Ab209775

RNEU_420-429 (PDSLRDLSVF), the immunodominant peptide of HER2/neu for MHC class I, and NP_118-126 (RPQASGVYM), a peptide derived from a nuclear protein as a control, were obtained from Hokkaido System Science (Sapporo, Japan). An APC-anti-mouse CD8a monoclonal antibody (mAb), PE-anti-mouse IFN-γ mAb, and Fixable Viability Stain 520 were obtained from BD Biosciences (Franklin Lakes, NJ, USA). A therapeutic anti-PD-1 mAb (clone: RMP1-14) and anti-CTLA-4 mAb (clone: 9H10) were obtained from BioXcell (Lebanon, NH, USA). IgG from rat serum, administered as a therapeutic control, was obtained from Sigma-Aldrich (St. Louis, MO, USA). The antibodies (Abs) used for immunostaining were as follows: anti-CD8 mAb (ab209775, 1:2000, Abcam, Cambridge, United Kingdom), anti-CD4 mAb (ab183685, 1:1000, Abcam), anti-FOXP3 Ab (polyclonal, NB100-39002, 1:800, Novus Biologicals, Centennial, CO, USA), and Histofine Simple Stain Mouse MAX-PO (Nichirei Biosciences, Tokyo, Japan).

Immunohistochemistry staining was performed by the Pathology Core at The Toronto Centre for Phenogenomics. Tissue sections were submitted to heat-induced epitope retrieval with citrate buffer (pH 6.0) or with Tris-EDTA buffer (pH 9.0) for 7 min, followed by the quenching of endogenous peroxidase with 0.3% hydrogen peroxide in methanol. Non-specific antibody binding was blocked with 2.5% normal horse or goat serum (Vector, Laboratories, Burlingame, CA, USA), followed by incubation for 1 h in rat anti-CD3 at a 1:150 dilution (ab11089, Abcam, Toronto, ON, Canada), rabbit anti-CD4 at a 1:500 dilution (ab183685, Abcam, Toronto, ON, Canada), rabbit anti-CD8 at a 1:1000 dilution (ab209775, Abcam, Toronto, ON Canada), or rat anti-B220 at a 1:200 dilution (14-0452-82, Invitrogen, Mississauga, ON Canada). After washes, the tissue sections were incubated for 30 min with an ImmPRESS HRP (horseradish Peroxidase) reagent anti-rabbit or anti-rat IgG polymer (Vector, Laboratories, Burlingame, CA, USA), followed by a DAB (3′,3′-Diaminobenzidine) reagent.

Paraffin-embedded brain slices were used for H&E staining and IHC analysis as previously described [18 (link)]. Briefly, 4 μm-thick brain slices were deparaffinized, rehydrated, endogenous peroxidase blocked, antigen-retrieved, blocked with 5% BSA, incubated with primary and corresponding secondary antibodies (Polink-1 HRP DAB Detection System, ZSGB-BIO, China), and the colorimetric end products were produced by applying diaminobenzidine tetrachloride. Primary antibodies were anti-Ki67 (1:100, ab16667, Abcam, UK), anti-CD3 (1:2000, ab237721, Abcam, UK), anti-CD4 (1:2000, ab183685, Abcam, UK), anti-CD8 (1:2000, ab209775, Abcam, UK), and anti-FoxP3 (1:100, #12,653, CST, USA). Whole brain images were obtained by scanning the brain sections at 200×magnification with an automatic slice scanning system-SV120 (Olympus, Japan). Statistical analysis used data from 6 slices from 6 mice brains per group. The data were expressed as mean number of positive cells per mm² microscopic field.

Following the MRI imaging, mice were anesthetized, and a cohort of mouse brains containing tumor were collected (Supplementary Material). Formalin-fixed paraffin embedded sections were stained with H&E to assess tumor extent and examine the ependymal and subependymal wall, and immunohistochemistry for beta-IV tubulin to assess ependymal cilia. 1:200 dilution Olig2 (ab109186, Abcam) and 1:100 dilution Ki67 (ab16667, Abcam) were used to confirm high-grade glioma (HGG) characteristics of the tumor, and 1:400 dilution GFAP (ab7260, Abcam), 1:100 dilution CD45 (ab10558, Abcam), 1:200 dilution CD163 (ab182422, Abcam), and 1:500 dilution CD8 (ab209775, Abcam) were used to examine the immune composition.

Genital tracts from the mice were removed following euthanasia and fixed at room temperature in 4% formaldehyde (VWR chemicals) and paraffin embedded.
Processing, sectioning and staining were done by the technical staff at BioSiteHisto (Finland). Four micrometer thick sections were collected on Superfrost Plus slides (Thermo Fischer) and stained with hematoxylin and eosin (H&E). Furthermore 4 µm thick sections were collected on TOMO adhesive coated slides for immunohistochemical (IHC) staining. Before IHC staining, epitope retrieval was performed as described previously.^{52 (link)} The IHC staining method was based on peroxidase and phosphatase reactions to detect CD8 and CD4 epitopes. Detection of CD8 was performed with a rabbit anti CD8 alpha monoclonal antibody (Abcam, ab209775), an HRP based detection system and a brown DAB chromogen. CD4 was detected with a rabbit-anti-CD4 monoclonal antibody (SinoBiological, 50134-R001) and an AP based detection system and a permanent red chromogen. The IHC slides were counterstained with Mayer Hematoxylin (Merck). Slides were digitalized as WSI in Mirax format with 3DHistech Panoramic MIDI scanner (3DHistech Ltd.). Slides were visualized and analyzed using Caseviewer version 2.3.

At the end of the experiment, excised MC38 tumor tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned with 4 µm thickness. After deparaffinization and rehydration, the sections were incubated overnight at 4 °C with primary antibodies for CD8 (Cat. ab209775, Abcam), CD4 (Cat. ab183685, Abcam), or FoxP3 (Cat. ab215206, Abcam). The tissue sections were incubated with the HRP-labeled secondary antibody for 30 min and counterstained with DAB. Finally, images from random fields were obtained using a Nikon fluorescence microscope (NI-U, NIKON). The expression levels of CD8, CD4, and FoxP3 were quantified by counting the numbers of positive particles under magnification.

The primary antibodies used in western blot analysis included PD‐L1 anti‐human (#13684, CST), PD‐L1 anti‐mouse (17952‐1‐AP, Proteintech), ALDH2 (15310‐1‐AP, Proteintech), Myc tag (#2278, CST), Flag tag (20543‐1‐AP, Proteintech), HA tag (51064‐2‐AP, Proteintech), SPOP (16750‐1‐AP, Proteintech), and GAPDH (60004‐1‐Ig, Proteintech). Antibodies used in IHC assay were anti‐mouse CD3 (ab16669, Abcam), anti‐mouse CD8 (ab209775, Abcam), anti‐mouse granzyme B (ab4059, Abcam), anti‐human CD3 (Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China), and anti‐human CD8 (Zhongshan Golden Bridge Biotechnology Co., Ltd.). The secondary antibodies used in immunofluorescence assay were AF488‐anti‐rabbit (#8878, CST) and AF594‐anti‐mouse (Biotech). MG‐132 (M8699) and chloroquine (C6628) were purchased from Sigma. CHX (HY12320) was purchased from MedChem Express.