Figure 4.

ShK-186 inhibits the pro-inflammatory cytokine production of CD4⁺T_EM cells. CD4⁺T cells subsets (i.e. CD4⁺T_NAIVE, CD4⁺T_CM, CD4⁺T_EM and CD4⁺T_TD cells) were isolated from peripheral blood mononuclear cells of HCs followed by stimulation with PMA and CaI with and without increasing concentrations of ShK-186. Intracellular IL-4, IL-17, IL-21, TNFα and IFNγ cytokine production in the CD4⁺T cell subsets was analysed using flow cytometry. (A) Representative flow cytometry dot plots of CD4⁺T cells subsets based on surface expression of CD45RO and CCR7 (centre dot plot), and the cytokine expression within CD4⁺T_NAIVE cells (upper left, red), CD4⁺T_CM cells (upper right, green), CD4⁺T_TD cells (lower left, purple) and CD4⁺T_EM cells (lower right, blue) after stimulation in the presence and absence of ShK-186 from a HC. (B) Percentages of intracellular cytokine production in CD4⁺T_NAIVE cells (red symbol and line), CD4⁺T_CM cells (green symbol and line), CD4⁺T_TD (purple symbol and line) and CD4⁺T_EM cells (blue symbol and line) after stimulation in the presence and absence of ShK-186 from HCs (n = 5). Data represent mean values ± SEM. ##P < 0.01 and ###P < 0.001 indicate CD4⁺T_EM cells vs CD4⁺T_NAIVE cells, CD4⁺T_CM cells and CD4⁺T_TD cells. *P < 0.05, **P < 0.01, ***P < 0.001 indicate CD4⁺T_EM cells with vs without ShK-186. ##: IL-21 production in CD4⁺ T_EM cells is only significantly different compared with CD4⁺T_NAIVE and CD4⁺T_TD cells and not significantly different compared with CD4⁺T_CM cells. GPA: granulomatosis with polyangiitis; HC: healthy control; PMA: phorbol myristate acetate; CaI: calcium ionophore; CD4⁺ T_CM: CD4⁺ central memory T cells; CD4⁺ T_EM: CD4⁺ effector memory T cells; CD4⁺ T_NAIVE: CD4⁺ naive T cells; CD4⁺ T_TD: CD4⁺ terminal differentiated T cells