Figure 1.

Janus kinase (JAK) inhibitors prevent cytokine-induced maturation of cytokine-activated T (Tck) cells and inhibit the production of IL-6, IL-15 and IL-1RA by activated macrophages. CD14⁺ monocytes (5×10⁵ cells/mL) were differentiated into macrophages by culturing them in the presence of macrophage-colony stimulating factor (MCSF; 50 ng/mL) for 6 days in a complete medium in a 96-well plate. Purified CD4⁺ T cells (1×10⁶ cells/mL) were stimulated with a cocktail of IL-2, IL-6 and TNF in the absence or presence of 0.001% DMSO (used as Vehicle), tofacitinib (1000 nM) or ruxolitinib (1000 nM). The cells were incubated for 6 days to generate Tck. The Tck populations were washed and then cocultured with monocyte-derived macrophages at a concentration of 4:1 Tck-macrophage ratio. The supernatants were harvested, and the level of TNF was measured by ELISA. (A) The comparison of TNF production between DMSO and Tofacitinib (Tofa)-treated or ruxolitinib-treated Tck. (B) The comparison of TNF production between medium and DMSO-treated Tck. In separate experiments, cytokine-generated Tck were cocultured with monocyte-derived macrophages (MФs) at 4:1 ratio in the absence or presence of 0.001% DMSO (Veh) or tofacitinib (100 nM, 300 nM and 1000 nM) or ruxolitinib (100 nM, 300 nM, 1000 nM, 50 µM). Culture supernatants were harvested, and the levels of (C, D) IL-6, (E, F) IL-15 and (G, H) IL-1RA were measured using Luminex. Data represent a mean of triplicate cultures±SE of the mean of at least five experiments. Statistically significant differences are indicated (*p< 0.05; **p<0.01; ***p<0.001). DMSO, dimethyl sulfoxide; ns, not significant; Veh, vehicle.