Earlier studies have recorded that selective delivery of protein antigens to

Earlier studies have recorded that selective delivery of protein antigens to cells expressing mannose receptor (MR) can result in enhanced immune system responses. December-205 or MR, and also have recently reported the info from the stage 1 trial of our MR-targeted oncofetal antigen, human being chorionic gonadotropin beta string (hCG).22 This vaccine, known as B11-hCG or CDX-1307, comprises hCG fused towards the MR-specific human being monoclonal antibody B11. distribution of the vaccine and the resulting immune responses. We found that GM-CSF upregulated the expression of MR and enhanced humoral but not Th1 responses to B11-hCG. Furthermore, poly-ICLC and CpG promoted the accumulation of B11-hCG-loaded DCs in the T-cell areas of draining lymph nodes, which correlated with strong Th1 immunity. Materials and CAY10505 methods Antibodies and reagents The anti-hMR antibody B11 was generated by immunizing human immunoglobulin transgenic mice with human mannose receptor. The monoclonal antibody (mAb) B11 binds human mannose receptor, but not mouse mannose receptor.11 The B11-hCG fusion protein was generated by genetically coupling hCG to the carboxyl terminus of the B11 heavy chain, and clinical grade material was manufactured from transfected Chinese hamster CAY10505 ovary cells.22,23 The labeling of B11-hCG with Alexa-647 was performed according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA, USA). Antibodies for staining of CD3 (145-2C11), CD4 (H129.19), CD8 (53-6.7), CD11c PRKD3 (HL3), MHC class II I-A/I-E (M5/114.15.2), F4/80 (BM8) and CD103 (2E7) were purchased from BD Biosciences (San Jose, CA, USA) or eBioscience (San Diego, CA, USA). hMR was stained with either B11 or 19.2 (BD Biosciences), mouse MR (mMR) with MR5D3, and DEC-205 with NLDC-145 (AbD Serotec, Raleigh, NC, USA and BMA Biomedicals, Augst, Switzerland). Mouse GM-CSF was from Peprotech (Rocky Hill, NJ, USA). Complete Freund’s adjuvant (CFA) was from Sigma-Aldrich (St. Louis, MO, USA). CpG (ODN1826) CAY10505 and polyinosinic-polycytidylic acid (poly-IC) were from InvivoGen (San Diego, CA, USA). Poly-ICLC (poly-IC stabilized with poly-lysine and carboxymethylcellulose) was supplied by Oncovir, Inc (Washington, DC, USA). Mice hMR-Tg mice on a C57BL/6 background were generated by BAC clone DNA microinjection and the transgene expression was directed by the native human promoter.19 Heterozygous hMR-Tg mice and age- and gender-matched wild-type (WT) mice between 6 and 15 weeks of age were used in all experiments. Mice were housed under specific pathogen-free conditions in our animal facilities and were treated and used in accordance with the guidelines established by the Institutional Animal Care and Use Committee at Celldex. Immunohistochemical (IHC) and immunofluorescence staining To visualize the location of the hCG+ cells in skin and lymphatic CAY10505 organs, hMR-Tg mice were injected subcutaneously (s.c.) at the tail base with 10 g of B11-hCG plus or minus adjuvant as indicated. Skin near the injection site (local skin) and draining lymph nodes (inguinal) were collected 24 h later. The organs were OCT-snap frozen, sectioned, fixed and stained with rabbit anti-hCG (Dako, USA, Carpinteria, CA, USA) after blocking Freceptors with -globulin. A Dako EnVision Kit was used to reveal the immuno-binding and hematoxylin for counterstain. To define T-cell and B-cell areas, consecutive sections of draining lymph nodes were stained with Texas red-labeled anti-CD3 and anti-B220 antibodies (BD Biosciences) as well as the fluorescent dye 4,6-diamidino-2-phenylindole for cellular nuclei. To examine the colocalization of hCG and hMR, local skin and draining lymph node sections were stained with rabbit anti-hCG and FITC-labeled mouse anti-hMR (clone 19.2; BD Biosciences), and followed with donkey anti-rabbit IgG-Cy3 (Jackson ImmunoResearch, West Grove, PA, USA) and Goat anti-FITC/Oregon green-Alexa488 (Invitrogen). For the colocalization of hCG and MHC class II, rabbit anti-hCG and biotinylated anti-mouse I-Ab (BD Biosciences) were used as primary antibodies, and followed by goat CAY10505 anti-rabbit IgG-Alexa488 and strepavidin-Cy3 (Jackson ImmunoResearch). Images were taken with a confocal microscope (Carl Zeiss, Cambridge, UK). MR regulation hMR-Tg mice and WT littermates were injected intraperitoneally (i.p.) with 1 ml of 3% thioglycolate on day 1, and s.c. with either 2 g of GM-CSF on days 2, 3 and 4, or 20 g of poly-ICLC or 25 g of CpG on day 4. Peritoneal exudate cells (PECs) and bone marrow (BM) had been collected on time 5. After BM was.