Transfection of imDCs with short hairpin RNA (shRNA) targeting CCR7

The sequences of the shRNA targeting CCR7 and the control sequences were as follows: shRNA-CCR7, 5′-TGGATCTTTGGTGCCTACCTGTGTA-3′; control shRNA, 5′-TTCTCCGAACGTGTCACGTAA-3′. shRNA-CCR7 and control shRNA was inserted into a pHBAd-U6-GFP backbone, which was packaged into an adenovirus. The 293A cells were transfected with the shCCR7 plasmid, and the adenovirus was harvested. The plasmid, shRNA and adenovirus were all supplied by Hangzhou Hibio Technology Co., Ltd. As control, an empty vector was used, and the infection efficiency was assessed by western blot analysis (Fig. S2). Then, 1×10⁶ imDCs were suspended in 1 ml adenoviral supernatant (MOI=50) with 1% FBS (Invitrogen; Thermo Fisher Scientific, Inc.), 10 ng/ml GM-CSF and 10 ng/ml IL-4 at 37°C for 72 h. Subsequently, cells were centrifuged at 300 × g at 37°C for 2 h. After infection, imDCs were washed twice in PBS and incubated with RPMI-1640 medium containing 10% fetal calf serum (FCS; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10 ng/ml GM-CSF and 10 ng/ml IL-4 at 37°C with 5% CO₂. The cells were harvested for injection 48 h after infection.