Las af acquisition program

Sections of 4 μm of representative tumor cryosections of resection specimens (Table 1) and of B-cell lymphoma control tissue were fixed in acetone at −20°C for 10 min (for IHC: supplemented with 0.3% hydrogen peroxide (Sigma-Aldrich, St. Louis, MO, USA) to inactivate endogeneous peroxidase), followed by incubation in 10% normal goat serum (Dako, Glostrup, Denmark) in PBS buffer to block non-specific antibody binding.
Immunohistochemical expression of CD70 was assessed using the mouse monoclonal anti-CD70 2 F2 (IgG1, 0.16 μg/ml) antibody followed by a polyclonal goat anti-mouse/rabbit/rat IgG HRP-linker antibody conjugate (Brightvision, DPVO-110HRP; Immunologic, Duiven, the Netherlands) and DAB + Substrate Chromogen System (Dako) detection. All sections were examined with an Olympus BX41 microscope and Cell^B acquisition software (Olympus, Tokyo, Japan).
Immunofluorescent double-staining for CD3 and CD70 or CD3 and CD27 was performed with rabbit polyclonal anti-human CD3 (2.4 μg/ml; Dako), CD70 2 F2 and mouse monoclonal anti-human CD27 137B4 (IgG1, 1:200; Novocastra, Leica Microsystems, Wetzlar, Germany) followed by goat anti-rabbit Alexa 488 or goat anti-mouse IgG1 Alexa 546 (1:300; Invitrogen, Carlsbad, CA, USA). All sections were examined with a Leica DM5000 fluorescence microscope and LAS-AF acquisition program (Leica, Solms, Germany).

Tumor cell lines were labeled with CFSE (1 μM; Invitrogen) and incubated overnight to allow leakage of excess CFSE. IL-10–stimulated M2-like Macrophages were co-cultured with CFSE-labeled HOS-143b cells for two hours at 1:1 ratio. All cells were harvested from the culture by cell scraping and macrophages were labeled with APC-labeled anti-CD32 antibodies. Cell conjugate formation between macrophages and tumor cells was analyzed by flow cytometry, assessing the percentage of CD32⁺ macrophages acquiring high CFSE fluorescence from tumor cells.
For an indication of phagocytosis, after the cell conjugate formation assay, CD32⁺ macrophages which have acquired the fluorescent signal of CFSE⁺ tumor cells were sorted by flow cytometry in one experiment. The cells were stained with mouse anti-human HLA-DR (TAL.1B5; Dako, Glostrup, Denmark) followed by the Alexa-Fluor-594 goat anti-mouse IgG1 secondary antibody (Invitrogen) and embedded in Vectashield mounting medium containing DAPI (Vectorlabs, Burlingame, CA, USA). Cell conjugates were examined with a Leica DM5000 fluorescence microscope and LAS-AF acquisition program (Leica, Solms, Germany), detecting nuclei in blue, HLA-DR⁺ macrophages in red and CFSE⁺ tumor cells in green.