2.1. Cell lines, conditioned media, reagents, and functional assays

The HL cell lines L428, KM‐H2, L1236, L540, and HDLM‐2 were provided by V. Diehl (Cologne). The DLBCL cell lines HBL‐1 and OCI‐LY3 were obtained from D. Krappmann (Munich) and the Deutsche Sammlung für Mikroorganismen und Zelllinien (DSMZ; Braunschweig, Germany), respectively. All cell lines were maintained in Roswell Park Memorial Institute (RPMI) 1640 supplemented with 2 mg·mL⁻¹ glutamine and 10% FCS. At regular intervals, we used a highly specific Ig PCR to test for cell identity. In addition, L428, L1236, KM‐H2, OCI‐Ly3, and HBL1 cells had been subjected recently to STR profiling by the DSMZ (Braunschweig, Germany).

For the production of lymphoma‐conditioned media (CM), cells were seeded at a density of 5 × 10⁵ cells per mL and incubated in complete RPMI 1640 medium for 2 days. Cell supernatants were centrifuged at 300 g for 10 min at 4 °C, sterile‐filtered, and stored at 4 °C for a maximum of 2 weeks.

Peripheral blood mononuclear cells (PBMCs) of healthy donors were isolated from fresh buffy coats by density‐gradient centrifugation over Biocoll separating solution (Biochrom, Berlin, Germany). CD14⁺ monocytes were obtained from PBMCs by magnetic cell separation using CD14 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. Purity of CD14⁺ cells after magnetic cell separation was determined by staining with specific markers and quantification by flow cytometry using a FACSCanto II (BD Biosciences, Franklin Lakes, NJ, USA).

Monocyte isolation and macrophage differentiation were performed as described previously (Menck et al., 2014). Monocytes were differentiated either in the presence of RPMI 1640 containing 10% (v/v) FCS, Pen/Strep, and 2.5 ng·mL⁻¹ recombinant M‐CSF (Immunotools, Friesoythe, Germany) or in lymphoma‐CM mixed in equal parts with complete RPMI1640. Briefly, M‐CSF or CM of the tested lymphoma cell lines was used to incubate primary human monocytes for 7 days in Teflon‐coated cell culture equipment. After 7 days, cells were counted, harvested, and transferred into cell culture dishes for additional functional analysis. Freshly isolated monocytes or Mφ were stained with cell‐specific antibodies or isotype controls. Expression was quantified by flow cytometry using a fluorescence‐activated cell sorting (FACS) Canto II and calculated by dividing the mean fluorescence intensity (MFI) by the MFI of the corresponding isotype control. For intracellular CD68 staining, cells were fixed and permeabilized using the Cytofix/Cytoperm Kit (BD BioSciences) following the manufacturer’s instructions.

For migration assays, a Boyden chamber with a 5‐µm porous membrane was used (Neuro Probe Inc., Gaithersburg, MD, USA). 5 × 10⁴ monocytes were seeded per well in 50 µL of RPMI 1640 medium and allowed to migrate for 2 h toward RPMI 1640 supplemented with 1% or 10% (v/v) FCS or lymphoma‐ CM. The cells that had migrated into the lower chamber were counted.

To analyze endocytosis, Mφ were allowed to adhere overnight on tissue culture dishes. Cells were washed twice with PBS and maintained in RPMI 1640 containing 10% (v/v) FCS and pen/strep. 10‐ and 70‐kDa FITC dextran (at 1 mg·mL⁻¹ for 2 h; Sigma Aldrich, Munich, Germany), respectively, or Gelatin Oregon Green™ 488 Conjugate (at 5 µg·mL⁻¹ for 30 min; Thermo Scientific, Waltham, MA, USA) were added to each plate and incubated at 37 °C or on ice. After incubation, cells were washed twice with PBS, harvested using trypsin/EDTA, and fixed in 2% formaldehyde solution in PBS. Uptake was analyzed by means of a FACS Canto II and calculated by dividing the MFI of the 37 °C sample by the corresponding MFI of the sample incubated on ice.

The activity of matrix metalloproteinase (MMP)‐9 was determined in cell culture supernatants by gelatin zymography. First, proteins were separated by SDS/PAGE. Gel mixes were used to prepare 8% acrylamide gels containing 1% gelatin and 5% acrylamide stacking gels. Fifteen microliters of cell culture supernatant has mixed with an equal volume of loading buffer (62.5 mm Tris/HCl, pH 6.8, 4% SDS, 25% glycerol, 0.01% bromophenol blue) and loaded onto the gel. Electrophoresis was performed under cooling using freezer packs. Afterward gels were incubated in wash buffer (50 mm Tris/HCl, pH 7.5, 5 mm CaCl₂, 2.5% Triton X‐100) for 1 h. Gels were transferred to renaturation buffer (2.5% Triton X‐100) and incubated 1 h. Afterward gels were covered in development buffer (50 mm Tris base, 150 mm NaCl, 10 mm CaCl₂) and incubated overnight at 37 °C with soft agitation. To visualize the gel degradation by MMP activity, the gels were then stained in staining buffer (0.5% Coomassie blue, 40% methanol, 10% acetic acid) for 1 h, followed by destaining in destaining buffer (40% methanol, 10% acetic acid) for 1.5 h. Gels were then fixed for 30 min in fixation buffer (5% glycerol, 30% methanol), placed between two cellophane membranes and dried overnight. Fixed and dried gels were scanned for image processing.

Recombinant M‐CSF (ImmunoTools) and IL‐13 (PeproTech, Hamburg, Germany) were used at 2.5 and 10 ng·mL⁻¹, respectively. Inhibitors: the pan‐JAK inhibitor pyridone‐6 was obtained from Merck (Darmstadt, Germany), while ruxolitinib, BKM120 (NVP‐BKM120), and ibrutinib ((PCI‐32765) were purchased from Absource Diagnostics GmbH (Munich, Germany).

FITC mouse anti‐CD1a (HI149), FITC mouse anti‐CD11b (LT11), FITC mouse anti CD11c (BU15), FITC mouse anti‐CD31 (MEM‐05), FITC mouse anti‐CD33 (HIM3‐4), FITC mouse anti‐CD40 (HI40a), FITC mouse anti‐CD44 (MEM‐85), FITC mouse anti‐CD54 (1H4), FITC mouse anti‐CD80 (MEM‐233), FITC mouse anti‐CD86 (BU63; ImmunoTools), FITC mouse anti‐CD14 (M5E2), FITC mouse anti‐HLA‐DR (G46‐6), phycoerythrin (PE) mouse anti‐CD68 (Y1/82A; BD Bioscience), allophycocyanin (APC) mouse anti‐CD163 (GHI/61), APC mouse anti‐CD206 (15‐2), APC mouse anti‐PDL1 (29E.2A3; BioLegend, San Diego, CA, USA) and isotype controls FITC mouse IgG1 (PPV‐06), FITC mouse IgG2b (PLRV219; ImmunoTools), FITC mouse IgG2a (G155‐178), PE mouse IgG2b (27‐35; BD BioScience), APC mouse IgG2b (MPC‐11; BioLegend) were used for flow cytometry.

Primary mouse anti‐CD30 (Ber‐H2) and mouse anti‐CD68 (KP1), anti‐Prox1, anti‐CD206 (D‐1; Santa Cruz Biotechnology Inc., Dallas, TX, USA), and secondary goat anti‐mouse horseradish peroxidase (HRP) polyclonal; Agilent, Santa Clara, CA, USA) were used for peroxidase and immunofluorescence staining.

ELISA kits for the detection of secreted M‐CSF and IL‐13 in cell culture supernatants were purchased from R&D Systems (Minneapolis, MN, USA). The sensitivity was 11.2 and 13.2 pg·mL⁻¹, respectively.

Conventional immunohistochemical staining of CD30, CD68, and CD206 was carried out according to standard procedures including endogen peroxidase blocking (10 min in methanol/H₂O₂) and antigen retrieval (3 min at 100 °C in 10 mm citrate buffer, pH 6.0) prior to incubation with the antibodies. Detection was performed using the ZytoChemPlus (HRP) Polymer Bulk Kit (Zytomed Systems GmbH, Berlin, Germany). The counterstaining was performed with Hemalaun after Meyer (1 : 4; Merck) for 3–5 min and rinsing with water for 10 min. After dehydration, the sections were covered with the mounting medium Pertex (Histolab Products AB; Göteborg, Sweden). Immunofluorescence detection of Prox1 (1 : 500, Reliatech, Wolfenbüttel, Germany), CD30, and CD68 (Dako/Agilent, Hamburg Germany) was performed on 12‐µm cryosections. Appropriate Alexa Fluor®‐conjugated secondary antibodies were used (1 : 200; Life Technologies, Eugene, OR, USA). Immunofluorescence staining of CD163 and CD206 in tonsil and HL tissue sections was performed using anti‐CD163 antibody (1 : 20, AM10011PU‐S rabbit, ACRIS/ OriGene Technologies GmbH, Herford, Germany) or the above‐mentioned anti‐CD206 antibody (1 : 100) in Antibody Diluent (Medac GmbH, Wedel, Germany), and subsequent staining with corresponding secondary antibody (Donkey anti‐mouse Alexa 555 and Donkey anti‐rabbit Alexa 488 [1 : 100, Invitrogen/Thermo Fisher Scientific GmbH, Dreieich, Germany)] and 4′,6‐diamidino‐2‐phenylindole (1 : 5000, Invitrogen/Thermo Fisher Scientific GmbH). For each staining step, 100 µL of the respective dilution pro object slide was used. The anti‐CD206 staining of TMAs was performed with the above‐mentioned anti‐CD206 antibody and Histofine anti‐Mouse Histofine anti‐mouse and anti‐rabbit (Medac GmbH) and stained with 3,3'‐diaminobenzidine (Dako/Agilent Germany). For tissue microarray (TMA) staining, 200 µL of each dilution was used.

Slides were scanned by a Hamamatsu Nanozoomer 2.0 RS slide scanner (Hamamatsu Photonics, Ammersee, Germany) with 40× magnification for the TMAs and a 20× magnification for the fluorescent‐labeled slides of HL and tonsils. Raw image data were saved in.ndpi format and handled by the software kit ndp.view 2 (Hamamatsu Photonics) to save details of the whole image in.jpeg format. Figures were made with Adobe Photoshop CS4 Extended (Adobe Systems Incorporated, San Jose, CA, USA). Chick chorioallantoic membrane (CAM) tissue slides were scanned with a 20× objective (UPlanSApo, NA 0.75) using the virtual slide microscope VS120‐L100 (Olympus, Hamburg, Germany) equipped with a VC‐50 camera.

The experiments and analysis were undertaken with the understanding and written consent of each subject. The study methodologies conformed to the standards set by the Declaration of Helsinki. The study methodologies were approved by the local ethics committee [Medical Faculty of the Georg‐August‐University Göttingen (No. 16/5/18An); Medical Faculty of the Christian‐Albrechts‐University Kiel, Germany (No. 447/10)].