Female C57BL/6J mice (8 weeks old; Jackson Laboratories Japan, Inc., Kanagawa, Japan) were used. All mice were fed ordinary laboratory chow and allowed free access to water under a consistent 12-h light/dark cycle. All animal procedures were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals at Chugai Pharmaceutical Co., Ltd., and all experimental protocols were approved by the Animal Care Committee of the institution and conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.

Torpedo AChR was purified from electroplaque tissue of Torpedo californica as described in Wu et al. (19), with minor modifications. On Day 0, mice were immunized by subcutaneous inoculation at a hind footpad and a shoulder with 100 μg of the purified AChR emulsified in complete Freund’s adjuvant (CFA) supplemented with Mycobacterium tuberculosis extract H37Ra (231141; Becton Dickinson, Franklin Lakes, NJ, United States). Control mice were treated with complete Freund’s adjuvant and phosphate-buffered saline (PBS) alone. Four weeks after the initial inoculation, the mice received a booster inoculation at a shoulder and the base of the tail with the same amount of AChR in incomplete Freund’s adjuvant (263910; Becton Dickinson). The control mice received a similar booster without AChR. Muscle strength was evaluated for 8 weeks after the first inoculation once a week. And tibialis anterior muscle, serum and inguinal lymph nodes were harvested for immunohistochemistry, electrochemiluminescence immunoassay, real-time PCR analysis 4 weeks after booster inoculation. On 1 week after booster inoculation, inguinal lymph nodes were harvested for flow cytometric analysis.

Rat anti-mouse IL-6 receptor antibody (MR16-1) was prepared using a hybridoma established in our laboratory (17). Mice were intraperitoneally administered MR16-1 (8 mg/mouse) or vehicle (PBS) just before the first and the booster inoculation. In addition, the mice were administered MR16-1 (0.5 mg/mouse) or vehicle once a week, excluding the immunization weeks, for the duration of the experiment.

Grip strength assessment was conducted in principle as described in Mori et al. (20). Forelimb muscle strength was measured with an MK-380CM/R grip-strength meter (Muromachi Kikai, Tokyo, Japan). Grip strength was evaluated after mice had made 30 consecutive paw grips to the top of their steel grid cage. Seven measurements were performed per mouse; five of the seven measurements (excluding the maximum and minimum) were used for statistical analysis.

Frozen slices of tibialis anterior muscle were prepared as follows. Mice were euthanized under anesthesia with isoflurane, and their tibialis anterior muscles were removed. Samples were embedded in optimal cutting temperature compound (45833; Sakura Finetek, Tokyo, Japan). Slices were fixed with 4% paraformaldehyde (09154-85; Nacalai Tesque Inc., Kyoto, Japan) for 10 min at room temperature and then stained by overnight incubation at 4°C with alpha-bungarotoxin conjugated to Alexa Fluor 555 (B35451; Thermo Fisher Scientific, Waltham, MA, United States) and with goat anti-mouse IgG H&L conjugated to Alexa Fluor 488 (ab150117; Abcam, Cambridge, United States).

Whole-slide images were obtained with a NanoZoomer S60 digital slide scanner (Hamamatsu Photonics, Shizuoka, Japan). Mouse IgG-positive and AChR-positive areas were quantified by using HALO AI imaging analysis software (v3.4.2986.209; Indica labs, NM, United States).

Mice were anesthetized with isoflurane and blood was collected from the inferior vena cava. Electrochemiluminescence (ECL) assay for the detection of anti-mouse AChR autoantibodies was conducted as follows. Mouse AChR was purified from the muscle of mice that had been used in other studies. Biotinylated alpha-bungarotoxin (B1196; Thermo Fisher Scientific) was immobilized onto 96-well streptavidin-coated plates (L15SA-1; Meso Scale Discovery, Rockville, MD, United States) by incubating for 1 h at room temperature. The plates were then washed 3 times with PBS-T (T9183; Takara Bio Inc., Shiga, Japan). After washing, purified mouse AChR was added to each well and incubated for 1 h at room temperature. After 3 washes, diluted serum was added to each well and incubated for 1 h at room temperature. After 3 washes, SULFO-TAG labeled goat anti mouse IgG H&L (R32AC-1; Meso Scale Discovery) was added and incubated for 1 h at room temperature. Staining with the following secondary antibodies was used to measure IgG isotypes: sulfo-tag conjugated goat anti-mouse IgG1 (D22JL-2; Meso Scale Discovery), sulfo-tag conjugated goat anti-mouse IgG2a (D22JM-2; Meso Scale Discovery), sulfo-tag conjugated goat anti-mouse IgG2b (D22JN-2; Meso Scale Discovery), and sulfo-tag conjugated goat anti-mouse IgG3 (D22JO-2; Meso Scale Discovery). After washing, Read Buffer T (R92TC-1; Meso Scale Discovery) was added. Light intensity was measured with a MESO QuickPlex SQ 120MM Reader (Meso Scale Discovery).

Lymph nodes were passed through 40 μm cell strainers to obtain single cell suspensions. For Th1 and Th17 analysis, cells were stimulated for 4 h in RPMI 1640 (R8758; SIGMA, St. Louis, MO, United States) containing 10% FBS (F2442; SIGMA), 55 μM 2-Mercaptoethanol (21985–023; Thermo Fisher Scientific), 10 mM HEPES (H0887; SIGMA), 1 mM Sodium pyruvate (11360–070; Thermo Fisher Scientific) and 100 U /ml Penicillin–Streptomycin (15140-122; Thermo Fisher Scientific) with cell stimulation cocktail containing phorbol 12-myristate 13-acetate and ionomycin (00-4970; Thermo Fisher Scientific) in the presence of 0.1% of BD GolgiPlug (555028; Becton Dickinson). Mononuclear cells were incubated with Mouse BD Fc Block (553142; Becton Dickinson) before staining. The cells were initially stained with the antibody of FITC-conjugated anti-CD4 (100510, BioLegend, San Diego, CA, United States), and then intracellular stained using the antibodies of PE-conjugated anti-IL-17A (506904, BioLegend), and APC-conjugated anti-IFN-γ (505810, BioLegend) performed with the Fixation/Permeabilization Solution Kit with BD GolgiPlug (555028; Becton Dickinson) according to the manufacturer’s protocol. For T_FH and Treg cell analysis, mononuclear cells were incubated with Mouse BD Fc Block before staining. The cells stained with the antibodies of FITC conjugated anti-CD4, biotinylated anti-CXCR5 (13-7185-82; Thermo Fisher Scientific), BV786-conjugated anti-CD25 (564023; Becton Dickinson), PE/Cyanine7 conjugated anti PD-1 (109110; BioLegend), and then APC-streptavidin (405207; BioLegend) was utilized to detect biotinylated primary antibody. Intracellular stained using the antibodies of PE-conjugated anti Foxp3 (12-5773-80B; Thermo Fisher Scientific) performed with the Anti-Mouse/Rat Foxp3 Staining Set (72-5775-40; Thermo Fisher Scientific) according to the manufacturer’s protocol. For B and plasma cells analysis, mononuclear cells were incubated with Mouse BD Fc Block before staining. The cells were stained with the antibody of FITC conjugated anti-B220 (553088; Becton Dickinson), BV605 conjugated anti-CD19 (115540; BioLegend), APC conjugated anti-CD138 (558626; Becton Dickinson), PE conjugated anti-TACI (133403, BioLegend). Data were acquired using BD LSRFortessa X-20 (Becton Dickinson) and analyzed using FlowJo 10.5.3 (FlowJo, Ashland, OR, United States).

Total RNA was isolated from the inguinal lymph nodes by using an RNeasy mini kit (74106; Qiagen, Venlo, Netherlands). TaqMan quantitative real-time PCR (qRT-PCR) was performed with TaqMan Gene Expression Assays for Il21 (Mm00517640_m1), Bcl6 (Mm00477633_m1), Prdm1 (Mm00476128_m1), Irf4 (Mm00516431_m1), and Xbp1 (Mm00457357_m1) in QuantStudio 7 Pro (Thermo Fisher Scientific). Gene expression was normalized to the endogenous control, GAPDH (4352661; Thermo Fisher Scientific).

All data are expressed as mean and SD. The n values refer to the number of individual animals in each group on which experiments were performed. The statistical significance of differences was determined by using Tukey’s multiple comparison test or Steel–Dwass’s multiple comparison test for comparison of multiple groups. Two-way ANOVA was used for comparison of time course data. Statistical significance was defined as a p-value of <0.05. Statistical analyses were performed using JMP version 15.0.0 software (SAS Institute, Cary, NC, United States).

Grip strength was significantly lower in AChR-immunized mice than in CFA-treated control mice. MR16-1 significantly improved muscle weakness in AChR-immunized mice compared with vehicle-treated AChR-immunized mice (Figure 1).

These results indicate that MR16-1 significantly improved muscle weakness in the EAMG mouse model.

Neuromuscular transmission failure in MG is caused by AChR autoantibodies binding to AChR at the neuromuscular junction (3). Therefore, we investigated whether IL-6R antibody treatment prevented the deposition of AChR autoantibodies in the muscle of EAMG mice. In the tibialis anterior muscle of CFA-treated control mice, there was no co-localization of AChR and mouse IgG (Figure 2A). In AChR-immunized mice, in contrast, co-localization of AChR and mouse IgG was detected, which suggests that AChR autoantibodies were deposited at the neuromuscular junction (Figure 2A). In AChR-immunized mice treated with MR16-1, the area of mouse IgG-positive tissue as a percentage of the area of AChR-positive tissue was lower than that in AChR-immunized mice treated with vehicle alone (Figure 2B).

These results suggest that IL-6R antibody treatment improved the muscle weakness in EAMG mice by decreasing the deposition of AChR autoantibodies in the neuromuscular junction.

Next, we investigated whether IL-6R antibody treatment suppressed the production of AChR autoantibodies. Serum samples were collected at 8 weeks after first inoculation. Levels of AChR autoantibodies measured by ECL were significantly higher in serum from AChR-immunized mice than from CFA-treated control mice. Administration of MR16-1 lowered the levels of AChR autoantibodies in serum of AChR-immunized mice (Figure 3A). Examination of IgG subclasses revealed that MR16-1 lowered the levels of IgG2b subclass in serum of AChR-immunized mice (Figure 3B).

These results indicate that MR16-1 significantly reduced production of AChR autoantibodies in EAMG mice.

T_FH cells are critical in germinal center formation and antibody production (21). It has been previously reported that the proportion of T_FH cells among CD4⁺ T cells is higher in EAMG rats than in control rats (22). In addition, it has been reported that IL-6 is involved in T_FH differentiation (10). Therefore, we investigated whether anti-IL-6R antibody suppressed T_FH cells related gene expression in lymph nodes. In MR16-1-treated mice, expression of B cell lymphoma 6 (Bcl6), a transcription factor for T_FH differentiation, and IL-21, a cytokine produced from T_FH, were lower than AChR-immunized mice. In addition, expression levels of transcription factors for formation of plasma cells, such as Prdm1, Irf4, and Xbp1 were also lower in the MR16-1-treated mice (Supplementary Figure S1). Accordingly, we investigated whether the treatment with IL-6R antibody could suppress T_FH cells and plasma cells in lymph nodes. At 1 week post the second inoculation, we harvested the inguinal lymph nodes and confirmed the population of T and B cells. In AChR-immunized mice, the percentage of T_FH, Th1, Th17, and Treg cells among CD4 positive cells were higher than in naive mice (Figures 4A–D). T_FH and Th17 cells were lower in MR16-1-treated mice compared with AChR-immunized mice (Figures 4A,C). However, the administration of MR16-1 did not affect Th1 cells (Figure 4B). Although Treg cells were lower in mice treated with MR16-1 compared with AChR-immunized mice, the Th17/Treg ratio also decreased (Figures 4D,E). We further investigated the effect of MR16-1 on B cells and plasma cells. In AChR-immunized mice, the percentage of B cells and plasma cells was higher than in naive mice (Figures 4F,G). The proportion of plasma cells was lowered in MR16-1-treated mice, although there was no difference in the proportion of B cells (Figures 4F,G).

In conclusion, our findings suggest that the administration of MR16-1 effectively reduces the proportion of T_FH, plasma cells and Th17/Treg balance in the lymph nodes, potentially elucidating a novel mechanism of immune modulation in MG.