Rmgm csf

Recombinant human (rh) M-CSF (a gift from Morinaga Milk Industry, Kanagawa, Japan), which is cross-reactive to murine cells^{31 (link)}, was used to expand and maintain self-renewing macrophages. Recombinant murine (rm) M-CSF (BioLegend) was also used in a selected experiment. Other cytokines used were as follows: rmGM-CSF (BioLegend), rmIL-4 (BioLegend), rmIL-13 (Peprotech), rmIL-33 (Peprotech), rmIL-34 (BioLegend), and rhRANKL (Oriental Yeast, Tokyo, Japan).

BMDMs and BMDCs were isolated from WT mice using well-established protocols (33 (link)). In brief, bone marrow cells were flushed from the femur and tibia using 60 mL of Hank’s Balanced Salt Solution, and then incubated with specific completed medium. BMDMs were incubated with Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 20% L929 cell culture medium, penicillin, streptomycin, 2-mercaptoethanol, glutamine, and 10% heat-inactivated fetal calf serum (FBS) (Gibco, Thermo Fisher Scientific). BMDCs were incubated with RPMI-1640 supplemented with 20 ng/ml rmGM-CSF (Biolegend #576302), penicillin, streptomycin, and 10% FBS. The medium was refreshed on day 3. Cells were harvested on day 7 for use in subsequent experiments.

BMDM cells from wild-type, STING1^−/− and hSTING knock-in mice were generated by culturing cells from the tibias and femurs in DMEM containing 10% FBS and 1% penicillin/streptomycin in the presence of rmGM-CSF (20 ng ml⁻¹; BioLegend) for 9 days at 37 °C and 5% CO₂.

Bone marrow was isolated from or WT C57BL/6J mice for generation of iDCs^{57 (link),58 (link)}. Following erythrocyte lysis, the bone marrow cells were resuspended in complete DC medium (RMPI 1640 + 25 mM HEPES + 10% FBS, 1% l-glutamine, 1% 200 mM sodium pyruvate, 1% MEM-NEAA, 1% penicillin–streptomycin, 0.5% sodium bicarbonate, 0.01% 55 mM 2-mercaptoethanol) supplemented with rmGM-CSF (50 ng/mL) and rmFlt3-L (200 ng/mL) (Biolegend, San Diego, CA). The culture medium was changed on day 9 of culture. After 16 days of culturing, non-adherent cells and loosely adherent cells were harvested and gently washed by DPBS for subsequent labeling experiments or for T-cell co-culture experiments. In the iDC-OT-I CD8+ T-cell interactions experiments, iDCs were activated overnight with class C ODN 2395 (InvivoGen, San Diego, CA) and then cultured with or without indicated antigen peptides at 37 °C for 30 min. Non-adherent cells in the culture supernatant and loosely adherent cells were harvested and gently washed with DPBS for subsequent experiments. iDCs were cultured with or without indicated antigen peptides or tumor lysates (tumor cell/DC ratio = 10:1) and with indicated T cells (T cell/iDC ratio 1:2) in a complete T-cell medium prior to endpoint analysis as described below.