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 F ) Bead-based multiplex assays were used to measure plasma concentration of IL-6, and IL-12p70 (A); TNF-α, IFN-γ, and IL-1β [B; HC ( n = 19), irAE ( n = 34), RAC ( n = 45), and ICI ( n = 9)]; IP-10 (CXCL10), CXCL11, and CXCL9 (C; HC, n = 17; irAE, n = 33; RAC, n = 46; ICI, n = 17); CCL20 (D); CX3CL1 (E); and CCL2 (F). ( G to J ) Human naïve B cells were isolated and cultured with anti–human CD40 (0.5 μg/ml), anti–human Ig (M + G + A) (2.5 μg/ml), and rhIL-21 (20 ng/ml) with IFN-α (100 ng/ml), IL-6 (100 ng/ml), IL-12 (100 ng/ml), control, or the combination of IFN-α, IL-6, and IL-12 for 7 days. Cells and culture supernatants were analyzed. (G) Representative flow plot of CD38 and CD138 expression on CD27 hi CD38 hi ASCs. Right: A summary of the percentage of CD138 + ASCs; n = 6. (H) Expression of CD11c and CD27 on CD27 − IgD − B cells. Right: Percentage of CD11c + IgD − CD27 − B cells; n = 6. (I) Expression of active-caspase-3 in B cells. Right: Percentage of active-caspase-3 + B cells from different groups; n = 3. (J) Different immunoglobulin isotype levels in the culture supernatants from (G) to (H) were measured by the multiplex assay; n = 6. Data in graphs represent mean ± SEM. Significance was tested by one-way ANOVA [(A) to (I)] and paired Student’s t test (J). [(A) to (F)] ICI, ICI control.

Article Snippet: Condition 1: anti–human Ig (M + G + A) (2.5 μg/ml; Jackson ImmunoResearch, catalog no. 109-006-064), CpG oligodeoxynucleotide (ODN) (2.5 μg/ml; Invivogen, catalog no. tlrl-2006-1), anti–human CD40 (10 μg/ml; Bio X Cell, catalog no. BE0189), rhIL-21 (20 ng/ml; Peprotech, catalog no. 200-21-50UG), rhIL-4 (10 ng/ml; BioLegend, catalog no. 574004), and rhIL-2 (10 ng/ml; Peprotech, catalog no. 200-02-250UG).

Techniques: Multiplex Assay, Clinical Proteomics, Concentration Assay, Isolation, Cell Culture, Control, Expressing

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 ) CD8 + T cells isolated from healthy donor were cultured in the plate coated with anti–human CD3/CD28 (10 μg/ml) for indicated days in the presence of vehicle control (control), IFN-α (100 ng/ml), IL-6 (100 ng/ml), IL-12 (100 ng/ml), or the combination. (A) Summaries of the percentage of CD38 + CD127 − CD8 + T cells at days 1 and 5. (B) MFI of CD69 (day 5). (C) MFI of CD25 (day 5). (D) Activated CD8 + T cells were restimulated with PMA, ionomycin, and monensin for 5 hours; expression of granzyme B and IFN-γ in CD8 + T cells was examined. [(E) to (G)] CD8 + T cells were cultured for 5 days. MFIs (all relative to those under control condition) of MTDR (E), MTG (F), and GluCy5 (G) were summarized. ( H to L ) CD4 + T cells were isolated from HC, cultured in the plate coated with anti–human CD3/CD28 (10 μg/ml) for indicated days in the presence of IFN-α (100 ng/ml), IL-6 (100 ng/ml), IL-12 (100 ng/ml), control, or the combination of them. (H) Activated CD4 + T cells were restimulated with PMA, ionomycin, and monensin for 5 hours, and IL-21 level was examined in the CD45RA − CD4 + T cells. (I) Activated CD4 + T cells were restimulated with plate-coated anti–human CD3/CD28 (10 μg/ml) with monensin for 6 hours, and expression of CXCL13 was measured in CD45RA − CD4 + T cells. (J) CD38 and CXCR5 were examined on CD4 + T cells at day 5. (K) Percentage of CXCL13 + cells in indicated T cells at day 5. (L) Percentage of IL-21 + cells in indicated T cells at day 5. Data in graphs represent mean ± SEM. Significance was tested by one-way ANOVA.

Article Snippet: Condition 1: anti–human Ig (M + G + A) (2.5 μg/ml; Jackson ImmunoResearch, catalog no. 109-006-064), CpG oligodeoxynucleotide (ODN) (2.5 μg/ml; Invivogen, catalog no. tlrl-2006-1), anti–human CD40 (10 μg/ml; Bio X Cell, catalog no. BE0189), rhIL-21 (20 ng/ml; Peprotech, catalog no. 200-21-50UG), rhIL-4 (10 ng/ml; BioLegend, catalog no. 574004), and rhIL-2 (10 ng/ml; Peprotech, catalog no. 200-02-250UG).

Techniques: Isolation, Cell Culture, Control, Expressing

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: Condition 1: anti–human Ig (M + G + A) (2.5 μg/ml; Jackson ImmunoResearch, catalog no. 109-006-064), CpG oligodeoxynucleotide (ODN) (2.5 μg/ml; Invivogen, catalog no. tlrl-2006-1), anti–human CD40 (10 μg/ml; Bio X Cell, catalog no. BE0189), rhIL-21 (20 ng/ml; Peprotech, catalog no. 200-21-50UG), rhIL-4 (10 ng/ml; BioLegend, catalog no. 574004), and rhIL-2 (10 ng/ml; Peprotech, catalog no. 200-02-250UG).

Techniques: Cell Culture, Control, Expressing