Ab245235

The IHC was performed as previously described [25 ]. The antibodies used for IHC were anti-CD27 (Abcam Cat# ab214043, 1:100), anti-CD8 (Abcam Cat# ab109228, 1:100), anti-CD19 (Abcam Cat# ab245235, 1:100), anti-PD-L1 (Abcam Cat# ab213480, 1:100), and anti-YY1 (Abcam Cat# ab109228, 1:250). The scores of the IHC results were blindly accessed by two pathologists [26 (link)]. Briefly, we defined the negative staining as score 0; weak staining (ex. light yellow) as score 1; moderate staining (ex. yellow-brown) as score 2; and strong staining (ex. brown) as score 3. The percentage of positively stained cells was defined as the proportion between 0 and 100%.

Dissected tumors from mice were fixed with 4% PFA in PBS for 24 h and then transferred to 30% sucrose in PBS for about 2 days until the tissue sank to the bottom of 15 ml Falcon tubes. Tumors were embedded in OCT compound, frozen on dry ice, and stored at −80 °C before cryosection. Ten micrometer thick sections were prepared and adhered to superfrost glass slides. After three washes with PBS, the tumor slices were incubated in 0.3 M glycine in PBS for 30 min and then blocked and permeabilized with IF buffer (5% donkey serum, 2% BSA, and 0.1% Triton X-100 in PBS) for 60 min at room temperature (RT). Tumors slices were incubated with anti-CD19 (abcam, Cat # ab245235, dilution 1:100, final concentration 4.6 µg/ml) and anti-CD68 (abcam, Cat # ab53444, dilution 1:300, final concentration 3.3 µg/ml) antibodies for 24 h at 4 °C. After three washes with PBS, the slices were incubated with donkey anti-rabbit IgG H&L Alexa Fluor 488 (Abcam, Cat # ab150073, dilution 1:500, final concentration 4 µg/ml) and donkey anti-rat IgG H&L Alexa Fluor 568 (Abcam, Cat # ab175475, dilution 1:500, final concentration 4 µg/ml) at RT for 2 h. After washing with PBS three times, slides were mounted with ProLong™ Diamond Antifade Mountant with DAPI (Invitrogen) and imaged using a Zeiss LSM 710 confocal microscope.

The hBMSCs were isolated from the bone marrows harvested in the pelvis of the healthy donors (15–85 years old) who underwent osteotomy for health reasons in Linyi People’s Hospital. In brief, under aseptic conditions, 10 mL of the bone marrow was extracted using a 20-mL syringe (containing 2000 IU heparin) and immediately mixed with heparin. The bone marrow was centrifuged at 1200 g for 10 min for the separation of adipose tissues. The bone marrow was then resuspended in 15 mL of DMEM and added into the centrifuge tube with the same volume of Ficoll-Paque™ Plus lymphocyte separation solution (at the density 1.077 g/mL), followed by centrifugation at 2000 g for 20 min. The supernatant containing nucleated cells was collected using a pipette and subsequently washed with phosphate buffer saline (PBS), followed by centrifugation at 1000 g for 8 min. Next, 10 μL of cell suspension was added into 490 μL of PBS. The cells were then seeded in culture flasks at a density of 1 × 10⁵ cells/flask and cultured in a 5-mL low-glucose medium at 37 °C in 5% CO₂ and saturated humidity. The relevant markers for hBMSCs (Abcam Inc., Cambridge, UK) CD90 (ab225), CD105 (ab227388), CD44 (ab25024), and CD73 (ab239246) as well as hemopoiesis markers (Abcam Inc., Cambridge, UK) CD19 (ab245235), CD34 (ab18224), CD45 (an27287), and HLA-DR (ab1182) were used in this study.

Pancreas tissue and cell pellets were fixed overnight at room temperature in 10% neutral-buffered formalin (KliniPath, Olen, Belgium). Next, samples were washed twice with PBS, dehydrated, and embedded in paraffin. Sections of 4 µm thickness were cut. Automated stainings were performed on the Leica Bond RX™. Primary antibodies used are: anti-EVI-1 (1/1000, C50E12, Cell Signaling Technology, Danvers, MA, USA), anti-cleaved caspase 3 (1/200, 9661, Cell Signaling Technology), anti-CD3 (1/50, A0452, Agilent), anti-Krt19 (1/100, TromaIII, Developmental Studies Hybridoma Bank, Iowa City, IA, USA), anti-F4/80 (1/100, APC clone BM8.1, Sigma-Aldrich), anti-CD142 (1/100, AF2419, Bio-techne, Minneapolis, MN, USA), anti-Krt19 (1/20, M0888, Agilent, Santa-Clara, CA, USA), anti-CD19 (1/500, ab245235, Abcam), anti-collagen IV (1/500, 1340-01, Southern Biotechnology Associates) Trichrome Masson’s staining was performed automated at the Pathology Department of UZ Brussel, Brussels, Belgium. DNA staining was performed with DAPI (Agilent) or Hoechst.

Multiplex immunohistochemistry (mIHC) was performed using a tyramide signal amplification (TSA) fluorescence staining kit (Servicebio, Woburn, MA, USA, G1226-100T) according to the manufacturer’s instructions. Briefly, sections were subjected to heat-induced epitope retrieval, deactivation of endogenous peroxidase, and incubated with a primary antibody for CD20 (Abcam, ab78237), pBTK (Bioss antibodies, bs-3069R), CD19 (Abcam, ab245235), or CD8 (Abcam, ab237709 for human tissues, ab217344 for mouse tissues) overnight at 4 °C. Secondary antibodies (Gene Tech, GK500710) were then used on the next day, followed by fluorophore-conjugated TSA buffer: iF488 and iF555. Heat-induced epitope retrieval and antibody incubation steps were repeated for a second primary antibody and TSA. Finally, the sections were stained with DAPI for 10 min at room temperature in the dark prior to coverslipping in the antifade mountant (Beyotime, P0131). Confocal images were photographed and investigated under a confocal microscope (LSM880, Zeiss, Oberkochen, Germany).

For histopathological analyses, lung fragments were fixed in 10% formaldehyde for seven days after collection. Samples were processed in alcohol and xylol using the PT05 TS tissue processor (LUPETEC, UK) and embedded in histological paraffin (Histosec®, Sigma). The 4 μm thick sections of tissues were performed using the microtome RM2125 RTS (Leica) and stained with hematoxylin and eosin.
Prior to initiating the IHC staining protocol, paraffin-embedded section tissue was deparaffinized and heat-mediated antigen retrieval with Tris/EDTA buffer pH = 9.0 was performed for 20 min at 60 °C. The endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 30 min. Slides were incubated with anti-mouse CD4 (Cat. # ab183685; 1:50; Abcam), anti-mouse CD8 (Cat. # ab217344; 1:200; Abcam) and anti-mouse CD19 (Cat. # ab245235; 1:200; Abcam) primary antibodies overnight at 4 °C. Next, the slides were incubated with MACH 1 Universal HRP-Polymer (Biocare Medical, USA) for 30 min at RT, according to the manufacturer’s recommendations. Finally, the slides were stained using 3,3′-diaminobenzidine (DAB) chromogen (Biocare Medical, USA) and counterstained with Mayer’s hematoxylin. The histopathological and immunohistochemical analyses were carried out by two independent pathologists.