Understanding: Lymphocyte alterations in MG

Link to paper: https://doi.org/10.1016/j.medj.2025.100987

Dodd, Katherine C., et al. “Lymphocyte Alterations and Elevated Complement Signaling Are Key Features of Refractory Myasthenia Gravis.” Med, vol. 7, no. 100987, Mar. 2026. Elsevier, https://doi.org/10.1016/j.medj.2025.100987.

The first thing to do is to always research and define anything that may seem intimidating, like those huge words in the title.
Lymphocytes are a category of white blood cells that form the backbone of the adaptive immune system. The two major types relevant here are B cells, which produce antibodies, and T cells, which regulate immune responses and directly attack infected or abnormal cells. “Lymphocyte alterations” means the researchers found that the composition and behavior of these cells were abnormal in certain patients.
Complement signaling refers to the complement system, a set of proteins that circulate in the blood and amplify the immune response. When activated, complement proteins tag foreign or damaged material for destruction, recruit inflammatory cells, and can directly punch holes in cell membranes through what is called the membrane attack complex (MAC). “Elevated complement signaling” means that certain complement proteins were found at higher-than-normal levels.
Refractory myasthenia gravis refers to patients with MG whose disease does not adequately respond to standard immunosuppressive treatments. These patients continue to experience significant weakness and disability despite being on corticosteroids, steroid-sparing agents, and sometimes multiple lines of therapy.
Altogether, the title reads: In patients with myasthenia gravis who do not respond to standard treatment, what changes are occurring in their immune cells and complement system that might explain why their disease is so difficult to control?
Background: Disease Characteristics
For the disease characteristics of Myasthenia Gravis, refer to the dedicated MG Disease Characteristics post.
How immune function is measured in practice (the methodology of this paper)
This study used several laboratory and clinical methods to characterize the immune profiles of MG patients at different stages of disease and treatment.

Flow Cytometry

Flow cytometry is the primary technique used throughout this study. It works by passing individual cells in a fluid stream through a laser beam. Before this, cells are labeled with fluorescent antibodies that bind to specific surface or intracellular proteins (called markers). When the laser hits a labeled cell, the fluorescent tags emit light at characteristic wavelengths, and detectors capture the signal. This allows researchers to identify and quantify different cell types and their activation states within a mixed population of blood cells.
In this study, the researchers isolated peripheral blood mononuclear cells (PBMCs) from blood samples. PBMCs include lymphocytes (B cells and T cells), monocytes, and dendritic cells, but exclude red blood cells and granulocytes. Using panels of fluorescent antibodies, they measured the frequencies and expression levels of dozens of markers across B cells, T cells, regulatory T cells (Tregs), dendritic cells, monocytes, and natural killer cells.
For example, to identify memory B cells, they looked at cells expressing CD19 (a general B cell marker), CD24, and CD38 in a specific pattern. To identify regulatory T cells, they looked for CD4+ T cells co-expressing CD25 and the transcription factor FoxP3. Each cell type has a characteristic “signature” of markers that allows researchers to distinguish it from other populations.

Cell Stimulation Assays

To understand not just what immune cells are present but how they behave when activated, the researchers stimulated PBMCs with specific agents that mimic immune activation. They used CD40L (which engages the CD40 receptor on B cells, simulating T cell help), CpG-B (a TLR9 agonist that mimics bacterial DNA), and R848 (a TLR7 agonist that mimics viral RNA). After 48 hours of stimulation, they measured the production of cytokines — signaling molecules that drive inflammation or regulation — including IL-6 (pro-inflammatory, promotes plasma cell differentiation), TNF-α (pro-inflammatory, promotes cell death and inflammation), and IL-10 (anti-inflammatory, associated with regulatory function).
This approach reveals whether the B cells in MG patients are functionally “primed” to produce more inflammatory signals than those in healthy people, even before accounting for their antibody production.

Complement Immunoassays (ELISA)

The researchers measured circulating levels of 15 complement proteins in plasma using enzyme-linked immunosorbent assays (ELISA). ELISA works by coating a plate with an antibody that captures the target protein from the plasma sample. A second detection antibody, linked to an enzyme, binds the captured protein. When a substrate is added, the enzyme produces a color change proportional to the amount of protein present. The intensity of the color is measured by a plate reader.
The complement proteins measured included components from the classical pathway (C1q, C3, C4), the alternative pathway (properdin, Ba, Factor H, and others), and the terminal pathway (C5, C9, clusterin, and the terminal complement complex). This allowed the researchers to build a comprehensive picture of complement activation across different arms of the system.

Clinical Severity Measures

Disease severity was assessed using the MG Composite score, a validated clinical tool that combines examiner-tested items (such as ptosis duration, diplopia, swallowing, speech, grip strength, and respiratory function) into a single numerical score. Quality of life was measured using the MG-QOL-15r, a patient-reported questionnaire covering the physical and psychosocial impact of the disease.

Patient Stratification

A critical feature of this study’s design is how the researchers divided patients into four cohorts based on their treatment status and disease activity, rather than treating all MG patients as a single group. The cohorts were: stable non-immunosuppressed (SNIS, patients well-controlled on only low-dose pyridostigmine), stable immunosuppressed (SIS, patients well-controlled on azathioprine or mycophenolate with low-dose prednisolone), refractory (patients still symptomatic despite immunosuppression, eligible for rituximab), and treatment-naive (recently diagnosed, not yet on immunosuppression). All patients were AChR-antibody positive, and those with thymoma were excluded. Comparing across these groups allowed the researchers to distinguish between immune changes caused by the disease itself versus those caused by treatment.

Principal Component Analysis and Unsupervised Clustering

To determine whether refractory MG patients have a truly distinct immune signature rather than just individual marker differences, the researchers applied principal component analysis (PCA) followed by unsupervised k-means clustering to their flow cytometry data. PCA reduces a large number of variables into a smaller set of “principal components” that capture the most variance in the data. K-means clustering then groups patients based on similarity in this reduced space, without knowing which clinical cohort they belong to. The fact that one cluster was enriched for refractory patients and defined by a consistent set of 16 immune markers provides strong evidence that refractory MG is associated with a distinct immune phenotype, not just random variation.
What the abstract is saying
This study investigated whether patients with refractory MG have a distinct circulating immune profile compared to treatment-responsive patients, treatment-naive patients, and healthy controls. The researchers performed comprehensive immune phenotyping of peripheral blood from 58 patients with AChR-antibody-positive MG and 20 healthy controls.
The key findings were that refractory MG patients had the highest frequency of memory B cells and produced significantly more of the pro-inflammatory cytokines IL-6 and TNF-α when their immune cells were stimulated in the lab. At the same time, these patients showed a dramatic reduction in regulatory T cells and dendritic cells — two cell populations that normally keep immune responses in check.
Refractory MG was also characterized by elevated circulating levels of the complement proteins C3, C5, and clusterin, and by increased expression of complement receptors on lymphocytes. These complement changes correlated with disease severity and quality of life.
In a subset of refractory patients treated with rituximab (a B cell-depleting therapy), the researchers found that patients with very low baseline B cell frequencies (under 3%) responded poorly. After B cell depletion, the remaining circulating B lineage cells were predominantly plasmablasts — antibody-secreting cells that lack the CD20 surface marker that rituximab targets, which is why they survive the treatment.
The authors concluded that refractory MG is associated with a distinct immune signature and suggested that therapies targeting plasma cells, IL-6, complement, or strategies to expand regulatory T cells may be more effective than current approaches for treatment-resistant disease.
What the introduction is saying
The introduction establishes that myasthenia gravis is a highly heterogeneous disease. While about 85% of cases involve antibodies against the acetylcholine receptor, the severity and treatment response vary enormously between patients. A significant proportion remain refractory to standard immunosuppressive therapy, placing a large burden on both patients and healthcare systems.
The authors note a fundamental gap in the field: there are no reliable biomarkers to guide treatment decisions or predict which patients will respond to which therapies. AChR antibody titers, for instance, do not correlate well with disease severity on an individual basis, though the rate of change in titers has shown some association with outcomes.
The introduction reviews what is already known about immune alterations in MG. On the B cell side, previous studies have reported expansions of class-switched memory B cells and circulating plasmablasts, but most of these studies compared unstratified MG patients to healthy controls without separating patients by treatment response. On the T cell side, functional impairments in regulatory T cells have been documented, and MG is known to be T cell dependent. On the innate immune side, dendritic cells and monocytes have been implicated in MG pathophysiology, but circulating frequencies remain poorly characterized.
The introduction also addresses the complement system’s dual role in MG. AChR antibodies activate complement at the neuromuscular junction, leading to membrane attack complex deposition and structural damage to the postsynaptic membrane. But complement also modulates lymphocyte function. The complement receptor CD21 on B cells promotes survival, activation, and antibody production, and has been found to be upregulated on AChR-antibody-producing B cells. Meanwhile, complement regulators on T cells (CD55, CD46, CD59) provide negative feedback to complement activation but also directly influence T cell function — a dimension that had not been previously characterized in MG.
The authors state that in an era of emerging high-cost targeted therapies (complement inhibitors, FcRn inhibitors, IL-6 inhibitors), identifying biomarkers that predict treatment resistance is of increasing clinical importance. This study was designed to address that need by defining the immune cell profiles specific to refractory MG, with the aim of informing personalized therapeutic strategies.
The study included 58 patients with AChR-MG and 20 controls. The refractory cohort contained only 10 patients, limiting the statistical power for subgroup analyses, particularly regarding rituximab response. The authors acknowledge this and note that larger independent cohorts are needed to validate these findings as predictive biomarkers.