Anti human cd8 clone c8 144b

Multiplexed IF was performed on a total of 45 FFPE samples (29 pre-BCG tumor and 16 post-BCG specimens) with the Opal system and images were acquired using the Vectra 3.0 Automated Quantitative Pathology Imaging System (Perkin Elmer) with 4′,6-diamidino-2-phenylindole (DAPI) as the nuclear marker as previously described (26 (link)). The antibodies used are anti-Human CD4 (clone EPR6855; Abcam), anti-Human CD8 (clone C8/144B; DAKO), anti-Human FOXP3 (clone 236A/E7; Abcam), and anti-Human PD-1 (clone NAT105; Abcam). From these 45 FFPE samples, another single-plexed staining of PD-L1, anti-human PD-L1 antibody (clone 22C3; DAKO), was separately performed on consecutive slide of 12 samples (six pairs of matched pre- and post-BCG tumor from non-responders). Full tissue sections were used for all samples. For specimens smaller than 1cmx0.5cm, whole tissue was imaged and quantified; whereas for specimens larger than 1cmx0.5cm, at least 10 areas of 2 mm x 3 mm with high infiltration of immune cells were imaged and quantified. Quantification was done by ImageJ and the mean was calculated as the cell density (number/mm²) for each patient sample, while area with PD-L1 positive staining was quantified and the mean was reported as PD-L1+area/mm². The median value of density for each immune subsets was used as the cutoff point to dichotomize the patients into two groups (low versus high).

In formalin-fixed, paraffin embedded tissues, the EnVision system (Dako) with anti-Human CD4 (Clone 4B12, Dako), anti-Human CD8 (Clone C8/144B, Dako), anti-Human CD14 (Clone TÜK4, Dako), anti-Human CD56 (Clone 123C3), and anti-Human CD68 (Clone PG-M1, Dako), was used. Briefly, the paraffin sections were deparaffinized and rehydrated in xylene and graded alcohols. After blocking with peroxidase in ChemMate peroxidase-blocking solution (Dako), the slides were incubated with the primary antibodies. After application of the peroxidase-labeled polymer, the slides were incubated with the diaminobenzidine substrate chromogen solution, counterstained with hematoxylin, washed again, dehydrated, and mounted. The immunohistochemical slides were observed using a Zeiss Axio Imager M2 light microscope equipped with a Zeiss Axio Cam HRc Camera to capture images of the placenta. Ten photos were collected per slide, with 40X objective lens. Subsequently, each photograph was analyzed and the number of cells counted for each photo. The number of positive cells was calculated and analyzed using the Image J software (Image J 1.46r Wayne Rasband National Institutes of Health, USA, https://imagej.nih.gov/ij/index.html).

p16 IHC on formalin-fixed, paraffin-embedded (FFPE) tumor tissue was performed as part of the routine diagnostics process (Supplementary Table 1). Additional IHC was performed on FFPE tissue using standard protocols for the automated platforms Dako PT Link for Heat Induced Epitope Retrieval and Dako Autostainer 48S Link (Agilent, Santa Clara, CA, USA) with staining of tumor-infiltrating lymphocytes using anti-CD8 antibodies (Anti-Human CD8, Clone C8, 144B, Dako, concentration: 1:100, Agilent, Santa Clara, CA, USA). An unsuccessful immunohistochemical approach for HPV16 E2 and E7 was performed with following antibodies [Anti-HPV16 E2, NBP2-53115, NovusBio (no literature), Centennial, CO, USA; Anti-HPV16 E7, ab20191, Abcam, Cambridge, UK (32 ); Anti-HPV16 E7 (716–325), sc-51951, Santa Cruz Biotechnology, Dallas, TX, USA (33 (link))]. Different concentrations and antigen retrieval techniques revealed still unspecific staining.
Frequency and density of CD8 positive T cells were determined as previously described (34 (link), 35 (link)). One case was excluded in the further IHC analysis due to non-specific IHC staining.