Autoantibodies to the muscle acetylcholine receptor (AChR) cause the symptoms of human and experimental myasthenia gravis (EMG). AChR-specific CD4+ T cells permit development of these diseases, but the role(s) of the Th1 and Th2 subsets is unclear. The STAT4 and STAT6 proteins, which mediate intracellular cytokine signaling, are important for differentiation of Th1 and Th2 cells, respectively. Wild-type (WT) BALB/c mice, which are prone to develop Th2 rather than Th1 responses to Ag, are resistant to EMG. We have examined the role of Th1 and Th2 cells in EMG using STAT4 (STAT4−/−)- or STAT6 (STAT6−/−)-deficient BALB/c mice. After AChR immunization, STAT6−/− mice were susceptible to EMG: they developed more serum anti-AChR Ab, and had more complement-fixing anti-AChR IgG2a and 2b and less IgG1 than WT or STAT4−/− mice. The susceptibility to EMG of STAT6−/− mice is most likely related to the Th1 cell-induced synthesis of anti-AChR Ab, which trigger complement-mediated destruction of the neuromuscular junction. CD4+ T cells of the STAT6−/− mice had proliferative responses to the AChR comparable to those of WT and STAT4−/− mice, and recognized similar AChR epitopes. STAT6−/− mice had abundant AChR-specific Th1 cells, which were nearly absent in WT and STAT4−/− mice. Spleen and lymph nodes from STAT6−/− mice contained cells that secreted IL-4 when cultured with AChR: these are most likely STAT6-independent cells, stimulated in a non-Ag-specific manner by the cytokines secreted by AChR-specific Th1 cells.
Myasthenia gravis (MG) 4 is an autoimmune disease characterized by excessive muscle weakness and fatigue (1). The target of this autoimmune response is the acetylcholine receptor (AChR) at the neuromuscular junction (NMJ): binding of high affinity anti-AChR Ab to the postsynaptic membrane causes AChR loss and complement-mediated destruction of the NMJ and impaired neuromuscular transmission (1, 2).
Experimental MG (EMG), the animal model of MG, can be induced in mice by multiple immunizations with AChR purified from the electric organ of Torpedo fish (TAChR): the anti-TAChR Ab cross-react with the mouse AChR and cause myasthenic symptoms (1, 2).
Synthesis of pathogenic high affinity anti-AChR Ab requires AChR-specific CD4+ T cells (1, 3, 4, 5, 6). However, the role of the different CD4+ T cell subclasses and cytokines in MG and EMG is complex, and still unclear. Th1 cells produce proinflammatory cytokines (e.g., IL-2, IL-18, and IFN-γ), which are important for cell-mediated immune responses and production of IgG subclasses that bind complement (7, 8). Th2 cells produce anti-inflammatory cytokines (e.g., IL-4 and IL-10), which may down-regulate immune responses, either directly or by acting as growth and differentiation factors for other subset of regulatory CD4+ T cells (7, 8). Th2 cells are important in the Ab responses, because they induce synthesis of IgA, IgE, and IgG subclasses that do not fix complement (7, 8). Th1 cells have a pathogenic role in MG and EMG (e.g.,1, 9, 10, 11, 12); in contrast, Th2 cells may have a protective role (13, 14, 15). However, IL-10 facilitate EMG development (16, 17).
The STAT proteins mediate the intracellular signaling triggered by the binding of cytokines to their receptors (18, 19, 20). STAT4 and STAT6 regulate the differentiation of Th1 and Th2 cells, respectively, after interaction of IL-12 or IL-4 with the naive CD4+ T cells (21). In STAT4−/− mice, all functions stimulated by IL-12 binding are impaired, including production of IFN-γ by Th1 cells and enhancement of NK cell cytotoxicity (21). In contrast, STAT6−/− mice have defective IL-4-mediated functions, such as B cell proliferation, Th2 cell development, and secretion of IgE (21).
BALB/c mice are resistant to EMG induction (22, 23, 24). Their resistance has been attributed to their H-2d haplotype, which appears to correlate with a reduced propensity to EMG. However, their propensity to develop strong Th2 and weak Th1 responses to Ag may have a role in their EMG resistance.
In this study, we have examined the effects of reduced Th1 or Th2 responses in the induction of EMG, using BALB/c mice genetically deficient in STAT4 and STAT6 proteins.
Materials and Methods
STAT4−/− (backcross generation N10), STAT6−/− (backcross generation N6), and wild-type (WT) BALB/c mice, purchased from The Jackson Laboratory (Bar Harbor, ME), were bred at the animal facility of the University of Minnesota.
TAChR, purified as described previously (25), was stored at −80°C. We diluted the TAChR in PBS for mice immunizations, and in RPMI 1640 and sterilized it by UV irradiation for proliferation assays. For the proliferation assays, we also used synthetic peptides, ∼20 residues long and overlapping by 5–8 residues, which spanned the sequence of the TAChR α subunit. The peptide codes include the symbol α and two numbers referring to the position of the first and last peptide residue on the α subunit sequence. We have described the synthesis and characterization of the peptides previously (25).
Female WT, STAT6−/−, and STAT4−/− BALB/c mice (8–10 wk old) were immunized by multiple s.c. injections with solubilized TAChR (25 μg/mouse) in 100 μl of PBS, emulsified in an equal volume of CFA (10, 13, 14, 15, 16). We boosted the mice 4 and 8 wk after the first immunization and 5–7 days before sacrifice, using the same amount of TAChR in IFA.
Evaluation of EMG symptoms
We tested the mice every 2 wk for appearance of myasthenic weakness, using a pancuronium-sensitized forced exercise test that we described previously (e.g.,10, 13, 14, 15, 16). The mice received 0.03 mg/kg pancuronium bromide i.p., and were forced to hang on an inverted grid: the time it takes for the mouse to fall three times (holding time) is a reliable indicator of the extent of their weakness. The myasthenic nature of the weakness is verified by administering edrophonium chloride (Reversol; Organon, West Orange, NJ), a cholinesterase inhibitor that causes an immediate increase in the holding time of mice with EMG. The average holding time of 285 naive B6 mice was 11.4 ± 1.55 min (10, 16), and it is comparable to that observed in this study for naive BALB/c mice. We considered healthy (clinical score = 0) mice with holding times of 8.3 min (the average holding time of healthy mice minus 2 SD) or more. Mild EMG (clinical score = 1) corresponded to holding times between 8.3 and 6.75 min (the average holding time of healthy mice minus 3 SD); moderate and severe EMG (clinical score = 2 and 3, respectively) corresponded to holding times between 6.75 and 5.2 min, or less than 5.2 min (the average holding time of healthy mice minus 4 SD), respectively. We assigned a holding time of 0 (clinical score = 4) to mice that died because of EMG, or that were paralyzed in the absence of pancuronium.
Assay of serum anti-TAChR and anti-mouse AChR Ab
We measured the anti-TAChR and anti-mouse AChR IgG in sera collected 12 wk after the first immunization, using a radioimmunoprecipitation assay. We used TAChR solubilized with Triton X-100 and labeled by the binding with 125I-labeled α bungarotoxin (125I-αBTX) to determine the concentration of anti-TAChR IgG. 125I-αBTX was radiolabeled, as described previously (26). We set up the assay in 96-well Immulon-4 Removawell plates (Dynex Technologies, Chantilly, VA), blocked with 2% BSA in PBS. Increasing amounts of the test serum (from 0.05 to 0.2 μl; each volume set up in triplicate samples) were incubated with the 125I-αBTX-labeled TAChR. Naive BALB/c mouse serum was added to each sample, to reach a final volume of 0.2 μl of mouse serum/well. Triplicate tubes containing only naive mouse serum were used to determine the background value. The TAChR/Ab complexes were precipitated by 20 μl Zysorbin (Zymed Laboratories, San Francisco, CA). The resulting pellets were counted for radioactivity in a gamma 5500 counter (Beckman Coulter, Palo Alto, CA). The linear part of the resulting binding curve was analyzed for linear regression fit, using the program Cricket Graph (Computer Associated International, Islandia, NY). Curves yielding a correlation coefficient lower than 0.8 were disregarded. The concentration of anti-TAChR Ab was calculated from the slope of the binding curve, as described previously (26). We express the Ab concentration as μM of precipitated 125I-αBTX binding sites (16). We measured the concentration of IgG that bound mouse AChR using the same assay, and Triton X-100 extract of muscle from naive BALB/c WT mice as the source of mouse AChR. We express the Ab concentration as nM of precipitated 125I-αBTX binding sites.
Assay of serum anti-TAChR IgG subclasses
We measured the anti-TAChR IgG subclasses in sera obtained 12 wk after the first immunization, using an ELISA that we described in detail previously (16). We used biotinylated anti-mouse IgG subclass Ab (BD PharMingen, San Jose, CA) and avidin HRP (Sigma-Aldrich, St. Louis, MO) as the revealing system. We measured the absorbance at 405 nm with an automated microplate reader ELx800 (Bio-Tek Instruments, Winooski, VT). We deduced the serum concentration of anti-TAChR IgG subclasses from standard curves obtained by coating ELISA plates with increasing amounts of purified mouse IgG1, IgG2a, and IgG2b (Sigma-Aldrich) in 100 μl/well of coating buffer, and detecting their presence using biotinylated anti-mouse IgG subclass and avidin HRP, as described above. Ideal standard curves would have required the use of purified anti-TAChR IgG subclasses, bound to the TAChR coating the plate. Thus, the serum concentrations of IgG subclasses reported in this study are not absolute values. However, our standard curves served well as internal standards, to compare results obtained in different mice and experiments.
We euthanized the mice 5–7 days after the last immunization. We pooled splenocytes and lymph node cells from three identically treated mice, and depleted from them the CD8+ T cells using rat anti-mouse CD8 Ab (BD PharMingen) and paramagnetic beads coated with goat anti-rat IgG, as described previously (e.g.,10, 15, 16). We used the resulting cell population, which is enriched in CD4+ T cells (CD4+-enriched cells) in 4-day proliferation assays, using as Ag the individual peptides spanning the TAChR α subunit sequence (10 μg/ml) and the TAChR (2.5 μg/ml). We used PHA (5 μg/ml) as a positive control. As a negative control, we used cells cultured without any Ag, or in the presence of a 20-residue peptide (10 μg/ml) synthesized by the same method as the AChR peptides, and unrelated to the AChR sequence. We determined the cell proliferation from the incorporation of [3H]thymidine, measured by liquid scintillation. When an Ag induced a significant (p < 0.05) increase in cell proliferation as compared with the negative controls, we calculated the stimulation index (SI: the ratio between the average cpm of cultures in the presence of the Ag, and the average basal proliferation of the same cells). The use of SI normalizes results, and allowed comparison of experiments conducted at different times, or with different mice.
Solid-phase ELISPOT assay to detect Ag-induced IL-4 and IFN-γ secretion
We euthanized the mice 12 wk after the first immunization. We used an ELISPOT assay, which we described previously (15), to test the TAChR- or ConA-induced production of IFN-γ and IL-4 by CD4+-enriched cells, or their purified CD4+ T cells, obtained using the mouse T cell CD4 subset column kit (R&D Systems, Minneapolis, MN). The cells (200,000 CD4+-enriched cells/well; or 80,000 pure CD4+ T cells/well plus 240,000 irradiated speloncytes from naive WT BALB/c mice as APC) were cultured for 48 h in ELISPOT plates (CTL, Cleveland, OH) coated with anti-mouse IL-4 or anti-mouse IFN-γ (BD PharMingen), in the presence of 2.5 μg/ml of TAChR, or of 5 μg/ml of Con A (positive control), or in the absence of any stimulus (negative control). We set up 12 replicate cultures for the TAChR, 3 for Con A, and 9 for the negative controls. We used a biotin-conjugated secondary Ab and streptavidin-conjugated HRP (Vector Laboratories, Burlingame, CA) as revealing system. The development solution contained 800 μl of 100 mg 3-amino-9-ethyl carbazole in 10 ml of N,N-dimethylformamide, 24 ml of 0.2 M acetic acid, 0.2 M sodium acetate, pH 5.0, and 12 μl of 30% H2O2.
We used a two-tailed Student’s t test (Excel; Microsoft, Redmond, WA) for analysis of the proliferative responses, and repeated measure ANOVA (STAT view 5.0.1; SAS Institute, Cary, NC) for analysis of the clinical symptoms. We considered a difference significant when p < 0.05.
STAT6−/− BALB/c mice are susceptive to EMG
We conducted two independent experiments. In each experiment, we immunized groups of WT, STAT6−/−, and STAT4−/− mice. We measured the mouse strength every 14 days for 12 wk, starting on the day before the first immunization (wk 0). In both experiments, the STAT6−/− mice developed more frequent and more severe myasthenic weakness than either the WT or the STAT4−/− mice. At the end of the observation period, in the first experiment all of the STAT6−/− mice, 80% of the WT mice, and 40% of the STAT4−/− mice had EMG; in the second experiment, 55% of the STAT6−/− mice had severe EMG (Fig. 1), whereas only 1 of the 10 WT mice and none of the STAT4−/− mice had myasthenic symptoms. Variable susceptibility to EMG of different groups of mice of the same strain has been reported previously for a variety of strains (1). Three STAT6−/− mice (one in the first and two in the second experiment), but none of the WT or the STAT4−/− mice, died of EMG. Fig. 2 shows the average clinical scores of all the mice used in the two experiments (WT, n = 15; STAT4−/−, n = 15; STAT6−/−, n = 14). STAT4−/− and WT mice had similar clinical score during all the observation period. The repeated measure ANOVA analysis showed a significant difference between the clinical scores of STAT6−/− mice and those of WT or STAT4−/− mice (p < 0.01).
Serum anti-TAChR and anti-mouse AChR Ab
We measured the serum concentration of anti-TAChR and anti-mouse AChR Ab at the end of the observation period (12 wk after the first immunization). The titer of anti-TAChR and anti-mouse AChR Ab did not correlate with the severity of the disease (data not shown). This agrees with several observations, that in BALB/c and in other mouse strains the anti-AChR Ab titer does not correlate with the severity of the symptoms (27). Fig. 3,A shows the serum anti-TAChR Ab concentration of the mice used in both the first and the second experiments: STAT6−/−mice had significantly higher concentrations than either the STAT4−/− or the WT mice (p < 0.001 and p < 0.002, respectively). Fig. 3 B shows the serum anti-mouse AChR Ab concentration of the WT mice used in both experiments, and of the STAT4−/− and STAT6−/− mice used in the second experiment (for the first experiment, we did not have enough serum from the STAT4−/− and STAT6−/− mice to carry out this assay). Seven of 15 WT mice, 3 of 7 STAT6−/− mice, and 1 of the 9 STAT4−/− mice had anti-mouse AChR Ab. The average serum concentration of anti-mouse AChR Ab of STAT6−/− mice was significantly higher than that of the STAT4−/− mice (p < 0.04).
Serum anti-TAChR IgG subclasses
We examined the presence of anti-TAChR IgG1, IgG2a, and IgG2b in the sera of TAChR-immunized STAT6−/−, STAT4−/−, and WT mice, 12 wk after the first immunization. We assayed the sera of all the WT mice used for the first and second experiment, and of the STAT4−/− and STAT6−/− mice used for the second experiment (Fig. 4). As expected from the important role of Th2 cells in inducing IgG1 synthesis, STAT6−/− mice had less serum anti-TAChR IgG1 than STAT4−/− or WT mice (p < 0.004 and p < 0.001, respectively). The concentration of Th1-induced IgG2a and IgG2b was significantly higher in STAT6−/− than in WT or STAT4−/− mice (for IgG2a, p < 0.03 and p < 0.002, respectively; for IgG2b, p < 0.03 and p < 0.003, respectively).
Proliferation of CD4+-enriched cells to TAChR and α subunit peptides
We used CD4+-enriched cells of mice euthanized 5–7 days after the last TAChR boost to carry out proliferation assays. As stimulants we used TAChR and the individual α subunit peptides. The CD4+-enriched cells responded to TAChR in all experiments: the intensity of the proliferative responses was similar in all strains (Fig. 5). The three strains recognized consistently the same α subunit peptides, α1–20 and α304–322. These results agree with previous studies, which identified the sequence α304–322 as the immunodominant epitope in TAChR-immunized BALB/c mice (28). A few other peptides elicited weak, yet significant responses. Fig. 6 shows the results of a representative experiment. Fig. 7 summarizes the average of the SI obtained in three to four independent proliferation assays for each strain: each assay was set up with quadruplicate cultures for each peptide.
CD4+ T cells that secrete IFN-γ or IL-4 in response to the presence of TAChR
We used an ELISPOT assay to examine the production in vitro of IFN-γ and IL-4 by the CD4+-enriched cells in response to the presence of TAChR. We chose IFN-γ and IL-4 as representative Th1 and Th2 cytokines, respectively. Cells grown in the absence of any stimulus served as the negative controls. Fig. 8 shows the results obtained with cells derived from five mice of each group, among the mice used for the second experiment, euthanized 12 wk after the first TAChR immunization. Each mouse was tested independently. All the STAT6−/− mice had abundant cells that secreted IFN-γ and, surprisingly, IL-4, when cultured with the TAChR.
All the WT mice and the STAT4−/− mice had cells that produced IL-4 when cultured with TAChR. We detected TAChR-induced IFN-γ production by the cells of only two WT mouse and three STAT4−/− mice.
We wished to determine whether the IL-4 production we noted in the above experiments when using CD4+-enriched cells from STAT6−/− mice was due to CD4+ T cells, or to other cell types. Toward this aim, we used ELISPOT assays to measure the secretion of IL-4 and IL-5 by a population of pure CD4+ T cells, obtained from STAT6−/− mice sacrificed 2 wk after a TAChR immunization (Fig. 9). The purified CD4+ T cells did not respond to stimulation with TAChR by secreting IL-4 or IL-5. They had increased IL-4 production in response to a nonspecific stimulus such as Con A. The IL-4-producing cells induced by Con A were fewer than those we observed in cultures of purified CD4+ T cells from naive WT mice, stimulated with Con A (data not shown). In cultures of purified CD4+ T cells from STAT6−/− mice, the presence of Con A or TAChR did not cause any increase in the number of cells that secreted IL-4 as compared with the numbers we observed in the absence of any stimulus, confirming the absence of functional Th2 cells. In contrast, purified CD4+ T cells from STAT6−/− mice, when cultured with TAChR or Con A, had significantly higher numbers of IFN-γ-producing cells than when cultured without any stimulus.
The factors that facilitate or hamper the development of MG and EMG are still unknown. Among the genetically determined factors, the MHC class II alleles correlate with susceptibility to MG and EMG, perhaps because they influence the repertoire of the anti-AChR CD4+ T cells. However, also the balance between anti-AChR Th1 and Th2 cells may affect the susceptibility to EMG. Environmental factors can modify the Th1/Th2 balance, and this may be the mechanism by which the environment influences development of autoimmunity. Cytokines have overlapping functions, and their effects on immune responses may differ, depending on the time and place of their action. This makes the elucidation of their role in autoimmune diseases a complicated task.
The BALB/c mouse strains we used in this study are a suitable system to examine the relative importance in the development of EMG of the MHC haplotype, the repertoire of the anti-AChR CD4+ T cells, and the Th1/Th2 balance. The WT mice are relatively resistant to EMG: after multiple TAChR immunizations, only 7–40% of WT mice develop EMG (22, 23). They express the H-2d haplotype, which correlates with resistance to EMG (23, 24, 29). The epitope repertoire of their anti-TAChR CD4+ T cells does not overlap with that of anti-TAChR CD4+ T cells of H-2b strains (like C57BL/6 mice), which usually are susceptible to EMG (1, 22, 23). The EMG resistance of BALB/c mice has been attributed to the constraints imposed by their H-2 class II molecules on the recognition of TAChR epitopes by the CD4+ cells (29, 30). However, BALB/c mice have a propensity to develop Th2 rather than Th1 immune responses, whereas the EMG-susceptible C57BL/6 mice are prone to develop Th1 responses (1, 31). The different EMG susceptibility of these two strains might be caused by their different Th1/Th2 balance, rather than by their different H-2 class II alleles and anti-AChR CD4+ T cell repertoires.
The present results suggest that an imbalance toward an anti-TAChR Th1 response overshadows any protective effect exerted by the H-2d haplotype: the susceptibility to EMG of STAT6−/− mice was much higher than in the parental WT strain or in the STAT4−/− mutants (Fig. 2), even though they all express the same H-2d alleles, and their anti-TAChR CD4+ T cells recognize the same epitope repertoire (Fig. 6 and 7). However, the STAT6−/− mice, but not the WT or the STAT4−/− mice, developed a Th1 sensitization to the TAChR. This is demonstrated by the presence of cells that secreted IFN-γ when challenged in ELISPOT assays with the TAChR, in cultures of CD4+-enriched cells or purified CD4+ T cells from STAT6−/− mice (Figs. 8 and 9). The similar resistance to EMG of STAT4−/− and WT mice suggests that the EMG resistance of WT mice is related to the Th2 polarization of their anti-TAChR responses. That such Th2 polarization occurs in vivo is well exemplified by the profile of the anti-IgG subclasses in the three strains we used; the WT and STAT4−/− strain had significantly higher concentrations of Th2-driven anti-TAChR IgG1 in the serum, and significantly lower serum concentrations of Th1-driven IgG2 anti-TAChR Ab, than the STAT6−/− mice (Fig. 4).
The EMG resistance of STAT4−/− and WT mice does not result from an inadequate TAChR presentation, which might result from a reduced secretion of IFN-γ by Th1 cells and reduced activation of APC, as compared with strains prone to Th1 responses. This is because the proliferative responses to the TAChR of CD4+-enriched cells from STAT4−/− and WT mice were comparable in their intensity to those we observed for the STAT6−/− mice.
These results confirm the importance of Th1-induced, complement-fixing Ab for the development of myasthenic symptoms. Two findings support this conclusion. First, STAT4−/− and WT mice developed a substantial anti-TAChR Ab response, although less intense than the STAT6−/− mice, yet they seldom had EMG symptoms. Second, the STAT6−/− mice synthesized much more complement-fixing IgG subclasses than the parental WT strain and the STAT4−/− mice. The finding of complement components at the NMJ of MG patients, and the altered morphology of the NMJ in MG and chronic EMG first suggested an important role of complement-mediated destruction in the pathogenesis of myasthenic symptoms. The more recent finding that IL-12−/− C57BL/6 mice, which are EMG resistant (10, 32), have IgG, but not complement at their NMJ (10) verified the importance of this mechanism in EMG pathogenesis.
The EMG resistance of STAT4−/− mice, which have a polarized anti-TAChR Th2 response, is consistent with a modest pathogenic potential of anti-TAChR Th2 cells. These results are consistent with those observed in IL-12-deficient C57BL/6 mice, which did not develop EMG despite their substantial CD4+ T cell and Ab response to the TAChR (10).
Although STAT4−/− mice do not have immune functions mediated by IL-12, they produce small amounts of IFN-γ (Fig. 8), which is induced by IL-18 (33, 34) and suffices to activate APC able to prime anti-AChR CD4+ T cells. However, the IFN-γ produced by the STAT4−/− and WT mice was not sufficient to induce a strong production of anti-TAChR IgG subclasses able to fix the complement (Fig. 4) and induce the development of EMG.
When challenged with TAChR, the CD4+-enriched cells from TAChR-immunized STAT6−/− mice included abundant IL-4-secreting cells (Fig. 8). These were not Th2 cells, which are absent in STAT6−/− mice (35): when we used purified CD4+ T cells in the same type of ELISPOT experiments, the presence of TAChR in the culture did not result in any increase in the number of IL-4- or IL-5-secreting cells (Fig. 9). STAT6-mediated signaling is not required for expression of the IL-4 gene: STAT6−/− mice have STAT6-independent cells able to secrete IL-4, such as NK T cells, mast cells, and others (36, 37). The IL-4-secreting cells we observed when STAT6−/− CD4+-enriched cells were cultured with TAChR (Fig. 8) should be non-TAChR specific. They should be cells able to secrete IL-4 by a STAT6-independent mechanism, perhaps by stimulation by the cytokines released by the TAChR-specific Th1 cells (38).
STAT6−/− and STAT4−/− mice have been used to study the role of Th1 and Th2 cytokines in other autoimmune models (39, 40, 41). A previous study showed that absence of STAT4 conferred resistance to insulin-dependent diabetes mellitus induced by infection with the lymphocytic choriomeningitis virus in mice expressing the lymphocytic choriomeningitis virus nucleoprotein in the pancreas β cells (39). STAT4 was necessary for induction of experimental autoimmune encephalomyelitis, whereas lack of STAT6 was associated with high susceptibility to this disease (40). The results of those studies, and the ones we report in this work, confirm the importance of Th1 cells in the pathogenesis of autoimmune diseases.
Also, the data reported in this work emphasize the importance of anti-AChR Th1 cells in the pathogenesis of EMG. They confirm that anti-AChR Th2 cells have a small pathogenic potential, despite their ability to secrete IL-10, a cytokine that might facilitate the pathogenic Ab response in EMG (16). Thus, the anti-AChR Th1 cells are a viable target for immunosuppressive treatment able to down-regulate those cells.
Supported by National Institute of Neurological Disorders and Stroke Grant NS23919 (to B.M.C.-F.).
Abbreviations used in this paper: MG, myasthenia gravis; AChR, acetylcholine receptor; EMG, experimental MG; 125I-αBTX, 125I-labeled α bungarotoxin; NMJ, neuromuscular junction; SI, stimulation index; TAChR, torpedo AChR; WT, wild type.