70 m nylon mesh

At indicated time points after inoculation of Vĸ*MYC cells, spleens and BM were harvested, mashed through a 70 µm nylon mesh (Corning) into single-cell suspension and the red blood cells were lysed using ACK solution (Thermo Fisher Scientific). For detection of cell surface antigens, cells were first stained with Zombie-NIR Fixable Viability Kit (BioLegend) according to manufacturer’s instructions, blocked on ice with 5% normal rat serum in FACS buffer (PBS; 1% BSA, 0.01% NaN₃) and then incubated for 30 min on ice with fluorochrome-conjugated antibodies (listed in Supplementary Table 1). When necessary, controls for background staining such as isotype or FMO (fluorescence minus one) controls were applied. After washing with FACS buffer, cells were immediately acquired on FACS Canto II (Becton Dickinson) instrument operated by FACSDiva v 8.0 software. For data analysis Flow Jo v7.6.5 software (Tree Star) was used^{22 (link)}. Gating strategies used for identification of MM cells, myeloid cells, macrophages, DCs, monocytes/monocytic- and granulocytes/granulocytic MDSCs, and subgating strategies for Ly6C⁺ and Ly6G⁺ cells are presented in Supplementary Figs. 1–6, respectively.

Tumor tissue samples were washed with 3× PBS, minced, and treated with digestive solution composing of 95 % RPMI 1640, 2 % FBS, 1 % collagenase IV solution, 1 % DNase I, and 1 % Dispase II at 37 °C. Following this, single cells obtained by centrifugation (200 rpm for 1 h) were filtered using a 70-µm nylon mesh (Corning) and stained with anti-mouse CD274, CD45, CD3, CD8, CD4, Granzyme B (GZMB), IFNγ, TNF-α, mouse Fc block (2.4G2), anti-human CD274, and human Fc block (BD Biosciences or BioLegend) separately, as directed by the corresponding manufacturers. A Beckman FACS flow cytometer was used for data analysis.

Human brain tumor samples were obtained from the Neurosurgery Department at Tian Tan Hospital in Beijing, China, after patients with glioma provided informed consent. Specimens were reviewed by a neuropathologist to assess the grade and tumor type before assays were performed. Human glioma samples were transferred to a Petri dish, washed three times in phosphate-buffered saline (PBS, Gibco, USA), and cut into 1-mm³ pieces. Next, 0.05% trypsin (Gibco, USA) was added to the tumor specimens. Then, the single pieces were digested for 10 min, filtered with a 70-µm nylon mesh (Corning, USA) and centrifuged at 1000 rpm for 5 min. Single cells were resuspended in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, USA) containing 10% fetal bovine serum (FBS; Invitrogen, China) and 100 U/ml penicillin/streptomycin (Gibco, USA), seeded into 25-cm² culture flasks (Corning, USA) and incubated at 37 °C and 5% CO₂ in a humidified chamber. The media was changed 2–3 times a week. Primary cells at 70–80% confluence were passaged using 0.05% trypsin (Gibco, USA) and were used for different experiments after purification. This line of primary GBM cells was named BT-01 and identified by short tandem repeat (STR), moreover, the biological characteristics of the primary GBM cells can be found in our previous study [27 (link)].