Anti cd8 antibody

IR780, HSA, zinc acetate, and sodium sulfide were purchased from Solarbio (Beijing, China). Fetal bovine serum (FBS) and Dulbecco’s modified Eagle medium (DMEM) were purchased from GIBCO (Grand Island, NY, USA). Mito-Tracker Green was purchased from Beyotime (Shanghai, China; #C1048). Annexin V-FITC Apoptosis Kit (BD, San Jose, CA, USA; #556557) was from Becton, Dickinson and Company (Franklin Lakes, NJ, USA). Anti-DFNA5/GSDME-N-terminal (Abcam, Cambridge, UK; #AB215191), anti-calreticulin antibody (Abcam, #ab92516), anti-high-mobility group box 1 (HMGB1) antibody (Abcam, #ab79823), anti-calreticulin antibody (Abcam, ab92516), goat anti-rabbit IgG H&L (Alexa Fluor 488) (Abcam, #ab150081), anti-CD8 antibody (Abcam, #ab217344), anti-FOXP3 (Abcam, #ab20034) were from Abcam. Cleaved caspase-3 (CST, Danvers, MA, USA; #9664T), cGAS (CST, #31659S), STING (CST, #50494S), and p-STING (CST, #50907) were from Cell Signaling Technology (Danvers, MA, USA). Atezolizumab (Selleck, Munich, Germany; #A2004) were from Selleck. Mouse IFN-β ELISA Kit (Beyotime, #PI568) and human IFN-β ELISA Kit (Beyotime, #PI572) were from Beyotime. SOSG9 (Meilunbio, Dalian, China; #MA0326) was from Meilunbio.

After deparaffinizing, the 2-μm glossopharyngeal tissue section was hydrated in gradient ethanol. For antigen retrieval, slides were immersed in 0.01 M sodium citrate buffer and heated for 30 min. After inactivating endogenous peroxidase with 3% hydrogen peroxide and blocking with goat serum albumin, the sections were incubated with anti-CD8 antibody (1:200, Abcam, United Kingdom) at 4°C for overnight. The slides were incubated with biotinylated secondary antibodies. Then the sections are incubated with peroxidase-streptavidin and stained with 3,3′-diaminobenzidine tetrahydrochloride (DAB) for 30 s. Finally, the nuclei were counterstained with hematoxylin. As a negative control, tissue sections were processed in parallel by incubating with PBS instead of primary antibody.
The semi-quantitative assessment of IHC staining was reviewed by three different pathologists and classified as negative, weak, medium, or strong. To determine the H score, antibody-stained tissue was scored by calculating the product of the percentage of cells staining for each intensity level and the intensity level (0, negative; 1+, weak; 2+, medium; 3+, strong). Then the individual intensity level scores are added together to calculate the H score (Sun et al., 2020 (link)).

The removed tumor tissues were fixed in 4% paraformaldehyde for 24 h. Then, the tissues were embedded in paraffin and sectioned. The paraffin-embedded tumor sections were dewaxed and treated with 3% H₂O₂ to deactivate endogenous peroxidase. After blocking with goat serum for 1 h, the sections were incubated with an anti-CD8antibody (1:500, Abcam, USA) at 4 °C overnight. After the sections were incubated with goat anti-mouse IgG H&L (HRP) at 37 °C for 30 min, they were stained with DAB and counterstained with hematoxylin. Images of stained sections were observed under a light microscope (Nikon, Japan).

All animal experiments were reviewed and approved by the Animal Care and Use Committee, Tohoku University School of Medicine and performed in accordance with institutional ethical guidelines. Six to eight-week-old, female, C57BL/6 mice and BALB/c nude mice were purchased from SLC Japan, Inc. (Shizuoka, Japan). The generation of OX40-knockout (OX40KO) mice was described previously [24 (link),25 (link)]. After anesthesia, each mouse was injected either with GL261 cells (1 × 10⁵ or 2 × 10⁵) or NSCL61 cells (1 × 10⁴) into the right striatum. A small number of NSCL61 cells were inoculated because of their aggressive growth potential. Details are described in the Additional file 1.
Mouse brain frozen sections were stained with either the anti-CD4 or the anti-CD8 antibody (Abcam, Cambridge, MA), followed by anti-rat IgG Zenon® Alexa 568 (Invitrogen, Carlsbad, CA). Details are in the Additional file 1.