Normalized circulating Tfh and Th17 associates with improvement in myasthenia gravis treated with ofatumumab

Objective To assess the effect of B cell depletion therapy (BCDT) on circulating T follicular helper (cTfh) and circulating T helper 17 (cTh17) cells and its relation to clinical improvement in patients with myasthenia gravis (MG). Methods 28 anti-AchR positive MG patients treated with ofatumumab and 28 healthy controls (HCs) were included. Frequencies of cTfh and cTh17 cells were monitored by flow cytometry at baseline and 4, and 12 weeks after the initial dose ofatumumab. Serum cytokines associated with cTfh and cTh17, including IL-6, IL-21, and IL-17, were also analyzed. Results The frequency of cTfh and cTh17 significantly increased in MG patients compared with HCs. Additionally, elevated levels of both T-cell subsets correlated with MG severity. During the follow-up, cTfh and cTh17 return to normal after BCDT. Furthermore, the decrease in cTfh and cTh17 was associated with MG scores improvement over time. Notably, cTfh- and cTh17-related cytokines, including IL-6, IL-21, and IL-17, exhibited a marked decrease following ofatumumab therapy. Conclusions Abnormal expansion of cTfh and cTh17 cells may be key features in the immunopathology of MG. Their levels returned to normal after BCDT, which was closely correlated with clinical amelioration. This result suggests that these two T-cell subsets may be targets for BCDT treatment of MG.


Introduction
Myasthenia gravis (MG) is a rare autoimmune disease characterized by fluctuating muscle weakness and fatigue (1).Autoantibodies against postsynaptic membrane components of the neuromuscular junction (NMJ) are present in most patients with MG, with acetylcholine receptor (AChR) antibodies being the most common (1).B-cell depletion therapy (BCDT) with rituximab has been successfully employed in treating generalized MG based on two recent randomized placebo-controlled clinical trials (2,3).However, rituximab does not impair the production of bone marrow B cells or antibodies secreted by long-lived plasma cells (4).Moreover, the anti-AchR antibody tiers are not always correlated with the clinical severity (5,6).Therefore, it is unlikely that disease remissions achieved through BCDT are solely a result of autoantibody depletion.
Increasing evidence showed that rituximab depletes B cells that could potentially interact with T cell subsets involved in autoimmune pathology (antibody-mediated autoimmune).For example, T follicular helper (Tfh) cells are a T cell subset distributed in the germinal center of secondary lymphoid tissues, which plays a central role in promoting B-cell maturation and antibody production (7).Patients with neuromyelitis optica spectrum disorder exhibit a disturbed circulating Tfh (cTfh) cell subset equilibrium that promotes B-cell differentiation (8).However, this imbalance can be rectified by treatment with rituximab (8).T helper (Th) 17 cells represent a distinct subgroup of CD4 + T helper cells known for their ability to produce the signature cytokine IL-17 (9).Research findings have consistently linked Th17 cells to tissue-specific autoimmune inflammatory disorders (9).BCDT was reported to decrease circulating Th17 (cTh17) cells in patients with primary Sjögren syndrome (10).Moreover, reduced levels of cTh17 cells and IL-17 are associated with disease amelioration and declined autoantibody titers (10).
In this study, we quantified the frequencies of cTfh and cTh17 in MG patients on ofatumumab monotherapies.Given that ofatumumab is a fast-acting B-cell depletion agent (11, 12), we hypothesized that the two T-cell subsets would change obviously after applying ofatumumab.Furthermore, we analyzed the relationship between the change in frequency cTfh and cTh17 and clinical improvement in patients with MG after treatment.

Patients
We retrospectively included 28 anti-AchR positive MG patients attending Peking University People's Hospital and 28 age-matched healthy controls (HCs) (Table 1).None of the HCs had a history of disease or received any treatment within the previous 6 months.Detailed past medical history and duration of follow-up for each MG patient was shown in Supplementary Table 1.In MG patients, the administration of ofatumumab involves a subcutaneous dose of 20 mg every 4 weeks following loading doses of 20 mg on days 1, 7, and 14.After starting ofatumumab monotherapy, all participants discontinued their previous immunosuppressive medications.Peripheral whole-blood samples were obtained at baseline, 4 weeks, and 12 weeks after the initial dose for antibody, cytokines, and flow cytometric analysis.At the same time, the clinical severity was evaluated with the Myasthenia Gravis Foundation of America quantitative myasthenia gravis (MGFA-QMG) score, the 15-item Myasthenia Gravis Quality of Life scale (MG-QOL15), and the MG-Related Activities of Daily Living (MG-ADL) score.

Measurement of IL-6, IL-17, IL-21, AChR antibody in serum
The serum was rapidly separated from whole blood by centrifugation at 10,000g for 10 minutes and stored at -80°C.Serum levels of IL-6, IL-17, and IL-21 were determined by an enzyme-linked immunosorbent assay (ELISA) kit (eBioscience) according to the manufacturer's instructions.Serum AChR autoantibody titers were measured using an ELISA kit from RSR Ltd, with positivity defined as ≥ 0.45 nmol/L.

Statistical analyses
Continuous variables are presented as mean [standard deviation (SD)] unless otherwise specified.Categorical variables were shown as numbers (percentages).Mann-Whitney U test was used for analyzing unpaired continuous data, and Pearson's chi-square for categorical data.Wilcoxon signed-rank test was used to assess the difference between before and after ofatumumab treatment.Spearman rank correlation was applied for correlation analysis.A p-value < 0.05 indicated statistical significance.All data were analyzed with IBM SPSS version 22.0 (IBM Corp, Armonk, New York) and GraphPad Prism 9.5 (GraphPad Software Inc., La Jolla CA, USA).

Clinical improvement after ofatumumab treatment
The clinical evaluations using MG scales indicated significant improvement at each visit (Figures 4A-C).Four weeks after the initial dose of ofatumumab, there were significant reductions in the mean values of MG scores: MGFA-QMG decreased by 90.6%, MG-QOL decreased by 87.9%, and MG-ADL decreased by 92.9%.By the 12-week mark, MGFA-QMG, MG-ADL, and MGQOL-15 were further declined compared to baseline, with reductions of 94.7%, 89.3%, and 96.0%, respectively.
Profound depletion of circulating CD19 + B was found in MG patients at both 4 weeks (-98.7%compared to baseline, P < 0.001) and 12 weeks (-98.8%compared to baseline, p < 0.001) after initiating ofatumumab therapy (Figure 4D).Compared with baseline, AChR antibody levels did not decrease significantly at 4 weeks (P = 0.075) but showed a meaningful reduction after 12 weeks (P=0.001)(Figure 4E).Notably, decreases in AChR antibody levels was not correlated with reductions in MG scales at 4 weeks, and it only moderately correlated with improvement in MG-QOL15 (r=0.378,p=0.047) at 12 weeks.

cTfh-and cTh17-related cytokines decreased after ofatumumab therapy
BCDT is associated with a reduction in the production of IL-6, which plays a role in the differentiation of Th17 and Tfh cells (18).Additionally, IL-17 and IL-21 are primarily produced by activated Th17 and Tfh cells, respectively (19, 20).Therefore, we examined the levels of these three cytokines in the serum of patients with MG and HCs.At baseline, there were noticeable differences in the serum levels of IL-6 (21.5 pg/mL vs 5.6 pg/mL, p < 0.001), IL-17 (16.5 pg/ mL vs 3.8 pg/mL, p < 0.001), and IL-21 (43.9 pg/mL vs 4.4 pg/mL, p < 0.001) between MG patients and HCs (Figures 6A, B).However, at the 4-week follow-up, the fold difference in the levels of IL-6 (14.5 pg/mL vs 5.6 pg/mL, p < 0.001), IL-17 (14.9 pg/mL vs 3.8 pg/mL, p < 0.001), and IL-21 (19.5 pg/mL vs 4.4 pg/mL, p < 0.001) between MG patients and HCs were minimized (Figures 6A, B).By the 12week mark, the difference in the levels of the IL-17 (6.5 pg/mL vs 3.8 pg/mL, p = 0.010), IL-21 (8.4 pg/mL vs 4.4 pg/mL, p = 0.010) in MG patients and HCs were further reduced (Figures 6A, B).At the same time point, no difference was found in the levels of IL-6 between MG patients and HCs (4.3 pg/mL vs 5.6 pg/mL, p = 0.198) (Figures 6A, B).

Discussion
In our study, two key points were identified: (1) Patients with MG exhibited abnormal expansion of cTfh and cTh17 cells.Their levels returned to normal after ofatumumab therapy; (2) The restoration of cTfh and cTh17 to normal levels was more associated with clinical improvement in this patient population.Therefore, cTfh and cTh17 cells are potential biomarkers for therapeutic efficacy in MG receiving BCDT.
The quantity of cTh17 cells is increased in thymoma-associated MG.It is positively correlated with the MGFA-QMG score and anti-AChR antibody titer (21).It has been proposed that Th17 cells affect autoantibody production by influencing the balance of Th1and Th2-related cytokines in MG patients (22).In addition, the frequency of cTfh was significantly elevated in patients with MG compared with HC (6,23).Functionally activated cTfh are key features in the immunopathology of MG and was correlated with disease severity (6) and levels of serum anti-AChR antibody (23).Our study also found similar results to those described above (Figures 2A, B, 3A-F).Furthermore, we demonstrated that decreases in cTfh and cTh17 cell percentages were more associated with clinical improvement in MG than the change in serum anti-AchR antibody titer.Taken together, the expansion of cTfh and cTh17 may contribute to the immunopathogenesis of MG.
Interestingly, 12 weeks after initiating ofatumumab therapy, MG patients showed a significant decrease in the frequencies of cTh17 and cTfh cells in our research.This supports the theory that anti-CD20 agents deplete B cells that have potential crosstalk with T cell subsets (10, 24, 25).A significant decrease in the frequency of cTfh cells has been observed in multiple sclerosis patients following BCDT treatment, and this reduction has been associated with disease remission (26).These observations suggest that BCDTs may indirectly deplete potentially pathogenic cTfh cells, highlighting their interdependence with B cells.In addition, ex vivo experiments (about NMOSD) showed that both IL-6 and direct B/cTfh cell contact were essential for the maintenance of cTfh cells.BCDT inhibited the expansion of cTfh cells through ablating IL-6secreting B cells and blocking direct contact (25).To be noted, IL-6 also plays a role in the differentiation of Th17 and Tfh cells (18).Consistently, our findings demonstrated that BCDT resulted in a significant reduction in the serum IL-6, IL-17, and IL-21, with the latter two cytokines being primarily generated by Th17 and Tfh cells.
However, we cannot entirely rule out the possibility that the decrease in cTfh and cTh17 cells was due to the direct clearance effect of the ofatumumab.It is worth noting that, in addition to B cells, there exists a small subset of T cells that express CD20 on their cell surface (27).This is supported by recent research, where ofatumumab demonstrated an effective reduction of peripheral CD20 + T cells in multiple sclerosis (28).Furthermore, in rheumatoid arthritis patients, it has been observed that a higher proportion of cTh17 cells exhibit a CD20 + phenotype compared to healthy controls (29).Overall, these observations suggest that the effects of ofatumumab on both B cells and CD20-expressing T cells should be taken into consideration when interpreting the changes in cTfh and cTh17 cell percentages in our study.Further research is warranted to better understand the specific mechanisms underlying these observations and their implications for clinical outcomes.
This study has several limitations.First, we focused on studying changes in circulating Tfh and Th17 cells in MG patients after BCDT.However, we were unable to detect Tfh cells in lymphoid tissue germinal centers because these cells are difficult to obtain in the routine clinical setting.Second, we did not assess the expression of CD20 markers on cTfh and cTh17 cells.As mentioned previously, it is possible that BCDT depletes specific subpopulations of cTfh and cTh17 cells by binding directly to their surfaces.Third, ficoll isolation was shown to decrease the expression of specific chemokine receptors (e.g., CCR5 and CXCR5) on PBMCs (30,31).By contrast, staining cells at 37°C following ficoll separation or staining whole blood prior to lysing red blood cells may minimized artifacts on the immunophenotyping (31).Thus, in our study, standard ficoll gradient density separation may have compromised the accuracy of identification of cTfh cells.Fourth, some patients had a short corticosteroid withdrawal period before starting BCDT treatment.As corticosteroids can reduce the proportion of circulating Tfh cells (32), we cannot fully eliminate their confounding effect on T-cell phenotype.Finally, this study had a small sample size and a relatively short follow-up period.Large-scale, long-term studies are needed to confirm the value of cTfh and cTh17 cells in monitoring BCDT efficiency in MG.

Conclusion
Abnormal expansion of cTfh and cTh17 cells may be key features in the immunopathology of MG.Their levels returned to normal after BCDT, which was closely correlated with clinical amelioration.This result suggests that these two T-cell subsets may be targets for BCDT treatment of MG.

6
FIGURE 6 Change of serum IL-6, IL-17, and IL-21 after ofatumumab therapy.(A) Comparison of serum levels of IL-6, IL-17 and IL-21 in MG and HC patients at each visit; (B) The heatmap demonstrates the relative changes in serum cytokine levels for each individual with MG, relative to the average cytokine levels observed in the HC group.Each column represents a specific cytokine at every visit, while each row corresponds to an MG patient.The varying intensity of color within each square on the heatmap represents the extent of fold change in cytokine levels.MG, myasthenia gravis; HC, healthy control."ns", "*", and "***" represent not significant, P < 0.05, and P < 0.001, respectively.

TABLE 1
Demographic characteristics of MG patients and HCs.