Cd8 clone 4b11

Formalin-fixed biopsies were deparaffinized, rehydrated, and subjected to antigen retrieval using citrate pH 6.0 (for HbcAg), Tris-EDTA pH 9.0 (for CD4 and CD8), pepsin (for CD68), or commercial CC1 buffer Ventana (for PD-1 and PD-L1). Sections were then incubated with antibodies to the following: HBsAg (clone A10F1, 1:100, Cell Marque, Rocklin, CA), HbcAg (1:800, Abcam, Cambridge, UK), CD4 (clone 4B12, 1:200, Leica, Buffalo Grove, IL), CD8 (clone 4B11, 1:1000, Leica, Buffalo Grove, IL), CD68 (clone PG-M1, 1:300, Dako, Santa Clara, CA), PD-1 (clone NAT105, 1:200, Abcam, Cambridge, UK), PD-L1 (clone 28-8, 1:200, Abcam, Cambridge, UK). Expression of the markers was quantified using the Visiopharm software, by dividing the number of strongly positive cells by the total stained area. PD-L1 staining on hepatocytes was scored on a semi-quantitative scale: 1+ 25% positive, 2+ as intermediate, and 3+ diffuse staining.

The following mouse monoclonal antibodies were used to stain serial sections (4 µm) of cryopreserved PDA tissues and/or FFPE 3D bioprints: CD3ϵ (clone ab16669, 1:100, Abcam, UK; RRID: AB_443425), CD4 (clone 4B12, 1:150, Leica, Germany; RRID: AB_563559), CD8 (clone 4B11, 1:100, Leica, Germany; RRID: AB_442068), CD20 (clone L26, 1:100,Leica, Germany; RRID: AB_442055), CD163 (clone EPR19518, 1:500, Abcam, UK; RRID: AB_2753196), NKp46 (clone 195314, 1:175, R&D Systems, USA; RRID: AB_2149153), FoxP3 (clone 236A/E7, 1:100, Thermo Fisher Scientific, Germany; RRID: AB_467555), IL9 (clone EPR23484-151, 1:100, Abcam, UK), IL18 (clone EPR19954-188, 1:100, Abcam, UK), Granzyme B (clone 23H8L20, 1:200, Thermo Fisher Scientific, USA), Ki67 (clone MIB-1, 1:200, Dako, USA), LCK (clone 3A5, 1:50, Santa Cruz Biotechnology, USA). The complete staining procedure was carried out on a fully automated staining system (Bond-Max, Leica, Germany).

FFPE blocks were obtained from the Pathology Department of Tokyo Metropolitan Bokutoh Hospital. Immunohistochemistry was performed using the Ventana BenchMark automated immunostainer (Ventana Medical Systems, Tucson, AZ, USA) with labeled streptavidin–biotin and visualized with 3,3′-diaminobenzidine. The primary antibodies used were anti-CD3 (clone LN10, Leica), -CD4 (clone SP35, Ventana), -CD8 (clone 4B11, Leica), -CD45RO (clone UCHL-1, Ventana), -FOXP3 (clone 236A/E7, Abcam), -CD20 (clone L26, Leica), -NKp46 (clone #195314, R&D), -CD68 (clone Kp-1, Dako), -CD163 (clone 10D6, Leica), -CD204 (clone SRA-E5, Transgenic), -Ki-67 (clone MIB-1, Dako), -PD-L1 (clone E1L3N, Cell Signaling), -MLH1 (clone ES05, Leica), -MSH2 (clone FE11, Dako), -MSH6 (clone Polyclonal (Rabbit), GeneTex) and -PMS2 (clone M0R4G, Leica). EBV-encoded small RNA in situ hybridization (EBER-ISH) was performed on paraffin sections using a fluorescein isothiocyanate (FITC)-labeled peptide nucleic acid probe (Y5200; Dako, Glostrup, Denmark) and anti-FITC antibody (V0403, Dako). Slides were digitized with a Nanozoomer 2.0-HT virtual slide scanner (Hamamatsu Photonics, Hamamatsu, Japan) and observed in the NDP.view2 software (Hamamatsu Photonics). The density of immune cells was analyzed by Tissue Studio 2.0 software (Definiens, Munich, Germany).