Female Wistar Furth rats were purchased from Charles River Laboratories (Wilmington, MA). DC-SIGN knockout (KO) mice were generously provided by the Consortium for Functional Glycomics (CFG, The Scripps Research Institute, La Jolla, CA). All animals were maintained under specific pathogen-free conditions. Animal care and experiments were conducted according to institutional guidelines of the Rockefeller University and Memorial Sloan-Kettering Cancer Center.
Hybridoma, Chinese hamster ovary (CHO), and 293TAg cells were cultured in DMEM (GIBCO Invitrogen, catalog number 11995) with 7 % FBS (Sigma) or 5 % Ultra-Low IgG FBS (GIBCO Invitrogen) supplemented with 1× solutions of 2-mercaptoethanol (GIBCO Invitrogen), Antibiotic-Antimycotic (GIBCO Invitrogen), and Non-Essential Amino Acids (GIBCO Invitrogen).
We purchased anti-rat IgG isotypes, anti-mouse IgG isotypes, and anti-rat IgM conjugated with HRP, PE, or PE/Cy5.5 from Southern Biotech (Birmingham, AL), and streptavidin conjugated with PE, APC, or Alexa fluorochromes from Invitrogen (Carlsbad, CA) and BD Biosciences (San Jose, CA). PE- or biotin-conjugated anti-mouse DC-SIGN MAbs, 5H10 and LWC06, were purchased or kindly provided by eBioscience (San Diego, CA). Rabbit polyclonal antibodies against the C-terminal 13-aa peptide of mouse SIGN-R1 (PAb-R1C13) and the16-aa peptide in the carbohydrate recognition domain (CRD) of mouse SIGN-R3 (PAb-R3CRD16) were described previously (Kang et al., 2003
; Kang et al., 2004
). Similarly, a rabbit polyclonal antibody against the 14-aa peptide (NH2–GKRQLRPLDEELLT-COOH) in the cytosolic domain of mouse DC-SIGN (PAb-DSCYT14) were generated by Invitrogen, as previously described (Kang et al., 2003
; Kang et al., 2004
2.4. Construction of vectors and expression of proteins
CHO cells (CHO-S cells, GIBCO Invitrogen) stably expressing DC-SIGN, SIGN-R1, and SIGN-R3 were described previously (Kang et al., 2003
; Zhang et al., 2008b
). CHO/DCIR2 cells were generated as follows. The cDNAs encoding the open reading frames (ORFs) of mouse DCIR2 were cloned from a mouse splenic cDNA library (Park et al., 1996
) by PCR, sequenced, and inserted into the pCMV mammalian expression vector (Clontech). Then, the stable CHO cells were generated by transfection with pCMV-DCIR2 by Lipofectamine™ 2000 reagent (Invitrogen) followed by selection of G418 (1.5 mg/ml) in DMEM culture medium.
Soluble human IgG1 Fc (S.hIgG1Fc) fusion protein of mouse DC-SIGN extracellular domain (ECD) was generated similarly to a method described previously (Galustian et al., 2004
). In brief, the synthetic DNA encoding mouse DC-SIGN ECD (GenBank accession number FJ168685) was generated by PCR, sequenced, and inserted into the pCMV mammalian expression vector carrying a soluble hIgG1 Fc fusion cassette. Stable CHO cells (CHO/ S.hIgG1Fc.DC-SIGN.ECD) were generated as described above. Then, soluble Fc-tagged DC-SIGN ECD protein was purified from culture supernatants of stable CHO cells by Protein A affinity column as described (Galustian et al., 2004
The His-tagged C-type lectin domains of mouse DC-SIGN (His.DC-SIGN.CRD; residues 98 to 239) and SIGN-R3 (His.SIGN-R3.CRD; residues 95 to 238) were cloned into pET22b(+) (Novagen). The identities of the cloned genes were verified by plasmid sequencing. The proteins were expressed in E. coli strain BL21(DE3) by inducing with 0.6–0.8 mM IPTG at 37 °C for 3 to 4 hrs. Both proteins were expressed in insoluble inclusion bodies. The cells were lysed by sonication and the inclusion bodies were washed for 3 times in a buffer containing 50 mM Tris, 100 mM NaCl, 5 mM EDTA, and 0.5% Triton-X100 at pH 8.0. Purified inclusion bodies were resuspended in water (~5 ml per 4 liter of culture) and dissolved in 7 M guanidine hydrochloride. The proteins were refolded by quick dilution into a refolding buffer containing 0.1 M Tris, 0.4 M arginine hydrochloride, 5 mM reduced glutathione, and 1 mM of oxidized glutathione at pH 8.3. About 100 to 200 mgs of denatured protein were diluted into 1 liter of refolding buffer. Refolded proteins were concentrated and purified by gel filtration chromatography on a Superdex200 (2.6×60) column (GE Healthcare) following elution with 20 mM Tris, 150 mM NaCl buffer at pH 7.5. The proteins were further purified by Ni affinity chromatography followed by gel filtration chromatography to remove the imidazole from the purified proteins. All the proteins purified were monomeric as analyzed by gel filtration chromatography.
2.5. Animal immunization and monoclonal antibody production
Wistar Furth rats or DC-SIGN KO mice at 4~6 wks of age were immunized subcutaneously (s.c.) with 50 µg of a protein mixture composed of S.hIgG1Fc.DC-SIGN. ECD and His.DC-SIGN.CRD in equal amounts following emulsification with TiterMax®
adjuvant (TiterMax USA, Inc., Norcross, GA) according to the manufacturer’s instruction. Rats were immunized 11 times and DC-SIGN KO mice were 6 times at monthly intervals on average. In addition, rats were boosted s.c. once with a cell lysate of 1×106
splenic low density cells enriched for DCs by floating in 30 % BSA (Inaba et al., 1998
), prepared by repeated freezing (−80 °C) and thawing (37 °C) in PBS, between the 5th and the 6th immunizations. Seven days after each immunization, sera were collected and screened by Western blot and ELISA as described below to determine which animal had the highest anti-DC-SIGN specific responses.
Rats, four months after the last round of immunization, and DC-SIGN KO mice, two months after the last round of immunization, were selected to receive intraperitoneally a final boost of 50 µg S.hIgG1Fc.DC-SIGN.ECD in the absence of adjuvant. After 5 days, spleen cells were used for hybridoma fusion at the Monoclonal Antibody Core Facility of the Rockefeller University and Memorial Sloan-Kettering Cancer Center. In brief, murine myelomas P3X63Ag8.653 (for rats) or SP2/0-Ag14 (for mice) cells (ATCC, Manassas, VA) were fused with spleen cells from the donor animal at a 1:4 ratio in 50 % polyethylene glycol (PEG; EM Science; Germany). Stable hybrids were selected by growth in Hybridoma-SFM (GIBCO Invitrogen) medium containing 15 % FBS, hypoxanthine, aminopterin and thymidine, according to standard protocols, and distributed into 96-well plates. Supernatants from the hybridoma cultures were screened by ELISA as described below.
Hybridoma cultures with supernatants showing antibody activity against mouse DC-SIGN were expanded and cloned twice by limiting dilution. Some hybridomas were grown in DMEM media containing 5 % ultra low IgG FBS. Culture supernatants were collected and the MAbs were purified with protein A/G (Pierce, Rockford, IL) or Protein G (GE Healthcare, Piscataway, NJ), according to manufacturer’s instructions. Purified MAbs were labeled with Alexa 488, Alexa 647 (Molecular Probes, Invitrogen) or EZ-Link Biotin (Pierce, Thermo Scientific) reagents following manufacturer’s instructions.
2.6. Detection of rat and mouse anti-mouse DC-SIGN MAb by ELISA
For the screening of hybridomas, the purified His.DC-SIGN.CRD protein was used for ELISA as described (Park et al., 2000
; Cheong et al., 2007
). In brief, ELISA plates (Microtest™ 96-Well ELISA Plate, BD Falcon, Bedford, MA) were coated overnight with 50 µl of 2 µg/ml of His.DC-SIGN.CRD (or His.SIGN-R3.CRD for a control) in PBS. Plates were then washed with PBS with 0.1 % Tween 20 (PBS-T) and blocked with 5 % normal goat serum (Sigma-Aldrich) in PBS-T (blocking solution) for 1 hr at 37 °C. Serial dilutions of the sera in blocking solution or undiluted hybridoma supernatants were incubated for 1 hr at 37 °C and were visualized with anti-rat or mouse IgG heavy (γ) chain conjugated to HRP (Southern Biotech), followed by colorimetric assay using OPD (o-phenylenediamine dihydrochloride; Sigma-Aldrich) in CPB solution, i.e. 10 mg OPD tablet in 25 ml CPB buffer (25 mM citric acid, 50 mM Na2
) with 10 µl of 30 % H2
solution, or TMB One Component HRP microwell substrate (BioFX Laboratories, Owings Mills, MD). Optical density (OD) at 450 nm was measured using an Opsys MR microplate reader (Thermo Labsystems, Chantilly, VA). The rat and mouse Ig isotypes of MAb heavy and light chains were determined by the ELISA using MAb supernatant as primary Ab and rat or mouse isotype specific HRP-conjugated Abs (Southern Biotech) as secondary Ab.
2.7. SDS-PAGE and Western blot analysis
Cell-lines expressing various C-type lectins were lysed with RIPA lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 8.0, 1 % NP-40, 0.5 % desoxycholate, 0.1 % SDS) including protease inhibitor cocktail (Sigma-Aldrich) or directly in sample buffers (Laemmli sample buffer without β-mercaptoethanol, Bio-Rad Laboratories; protein loading buffer blue with β-mercaptoethanol, National Diagnostics). Cell lysates, mixed with sample buffers and boiled for 5 min, were separated on 10 %, 12 %, or 15 % SDS-PAGE and blotted onto Hybond™-P polyvinylidine difluoride (PVDF) membrane (GE Healthcare, Piscataway, NJ). Blotted membranes were blocked with 10 % non-fat dry milk in PBS-T, incubated with serum/supernatant/antibody samples, detected by anti-rat, anti-mouse, or anti-rabbit IgG secondary antibodies conjugated with HRP (Southern Biotech, Birmingham, AL), and visualized with ECL Plus™ reagents (GE Healthcare).
2.8. Immunolabeling by flow cytometry and immunocytochemistry
After detaching with 1 mM EDTA in PBS for 10 min, stable CHO cells were incubated with primary Abs for 30 min at 4 °C. Cells were washed, detected by fluorochrome-conjugated secondary Abs or streptavidin for 30 min at 4 °C, and analyzed with FACSCalibur™ flow cytometer or LSR-II (BD Biosciences) at the Rockefeller University Flow Cytometry Resource Center. For competition assays, an excess amount of unlabeled, competing MAb was incubated with CHO/DC-SIGN cells for 10 min at 4 °C prior to adding the fluorochrome-conjugated MAb for additional 30 min incubation at 4 °C, followed by flow cytometric analysis.
For immunocytochemistry, stable transfectant CHO cells were grown on cover slides for 24 hrs. The slides were fixed with acetone for 10 min and then stained as above, followed by microscopic examination by deconvolution microscopy (Olympus, Melville, NY) or a Zeiss LSM 510 system (Carl Zeiss MicroImaging, Thornwood, NY) at the Rockefeller University Bio-Imaging Resource Center.
2.9. Collagenase treatment
CHO cells expressing mouse DC-SIGN, DCIR2, or control Neomycin were incubated in Hanks’ Balanced Salt Solution (HBSS) supplemented without or with 400 units/ml collagenase D (Roche Applied Science, Indianapolis, IN) at 37 °C for 30 or 60 min. EDTA (10 mM final concentration) was added to stop the reaction before the samples were divided into halves to be analyzed by FACS and Western blots.