Assay, Transfection, Control, Western Blot, Co-Immunoprecipitation Assay, Immunoprecipitation

Journal: Molecular Medicine

Article Title: Dysfunctional glycolysis-UCP2-fatty acid oxidation promotes CTLA4 int FOXP3 int regulatory T-cell production in rheumatoid arthritis

doi: 10.1186/s10020-025-01372-6

Figure Lengend Snippet: UCP2 expression in RA T-cells induces synovial tissue inflammation. Pairs of NSG mice were engrafted with synovial tissue from RA patients, and CD45RO − PBMCs from HCs were transferred to the chimeric mice. The mice were divided into two groups, vehicle (DMSO) and Genipie groups. A - B CTLA4 surface expression was detected by FACS in the CD4 + T cells from PBMCs. C - D CTLA4 surface expression was detected by FACS in the CD4 + T cells from spleen. E Representative H&E images. F - G The intensity of synovial inflammation was compared by qPCR to assess TRB and TNFSF11 gene expression in each group. H - I RANKL staining. J Representative images of anti-CD3 (red) and anti-CTLA4 staining (green) of the vehicle and UCP2 inhibitor-treated mice. Bar, 20 μm. K Co-immunostaining of CPT2 (green) and CD3 (red). Representative images were from one of three synovial tissue from each group. Bar, 20 μm. L - V UCP2 overexpression. CD45RO − PBMCs from RA were transfected with either control or mCherry-UCP2 plasmids and adoptively transferred to the chimeric mice. L - M CTLA4 surface expression was detected by FACS in the CD4 + T cells from PBMCs. N – O CTLA4 surface expression were detected by FACS in the CD4. + T cells from spleen. P Representative H&E images. Q - R TRB and TNFSF11 gene expression was compared by qPCR. S - T RANKL staining. U Representative images of anti-CD3 (red) and anti-CTLA4 staining (green). Bar, 20 μm. V Co-immunostaining of CPT2 (green) and CD3 (red). Bar, 20 μm. All data were presented as the mean ± SEM. ** p < 0.01; *** p < 0.001

Article Snippet: To sort CD4 + CD45RO − cells, Peripheral blood mononuclear cells (PBMCs) were negatively selected with CD45RO microbeads (130–046-001, Miltenyi Biotec Inc., Auburn, USA), followed by positive selection with CD4 micro-beads (130–097-048, Miltenyi Biotec Inc.) using autoMACS (130–097-048, Miltenyi Biotec Inc.).

Techniques: Expressing, Gene Expression, Staining, Immunostaining, Over Expression, Transfection, Control