Cd163

Macrophage purity, differentiation and Tie2 expression was assessed by flow cytometry (FACS Canto Flow Cytometer, BD Biosciences). Fluorochrome-labeled monoclonal antibodies against CD16 (clone 61D3, eBiosciences), CD64 (Biolegend), CD163, CD200R (both from BD Pharmingen) and Tie2 (R&D Systems), and equivalent concentrations of isotype control antibodies, were used. Before staining, Fc receptors were blocked with 10% human serum (Lonza). Data were analyzed with Flow Jo Flow Cytometry Analysis software (Tree Star). Values were expressed as the ratio of the geometric mean fluorescence intensity (geomean) of the marker of interest over that of the isotype control.

Tumor specimens with adjacent tissues, relevant internal organs and inguinal sentinel lymph nodes (SLN) were harvested and fixed in 4% neutral buffered formalin solution for paraffin sectioning and staining. Immunohistochemical test for in situ detection were performed with anti-MUC-1 (BD), CD49b/NK1.1 (Biolegend), anti-CD4, CD8, CD25, Foxp3, β-catenin, TERT (all abcam), and anti-CD44, CD133 (all Chemicon) monoclonal antibody severally. Imaging of thymic lobules for renewal hotspots were captured using Leica scanning confocal microscope. Thymic and tumor tissue/cells were stained with thymosin-α1(abcam)/-β4(Millipore) for immunofluorescence or with TCRαβ, TCRγδ, CD3, CD38, CD80, CD163, CD45RA (all BD) for positive subsets via FACS-Aria III cell sorting system. Successive subsets were incubated with 3D-CSC-subsets from in vivo tumor at a ratio of 120:1 (thymocyte: spheroid) in DMEM/F12 with 2% normal serum for over 72 hours of reactivity between renovated T-subsets and CSC pool.

Immunohistochemistry was conducted on deparaffinized and rehydrated slides with primary antibodies against the M1 markers CCR-7 (1:500, BD Pharmingen) and CD11c (1:400, BD Pharmingen) and the M2 marker CD163 (1:500, BD Pharmingen). The stained slides were imaged using a light microscope (Nikon).

After treatment, macrophages were collected for flow cytometry analysis. The cells were stained for macrophage surface markers to detect potential subtype alterations. The macrophage markers used were: CD68 (333809, BioLegend, San Diego, CA, USA) as a general macrophage marker, CD80 (305213, BioLegend, San Diego, CA, USA) as an M1 macrophage marker, and CD163 (555749, BD Biosciences, San Diego, CA, USA) and CD206 (321119, BioLegend, San Diego, CA, USA) as M2 macrophage markers. To exclude non-viable cells on this panel, 7-AAD marker (420403, BioLegend, San Diego, CA, USA) was used. Staining was done on ice with 30 min incubation time. At least 10,000 gated single cells were recorded. The instrument used was an LSR-II FACS from BD and the data analysis was done in FlowJo (FlowJo LLC, Ashland, OR, USA).

Using standardized flow cytometry protocols as described previously gmMφ and mMφ were phenotyped using antibodies against CD14, CD163 (both BD Biosciences, Franklin Lakes, New Jersey, USA) and MHC-II DR/DP (clone Q1514) [23] (link). FcγR expression was determined with antibodies against FcγRI (CD64, PE labeled, clone 10.1; Dako, Glostrup, Denmark), FcγRIII (CD16, PE labeled, clone DJ130c; Dako), clone IV.3 which preferentially binds to FcγRIIa (StemCell Technologies, Vancouver, Canada) and the FcγRIIb specific antibody 2B6 (Alexa488 labeled; MacroGenics, Rockville, Maryland, USA). Expression of unlabeled markers was visualized via a FITC labeled goat-anti-mouse secondary antibody. Cell fluorescence was measured on a FACS Calibur (BD) and analyzed using Flowjo software for the mean fluorescence intensity (MFI) and the proportion of positive cells relative to cells stained with the appropriate IgG isotypes.

Anti-human CD66bFITC, CD3, CD19, CD14, CD163,CD138, CD56, CD15 all PE conjugated, HLADRV450, CD45V500, CD16APC-H7, CD14PE-Cy7, CD38PE-Cy7, CD 206APC were obtained (BD Pharmingen, Stockholm, Sweden). CD34PE, CD235aPE, CD11bPerCp-eFlour710 (eBioscience, San Diego, CA, USA), CD192APC, and CX3CR1AlexaFlour647 were from BioLegend, San Diego, CA, USA. Human FcReceptor binding inhibitor, 7AAD, and Annexin V-Alexa647, were obtained from eBioscience, San Diego, CA, USA, Sigma–Aldrich, (St. Louis, MO, USA), and Molecular Probes, Eugene, OR, USA, respectively.