Supplementary Figure 2: Characterization in vivo of DCs expressing IL-6 (Thy1.1).

(a) Il6^RD/wt mice were crossed to CD11c-Cre and R26-Stop^flox/flox-YFP mice to generate compound heterozygous mice with DC conditional expression of an IL-6 reporter allele and YFP. In order to visualize large amounts of IL-6 producing DCs ex vivo, we injected Flt3L producing melanoma cells s.c. to expand DCs in vivo and 6 days later, treated the animals with LPS (3 mg/kg LPS [E. coli 0111:B4]) i.p. to stimulate IL-6 production. Two days after LPS injection, lymph nodes (LN) and spleen (SPL) were prepared and stained for Thy1.1 to visualize IL-6⁺ DCs by flow cytometry directly ex vivo. In order to analyze whether Thy1.1 (IL-6)⁺ DCs segregate into a specific DC subset, the indicated surface molecules were co-stained. (b) IL-6 Reporter mice allow for IL-6 conditional deletion of DCs in vivo. CD11c-Cre x Il6^RD/wt x R26-Stop^flox/flox-YFP mice were treated with Flt3L producing melanoma cells and LPS. The mice were then assigned to treatment with either Isotype (mouse IgG2a, C1.18.4) or anti-Thy1.1 (19E12) antibody treatment in order to deplete IL-6⁺ DCs. One day later, lymph nodes (LN) and spleen (SPL) were prepared and stained for Thy1.1 (OX-7) to visualize IL-6⁺ DCs by flow cytometry directly ex vivo. CD11c-Cre x R26-Stop^flox/flox-YFP x Il6^wt/wt mice, which were treated identically as the DC conditional IL-6 reporter mice, are shown as a "negative" staining control for Thy1.1 (left).