library_construction_protocol: Single cell suspension of glioma-005 cell line was stereotactically injected into the hippocampus of 8- to 12-week-old C57BL/6J mice. 7 days after tumor implantation, mice were randomly divided into groups and injected with antibodies αCTLA-4 (Syrian hamster clone 9H10), Isotype control antibodies rat IgG2a (clone LTF-2) and depletion antibody aCD4 (clone GK1.5) were administered intraperitoneally (i.p.) . All mice were perfused with ice-cold PBS after euthanasia to reduce the influence of peripheral blood derived cells. All the brains were collected in ice-cold PBS, and then mechanically and enzymatically dissociated using a papain-based neural tissue dissociation kit P (Miltenyi Biotec) supplemented with 0.1% type I collagenase (Thermo Fisher Scientific) in a C-tube (Miltenyi Biotec), and then dissociated with a gentleMACS Qcto Dissociator, followed by filtration with 70 µm cell strainers. Filtered cells were incubated with ACK lysis buffer (Invitrogen) to lyse red blood cells. For immune cell suspension, filtered cells were resuspended in 5 mL 37% Percoll, and slowly added beneath the 70% Percoll, and then centrifuged with brakes off. The immune cells were isolated from the interface of 37% and 70% Percoll and transferred into excessive RPMI 1640 medium containing 10% fetal bovine serum.Microglia (CD45intCX3CR1+), myeloid cells (CD45hiCD11b+), T cells (CD45hiCD3+) and all remaining immune cells (CD45hiCD11b-CD3-) were sorted from normal brain and 005 glioma-bearing brains treated with IgG, αCTLA-4, or αCTLA-4 combined with CD4 depletion. Cells from each treatment group were pooled at equal ratios and then subjected to scRNA-seq. Sorted cells were partitioned into an emulsion of nanoliter-sized droplets using a 10x Genomics Chromium Single Cell Controller and RNA sequencing libraries were constructed using the Chromium Single Cell 3′ Library & Gel Bead Kit v2 (10X Genomics, Cat# PN-120237) 10x Chromium Single cell 3'