Absence of IFN-γ or IL-12 Has Different Effects on Experimental Myasthenia Gravis in C57BL/6 Mice

Mice

We bought WT, IFN-γ^−/−, and IL-12^−/− B6 mice from The Jackson Laboratory (Bar Harbor, ME) and bred them at the animal facility of the University of Minnesota. Because of the unexpected sensitivity to EMG found for the IFN-γ^−/− mice (see below), we verified the genotype of 10 randomly selected IFN^−/− and 5 WT mice by PCR using DNA from 3 to 5 μl of blood from each mouse, prepared using Chelex 100 resin (Bio-Rad, Hercules, CA) (32). One microliter of supernatant of the Chelex extract was used as a template for PCR. Each reaction mixture (total volume, 10 μl) included 1 μl 10× PCR buffer II (Perkin-Elmer, Foster City, CA), 1 μl of 10× dNTPs (10× dNTPs: 2 mM each dATP, dCTP, dGTP, and dUTP (Pharmacia, Piscataway, NJ)), 0.8 μl of 25 mM MgCl₂ solution (Perkin-Elmer), 1 μl of 10 μM stock solutions for each of the primers of the pairs listed below, 2 μl of red sucrose PCR-compatible loading dye (60% sucrose and 1 mM cresol red), 0.1 μl of 5 U/μl Ampli Taq Gold DNA polymerase (Perkin-Elmer), and 3.1 μl of sterile double-distilled H₂O. Each sample was covered with 30 μl of mineral oil. For PCR, the samples were hot started for 10 min at 95°C, followed by a three-step cycling profile using 35 cycles of 30 s at 95°C, 30 s at 53°C, and 45 s at 72°C in a Perkin-Elmer model 9600 DNA thermal cycler. We used the following pairs of primers, as recommended by The Jackson Laboratory: for the WT IFN-γ allele, IMR126 −5′-AGA AGT AAG TGG AAG GGC CCA GAA G-3′ and IMR127 −5′-AGG GAA ACT GGG AGA GGA GAA ATA T-3′ (the resulting product is 220 bp); and for the disrupted IFN-γ allele, IMR128 −5′-TCA GCG CAG GGG CGC CCG GTT CTT T-3′ and IMR129 −5′-ATC GAC AAG ACC GGC TTC CAT CCG A-3′ (the resulting product is 375 bp). We used both pairs of primers for each mouse DNA: each pair was used in a separate tube, so that only one product could be amplified for each reaction. We assessed the size of the products by 1.5% agarose (Life Technologies, Gaithersburg, MD) gel electrophoresis, stained with 0.001% ethidium bromide (Sigma, St. Louis, MO). When we used DNA from WT mice, we obtained a PCR product of 220 bp only when we used the primers corresponding to the unmutated IFN-γ sequence. When we used DNA from IFN-γ^−/− mice, we obtained a PCR product of 375 bp only when we used the primers corresponding to the mutated IFN-γ sequence.

Purification of Torpedo AChR (TAChR)

We purified AChR from the electric tissue of Torpedo californica fish as alkali-stripped AChR-rich membrane fragments (TAChR)⁵ (33). The AChR structure is highly conserved along evolution: TAChR is quite similar to mammalian muscle AChR and suitable to induce EMG (9). We measured the protein concentration by the Lowry assay (34), the TAChR concentration as α-bungarotoxin (α-BTX) binding sites (33), and we assessed the protein composition by SDS-PAGE (35). The TAChR preparations we used contained 3.8–5.8 nmol of sites/mg protein and contained only the four TAChR subunits as the main protein bands. For use in cell cultures, we diluted the TAChR-rich membrane fragments in RPMI 1640 as needed and sterilized them by UV irradiation. For immunization and Ab assay, we solubilized the membranes in 1% Triton X-100 (33), diluted them to 0.5 mg/ml in PBS, and stored them at −80°C.

Peptides

To determine the epitope repertoire on the TAChR α subunit, which dominates the sensitization of anti-TAChR CD4⁺ cells in B6 mice (33), we used a panel of synthetic (36) peptides, ∼20-residue long and overlapping by ∼5 residues that spanned the sequence of the TAChR α subunit. We reported their characterization previously (33). We used solutions of the individual peptides in PBS sterilized by UV irradiation and stored frozen.

TAChR immunization

We immunized 8- to 10-wk-old female mice by s.c. injections, along the back and at the base of the tail, with solubilized TAChR (20–40 μg in 100 μl PBS) emulsified in an equal volume of CFA. We boosted them twice at 4-wk intervals with the same amount of TAChR in incomplete Freund’s adjuvant and a third time 5–7 days before being euthanized at the end of week 15.

Evaluation of clinical symptoms of EMG

We quantified the EMG symptoms using an inverted hang-forced exercise, sensitized by a minute amount of pancuronium bromide (0.03 mg/kg i.p.), given just before the test (37). The mice hang from a grid: we measured the time it took for the mouse to release its hold and fall three times (“holding time”). The test is parametric and gives a quantitative assessment of the severity of the weakness of the mouse. To verify the myasthenic nature of the weakness, we injected edrophonium chloride (Reversol; Organon, West Orange, NJ) intraperitoneum. Reversol is a cholinesterase inhibitor which immediately increased the strength of mice that have EMG. We tested the mice every 1–3 wk, starting on the day of the first immunization. We performed the test without knowledge of the treatment that the mouse had received.

The average holding time of 285 naive WT mice was 11.4 ± 1.55 min. Naive IL-12^−/− and IFN-γ^−/− mice had holding times indistinguishable from WT mice. We considered myasthenic mice with holding times of 8.3 min (the holding time of normal mice minus 2 SD) or less. Paralyzed mice or mice that died of respiratory paralysis are represented in the figures as having holding time of zero.

Anti-AChR Ab assay

We obtained sera after each clinical testing. We measured the serum concentration of anti-TAChR Ab by radioimmunoprecipitation assay (RIPA) using TAChR solubilized in Triton X-100 and labeled by the binding of ¹²⁵I-α-BTX (38). This assay uses as precipitating Ab a polyclonal serum raised against mouse Ig that include the κ and λ light chains shared by all Ig; therefore, the serum precipitates all Ig isotypes and IgG subclasses. We express the Ab concentration as micromolar precipitated ¹²⁵I-α-BTX binding sites.

Assay of anti-TAChR IgM, IgE, IgG, and IgG subclasses

We measured by ELISA the anti-TAChR IgM, IgE, IgG, and the IgG subclasses in mouse sera obtained at 8 and 14 wk after the first TAChR immunization. We coated ELISA plates (Nunc, Karstrup, Denmark) by overnight incubation at 4°C with 100 μl/well of 25 μg/ml TAChR in 10 mM NaHCO₃ (pH 9.6). We washed the plates four times with PBS containing 0.05% Tween 20 (PBS-T) and blocked them with 200 μl/well of 2% BSA in PBS-T (PBS-TB) for 1 h at 37°C. After four washings with PBS-T, we added 100 μl/well of three appropriate serum dilutions (in duplicate) in PBS-TB and incubated the plates overnight at 4°C. We used serum from normal untreated mice (NMS) as a negative control. We washed the plates six times with PBS-T, added 100 μl/well of 1 μg/ml biotinylated anti-mouse IgM, IgE, total IgG, or IgG subclass Ab (PharMingen, San Diego, CA) in PBS-TB, and incubated the plates for 1 h at 37°C. Mice carrying the Igh-1^b allele, like B6 mice, do not express IgG2a, but rather a related isotype, termed IgG_2a^b or IgG2c (39, 40). To detect anti-TAChR IgG2c in the mouse sera, we used the mAb biotin anti-mouse IgG_2a^b (Igh-1^b) (PharMingen).

After eight washings with PBS-T, we added 100 μl/well of 1 μg/ml avidin-peroxidase (Sigma, St. Louis, MO) and incubated the plates for 1 h at 37°C. We washed the plates eight times with PBS-T and added 100 μl/well of 0.3 mg/ml 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) in 0.01% H₂O₂/citric acid buffer (ABTS peroxidase substrate system; Kirkegaard & Perry Laboratories, Gaithersburg, MD). After development of the blue-green color at room temperature, we stopped the enzymatic reaction by adding 100 μl/well of 1% SDS. We measured the absorbance at 405 nm with an automated microplate reader ELx800 (Bio-Tek Instruments, Winooski, VT). We determined the specific absorbance by subtracting the absorbance of wells incubated with NMS dilutions from that of wells incubated with the sera from TAChR-immunized mice. The serum concentration of anti-TAChR Ig isotypes and IgG subclasses was inferred from standard curves obtained by coating ELISA plates directly with 100 μl/well of purified murine total IgG (Sigma), IgM, and IgE (PharMingen) or IgG1, Ig2a, and IgG2b subclasses (Sigma) from 100 to 0.097 ng/ml in 10 mM NaHCO₃ (pH 9.6) and detecting their presence with the biotinylated anti-mouse Ig class or IgG subclass/avidin-peroxidase system, as described above. An ideal standard curve would have required the use of purified anti-AChR IgG subclasses binding to the TAChR coating the plate. Therefore, the values of Ig and IgG subclass concentrations in serum we report here should not be considered as absolute concentrations. However, since our goal was to assess the relative amounts of the different anti-AChR Ig classes and IgG subclasses synthesized in IFN-γ^−/− or IL-12^−/− mice as compared with WT mice, we considered our standard curve a suitable reference for our evaluations, because it represents an internal standard that allows comparison of results for the individual isotypes and subclasses obtained in different experiments. We could not construct a standard curve for IgG2c because purified IgG2c is not commercially available. Thus, for IgG2c we simply report the values of OD, expressed as OD units at a wavelength of 405 nm, obtained for sera of the three mouse strains assayed simultaneously. We obtained the results reported here doing duplicate determinations of three serum dilutions (1:30,000, 1:90,000, and 1:270,000): the OD values in the linear range were normalized as to represent an arbitrary dilution of 1:90,000 and were averaged.

Detection of IgG, IgM, and IgG subclasses and complement at the muscle synapses by immunofluorescence microscopy

Hind limb muscle tissue of WT, IFN-γ^−/−, and IL-12^−/− mice, frozen in liquid nitrogen and stored at −70°C, was embedded in OCT Compound Tissue-Tek (Miles Laboratories, Elkhart, IN) and sectioned it in the transverse direction into 10-μm sections using a Jung Frigout 2800E Kryostat (Leica, Nublach, Germany). To detect simultaneously the presence of mouse IgG and complement at the neuromuscular junction, we incubated the sections at room temperature for 10 min in PBS and for 1 h with a 1:200 dilution of biotin-conjugated goat anti-mouse IgG polyclonal Ab (Sigma) in PBS containing 3% BSA. We washed the sections with PBS for 15 min three times and stained them for 1 h at room temperature with Texas Red-labeled α-BTX (Molecular Probes, Eugene, OR), FITC-labeled goat anti-mouse C3 Ab (Nordic Immunological Laboratories, Capistrano Beach, CA), and 6-((7-amino-4-methylcoumarin-3hexanoicacetyl)amino) acid, succinimidyl ester-labeled streptavidin (Molecular Probes) diluted in PBS containing 3% BSA at 1:4000, 1:100, and 1:200 dilutions, respectively. For detection of mouse IgM and different IgG subclasses, we incubated the sections for 1 h with 1:20 dilutions in PBS of goat anti-mouse IgM Ab (Sigma) or mAbs specific for mouse IgG1 (a rat mAb; Sigma), IgG2b (a rat mAb; Sigma), and IgG2c (PharMingen mAb anti-mouse IgG_2a^b (Igh-1^b); a mouse IgG3 mAb). The sections were washed with PBS for 15 min three times, followed by incubation with a biotin-conjugated secondary Ab, which was rabbit anti-goat IgG polyclonal Ab (Sigma) for IgM, goat anti-rat IgG polyclonal Ab (Sigma) for IgG1 and IgG2b, and a mAb specific for mouse IgG3 (PharMingen) for IgG2c. This was followed by staining with Texas Red-labeled α-BTX (Molecular Probes), FITC-labeled goat anti-mouse C3 Ab (Nordic Immunological Laboratories), and ALEXA 350-labeled streptavidin (Molecular Probes) as described above. We washed the sections three times for 15 min with PBS and viewed them in a fluorescence microscope (Nikon Eclipse E800; Nikon Diaphot, Melville, NJ). We collected digital images using the program Image Pro Plus (Media Cybernetics, Silver Spring, MD).

Assay of Ab bound to mouse muscle AChR

We measured the Ab bound to mouse muscle AChR by RIPA (33) as follows. Carcasses from three to five mice were skinned, eviscerated, and homogenized individually in two volumes of 50 mM Tris-HCl, 50 mM NaCl, 5 mM EDTA, 20 mM PMSF, and 1.2 mM EGTA (pH 7.4, extraction buffer). The homogenates were centrifuged at 30,000 rpm for 60 min. The pellets were suspended in 1 volume of extraction buffer and extracted with 2% Triton X-100 for 4 h under shaking. We centrifuged the extract at 30,000 rpm for 60 min and collected the supernatant. We added 4 pmol of I¹²⁵-α-BTX to each quadruplicate of 1-ml aliquots of Triton X-100 extract of each mouse carcass and incubated them for 24 h. Five microliters of NMS was added to each aliquot. One hundred microliters per sample anti-mouse IgG Ab were added. The samples were incubated overnight, centrifuged at 12,000 rpm, washed four times with 10 mM PBS (pH 7.4) containing 0.1% Triton X-100, and counted in a gamma 5500 counter (Beckman, Irvine, CA). All procedures were conducted at 4°C.

Proliferation assay

Five to 7 days after the last immunization, we obtained spleen cells (33) from two to three identically treated mice and pooled them. This was necessary to obtain enough cells for testing all of the Ag listed below. We depleted the splenocytes in CD8⁺ cells using paramagnetic beads and rat anti-mouse CD8 Ab (PharMingen) (33, 37, 38).

For proliferation assays, we suspended the cells in RPMI 1640 (Life Technologies) supplemented with 10% heat-inactivated FCS (Life Technologies), 50 μM 2-ME, 1 mM l-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 μg/ml streptomycin (1 × 10⁶ cells/ml). We seeded the cells in triplicate in 96-well flat-bottom plates (200 μl/well). We added one of the following Ag or stimulants: 10 μg/ml PHA (Sigma), 10 μg/ml TAChR, and 10 μg/ml of the individual α subunit peptides. Controls were triplicate wells cultivated without any Ag or with a 20-residue control peptide synthesized by the same method, unrelated to the TAChR sequence (10 μg/ml). After 4 days, we labeled the cells for 16 h with [³H]thymidine (1 μCi/well, sp. act., 6.7 Ci/mmol; DuPont, Boston, MA) and harvested them (Titertek; Skatron, Sterling, VA). We measured the [³H]thymidine incorporation by liquid scintillation.

Cytokine secretion by CD8⁺-depleted splenocytes in response to stimulation with TAChR

Five to 7 days after a last boost with TAChR, we prepared pooled CD8⁺-depleted splenocytes from three identically treated mice. We resuspended the cells at 5 × 10⁶ cells/ml and cultured them with and without 10 μg/ml TAChR in 24-well plates. In some experiments, we set up two independent cultures for each Ag. Cells cultivated without any Ag served as controls for spontaneous secretion of cytokines. We harvested the culture supernatants after 24 and 96 h. We measured the concentrations of IFN-γ, IL-2, IL-4, and IL-10 by capture ELISA using duplicate samples. We used anti-IFN-γ, anti-IL-2, anti-IL-4, and anti-IL-10 monoclonal and polyclonal Ab (PharMingen) and recombinant IFN-γ, IL-2, IL-4, and IL-10 (PharMingen) as standards according to the manufacturer’s instructions.

Statistical analysis

We determined the significance of the differences of the average responses of two groups using a two-tailed Student’s t test. We considered a difference significant when p < 0.05. To determine the significance of the differences between two curves, such as those describing the time course of the appearance of anti-AChR Ab, we used the program EXCEL-ANOVA. We considered a difference to be significant when p < 0.05.

IL-12-deficient mice are resistant to EMG, whereas IFN-γ-deficient mice are susceptible

In the first set of experiments, we immunized five WT, five IFN-γ^−/−, and four IL-12^−/− mice with TAChR. We measured the strength of the individual mice every 2 wk for 14 wk, starting just before the first TAChR injection (time 0, Fig. 1⇓). The horizontal line in Fig. 1⇓ corresponds to the holding time (8.3 min) below which we considered the mice to have EMG. Two WT mice developed persistent myasthenic weakness from week 10 onward. The other three mice had holding times indicative of EMG between 11 and 12 wk and recovered by week 14 (i.e., 6 wk after the third TAChR immunization). None of the IL-12^−/− mice developed stable EMG weakness, although two mice had a transient reduction of the holding time to values slightly below 8.3 min, indicating a modest reduction of the function of the neuromuscular transmission. All IFN-γ^−/− mice developed EMG. Three had abnormally reduced holding time after the second TAChR injection when the WT mice still had normal holding times. From week 9 onward, four IFN-γ^−/− mice had stable EMG and one had a transient reduction of the holding time to values slightly below 8.3 min, but recovered by week 14.

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FIGURE 1.

IFN-γ^−/− mice are susceptible to EMG, whereas IL-12^−/− mice are resistant. Strength of five WT, five IFN-γ^−/−, and four IL-12^−/−, as indicated inside the panels, measured by the pancuronium-sensitized hanging test at the times indicated. The mice received three TAChR injections at the times indicated with arrows (0, 4, and 8 wk). The tests at 0, 4, and 8 wk were conducted just before the TAChR-immunizing injections. The horizontal lines indicate a holding time of 8.3 min (holding time of normal mice minus 2 SD). Mice with a holding time of 8.3 min or less were considered to have EMG. See text for experimental details.

To verify the susceptibility of IFN-γ^−/− mice to EMG, we immunized a second set of mice that included eight IFN-γ^−/− mice and 19 WT mice. We tested the mouse strength just before the first TAChR immunization and every 2 wk for 10 wk. The results of the tests conducted 4, 8, and 10 wk after the beginning of the immunization are reported in Fig. 2⇓. IFN-γ^−/− and WT mice developed EMG with similar frequency and severity.

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FIGURE 2.

IFN-γ^−/− mice are as susceptible to EMG as WT mice. Strength of IFN-γ^−/− and WT mice, as indicated at the right side of the panels, measured by the pancuronium-sensitized hanging test. The tests were conducted for 4, 8, and 10 wk after beginning the immunization as indicated below the panels. The mice had received three TAChR injections (at weeks 0, 4, and 8). The horizontal lines indicate a holding time of 8.3 min. See text for experimental details.

WT, IFN-γ^−/−, and IL-12γ^−/− mice develop comparable amounts of anti-TAChR Ab

We measured the serum anti-TAChR Ab of WT, IFN-γ^−/−, and IL-12γ^/- mice using the RIPA. Fig. 3⇓ shows the anti-TAChR Ab concentration in the sera of the mice shown in Fig. 1⇑ that was obtained 4, 8, and 14 wk after the beginning of the TAChR immunization. In agreement with previous reports (9), we found substantial variations in the concentration of anti-TAChR Ab from mouse to mouse that are reflected in the large SDs of the values in Fig. 3⇓. The average concentration of serum anti-TAChR Ab was similar for all three groups. We obtained similar results when we used the sera of the IFN-γ^−/− and WT mice illustrated in Fig. 2⇑. After three immunizations with TAChR, WT and IFN-γ^−/− mice had average serum concentrations of anti-TAChR Ab of 2.8 ± 1.1 and 2.1 ± 0.7 μM: the difference was not statistically significant.

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FIGURE 3.

IFN-γ^−/− and IL-12^−/− mutants had similar concentrations of anti-TAChR Ab as WT mice. Average anti-TAChR Ab concentration (±SD) measured by RIPA in the sera of five WT mice, five IFN-γ^−/−, and four IL-12^−/− mutants immunized with TAChR. We obtained the sera at 4, 8, and 14 weeks after the beginning of the TAChR immunization, as indicated along the abscissa. The mice had received three TAChR injections (at weeks 0, 4, and 8). See text for experimental details.

Differential effects of IFN-γ and IL-12 deficiency on the synthesis of Th1-driven IgG subclasses

We measured by ELISA the serum concentration of anti-TAChR IgG subclasses driven by different cytokines. WT mice synthesized primarily anti-TAChR IgG2b and IgG2c, to a lesser extent IgG1 (Fig. 4⇓), while IgG3 was undetectable (data not shown); the anti-IgG2a mAb yielded a very small signal that likely resulted from cross-reactivity with the highly homologous IgG2c (Fig. 4⇓). IFN-γ^−/− and IL-12^−/− mice had anti-TAChR IgG1 in amounts similar to those of the WT mice, whereas their anti-TAChR IgG2c (and IgG2a) was strongly reduced: in both strains, they were almost undetectable even 14 wk after beginning the TAChR immunization (Fig. 4⇓). Absence of IFN-γ or IL-12 had different effects on synthesis of anti-TAChR IgG2b. They were significantly reduced in IL-12^−/− mice, whereas in IFN-γ^−/− mice their serum concentration was comparable to that of WT mice. IFN-γ^−/− mice had a slight reduction in anti-TAChR IgG2b at week 14, which was not significant as compared with WT mice.

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FIGURE 4.

Concentration of different anti-TAChR IgG subclasses in the sera of WT, IFN-γ^−/−, and IL-12^−/− mice. The mice had received three TAChR injections (at weeks 0, 4, and 8). The sera were obtained 8 and 14 wk after beginning of the TAChR immunization, as indicated below the panels. The columns represent the average ± SD of triplicate ELISA determinations using sera from individual mice. The asterisks represent significant decreases in the average concentration in a given group as compared with the WT mice (∗, p < 0.025; ∗∗, p < 0.005, ∗∗∗, p < 0.00001). See text for experimental details.

The data reported in Fig. 4⇑ suggest that the overall serum concentration of anti-TAChR IgG was higher in WT mice than in the cytokine mutants. On the other hand, the RIPA experiments indicated that all strains had similar amounts of serum anti-TAChR Ab. To identify reasons for this discrepancy, we determined by ELISA the concentration of anti-TAChR IgG, IgM, and IgE in the mouse sera. They all had large amounts of anti-TAChR IgG, although IFN-γ^−/− and IL-12^−/− mice had less IgG than WT mice: after three TAChR injections, the average total IgG in IFN-γ^−/− and IL-12^−/− mice was 50 and 40%, respectively, of the average total IgG in WT mice. None of the strains had measurable serum anti-TAChR IgM or IgE.

IFN-γ^−/− and WT mice immunized with TAChR have IgG2b and complement at the neuromuscular junction, whereas IL-12^−/− mice do not

We investigated whether the different susceptibility to EMG of these strains was related to the ability of the anti-TAChR Ab to bind to muscle and fix complement.

We measured by RIPA the amounts of mouse muscle AChR that was bound to Ab in three WT mice, four IFN-γ^−/− mice, and three IL-12^−/− mice immunized three times with TAChR and boosted with a fourth injection 5–7 days before euthanasia. We found comparable amounts of Ab-complexed AChR in all three groups (Fig. 5⇓A). Thus, the EMG resistance of the IL-12^−/− mice was not due to inability of their anti-TAChR Ab to bind mouse muscle AChR.

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FIGURE 5.

A, Concentration of muscle AChR complexed by Ab in the muscle of mice that had been immunized four times with TAChR as described above (at weeks 0, 4, 8, and 14), measured by RIPA. The mice had been sacrificed 5–7 days after the last TAChR immunization. See text for experimental details. B, Presence of mouse IgG and complement at the neuromuscular junction of IFN-γ^{−[supb]/[supb]−} and IL-12^−/− and WT mice. Muscle sections from mice of the different strains, as indicated at the right, were immunized with TAChR as described above and sacrificed 5–7 days after the last TAChR immunization. The sections were stained for mouse IgG (blue fluorescence) and the C3 component of complement (green fluorescence) as indicated below the panels. We localized the AChR at the neuromuscular synapses using triple immunofluorescence staining with α-BTX (red fluorescence) as indicated below the panels. Original magnification, ×1000. See text for experimental details. C, Presence of IgG2b and 2c at the neuromuscular junction of IFN-γ^−/− and IL-12^−/− and WT mice. Muscle sections from mice of the different strains, as indicated at the right, were immunized with TAChR as described above and sacrificed 5–7 days after the last TAChR immunization. The sections were stained for mouse IgG2b or IgG2c (blue fluorescence) as indicated below the panels. We localized the AChR neuromuscular synapses using binding of α-BTX (red fluorescence) as indicated below the panels. Original magnification, ×1000. See text for experimental details.

We used immunofluorescence to determine the presence of mouse IgG and complement at the neuromuscular junctions of three WT, three IFN-γ^−/−, and three IL-12^−/− mice immunized with TAChR. For each mouse, we analyzed at least 10 muscle sections in which we could identify neuromuscular junctions by α-BTX binding (red fluorescence). We identified mouse IgG and the C3 complement component by specific fluorescent Ab (blue and green fluorescence, respectively). Fig. 5⇑B shows the results of representative sections for each strain. All mice had IgG bound to the neuromuscular junctions. All WT and IFN-γ^−/− mice also had complement, whereas we could not detect the presence of complement in any muscle section from the IL-12^−/− mice.

We investigated whether the presence of complement at the neuromuscular junctions correlated with binding of complement-fixing IgG subclasses. We stained muscle sections of a mouse from each strain with α-BTX and with mAbs specific for IgG2b and IgG2c. Other sections of the same mice were stained for total IgG, IgM, and complement. All mice had IgG and none had IgM bound to the junctions; only WT and IFN-γ mice had complement. When stained for the different IgG2 subclasses, the neuromuscular junctions of the WT mouse stained for both IgG2b and IgG2c, those of the IFN-γ^−/− mouse stained only for IgG2b, whereas those of the IL-12^−/− mouse did not stain for either IgG2b or IgG2c (Fig. 5⇑C).

CD4⁺ T cell response to TAChR and TAChR epitopes in WT, IFN-γ^−/−, and IL-12^−/− mice

CD4⁺ cells of mice of B6 background immunized with TAChR recognize primarily an epitope or overlapping epitopes within the sequence region 146–169 of the TAChR α subunit (15, 33, 37, 38, 41, 42), and less intensely and consistently epitopes within the sequences 181–200 and 360–378 of the TAChR α subunit (33) and other minor epitopes (38). We used CD8⁺-depleted splenocytes from WT, IFN-γ^−/−, and IL-12^−/− mice immunized three times with TAChR and boosted with another TAChR injection 5–7 days before being euthanized to determine their proliferative response to the TAChR and to overlapping synthetic peptides spanning the sequence of the TAChR α subunit. We conducted two independent experiments that yielded consistent results. Fig. 6⇓ illustrates the results of one experiment. All strains recognized the TAChR vigorously and to comparable extents. They all recognized the peptides spanning the sequence region 146–169 and peptide 360–378. Other peptides were recognized inconsistently. In the experiment depicted in Fig. 6⇓, WT mice also recognized the sequence α30–47, IFN-γ^−/− mice also recognized peptides α106–122 and α181–200, and IL-12^−/− mice also recognized the peptides spanning the sequence α30–60 and α420–438.

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FIGURE 6.

CD8⁺-depleted spleen cells from WT mice and from the IFN-γ^−/− and IL-12^−/− mutants immunized with TAChR have similar proliferative responses to the TAChR and recognize a similar epitope repertoire on the TAChR α subunit. Responses of CD8⁺-depleted spleen cells from mice of the different strains, as indicated inside the graphs, to the TAChR and individual overlapping synthetic peptides screening the TAChR α subunit sequence, as indicated at the bottom of the panels. The mice had been immunized with TAChR at weeks 0, 4, and 8 and had received a further boost 5–7 days before being sacrificed. The columns represent average cpm ± SD of triplicate cultures. The baseline values obtained in the absence of any stimulus or in the presence of a synthetic 20-residue peptide unrelated to the TAChR sequence have been subtracted and they were: for the WT mice, 8602 ± 503 cpm; for the IFN-γ^−/− mice, 4247 ± 1717 cpm; and for the IL-12^−/− mice, 1679 ± 222 cpm. See text for experimental details.

Cytokine secretion by anti-TAChR CD8⁺-depleted spleen cells

Stimulation of modulatory Th2 or Th3 cells, which depend upon IL-4 for their development, is a possible mechanism of protection from EMG. We investigated whether an increased anti-TAChR sensitization of Th2 cells correlated with resistance to EMG by measuring the IL-4 and IL-10 secreted by CD8⁺-depleted spleen cells challenged in vitro with TAChR. We determined also the secretion of IFN-γ and IL-2 in the same cultures. We used CD8⁺-depleted splenocytes from mice immunized three times with TAChR and boosted with a fourth injection 5–7 days before the experiments. We cultured the cells with 10 μg/ml of TAChR and measured the cytokine concentrations in the culture supernatants. Fig. 7⇓ summarizes the results of one of two consistent experiments. The cells from IFN-γ^−/− mice secreted IL-4 and IL-10 in amounts comparable to those observed for the cells from the WT mice; they did not secrete IFN-γ and secreted moderate, yet significantly reduced amounts of IL-2 as compared with WT mice. The cells from IL-12^−/− mice secreted much more IL-4 and IL-10 than those from either WT or IFN-γ^−/− mice; they secreted IL-2 in amounts that were slightly, but not significantly, reduced as compared with WT mice and significantly less IFN-γ than the WT mice.

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FIGURE 7.

Cytokine secretion by CD8⁺-depleted splenocytes from WT, IFN-γ^−/−, and IL-12^−/− mice immunized with TAChR after challenge in vitro with TAChR. IFN-γ and IL-2 (top panels) and IL-4 and IL-10 (bottom panels) in the supernatant of cultures of CD8⁺-depleted splenocytes from WT (□), IFN-γ^−/− (▨), and IL-12^−/− (▪) mice. The mice had been immunized with TAChR at weeks 0, 4, and 8 and had received a further boost 5–7 days before being sacrificed. The columns represent the average ± SD of duplicate ELISA determinations in the supernatant of cells cultivated in the presence of 10 μg/ml TAChR. The spontaneous secretion of the different cytokines by cells cultivated in the absence of any stimulus was subtracted. The asterisks indicate a significant difference in the secretion of cytokines by the cells of the knockout mutants as compared with the secretion by the cells from WT mice (∗, p < 0.03; ∗∗, p < 0.02; ∗∗∗, p < 0.01, ∗∗∗∗, p < 0.002). See text for experimental details.