plate coated anti cd3  (Bio X Cell)

Journal: Science Advances

Article Title: Inflammatory arthritis irAE may represent a unique autoimmune disease primarily driven by T cells but likely not autoantibodies

doi: 10.1126/sciadv.aea4262

Figure Lengend Snippet: ( A ) Circos plots showing the percentage of cells from HC (Healthy control, n = 6), ICI ( n = 2), RAC (RA control, n = 5), or irAE ( n = 5). ( B ) Expression of CD45RA and CCR7 on CD8 + T cells. Right: Summaries of the percentage of cells from HC ( n = 53), irAE ( n = 29), RAC ( n = 41), and ICI ( n = 26). ( C ) Expression of CXCR3 and CCR6 on CD8 + T cells. Right: Summaries of T cell subsets. HC ( n = 45), irAE ( n = 27), RAC ( n = 31), and ICI ( n = 26). ( D ) The cytotoxic score was evaluated using the gene list identified previously . ( E ) Specific genes were evaluated on CD8 + T cells. ( F ) Pathways that were significantly enriched in the CD8 + T cells between irAE and ICI. NF-κB, nuclear factor κB; STAT5, signal transducer and activator of transcription 5; DN, down. Gene set enrichment analysis (GSEA) plots of the allograft rejection ( G ), oxidative phosphorylation ( H ), IFN-α response ( I ), and IFN-γ response ( J ) between irAE and ICI. NES, normalized enrichment score ( K to M ) PBMCs were stimulated with plate-coated anti-CD3 and anti-CD28 (10 μg/ml) for 5 days. Mean fluorescence intensities (MFIs) of MitoTracker Green (MTG) (K), MitoTracker deep red (MTDR) (L) [HC ( n = 31), irAE ( n = 18), RAC ( n = 32), and ICI ( n = 16)] or Cy5-linked-1-amino-glucose (GluCy5) (M) [HC ( n = 35), irAE ( n = 20), RAC ( n = 36), and ICI ( n = 18)] in CD8 + T cells were presented. Expression was normalized to the HC in each experiment. ( N ) UMAP shows the presence or absence of T cell receptor (TCR) in the major immune cells across all the samples. ( O ) Pie charts showing the distribution of the top 100 TCR clones across different T cell subsets. Data in graphs represent mean ± SEM. Significance was tested by one-way analysis of variance (ANOVA). [(A) to (E) and (G) to (O)] ICI, ICI control.

Article Snippet: A total of 0.5 million isolated CD4 + T cells or CD8 + T cells was stimulated with plate-coated anti-CD3 (10 μg/ml; BioXCell, catalog no. BE0001-2) and anti-CD28 (BioXCell, catalog no. BE0291), rhIL-6 (100 ng/ml), rhIFN-α 2 (100 ng/ml), rhIL-12 (100 ng/ml), or the combination of rhIL-6, rhIFN-α 2 , and rhIL-12 for 5 days.

Techniques: Control, Expressing, Phospho-proteomics, Fluorescence, Clone Assay

Journal: Science Advances

Article Title: Inflammatory arthritis irAE may represent a unique autoimmune disease primarily driven by T cells but likely not autoantibodies

doi: 10.1126/sciadv.aea4262

Figure Lengend Snippet: ( A ) The CD4/CD8 ratio in PBMCs. HC ( n = 53), irAE ( n = 23), RAC ( n = 42), and ICI ( n = 17). ( B ) Expression of CD25 and CD127 on CD4 + T cells. HC ( n = 53), irAE ( n = 27), RAC ( n = 42), and ICI ( n = 17). ( C ) Expression of CXCR5 and CD4 on the CD45RA − CD4 + T cells. HC ( n = 53), irAE ( n = 27), RAC ( n = 42), and ICI ( n = 17). ( D ) Expression of CCR6 and CXCR3 on CD45RA − CD4 + T cells. HC ( n = 41), irAE ( n = 26), RAC ( n = 36), and ICI ( n = 12). ( E ) Significantly enriched pathways in CD4 + T cells between irAE and ICI. UV, ultraviolet. GSEA plots of IFN-α and IFN-γ response ( F ), and oxidative phosphorylation ( G ). ( H to M ) PBMCs were stimulated with plate-coated anti-CD3/CD28 (10 μg/ml) for 5 days; MFI of MTDR (H; HC, n = 31; irAE, n = 18; RAC, n = 32; ICI, n = 16), tetramethylrhodamine methyl ester (TMRM) (I; HC, n = 34; irAE, n = 19; RAC, n = 35; ICI, n = 17), or GluCy5 (J; HC, n = 35; irAE, n = 20; RAC, n = 36; ICI, n = 18) in CD4 + T cells were presented. Expression was normalized to the HC in each experiment. [(K) to (M)] Fresh PBMCs were stimulated with phorbol 12-myristate 13-acetate (PMA), ionomycin, and monensin for 5 hours, and the frequencies of perforin + [(K); HC, n = 27; irAE, n = 13; RAC, n = 12; ICI, n = 8], [TNF-α + (L), and IL-2 + (M) (HC, n = 44; irAE, n = 25; RAC, n = 27; ICI, n = 16] CD4 + T cells were examined. Data in graphs represent mean ± SEM. Significance was tested by one-way ANOVA. [(A) to (D) and (F) to (M)] ICI, ICI control.

Article Snippet: A total of 0.5 million isolated CD4 + T cells or CD8 + T cells was stimulated with plate-coated anti-CD3 (10 μg/ml; BioXCell, catalog no. BE0001-2) and anti-CD28 (BioXCell, catalog no. BE0291), rhIL-6 (100 ng/ml), rhIFN-α 2 (100 ng/ml), rhIL-12 (100 ng/ml), or the combination of rhIL-6, rhIFN-α 2 , and rhIL-12 for 5 days.

Techniques: Expressing, Phospho-proteomics, Control

Journal: Science Advances

Article Title: Inflammatory arthritis irAE may represent a unique autoimmune disease primarily driven by T cells but likely not autoantibodies

doi: 10.1126/sciadv.aea4262

Figure Lengend Snippet: ( A to G ) PBMCs from the patients with irAE were cultured in the plate coated with anti-CD3 and anti-CD28 (10 μg/ml) in the presence of IgG1 isotype control or anti–human IL-6R (50 μg/ml), anti–human IL-12p40 (50 μg/ml), and anti–human IFNAR1 (50 μg/ml) for 3 days; n = 9. (A) Expression of CD38 and CD127 on CD8 + T cells. Right: Percentage of CD38 + CD127 − CD8 + T cells. (B and C) CD8 + T cells activated for 5 days were restimulated with PMA, ionomycin, and monensin for 5 hours. (B) Expression of granzyme B and IFN-γ. Right: Percentage of granzyme B + IFN-γ + CD8 + T cells. (C) Expression of perforin and IFN-γ. Right: Percentage of perforin + IFN-γ + CD8 + T cells. [(D) and (E)] MFIs of GluCy5 (D), TMRM, and MTDR (E) in CD8 + T cells were presented. [(F) and (G)] CD4 + T cells activated for 5 days were restimulated with PMA, ionomycin, and monensin for 5 hours. (F) Expression of IL-21 and IL-2. Right: Percentage of IL-21 + IL-2 + CD4 + T cells. (G) Expression of CD38 and CXCR5 on CD4 + T cells. Right: Percentage of CD38 + CXCR5 − CD4 + T cells. Data in graphs represent mean ± SEM. Significance was tested paired Student’s t test [(A) to (G)].

Article Snippet: A total of 0.5 million isolated CD4 + T cells or CD8 + T cells was stimulated with plate-coated anti-CD3 (10 μg/ml; BioXCell, catalog no. BE0001-2) and anti-CD28 (BioXCell, catalog no. BE0291), rhIL-6 (100 ng/ml), rhIFN-α 2 (100 ng/ml), rhIL-12 (100 ng/ml), or the combination of rhIL-6, rhIFN-α 2 , and rhIL-12 for 5 days.

Techniques: Cell Culture, Control, Expressing

Journal: Frontiers in Immunology

Article Title: Supersulfide controls intestinal inflammation by suppressing CD4 + T cell proliferation

doi: 10.3389/fimmu.2025.1506580

Figure Lengend Snippet: Cars2 +/- CD4 + T cells trigger exacerbated colitis in Rag2 -/- mice. (A) Cars2 +/- versus WT naïve CD4 + T lymphocytes induce exaggerated body weight loss in Rag2 -/- mice. Naïve CD4 + T cells derived from WT or Cars2 +/- mice were transferred into Rag2 -/- animals, and the hosts analyzed at different time points. The graph shows relative body weight of the recipient mice (n = 7 to 8). (B, C) Cars2 +/- CD4 + T cells trigger severe histological colitis. Donor cells were transferred into Rag2 -/- mice as described above, and the hosts analyzed 4 weeks later. The representative microscopic images of colonic sections display (B) H&E and (C) CD4-directed immunohistochemical staining while the bar graphs indicate (B) the histological scores as well as (C) quantification of CD4-positive cells in each group (n = 4 to 5). (D) Cars2 +/- CD4 + T lymphocytes accumulate in the intestine to a greater degree than do WT controls. The bar graph shows the absolute number of intestinal donor cells (CD3 + CD4 + Foxp3 - CD44 hi CD62L lo ) at the indicated time points (n = 3 to 5). (E) Th1 as well as Th17 differentiation rate is essentially the same between WT and Cars2 +/- donor cells. Several weeks after transfer into Rag2 -/- mice, donor cells were measured for IFN-γ as well as IL-17A expression. Bar graphs indicating (top) the frequency and (bottom) the absolute number of cytokine-producing cells among the total donor population accumulating in the colon are depicted (n = 3 to 5). (F) The rate of cell death is comparable between WT and Cars2 +/- donor cells. In the above experiments donor cells were stained with amine-reactive dye. The bar graph indicates the frequency of amine-reactive dye + (dead) cells among total donor population in the colon (n = 3 to 5). Data are (A, D-F) pooled from and (B, C) representative of two independent experiments performed. The data are shown as the mean ± standard deviation. Scale bars, 50 μm. * p < 0.05, ** p < 0.01.

Article Snippet: In and , naïve CD4 + T cells (5 x 10 4 cells per well) isolated by Naïve CD4 + T cell isolation Kit (Miltenyi Biotec) were stimulated with antibodies against plate-coated CD3 (1 μg/ml) and soluble CD28 (1 μg/ml) (both from Biolegend) in RPMI complete medium supplemented with 50 μM β-mercaptoethanol for 2 to 3 days.

Techniques: Derivative Assay, Immunohistochemical staining, Staining, Expressing, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Supersulfide controls intestinal inflammation by suppressing CD4 + T cell proliferation

doi: 10.3389/fimmu.2025.1506580

Figure Lengend Snippet: Cell cycle entry is accelerated in Cars2 +/- CD4 + T cells at an early phase of colitis. (A, B) Cars2 +/- CD4 + T cells more efficiently enter the cell cycle than do WT controls at an early stage of colitis. Naïve CD4 + T cells derived from WT or Cars2 +/- animals were transferred to Rag2 -/- host mice as described in Figure 3 , and the donor cells analyzed at the indicated time points. The representative dot plots display expression levels of Ki67 and total amounts of DNA in donor cells while the bar graphs show the frequency of cells within the indicated cell cycle phases among total donor populations at (A) 1 week and (B) 4 weeks post transfer (n = 4 to 6). A representative dot plot depicting gating strategies for G0, G1, S, and G2/M cells is also included. (C) Colonic Cars2 +/- T cells express lower levels of Trp53 . Real-time PCR was performed to detect Trp53 expression. The bar graph shows relative expression of Trp53 in donor cells (n = 11 to 12). (D) Cars2 +/- CD4 + T lymphocytes enter cell cycle more efficiently in vitro . Cars2 +/- naïve CD4 + T cells were stimulated with CD3 and CD28, and cell cycle phases analyzed 3 days later. The representative dot plots show the expression levels of Ki67 and the total amounts of DNA in CD4 + T cells, while the bar graphs show the frequency of cells within the indicated cell cycle phases (n = 8 to 13). Data shown are (A, B) representative of two independent experiments and pooled from (D) three and (C) four independent experiments. The data are shown as the mean ± standard deviation. * p < 0.05, ** p < 0.01.

Article Snippet: In and , naïve CD4 + T cells (5 x 10 4 cells per well) isolated by Naïve CD4 + T cell isolation Kit (Miltenyi Biotec) were stimulated with antibodies against plate-coated CD3 (1 μg/ml) and soluble CD28 (1 μg/ml) (both from Biolegend) in RPMI complete medium supplemented with 50 μM β-mercaptoethanol for 2 to 3 days.

Techniques: Derivative Assay, Expressing, Real-time Polymerase Chain Reaction, In Vitro, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Supersulfide controls intestinal inflammation by suppressing CD4 + T cell proliferation

doi: 10.3389/fimmu.2025.1506580

Figure Lengend Snippet: Treatment with GSSSG ameliorates colitis in Rag2 -/- mice that received Cars2 +/- CD4 + T cells. (A) GSSSG treatment inhibits body weight reduction of Rag2 -/- mice that have received Cars2 +/- naïve CD4 + T lymphocytes. Cars2 +/- naïve CD4 + T cells were transferred into Rag2 -/- hosts that were then daily treated with phosphate-buffered saline (PBS) or GSSSG. The graph shows relative body weight of the recipient mice at the indicated time points (n = 6 to 8). (B, C) Histological colitis triggered by Cars2 +/- naïve CD4 + T cells is ameliorated by supplementation with GSSSG. Representative microscopic images of colonic sections showing (B) H&E and (C) CD4-directed immunohistochemical staining together with bar graphs indicating (B) histological scores as well as (C) quantification of CD4-positive cells are displayed (n = 6 to 8). (D) GSSSG treatment inhibits accumulation of Cars2 +/- CD4 + T lymphocytes in the colon. The bar graph shows the absolute number of intestinal donor cells at 1 month post transfer (n = 6). (E) GSSSG inoculation does not affect differentiation of Th1 or Th17 cells. Four weeks after transfer into Rag2 -/- mice supplemented with PBS or GSSSG, donor cells were measured for IFN-γ as well as IL-17A expression. Bar graphs indicating the frequency and the absolute number of cytokine-producing cells among the total donor population accumulating in the colon are depicted (n = 6). (F) The rate of cell death in Cars2 +/- donor cells are comparable between PBS and GSSSG treated groups. In the above experiments donor cells were stained with amine-reactive dye. The bar graph indicates the frequency of amine-reactive dye + (dead) cells among total donor population in the colon (n = 6). (G) GSSSG administration inhibits cell cycle entry of Cars2 +/- CD4 + T cells at an early phase of colitis. The representative dot plots show expression levels of Ki67 and total amounts of DNA in donor cells while the bar graphs show the frequency of cells within the indicated cell cycle phases among total donor populations at 1 week and 4 weeks post transfer (n = 3 to 6). (H) GSSSG administration upregulates Trp53 expression in colonic Cars2 +/- T cells. A bar graph showing relative expression of Trp53 in donor cells at 1 week after transfer is displayed (n = 5 to 7). (I) GSSSG suppresses cell cycle entry of Cars2 +/- CD4 + T lymphocytes in vitro . Cars2 +/- naïve CD4 + T cells were stimulated with CD3 and CD28 in the presence or absence of GSSSG, and the cell cycle phases analyzed 3 days later. The representative dot plots show expression levels of Ki67 and total amounts of DNA in CD4 + T cells while the bar graphs show the frequency of cells within the indicated cell cycle phases (n = 4). Data are pooled from two or three independent experiments. The data are shown as the mean ± standard deviation. Scale bars, 50 μm. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: In and , naïve CD4 + T cells (5 x 10 4 cells per well) isolated by Naïve CD4 + T cell isolation Kit (Miltenyi Biotec) were stimulated with antibodies against plate-coated CD3 (1 μg/ml) and soluble CD28 (1 μg/ml) (both from Biolegend) in RPMI complete medium supplemented with 50 μM β-mercaptoethanol for 2 to 3 days.

Techniques: Saline, Immunohistochemical staining, Staining, Expressing, In Vitro, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Supersulfide controls intestinal inflammation by suppressing CD4 + T cell proliferation

doi: 10.3389/fimmu.2025.1506580

Figure Lengend Snippet: CARS2-dependent regulation of CD4 + T cell proliferation is operative in humans. (A-N) Intestinal CD4 + T cells derived from CD patients exhibit lower expression of CARS2 with highly proliferative signatures. Publicly available gene expression datasets of gut-infiltrating T lymphocytes from CD patients and controls were re-analyzed as described in Materials and Methods. (A) The plot displays re-clustered, CD3E -expressing cells, determined by the Uniform Manifold Approximation and Projection (UMAP) algorithm. Each dot represents a cell, and colors highlight unsupervised cell clusters. (B) The UMAP plots represent expression of CD3E and CD4 . (C) The dot plot shows the frequency of CARS2 -expressing cells and the relative expression level in CD4 + T cells. P values for expression level of signature gene are shown. (D) The box plot indicates the frequency of CARS2 -expressing cells among CD4 + T cells (control, n = 10; CD, n = 7). (E, F) The dot plots show the frequency of gene-expressing cells and the relative expression levels in CD4 + T cells. P value for the expression levels of signature gene is shown. (G) Correlation between the frequency of CARS2 -positive cells and those of TP53 -expressing CD4 + T cells (control, n = 10; CD, n = 7). (H) The UMAP plot displays CD4 -expressing cells extracted from CD3E -expressing cells. Each dot represents a cell, and colors highlight individual cell subsets. (I) The stacked bar graph shows the proportion of the indicated cell types. (J) The dot plot shows the frequency of CARS2 -expressing cells and the relative expression level in effector CD4 + T cells. P value for the expression level of the signature gene is shown. (K) The box plot indicates the frequency of CARS2 -expressing cells among effector CD4 + T cells (control, n = 10; CD, n = 7). (L, M) Dot plots show the frequency of gene-expressing cells and the relative expression levels in effector CD4 + T cells. P values for expression levels of signature genes are shown. (N) Correlation between the frequency of CARS2 -positive cells and that of TP53 -expressing effector CD4 + T cells (control, n = 10; CD, n = 7). (O) GSSSG inhibits cell cycle entry of activated CD4 + T lymphocytes in humans. Human naïve CD4 + T cells were stimulated with CD3 and CD28 in the presence or absence of GSSSG, and cell cycle phases analyzed 48 hours later. The representative dot plots display expression levels of Ki67 and total amounts of DNA while the graphs show the frequency of cells within the indicated cell cycle phases among CD4 + T lymphocytes (n = 6). Data presented in (O) are pooled from three independent experiments. The data are shown as mean ± standard deviation. R 2 , Pearson’s correlation coefficient. * p < 0.05.

Article Snippet: In and , naïve CD4 + T cells (5 x 10 4 cells per well) isolated by Naïve CD4 + T cell isolation Kit (Miltenyi Biotec) were stimulated with antibodies against plate-coated CD3 (1 μg/ml) and soluble CD28 (1 μg/ml) (both from Biolegend) in RPMI complete medium supplemented with 50 μM β-mercaptoethanol for 2 to 3 days.

Techniques: Derivative Assay, Expressing, Gene Expression, Control, Standard Deviation