Fig. 2.

IL-22 is secreted from memory CD4⁺ T cells in an AhR- and RORγt-dependent manner, and in vivo neutralization of IL-1 reduces tumor growth. (A and B) Splenocytes (2 × 10⁶/mL) were stimulated with 4T1 supernatant (A) or Line-1 supernatant (B) in the presence or absence of 10 µM CH-223191 (AhR antagonist) or 5 µM SR-2211 (RORγt antagonist) for 6 d. Values in A are the mean of three different experiments performed in triplicate. Values in B are representative of five different experiments performed in triplicate. IL-22 production was quantified by ELISA. (C and D) MACS-enriched CD4⁺ T cells and CD4⁻ splenocytes or total splenocytes were stimulated with 4T1 tumor supernatant (C) or 100 ng/mL recombinant IL-1α and IL-23 (D) for 6 d. Mean values of a minimum of four independent experiments are shown. (E) MACS-enriched CD4⁺ T cells were stimulated with 20 ng/mL recombinant IL-1α for 4 d. IL-22 production by Th1 (CD3⁺CD4⁺IFN-γ⁺), Th17 (CD3⁺CD4⁺IFN-γ⁻IL-17⁺), and Th22 (CD3⁺CD4⁺IFN-γ⁻IL-17⁻) T cells was analyzed by flow cytometry (data represent two independent experiments with five different mice). (F) MACS-enriched naive CD4⁺ T cells and CD4⁻ splenocytes including CD4⁺ CD44⁺ memory T cells were stimulated with 4T1 supernatant for 6 d. Mean values of three different experiments with eight replicates of supernatants are shown. (G) BALB/c mice bearing 1.25 × 10⁵ 4T1 tumor cells s.c. (n = 10 mice per group) were treated i.p. with 300 µg anti-mouse IL-1R antibody or isotype control every second day beginning on day 0. (H) C57BL/6 mice were injected s.c. in the right flank with 2.5 × 10⁵ E0771 tumor cells (n = 15 mice per group). Mice were treated with 1 mg anakinra or PBS i. p. every day beginning on day 0. In A–F error bars represent the SEM, and P values by two-sided Student’s t test are shown. In G and H, statistical significance was analyzed by two-way ANOVA with correction for multiple testing; n.d., not detectable; n.s., not significant; rec., recombinant.