Nkp46 clone 195314

Briefly, 4 μm tissue slices were prepared from cryopreserved tissues and mounted on glass slides. Immunohistochemistry was performed according to the manufacturer’s instructions using an automated staining facility (Bond Max, Leica, Wetzlar, Germany). The following mouse monoclonal antibodies were utilized: CD3ϵ (clone ab16669, 1:100, Abcam, Cambridge, UK; RRID: AB_443425), CD4 (clone 4B12, 1:150, Leica, Wetzlar, Germany; RRID: AB_563559), CD8 (clone 4B11, 1:100, Leica, Wetzlar, Germany; RRID: AB_442068), CD20 (clone L26, 1:100, Leica, Wetzlar, Germany; RRID: AB_442055), CD163 (clone EPR19518, 1:500, Abcam, Cambridge, UK; RRID: AB_2753196), NKp46 (clone 195314, 1:175, R&D Systems, Minneapolis, MN, USA; RRID: AB_2149153) and FoxP3 (clone 236A/E7, 1:100, Thermo Fisher Scientific, Waltham, MA, USA; RRID: AB_467555).

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).