Journal: medRxiv
Article Title: Inflammatory arthritis immune related adverse events represent a unique autoimmune disease entity primarily driven by T cells, but likely not autoantibodies
doi: 10.1101/2025.06.06.25328991
Figure Lengend Snippet: Inflammatory cytokines enriched in irAE patients promote both CD4 + and CD8 + T cell functions (A-G) CD8 + T cells were isolated from healthy donor PBMCs and cultured in the plate coated with 10 μg/mL anti-human CD3 and anti-human CD28 for indicated days in the presence of vehicle control (control), 100 ng/mL IFN-α, 100 ng/mL IL-6, 100 ng/mL IL-12, or the combination of IFN-α, IL-6 and IL-12. (A) Summaries of the percentage of CD38 + CD127 - CD8 + T cells at day 1 and day 5, n = 5. (B) CD69 expression was measured on day 5, and the mean fluorescence intensity (MFI) of CD69 was summarized on the right, n = 4. (C) CD25 expression was measured on day 5, and the MFI of CD25 was summarized on the right, n = 4. (D) CD8 + T cells activated for 5 days were restimulated with PMA/Ionomycin/Monensin for 5 h, expression of granzyme B and IFN-γ in CD8 + T cells were examined by flow cytometry. Right, percentage of granzyme B + IFN-γ + CD8 + T cells, n = 5. (E-G) CD8 + T cells were cultured for 5 days. MFIs (all relative to those in control condition) of MTDR (E), MTG (F), and GluCy5 (G) were summarized, n = 5. (H-L) CD4 + T cells were isolated from the PBMCs of HC, cultured in the plate coated with 10 μg/mL anti-human CD3 and anti-human CD28 for indicated days in the presence of 100 ng/mL IFN-α, 100 ng/mL IL-6, 100 ng/mL IL-12, control or the combination of them. (H) CD4 + T cells activated for 5 days were restimulated with PMA/Ionomycin/Monensin for 5 h, and IL-21 level was examined in the CD45RA - CD4 + T cells, n =3. (I) Activated CD4 + T cells were restimulated with 10 μg/mL plate-coated anti-human CD3 and anti-human CD28 with monensin for 6 h, and expression of CXCL13 was measured in CD45RA - CD4 + T cells, n = 4. (J) CD38 + CXCR5 - or CD38 - CXCR5 + CD4 + T cells were examined at day 5, and the percentages were summarized, n = 4. (K) Percentage of CXCL13 + cells in CD38 + CXCR5 - or CD38 - CXCR5 + CD4 + T cells at day 5, n = 4. (L) Percentage of IL-21 + cells in CD38 + CXCR5 - or CD38 - CXCR5 + CD4 + T cells at day 5, n = 3. (M-S) PBMCs from the irAE patients were cultured in the plate coated with 10 μg/mL anti-CD3 and anti-CD28 in the presence of IgG1 isotype control or 50 μg/mL anti-human IL-6R, 50 μg/mL anti-human IL-12p40 and 50 μg/mL anti-human IFNAR1 for 3 days, n = 9. (M) Expression of CD38 and CD127 on CD8 + T cells. Right, percentage of CD38 + CD127 - CD8 + T cells. (N, O) CD8 + T cells activated for 5 days were restimulated with PMA/Ionomycin/Monensin for 5 h, (N) Expression of Granzyme B and IFN-γ. Right, percentage of granzyme B + IFN-γ + CD8 + T cells, (O) Expression of perforin and IFN-γ. Right, percentage of perforin + IFN-γ + CD8 + T cells. (P, Q) MFIs of GluCy5 (P), TMRM, and MTDR (Q) in CD8 + T cells were presented. (R, S) CD4 + T cells activated for 5 days were restimulated with PMA/Ionomycin/Monensin for 5 h, (R) Expression of IL-21 and IL-2. Right, percentage of IL-21 + IL-2 + CD4 + T cells. (S) 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 by One-way ANOVA (A-L), and paired Student’s t-test (M-S).
Article Snippet: PBMCs from irAE patients were thawed and rested in a complete medium for 1 h, then activated with 10 μg/mL plate-coated anti-human CD3 and anti-human CD28 with IgG1 isotype control (BioXcell, Cat# CP174) or a combination of 50 μg/mL anti-human IL-6R (BioXcell, Cat# SIM0014), 50 μg/mL anti-human IL-12p40 (BioXcell, Cat# SIM0020) and 50 μg/mL anti-human IFNAR1 (BioXcell, Cat# SIM0022) for 3 days.
Techniques: Isolation, Cell Culture, Control, Expressing, Fluorescence, Flow Cytometry
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