Anti cd4 clone 4b12

Formalin-fixed paraffin-embedded blocks from 172 of the 456 patients in our cohort were sectioned and stained for immunohistochemistry from one of our cohorts. Anti-CD8 (clone 4B11; Leica Biosystems, Newcastle, UK) and anti-CD4 (clone 4B12; Leica Biosystems) antibodies were detected using the Bond Polymer Refine Detection System (Leica Biosystems) according to the manufacturer’s instructions. CD8+ T cell and CD4+ T cell counts were determined avoiding areas of necrosis. In cases of heterogeneity, CD8+ T cell and CD4+ T cell counts were estimated at the tumor front within the area of deepest invasion. A minimum of three random fields were examined to assess both the tumor center and the intraepithelial compartment. In cases of heterogeneity, the count that best represented the entire section was assigned, as previously described [22 (link)].

We obtained a total of 1,149 glioma cases of the brain (619 GBM cases and 530 low-grade glioma [LGG] cases) with known mRNA expression data from TCGA database (https://gdc.cancer.gov/about-data/publications/pancanatlas and https://www.cbioportal.org/) [12 (link)]. Normal samples as well as tumor samples with missing data were excluded from analysis. The analysis was finally performed on 525 cases with both virtual histopathological slides and clinical data (from a total of 619 GBM samples). We present the raw data of our study in Supplementary Data 1.
Immunohistochemical staining was performed to evaluate the presence or absence of tumor-infiltrating lymphocytes (TILs) in GBM human tissue diagnosed at Hanyang University Guri Hospital. Haematoxylin and eosin (H and E)-stained slides were reviewed by at least two pathologists for each case (Min and Kim). In non-necrotic tissue with TILs, immunostaining for anti-CD3 (clone LN10 Leica Biosystems, Newcastle, UK), anti-CD8 (clone 4B11 Leica Biosystems, Newcastle, UK) and anti-CD4 (clone 4B12 Leica Biosystems was performed using the Dako Autostainer Universal Staining System (DakoCytomation, Carpinteria, CA, USA) and the ChemMate™ Dako EnVision™ Detection Kit.

Bone marrow samples obtained from six OA patients and six RA patients were examined histopathologically. BM samples were fixed in Oxford fixative (formaldehyde 40%, glacial acetic acid 2%, sodium chloride 8.7%, distilled water), routinely processed and embedded in paraffin wax. Sections 3 μm thick were cut and stained with haematoxylin and eosin. The following antibodies were then used for further staining: anti-CD8 (polyclonal Ab, dilution 1:50; Dako, Glostrup, Denmark), anti-CD4 (clone 4B12, dilution 1:10; Novocastra, now part of Leica Microsystems, Wetzlar, Germany) and anti-IL-17A (dilution 1:50; Santa Cruz). Staining was performed according to the manufacturer’s instructions. The EnVision Detection System (Dako Denmark A/S, Glostrup, Denmark) was used for detection. Positive controls were performed on human tonsils. Negative (isotype) controls were performed using ready-to-use FLEX Negative Control Mouse (cocktail of murine IgG1, IgG2a, IgG2b, IgG3 and IgM, code number IR750; Dako Denmark A/S). Samples were reviewed for expression of these proteins by a qualified histopathologist who was blinded to outcome. Appropriate cellular localization for immunostaining was membrane for CD8 and CD4 and cytoplasmatic for IL-17A. All photographs were taken using Olympus microscope cameras: DP72 Olympus BX63 and DP12 Olympus BX (Olympus, Tokyo, Japan).