Animal and parasite
Six- to 10-week-old female BALB/c mice were obtained from Vital River Laboratory (Beijing, China). DO11.10 ovalbumin (OVA) -specific T-cell receptor (TCR) transgenic mice (on BALB/c background) were purchased from the Nanjing University Model Animal Research Centre (Nanjing, China). Mice were housed in the animal facility of the Guangzhou Institutes of Biomedicine and Health under specific pathogen-free conditions. All animal experiments were carried out in accordance with the national animal protection guidelines and approved by the Institutional Animal Care and Use Committee. The H. polygyrus parasites were kindly provided by Dr M. Scott (McGill University, Montreal, Canada) and maintained in BALB/c mice as previously described. To prepare ES products from the parasite, BALB/c mice were infected by oral inoculation with 400 third-stage larvae (L3) and killed 20 days after infection. The H. polygyrus adult worms were collected from the small intestine, washed extensively with sterile endotoxin-free PBS (Ginuo, Hangzhou, China) containing 200 U/ml penicillin and 200 mg/ml streptomycin (HyClone, Beijing, China) and cultured at a density of approximately 1000 worms/ml of RPMI-1640 medium (Invitrogen, Shanghai, China) supplemented with 2% glucose (Sigma-Aldrich, Rockville, MD) and antibiotics for 36 hr at 37°. The supernatant was harvested, centrifuged to remove eggs and worm debris, and stored at −80° until used.
Cloning, expression and purification rHp-CPI
Heligmosomoides polygyrus adult worms were collected from the intestines of mice 3 weeks after H. polygyrus L3 infection. Total RNA was isolated from adult worm homogenate using an RNA isolation kit (Omega Bio-Tek, Guangzhou, China) and reverse transcribed (Promega Corporation, Madison, WI). The cDNA fragment of CPI was amplified with Taq DNA polymerase (TaKaRa, Dalian, China). The sense 5′-TCA TCT CAA GTT GTT GCT GG-3′ and antisense 5′-AAT CTT CCC ATG GCT TCT-3′ primer sequences used for amplification were based on conserved sequences of cystatins previously described for Nippostrongylus brasiliensis, Onchocerca volvulus, Brugia malayi, Haemonchus contortus and Caenorhabditis elegans in GenBank. Based on the nucleotide sequence of cDNA fragments, specific primers were synthesized for 3′- and 5′-rapid amplification of cDNA ends (RACE; TaKaRa Biotechnology, Dalian, China) and used to determine the transcriptional start and terminal sites of CPI transcripts. The full-length cystatin cDNA obtained by RACE was subcloned into expression plasmid vector pET32a and expressed in Escherichia coli (Origami) as a protein fused to a leader sequence of Tobacco Etch virus (TEV) protease and six histidines. The recombinant fusion protein was purified from E. coli lysate by affinity chromatography using chelating Sepharose FF resin (GE Healthcare, Uppsala, Sweden). The His-peptide in the fusion protein was cut off by TEV protease (kindly provided by Dr J. Liu, Guangzhou Institutes of Biomedicine and Health, Guangzhou, China). The purity of the protein obtained was determined by SDS–PAGE and silver staining.
Measurement of protease inhibition activity of rHp-CPI
The activities of cysteine proteases, cathepsin B, C, L and S, was measured following the methods as described by others with some modifications. Bovine cathepsin B and C were purchased from Sigma and human cathepsin L and S were purchased from Calbiochem (Shanghai, China) and Enzo (New York, NY), respectively. The fluorogenic substrates for cathepthin B (Z-Arg-Arg-AMC; Sigma–Aldrich), cathepsin C (Gly-PhE-naphthylamide; Sigma-Aldrich), cathepsin S (Z-Phe-Arg-7-amido-4- methylcoumarin; Calbiochem) and cathepsin L (Z-Phe-Arg-7-amido-4-methyl coumarin; Calbiochem) were obtained from individual suppliers. To measure the inhibition activity of rHp-CPI, the protease was incubated with substrate in the absence or presence of serially diluted rHp-CPI in appropriate buffer for 15 min. The amount of product was measured fluorometrically with excitation at 360 nm and emission at 460 nm using a multiwall fluorescence spectrometer (Bio-Tek, Synergy HT, Corning, NY).
Generation of Hp-CPI monoclonal antibody
Monoclonal antibody (mAb) against rHp-CPI was generated following the standard protocol. Briefly, female BALB/c mice were immunized subcutaneously with 40 μg rHp-CPI emulsified in complete Freund's adjuvant (Sigma-Aldrich) and boosted twice at 4-week interval with 20 μg rHp-CPI in incomplete Freund's adjuvant. Spleen cells were isolated from the immunized BALB/c mice 1 week after final boosting, and fused with logarithmically growing SP2/0 myeloma cells at a ratio of 1 : 1 in the presence of polyethylene glycol 1500 (Roche, Basle, Switzerland). The treated cells were re-suspended in RPMI-1640 medium supplemented with 20% fetal calf serum, OPI (oxaloacetate, pyruvate, insulin) and HAT (hypoxanthine, aminopterin, thymidine) media supplements (Sigma-Aldrich) and plated into 96-well tissue culture plates at a density of 2·0 × 105 cells per well in a volume of 200 μl. After culturing at 37° with 5·0% CO2 for 7–10 days, the culture wells were screened using indirect ELISA for the presence of anti-rHp-CPI antibody. The cells in positive wells were collected and subjected to cloning by limited dilution. The cloned hybridoma cells were injected into the peritoneal cavity of naive mice. Ascites was collected 10 days after cell implantation and centrifuged at 10 000 g. to remove cells and debris and stored at −20°.
Generation of bone-marrow-derived DC
Bone marrow-derived dendritic cells (BMDC) were generated by culture of bone marrow cells following the method described by Lutz et al. Briefly, total bone marrow cells were collected from the femurs and tibias of BALB/c mice, suspended in RPMI-1640 medium (Invitrogen) supplemented with 10% heat-inactivated fetal calf serum (HyClone), 100 U penicillin/ml, 100 mg streptomycin/ml and 50 μm β-mercaptoethanol (Sigma–Aldrich) (complete medium). After lysing red blood cells with ammonium chloride buffer (0·15 m NH4Cl, 10 mm KHCO3 and 0·1 mm Na2 EDTA) and washing with complete medium, bone marrow cells were re-suspended in complete medium that was further supplemented with 10% supernatant from a mouse granulocyte–macrophage colony-stimulating factor (GM-CSF) -transfected cell line (Ag8653, kindly provided by Dr B. Stockinger, National Institute for Medical Research, London, UK) as a source of GM-CSF. Cells were cultured at 4 × 106/well in six-well plates (Greiner Bio-one, Frickenhausen, Germany) at 37° for 7–9 days in a humidified CO2 incubator. Cells were fed on days 3, 5 and 7 with complete medium containing GM-CSF supernatant. On day 9, non-adherent cells were collected, washed and used as immature BMDC. Cell viability was determined by trypan blue exclusion test and was 90–94% for the two groups of BMDC. The purity of BMDC was about 70–80% CD11c+ cells as determined by flow cytometry.
To analyse the effects of rHp-CPI on DC differentiation, rHp-CPI (50 μg/ml) were added in appropriate wells beginning at day 3 of culture and the cells were harvested on day 9 and analysed for cell surface molecule expression. In the preliminary experiments, graded doses of rHp-CPI were tested and the dose of 50 μg/ml rHp-CPI was found to be optimum. To investigate the effects of rHp-CPI on DC maturation, the bone marrow cells were cultured in the absence of rHp-CPI as described above for 7 days. The differentiated CD11c+ DC were harvested and activated with 1 μg/ml lipopolysaccharide (LPS; Sigma–Aldrich) or 1 μm CpG oligonucleotide (Invitrogen) with or without rHp-CPI for 18 hr.[15, 29] Control DC were cultured in complete medium alone. The DC were harvested and analysed for the expression of surface molecules and the cell culture supernatants were collected and stored at −20° for determination of cytokines.
DC and CD4+ T-cell isolation and co-culture
Bone marrow-derived dendritic cells were enriched by positive selection with anti-CD11c magnetic beads (Stemcell Technologies Inc., Vancouver, BC, Canada) according to the manufacturer's instructions. The enriched DC were typically of > 90% purity as determined by flow cytometry. CD4+ T cells in spleen were enriched by magnetic sorting using anti-CD4 magnetic beads (Miltenyi Biotec, Auburn, CA). The enriched CD4+ T cells had > 95% purity. To determine CD4+ T-cell proliferation induced by DC, the enriched BMDC were incubated with rHp-CPI (50 μg/ml) for 2 hr and OVA (1 mg/ml; Calbiochem) was added and incubated for another 2 hr. The DC were then treated with 50 μg/ml mitomycin (Sigma–Aldrich) for 20 min and washed with a sufficient amount of complete medium to remove the mitomycin. Dendritic cells (2 × 104/well) were co-cultured with CD4+ T cells (4 × 104/well) in a 96-well U-bottom plate in the presence of 1 mg/ml OVA for 72 hr. During the last 18 hr, 1 μCi/well of [3H]thymidine was added. Incorporation of [3H]thymidine by the cells was determined by scintillation counting. For determination of cytokine production in DC and CD4+ T-cell co-culture, 2 × 105 CD4+ T cells were co-cultured with 1 × 105 DC in U-bottom plates in the presence of 1 mg/ml OVA for 72 hr. Supernatants were harvested for cytokine analysis by ELISA.
BMDC in vivo transfer
The modulatory effect of rHp-CPI on DC function was analysed by DC transfer experiment. The BMDC were re-suspended at 2 × 106 cells/ml in complete medium and treated with rHp-CPI (50 μg/ml) for 3 hr before pulsing with 1 mg/ml OVA for 4 hr at 37°. After pulsing, cells were harvested, washed extensively with sterile endotoxin-free PBS and re-suspended in RPMI-1640 medium with 5% BALB/c mouse serum. Mice were injected intravenously with 5 × 105 BMDC. Four weeks after DC injection, BALB/c mice were injected intraperitoneally with 10 μg OVA protein emulsified in incomplete Freund's adjuvant (Sigma-Aldrich). Sera were collected 4 weeks after OVA injection and OVA-specific antibody levels were determined by ELISA.
Flow cytometric analysis
For cell surface staining, 106 cells were first incubated with FcR-blocking reagent (BD Biosciences, New York, NY) in sorting buffer (PBS with 1% BSA) on ice for 15 min. The cells were then washed and stained with anti-CD11c-FITC, anti-CD40-phycoerythrin (PE), anti-CD80-PE, anti-CD86-PE and anti-MHC-II-PE fluorescent mAbs (all from eBiosciences, San Diego, CA) following standard protocols. Isotype-matched mAbs were used for control staining. Cells were then washed and re-suspended in sorting buffer and analysed by flow cytometry using FACS Calibur (BD Biosciences). At least 10 000 events were acquired per sample, and the data analysis was performed using Flowjo software (TreeStar, Ashland, OR).
Cytokine and antibody determination by ELISA
Cytokine levels in cell culture supernatants were determined using ELISA kits for IL-12p40, TNF-α, IL-6 and interferon-γ (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. Serum levels of OVA-specific antibodies were determined by ELISA. Briefly, ELISA plates were coated with OVA antigen overnight at 4° and subsequently blocked with 1% BSA in PBS for 1·5 hr. After washing, serially diluted serum samples were added and incubated for 1 hr at room temperature. After extensive washing, horseradish peroxidase-conjugated goat anti-mouse total immunoglobulin, IgG1 and IgG2a antibodies (Southern Biotechnology Associates, Birmingham, AL) were added and incubated at room temperature for 1 hr. Reactivity was visualized by addition of substrate and optical density values were read in a microplate reader. Antibody levels in serum are expressed as endpoint titres, the reciprocal of the lowest dilution that yields the background optical density.
For immunoblotting, proteins were separated by SDS–PAGE and the gels were electroblotted onto a PVDF membrane (Pall Corporation, East Hills, NY). Anti-rHp-CPI mAb was used as the primary antibody, and horseradish peroxidase-conjugated anti-mouse IgG (Thermo Fisher Scientific, Guangzhou, China) diluted 1 : 50 000 was used as the secondary antibody. Bound antibody was detected using enhanced chemiluminescence reagents (Thermo Fisher Scientific).
Statistical analyses were performed with GraphPad Prism 5 software (GraphPad Software Inc., La Jolla, CA). Significance of differences between groups was analysed using the Student's t-test. Data are presented as mean ± SD. A P-value < 0·05 was considered significant.