Preparation of tissue-derived CII and synthetic peptides
Native CII was solubilized from fetal calf articular cartilage by limited pepsin-digestion and purified as described earlier . The purified collagen was dissolved in cold 20 mM acetic acid at 4 mg/ml and stored frozen at -70°C until used. The synthetic peptides were supplied by Biomolecules Midwest Inc. (Waterloo, IL, USA). A peptide containing the immunodominant determinant sequence of CII (GIAGFKGEQGPKGEB) is referred to as A2 or wild-type (WT) IEDBID 109115; a synthetic peptide representing the sequence (GIAGNKGDQGPKGEB) is designated A12.
Generation of Tg mice expressing DR
Mice expressing the chimeric (human/mouse) DRB1*0101 construct were maintained in our onsite pathogen-free facility. The chimeric DRB1*0101 construct has been previously described, as has the production of Tg mice expressing these constructs .
Generation of DR1 mice that are IL4 or IL10 knockout
The IL-4 (C57BL/6-IL4tm1cgnIL-4 and C57BL/6-IL10tm1cgnknockout) mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA) and backcrossed onto the arthritis-susceptible DR1 C57BL/10 background for 12 generations before they were intercrossed. Genomic DNA was obtained from blood samples, and polymerase chain reaction (PCR) was used to identify mice homozygous for the IL-4-/- and IL-10-/- phenotypes.
All mice were fed standard rodent chow (Ralston Purina Co., St. Louis, MO, USA) and water ad libitum. To ensure that the environment was pathogen free, sentinel mice were routinely tested for hepatitis and Sendai viruses. All animals were kept until the age of 7 to 10 weeks before being used for experiments, which were conducted in accordance with approved IACUC protocols.
CII was dissolved in 10 mM acetic acid at a concentration of 4 mg/ml and emulsified with an equal volume of CFA containing 4 mg/ml of Mycobacterium tuberculosis strain H37 Ra (Difco Microbiology Products, Becton Dickinson, Franklin Lakes, NJ, USA) . For the induction of arthritis, each mouse received 100 μg of CII emulsified in CFA subcutaneously at the base of the tail. For other immunizations, each mouse received subcutaneously 100 μg of either peptide A12 or Ova emulsified with CFA.
Measurement of the severity of arthritis
The severity of arthritis were determined by visually examining each forepaw and hindpaw and scoring them on a scale of 0 to 4, as described previously . Scoring was conducted by two examiners, one of whom was unaware of the identity of the treatment groups. Each mouse was scored thrice weekly, beginning 3 weeks after immunization and continuing for 8 weeks. The mean severity score (sum of the severity scores for the group on each day/total number of animals in the group) was recorded at each time point.
Production of soluble HLA-DR protein
Soluble DR1 was purified from culture supernatants of S2 Drosophila cells previously transfected with DRB1*0101 and DRA1*0101. Both the α- and β-chains of the DR1 constructs contain murine I-E leader sequences, followed by DRα or DRβ first domains and murine I-Eα and I-Eβ second domains. The analog (CII259-273, 263F, 266D), was inserted after the third residue of the β-chain, and a flexible ((Gly)4-Ser)3 linker was added to allow the peptide to fold into the binding groove of the DR molecule. The sequences encoding the cytoplasmic and transmembrane portions of the molecules were deleted from the cDNA by PCR, and a new stop codon was inserted at the 3' end of the second domain of the α-chain. The β-chains were altered by adding a linker and a biotinylation site 3' to the Eβ second domain, to allow site-specific addition of biotin and tetramerization by using streptavidin. The resulting cDNA was cloned into the Drosophila expression vector pRmHA-3 (gift from Dr. D. Zaller, Merck, Rahway, NJ, USA). S2 cells were transfected with a 10:10:1 ratio of chimeric DRB1 and DRA1 to pUChsNeo by using calcium phosphate precipitation. Soluble DR production was induced by 1 mM CuSO4, and 5 days later, the culture supernatant was collected and adjusted to 0.05% octyl glucoside (OcG). The DR molecules were purified by passage of the culture supernatant over an affinity column coupled with the anti-DR Ab LB3.1. The column was washed with 0.05% OcG and 0.15 M NaCl in phosphate buffer, pH 7.5, followed by 0.05% OcG and 0.5 M NaCl in phosphate buffer, pH 7.5, and a final wash with 10 mM Tris in 0.5 M NaCl, pH 7.5. The DR molecules were eluted with 100 mM Tris and 0.5 M NaCl, pH 11.2, and the fractions were immediately neutralized with acetic acid. The DR recovered was concentrated by using an Amicon Stirred Cell, quantitated by OD280 absorption and ELISA (PathScan Total DDR1 Sandwich ELISA Kit; Cell Signaling, Danvers, MA, USA), and was evaluated with SDS-PAGE before use. Biotinylation of the proteins was performed with BirA (Avidity, Denver, CO, USA) and incorporated into saturated complexes with phycoerythrin (PE)-streptavidin (BioSource International, Camarillo, CA, USA) before use.
For tetramer staining of T cells, lymph node cells were diluted to a concentration of 2.5 × 107/ml in DMEM medium (Gibco) supplemented with 50 U/ml penicillin G sodium, 50 μg/ml streptomycin sulfate, 0.05 mM 2-ME, 2 mM L-glutamine, and 0.1% BSA, and T-hybridoma cells were diluted to a concentration of 2.5 × 106/ml in DMEM complete. One million lymph node cells or 105 T-hybridoma cells were then aliquoted into a 96-well plate and were incubated with 1 μg of tetramer in 10 μl of complete medium supplemented with 5 mM NaN3. Cells were incubated at 37°C for 2.5 hours, at which time, antibodies to cell-surface markers were added, and cells were incubated for an additional 30 minutes at 4°C. After incubation with Abs to cell-surface markers, samples were washed 3 times with 200 μl of PBS supplemented with 0.1% NaN3 and 2% FBS, resuspended in 200 μl of PBS supplemented with 0.1% NaN3 and 2% FBS, and analyzed with flow cytometry by using a FACSCalibur (BD Biosciences). In some experiments, the draining lymph nodes of DR1 mice were harvested 10 days after immunization with CII and were incubated with tetramer and subjected to magnetic sorting to obtain a purified population of tetramer-positive cells (Miltenyi Biotec).
Flow cytometry analysis of TCR repertoire
To determine the frequency of individual Vβ subfamilies expressed by tetramer+ T cells in DR1 mice, draining inguinal cells from DR1 mice immunized 10 days earlier with A12/CFA were incubated with tetramer labeled with PE, anti-CD4-APC, anti-α/βTCR-PerCP (BD Biosciences, San Jose, CA, USA), and an anti-TCR-Vβ-specific Ab conjugated with FITC for 30 minutes at 4°C (Mouse Vβ TCR Screening Panel; BD Biosciences). Labeled cells were washed with PBS, and a minimum of 10,000 cells was analyzed from each sample with flow cytometry by using a FACSCalibur or LSR II (BD Biosciences). The final analysis was performed by using FlowJo software (Tree Star, Ashland, OR, USA).
Measurement of serum antibody titers
Mice were bled at 6 weeks after immunization, and sera were analyzed for antibodies reactive with native CII by using a modification of an enzyme-linked immunoassay (ELISA) previously described . Serial dilutions of a standard serum were added to each plate. From these values, a standard curve was derived by computer analysis by using a four-parameter logistic curve. Results are reported as units of activity, derived by comparison of test sera with the curve derived from the standard serum, which was arbitrarily defined as having 50 units of activity. Reactivity to CII was not detected in sera obtained from normal mice.
Phenotypic characterization of the cells
Inguinal lymph nodes from immunized mice were isolated, and the phenotype induced by A12 was determined by multiparameter flow cytometry with an LSRII flow cytometer (BD Biosciences). Cells were cultured with fluorochrome-labeled antibodies specific for CD4, CD25, or CD44 (BD Biosciences) as well as tetramer. In some experiments, intracellular labeling was performed by using antibodies specific for FoxP3 (FJK-16s; eBioscience, San Diego, CA, USA), per the manufacturer's protocol. In the experiments involving intracellular staining, specific gating was performed on both the CD4+ and the Vβ8+, Vβ14+ population of cells.
In transfer experiments, inguinal lymph node cells from DR1 mice previously immunized with the A12 analog peptide in CFA were collected 8 days after immunization, and various cell subsets were fractionated by using PE-labeled tetramers and ferromagnetic beads (Miltenyi Biotec, Gladbach, Germany), according to the manufacturer's protocol. The purity of each cell population was confirmed by flow cytometry to have > 95% purity. Recipient mice were given cells intravenously by using two protocols (a) 2 × 105 either tetramer+ or tetramer- T cell or (b) 5 × 105 of VB8+, VB14+ T cells. All recipient mice were immunized with CII either on the day of the cell transfer (prevention protocol) or 25 days before cell transfer (therapy protocol) and observed for arthritis.
Measurement of cytokines
To measure cytokines, inguinal CD4+ lymph node cells or purified tetramer+ T cells were cultured (5 × 105 CD4+ T cells/ml) with wild-type APCs (DR1-positive splenocytes) (1:2 ratio), which had been prepulsed with 100 μg/ml of the various peptides (A2 or A12). The CD4+ cells were collected 8 days after immunization (with either OVA, CII, or A12 emulsified in CFA) by negative selection by using ferromagnetic beads, according to the manufacturer's protocol (Miltinyi Biotech). (Each cell population was confirmed by flow cytometry to have > 95% purity). Each culture was set up by using pooled cells from three mice run in triplicates, and supernatants were collected at 72 hours and analyzed for the presence of IL-4, IL-10, IL-2, INF-γ, and IL-17 by using a Bio-plex mouse cytokine assay (Bio-Rad, Hercules, CA, USA) according to the manufacturer's protocol. Values are expressed as picograms per milliliter and represent the mean values for each group taken from three separate experiments.
Mean severity scores, antibody titers, and cytokine levels were compared by using the Mann-Whitney test.