Fig. 6
Inhibitory effects of IL-1Ra on T cell signaling and cytotoxic activity of OT-1 splenocytes. (A) Inhibition of IL-1Ra on a CD4 T cell line (Jurkat/NFAT-Luc) containing a luciferase reporter in response to NFAT signaling. Jurkat/NFAT-Luc cells were treated with PMA (20 ng/ml) and ionomycin (iono., 1 mg/ml), and relative luciferase light units (RLUs) were monitored in response to a dosage of IL-1Ra. Student’s t-test was performed. (B) Correlation analysis between IL1RN mRNA level and a cytolytic T cell index consisting of an average of GZMA and PRF1 mRNA level. r: Pearson’s correlation coefficient. (C) A flowchart of testing the inhibitory effect of IL-1Ra on cytolytic activity of OT-1 splenocytes. A melanoma cell line expressing full-length ovalbumin (B16F10-OVA) was labeled with CFSE and then incubated with OT-1 splenocytes. The effect of IL-1Ra was monitored by measuring the level of 7AAD staining. (D) Monitoring 7AAD and CFSE staining of B16F10-OVA cells in different conditions (No OT-1, +OT-1, +OT-1 + IL-1Ra) by flow cytometry. Percentages of cell populations were labeled red. (E) Inhibitory effects of IL-1Ra on cytolytic activities of OT-1 splenocytes. Changes of CFSE-positive (CFSE+) or 7AAD-positive (7AAD+) B16F10-OVA cells incubated with OT-1 splenocytes in response to IL-1Ra were analyzed (upper panel). The ratio of double-positive B16F10-OVA cells (7AAD+CFSE+) over CFSE+ ones was compared in response to IL-1Ra (lower panel). Student’s t-test was performed. * p < 0.05. **** p < 0.0001