Mouse myelin oligodendrocyte glycoprotein peptide (MOGp) 35–55 [MEVGWYRSPFSRVVHLYRNGK] was synthesized by solid-phase Fmoc chemistry by QCB, Inc. (Hopkinton, MA, USA) and CS Bio (Menlo Park, CA, USA).
C57BL/6 and B10.PL wild-type (wt) mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Vα2.3/Vβ8.2 TCR-transgenic mice provided by Dr Joan Goverman were bred in a specific pathogen-free facility at the University of Texas (UT) Southwestern. All protocols involving mice handling were approved by the UT Southwestern animal care facility.
Induction of experimental autoimmune encephalomyelitis
To induce active EAE, C57BL/6 female mice were immunized subcutaneously with MOGp35-55 emulsified in an equal volume of complete Freund adjuvant (CFA) (DIFCO Laboratories, Detroit, MI, USA) in each flank. Mice were 4 days, 1, 2, 3, 4, 5, 6, 7, 8, and 20 weeks of age. Immediately after the immunization, and again 48 hours later, mice received an intravenous injection of pertussis toxin (Ptx) in PBS. All animals received an equivalent dose of Ag, adjuvants, and Ptx on a dose per weight basis: Per 20 g bodyweight, 100 μl of vaccine, containing 100 μg MOGp35-55 and 2 mg/ml mycobacterium, as well as 400 ng Ptx were administered.
For the induction of EAE by adoptive transfer, spleens from either 2-week-old or 8-week-old Vα2.3/Vβ8.2 TCR-transgenic mice , were removed and single-cell suspensions were prepared. Splenocytes were cultured in 24-well plates at 1 × 106 cells/well with wt B10PL adult or neonatal irradiated splenocytes (3K Rads) at a ratio of 1:4. The cells were cultured in Roswell Park Memorial Institute (RPMI) medium 1640 with 10% FCS and stimulated with 6µg/ml myelin basic protein (MBP)Ac1-11 and 0.5ng/ml IL-12 in a 24-well plate for 72 hrs. Cells were washed with PBS and injected intraperitoneally (i.p.) into naive B10PL, adult (5 × 106/200 µls) or neonatal (1 × 106/50 µls) mice. Two independent experiments were conducted with a minimum of ten mice per group.
For all EAE experiments, individual animals were observed daily and clinical scores were determined as follows: 0 = no clinical disease, 1 = loss of tail tone, 2 = mild paraparesis, 3 = paraplegia, 4 = hindlimb and forelimb paralysis, 5 = moribund or death. Three independent experiments were conducted with a minimum of five mice per group.
Splenocytes (5 × 105 cells/well) from mice that had been immunized with MOGp35-55 10 days prior to sacrifice, were cultured in the presence of MOGp35-55 in RPMI 1640, supplemented with 5 × 10-5 M 2-mercaptoethanol, 2 mM glutamine, 100 μg/ml penicillin, 100 μg/ml streptomycin, 10% FCS (HyClone, Logan, UT, USA). After 72 hrs of culture, cells were pulsed with 1 μCi (3H)-thymidine and harvested 16 hrs later.
For assessment of Ag presentation by APC of various ages, splenocytes were γ-irradiated (33 Gy), and plated at 5 × 105 cells/well with 1 × 104 MOGp35-55-specific CD4+ T cells, and increasing doses of MOGp35-55. For generation of MOGp35-55-specific T cells, spleens were removed 0 to 14 days after immunization, and splenocytes were cultured in the presence of MOGp35-55 (25 μg/ml) and IL-2 (25 IU q3d). Every 14 days, T cells were washed, counted, and re-stimulated with APC and Ag for a minimum of three times. The purity of CD4+ T cells was analyzed by flow cytometry prior to each experiment, and approached 95% (data not shown). Cultures were pulsed with (3H) thymidine at 48 hrs and harvested 16 hrs later. The mean cpm ± SD of (3H) thymidine incorporation was calculated for triplicate cultures.
B cell adoptive transfer experiments
CD19+ B cells were negatively sorted by magnetic separation (Stem Cell Technologies, BC, Canada) with a purity of >85% from spleens of 8- to 12-week-old donor mice that had been actively immunized for EAE with 50 µg of mouse rMOG in CFA 10 days previously. Then, adjusting for body weight, 1 × 106 B cells were transferred i.p. into 4-day-old neonates and 5 × 106 B cells into 8-week-old adult recipients. At this time recipient mice were immunized with CFA/MOGp35-55. A week later, 3 × 106 B cells from adult donors immunized as described above were transferred i.p. to both neonates and adult recipients. Mice were monitored daily for clinical signs of disease as described previously.
In vitro T helper cell differentiation
Splenocytes were prepared from naïve 2- and 8-week-old mice and CD4+CD62L+ T cells were sorted on the FACSAria (purity was > 98%). In vitro polarization of T cells (0.25 × 106 cells/well in 2ml complete RPMI) was done in 24-well plates coated with anti-CD3 (1 mg/ml) and anti-CD28 (10 mg/ml) (BD Biosciences, San Jose, CA, USA) as previously described . For T cell polarization RPMI was supplemented as follows: 2 mg/ml anti-IFN-γ (R46A2) for Th0, 5 ng/ml IL-12 for Th1, 10 ng/ml IL-4, and 5 mg/ml anti-IFNγ for Th2 and 25 ng/ml IL-6, 0.5 ng/ml TGF-β, 10 ng/ml IL-1β and 10 ng/ml TNF-α for Th17. On day 3 cells were split into fresh antibody-coated plates and 1 ml of fresh RPMI supplemented with cytokines was added to the appropriate wells: 10 U/ml IL-2 and 2 μg/ml anti-IFNγ (R46A2) for Th0, 10 U/ml IL-2 and 5 ng/ml IL-12 for Th1, 10 ng/ml IL-4 for Th2 and 25 ng/ml IL-6, 0.5 ng/ml TGF-β, 10 ng/ml IL-1β and 10 ng/ml TNF-α for Th17. At 48 and 72 hrs of the second stimulation culture supernatants were collected and cytokine ELISA performed as described below. All monoclonal antibodies (mAb) and cytokines were purchased from R & D Systems (Minneapolis, MN, USA).
Enzyme-linked immunosorbent assay
Cell culture supernatants from experiments described above were collected at 48- and 72-hr time points for cytokine analysis as previously described [9, 10]. Quantitative ELISA for IL-17 and IFNγ was performed using paired mAb specific for corresponding cytokines as per manufacturer’s recommendations (BD Biosciences or R&D Systems). The results of ELISA assays are expressed as an average of triplicate wells ± SD. The SOFTmax ELISA plate reader and software was used for data analysis (Molecular Devices Corporation, Sunnyvale, CA, USA).
Mice were perfused via the left ventricle with cold PBS and brains, spinal cords, and spleens were harvested. Tissues were pressed through a 70-μm nylon mesh cell strainer. Splenocytes were treated with RBC lysing buffer (Sigma-Aldrich, St. Louis, MO, USA). CNS cells from all mice in each experimental group were pooled and processed as previously described . In brief, CNS cells were washed twice in 37% Percoll and CNS mononuclear cells were isolated by centrifugation at 2118 × g for 15 minutes at 22°C, over a 30/70% Percoll gradient. The interphase cells were collected, washed with 0.5% BSA/PBS, re-suspended in complete RPMI 1640, and counted. For flow cytometry, the following mAb were used: anti-CD3-Pacific Blue (500A2), anti-B220-PE (RA3-6B2), anti-CD11c-APC (HL3), anti-Gr1-APC-Cy7 (RB6-8C5), all from BD Biosciences; anti-CD11b PerCp-Cy5.5 (M1/70), anti I-Ab PE-Cy5 (M5/114-152), anti-CD45-PE-Cy7 (30-F11), anti-CD19-Alexa Fluor 700(1D3), biotinylated anti-pan NK (DX5), all from eBiosciences (San Diego, CA, USA); anti-CD4-PE-Texas Red (MCD0417) and anti-CD8-Pacific Orange (MCD0830), both from Invitrogen (Grand Island, NY, USA); and biotinylated anti-PDCA-1 from Miltenyi (Auburn, CA, USA). Biotinylated mAb were revealed with SA-Q Dot 655 from Invitrogen. Cells were re-suspended in staining buffer (4% FCS and 0.1% sodium azide in PBS) and Fc receptors blocked with anti-CD16/32 (BD Biosciences) for 15 minutes at 4°C before staining with mAb for 30 minutes at 4°C. Cells were washed, re-suspended in staining buffer, and fixed in 1% paraformaldehyde. Up to 500,000 events were acquired on a FACSAria (BD Biosciences) and analyzed using FlowJo software (Tree Star, Ashland, OR, USA).
RNA isolation and quantitative real-time PCR (QRT-PCR)
Neonatal and adult animals were sacrificed on day 10 post immunization with CFA/MOGp35-55. Following 1.5% Avertin overdose (400 mg/kg for adults), animals were transcardially perfused with 20 ml ice cold PBS and the CNS was removed. Total RNA was extracted from CNS homogenates using standard techniques . Taqman gene expression assays and the Step One Plus (Applied Biosystems, Foster City, CA, USA) were used to detect IFNγ, IL-10, IL-12 and IL-23. Relative gene expression between unimmunized mice (naïve) and mice immunized for EAE was determined using the ddCT method (Applied Biosystems User Bulletin #2). The dCt was normalized to the housekeeping gene ribosomal 18s rRNA, and the ddCt was normalized to the average of the naïve neonatal CNS dCts.
Ki-67 staining was performed to determine the number of proliferating cells in the CNS. Following fixation in 10% formalin, brains and spinal cords from naïve and immunized mice were serially sectioned. Tissues were embedded in paraffin, cut at 4 μm thickness, mounted on Fisher Brand Superfrost Plus glass slides, and stained with H&E (Fisher Scientific, Pittsburgh, PA, USA). Deparaffinization and staining with rabbit monoclonal Ventana anti-Ki-67 clone 30–9 was performed on the Ventana Benchmark XT automatic immunostainer with an ethylenediaminetetraacetic acid (EDTA)-based retrieval system (Ventana Medical Systems, Tucson, AZ, USA). The antibody was used at a concentration of 2 μg/ml, and immunoreactivity was detected with Ultraview horseradish peroxidase/diaminobenzidine. For analysis, the percentage of Ki-67-immunoreactive cells was calculated for selected mice using a Glasgow cell-counting graticule at 20× magnification with a Nikon Labophot-2 at a magnification of 20×. The thalamus location was chosen for analysis to avoid counting proliferating neuroglial precursors present in brains of immature mice. Ten fields were quantified for each mouse, and the mean was used for comparison.
Following fixation in 4% paraformaldehyde for at least 2 hrs, tissues were stored in 2% sucrose at 4°C until cutting. The brains were coronally sectioned and after embedding in Tissue-tek OCT Compound, the tissues were snap frozen in liquid nitrogen. Two 6 μm-thick sections were cut from each brain with a freezing microtome and mounted on Fisherbrand Superfrost Plus glass slides (Fisher Scientific, Pittsburgh, PA, USA). Tissue sections were then fixed in 10% formalin and rinsed in tap water. One set of tissues was stained with H&E and prepared for light microscopy, and the other set was prepared without staining for fluorescent microscopy. Areas of interest were captured on digitized images using a Leica TCS SP5 confocal microscope with the 63× objective and analyzed by ImageJ 1.35s software (public domain; http://rsbweb.nih.gov/ij/).
All experiments were repeated at least twice. Correlations between continuous and categorical variables were assessed using the Mann-Whitney U-test. The means of two normally distributed samples were compared by Student t-test. All other statistical comparisons between groups were examined using one-way multiple range analysis of variance (ANOVA) for multiple comparisons or Student-Newman-Keuls multiple comparison test. A P-value <0.05 was considered significant. Data are given as mean ± standard error.
It is generally accepted that 10 mice per treatment group are required to test the effect of reagents in active immunization EAE. This number was determined through power analysis. Through testing the null hypothesis (that the mean disease severity will be equal among the two treatment groups), it is possible to attain >80% power with the following assumptions: the criterion for significance (alpha) has been set at 0.050; the test is two-tailed, which means that an effect in either direction will be interpreted; this computation assumes that the mean difference is 1.7 (corresponding to mean EAE scores of 1.7 versus 0.0; or 2 versus 3.7) and the common intra-group SD is 1.0. This minimum significant difference of 1.7 was selected from prior observations that repeatable effects were associated with changes in mean disease score of between 1.5 and 2.0. Thus, this work assumes that effects smaller than 1.7 would not be of clinical or substantive significance and that differences in mean clinical score of 1.7 can be anticipated. A second goal of this study was to estimate the mean difference between the two populations. On average, a study of this design would enable us to report the mean difference with a precision (95.0% confidence level) of plus/minus 1.13 points. For example, an observed difference of 1.7 between treatment groups would be reported with a 95.0% CI of 0.57 to 2.83. The precision estimated here is the median precision. Precision will vary as a function of the observed SD (as well as sample size), and in any single study will be narrower or wider than this estimate. All analyses were performed with Prism 5 for Windows (Graphpad, La Jolla, CA, USA).