Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Nod2-deficiency augments Th17-responses and exacerbates autoimmune arthritis

doi: 10.4049/jimmunol.1700507

Figure Lengend Snippet: The T cellular response was evaluated 8 wk following injection of zymosan into SKG or Nod2−/−SKG mice. (A) Combined weight of popliteal lymph nodes (2/mouse). (B) Quantification of the total number of live T cell subsets from the dLN by flow cytometry. (C) The number of live T cell subsets from synovial fluid aspirated from the sub-capsular space of ankle joints was quantified by flow cytometry. (D-G) Single cell suspensions from combined popliteal lymph nodes were stimulated in vitro with PMA/Ionomycin (PMA/Io), stained for intracellular cytokines, then analyzed by flow cytometry. (D) Dot plots showing frequency of IL-17-producing CD4+ T cells. Th17 cells were quantified and are shown as (E) percentage and (F) total number of live CD4+ T cells. (G) The Th17 cell population was gated and the frequency of Th17 cells co-expressing cytokines (IFNγ, TNF, GM-CSF, and IL-22) was quantified. (H) IL-17A protein in synovial fluid from the sub-capsular space of ankle joints 8 wk post-zymosan was quantified by ELISA. Data are graphed as median with interquartile range. All data (expect panel H) are graphed as box and whisker plots demarcating the median with min to max whiskers and are combined from 2 independently performed studies. * p< 0.05 (Mann-Whitney U). Data in A-F are combined data from 2 independent experiments and data in (H) are representative of 3 independently performed experiments (n=10–12 mice/group).

Article Snippet: Cells were treated with 20 ng/ml PMA (LC Laboratories) and 1 1⁄4g/ml ionomycin (LKT Laboratories) vs. media for 5 h in the presence of Brefeldin A (1 1⁄4g/ml; BD Biosciences).

Techniques: Injection, Flow Cytometry, In Vitro, Staining, Expressing, Enzyme-linked Immunosorbent Assay, Whisker Assay, MANN-WHITNEY