Figure 3

Increase in Th17 cells after tremelimumab-based therapy by intracellular cytokine staining. A) Gating strategy for IL-17 intracellular staining. Starting from either whole PBMC or CD-4 sorted cells (as depicted here), the lymphocyte population was gated on by FSC-H and SSC-H dot plot. Live cells were gated in the same graphic. A second gate was performed in CD3 and SSC-H dot plot. We analyzed for IL-17-producing cells among CD4+ T cells after gating. B) Example of IL-17 intracellular staining. After 4-day activation of CD4-sorted cells with anti-CD3 and anti-CD28, cells were additionally stimulated with PMA and ionomycin while inhibiting protein transport, and the number of Th17 cells was determined by flow cytometry. Depicted are the plots of gated Th17 cells from patient NRA12. The left column is the baseline pre-dosing sample, and the right column the post-dosing sample. C) Th17 quantification by flow cytometry. Pre- and post-dosing whole PBMC (left graph) or CD4+ cells (right graph) analyzed by flow cytometry for Th17 cells as described above (p values by pairwise t-test).