Anti human pd 1 clone nat105

The biopsies were fixed in formalin and paraffin-embedded. Paraffin blocks containing sufficient material were selected for immunohistochemical (IHC) staining with the following anti-human monoclonal Abs: eosinophil peroxidase EPX-mAb (clone MM 25-82.2, kindly provided by Mayo Clinic, Scottsdale, USA), GATA-3 (clone L50-823, Cell Marque, Rocklin, USA), T-Bet (clone EPR9302 RabMab, Abcam, Cambridge, UK), and desmin to confirm muscle indemnity (clone D33, Dako, Glostrup, Denmark). The sections were stained with anti-human PD-L1 (clone SP263, Ventana, Bend USA) and anti-human PD-1 (clone NAT105, Abcam, Cambridge, UK). All markers, except for PD-L1, were determined using standardized automated protocols for LEICA BOND MAX II. PD-L1 expression was determined using the Benchmark ULTRA (Roche, Basel, Switzerland). Sections were examined by optical microscopy (Olympus BX40 microscope, DP2-BSW software), and digitalized images were analyzed using ImageJ software (NIH).

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).