Immune stimulatory monoclonal antibodies are currently evaluated as anti tumor agents.

Immune stimulatory monoclonal antibodies are currently evaluated as anti tumor agents. Rabbit polyclonal to ECE2. bone marrow chimeric mice were used to study the mechanism by which agonistic anti-CD40 mediates its effects (purchased from Jackson Laboratories Bar Harbor USA) and mice (a kind gift from Robert Mumford NCI) were bred at NCI/Frederick. Bone marrow chimeric mice were generated as previously described (27). Bone marrow chimerism was confirmed 4 weeks after bone marrow transplant and was above 80%. EL4 and B16 GM-CSF cells were a kind gift of Dr. Drew Pardoll (The Johns Hopkins University Baltimore USA) and previously used (27). 4T1 cells were kindly provided by Christopher A. Klebanoff (National Cancer Institute Bethesda USA). RIL-175 hepatocellular carcinoma cell line was obtained from Dr. Lars Zender (University Hospital of Tübingen Germany) and used recently (13 39 All tumor cell lines used were tested negative for using MycoAlert Plus kit (Lonza USA) routinely. Last test was performed on December 2014. Mice were injected subcutaneously in the flank with 1×106 AMG-8718 tumor cells. Tumor size was measured twice a week. Metastatic tumors were established in the liver by intrasplenic injection of 3×105 EL4 cells (28). Mice received antibody treatment 3 weeks after tumor cell inoculation into the spleen. All mice were handled fed and housed in accordance with the U.S. Department of Health and Human Services institutional guidelines. antibody treatment Tumor-free littermates or mice bearing subcutaneous tumors between 10 and 15 millimeters maximum diameter were inoculated intra-peritoneally with 100 μg of rat anti-mouse agonist CD40 antibody (clone FGK-45 BioXCell USA) or irrelevant rat IgG2a (2A3 BioXCell USA). Mice were sacrificed 24 hours after injection. Alanine/aspartate aminotransferase (ALT/AST) levels were determined in mouse sera by biochemistry analysis in the Department of Laboratory Medicine (NCI). Serum TNF-α levels were quantified by ELISA following manufacturer’s instructions (eBioscience USA). Hematoxilin-eosin stained liver tissues analyzed by a pathologist (D.K.) in a blinded fashion. Flow cytometry analysis Liver mononuclear cells were obtained as previously described (13). Mouse AMG-8718 cell samples were stained using antibodies from BD Biosciences and eBioscience (available upon request). When indicated tumor-induced hepatic myeloid cells were isolated using CD11b beads followed by MACS separation (Miltenyi Biotec AMG-8718 USA). Purity after enrichment was above 90%. Flow cytometry was performed on BD FACS Calibur or LSRII using CellQuest Pro or FACS Diva acquisition software respectively (Becton Dickinson USA). Data were analyzed using FlowJo software (Tree Star USA). Functional assays (29). DCFDA expression was quantified on gated mouse CD11b+Gr-1+ cells from liver mononuclear cells 3 hours after injection of 100 μg of either isotype or anti-mouse CD40 antibody. In another setting DCFDA expression was determined on gated human CD14+HLA-DRhigh and CD14+HLA-DRlow cells after incubation of healthy donor peripheral blood mononuclear cells in the presence or absence of 0.1 μg/ml megaCD40L (Enzo Life Sciences USA) for 2 hours. For arginase activity and TNF-α determination hepatic CD11b+ cells were isolated from AMG-8718 TB mice and cultured overnight alone or in the presence of 0.1 μg anti-mouse CD40 antibody. Supernatants were collected and TNF-α was quantified by ELISA following manufacturer’s instructions (eBioscience USA). Arginase activity in cell lysates was determined as described (30). For OVA cross-presentation 1×105 CD11b+ cells were cultured for 24 hours alone or in the presence of 0.1 μg of rat anti-mouse CD40 antibody. Cells were washed twice with PBS OT-I CD8+ T cells were MACS-sorted using mouse CD8+ T cell isolation kit (Miltenyi Biotec USA) added to the culture in a 1:1 ratio and stimulated with 0.1 μg/ml OVA-derived SIINFEKL peptide overnight. IFN-γ production by OT-I CD8+ T cells was determined by intracellular staining. Determination of hepatocyte cytotoxicity by hepatic CD11b+ cells Luciferase -expressing RIL-175 hepatoma target cells were cultured at.