A Novel MSN Mutation Impairs CD4⁺ T cell Differentiation and Drives Autoantibody Production

Abstract

Purpose

To report a patient with a novel MSN mutation causing X-linked moesin-associated immunodeficiency (X-MAID) and investigate its pathogenic mechanisms.

Methods

Clinical and immunological data of the patient were collected. Autoantibody levels were measured using antigen microarrays. Whole-exome sequencing was performed to identify gene mutations. The stability of the mutant MSN protein was analyzed via transient transfection and cycloheximide chase assay in HEK293T cells. MSN protein expression, immunophenotyping, and T cell activation and apoptosis were assessed by flow cytometry.

Results

The patient was a 2-year-old boy, presented with recurrent infections, Kawasaki disease, and early-onset broad-spectrum autoantibody production. A novel hemizygous MSN mutation (c.344T > C, p.I115T) was identified in the patient. The MSN p.I115T mutant showed reduced protein stability and decreased expression in multiple immune cell types. Immunophenotyping revealed follicular regulatory T (Tfr) cell reduction, Th1/Th17 imbalance, enhanced CD4⁺ T cell activation and proliferation, and impaired B cell maturation.

Conclusion

We report a novel MSN mutation associated with recurrent infections, early-onset broad-spectrum autoantibody production, and Kawasaki disease. MSN deficiency may contribute to autoimmunity through Tfr cell reduction and Th1/Th17 imbalance.

Introduction

Moesin (MSN) is one of three highly conserved members of the ezrin–radixin–moesin (ERM) family, which connects the actin cytoskeleton to the plasma membrane. It is composed of an N-terminal FERM domain, a central α-helical region, and a C-terminal actin-binding domain, and exists in inactive (closed) and active (open) conformations. In the closed state, the FERM domain associates with the C-terminal tail, masking the actin-binding site, whereas phosphorylation triggers the open conformation [1]. These transitions enable MSN to dynamically link the plasma membrane with F-actin, regulating cell shape, adhesion, motility and signal transduction [2–4]. MSN is highly expressed in hematopoietic cells, particularly in lymphocytes, where it plays a non-redundant role in maintaining T and B cell homeostasis [5, 6]. In vitro studies indicate that MSN deficiency impairs T cell activation, adhesion, and migration, as well as B cell receptor clustering and differentiation [7–9].

Moesin deficiency was first reported in 2016 as a cause of X-linked immunodeficiency, termed X-linked moesin-associated immunodeficiency (X-MAID) [10]. Since then, fewer than 20 patients have been described worldwide, carrying a variety of hemizygous MSN variants, including N23S, R171W, W217X, E312X, and R553X, among which R171W has been identified as a hotspot mutation [11]. Reported cases commonly present with recurrent bacterial and viral infections, particularly varicella-zoster virus (VZV), and some individuals develop autoimmune manifestations, including thrombocytopenia and antiphospholipid syndrome. Characteristic laboratory abnormalities include persistent lymphocytopenia, intermittent neutropenia, impaired T cell proliferative responses, and hypogammaglobulinemia. Nevertheless, the clinical spectrum is highly heterogeneous, with patients ranging from infancy [12] to adulthood [13] and exhibiting outcomes that span from early severe infections and malignancy [11] to relatively stable immune function [10]. This considerable variability highlights the need for additional patient cases to better define the phenotypic spectrum of X-MAID, as well as for mechanistic studies to elucidate how moesin dysfunction perturbs immune cell development and function.

Here, we describe a male patient carrying a novel hemizygous MSN mutant (c.344T > C, p.I115T), who presented with recurrent infections, early-onset broad-spectrum autoantibody production, and Kawasaki disease. Experimental analyses confirmed the pathogenicity of this mutation and demonstrated its impact on CD4⁺ T cell differentiation and function, providing new insights into the clinical manifestations and underlying immunological mechanisms of MSN-related immunodeficiency.

Methods

Patients and Samples

The patient included in this study was recruited from the Children’s Hospital of Fudan University. Clinical information was obtained through interviews with the patient’ legal guardians and review of electronic medical records. Peripheral blood samples were collected from the patient and age-matched healthy controls (HCs), and peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density-gradient centrifugation (Cytiva, USA). Written informed consent was obtained from all participants. The study was approved by the Ethics Committee of the Children’s Hospital of Fudan University and conducted in accordance with the Declaration of Helsinki.

Genetic Analysis

Genomic DNA was extracted from peripheral blood using the QIAamp DNA Blood Mini Kit (Qiagen, Germany). Whole-exome sequencing (WES) was performed with the IDT xGen Exome Research Panel V2 and sequenced on the Illumina NovaSeq 6000 platform. Raw reads were quality-filtered and aligned to the human reference genome (hg19) using BWA, followed by sorting with SAMtools and duplicate removal using Picard. Variant calling was performed with Sentieon, and annotations were generated using ANNOVAR, VEP, Human Gene Mutation Database (HGMD), and ClinVar. Variant filtering and prioritization were conducted following previously described pipelines [10], including exclusion of variants with MAF > 1% in gnomAD and those not fitting established disease-association criteria. The identified MSN variant was subsequently validated by Sanger sequencing. The potential functional impact of candidate variant was further evaluated using in silico prediction tools, including MutationTaster, SIFT, Provean, and PolyPhen-2.

Antigen Microarray Analysis

Plasma samples from the patient and three HCs were analyzed using an antigen microarray platform (GeneCopoeia, USA). The array contained 120 self-antigens for IgG detection. Serum samples were heat-inactivated and treated with DNase I. Samples were then diluted 1:100 in PBS and applied to the antigen-coated slides. IgG binding was detected using a Cy3-labeled anti-human IgG secondary antibody (1:1000). Slides were scanned with a LuxScan 10K-A fluorescence scanner (CapitalBio Corporation, China) at an excitation wavelength of 532 nm. Image files were processed using LuxScan_v3.0 to extract raw fluorescence values. For each antigen spot, net fluorescence intensity (NFI) was obtained by subtracting both the local background and the PBS negative control signal. The signal-to-noise ratio was calculated as (median foreground − median background) divided by the standard deviation of the background. To correct for technical variation, NFI values were normalized using a robust linear model based on internal positive controls included at multiple dilutions.

HEK293T Cell Culture, Transfection, and Cycloheximide Chase Assay

HEK293T cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Cytiva, USA) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin. Cells were cultured at 37 °C with 5% CO₂ and passaged at approximately 80% confluency. For transient transfection, the full-length human MSN coding sequence (NM_002444) was cloned into the pCMV-3×Flag vector, and the c.344T > C (p.I115T) variant was introduced by site-directed mutagenesis. All plasmids were confirmed by Sanger sequencing. Cells were seeded into 6-well plates one day before the experiment and transfected with 1.5 µg of wild-type or p.I115T MSN plasmids using Lipofectamine 3000 (Thermo Fisher, USA) following the manufacturer’s protocol. After 24 h, cycloheximide (CHX) was added to a final concentration of 100 µg/mL, and cells were collected at the indicated time points (0, 6, 12, and 24 h) to assess protein stability.

Western Blotting

Cells were lysed in RIPA buffer (Beyotime, China) supplemented with protease and phosphatase inhibitors (Roche, Switzerland). Lysates were cleared by centrifugation, and equal amounts of protein were separated on 10% SDS–PAGE gels (Yeasen, China). Proteins were transferred onto PVDF membranes and blocked with 5% BSA in TBST for 1 h at room temperature. Membranes were incubated overnight at 4 °C with primary antibodies against MSN and GAPDH (Cell Signaling Technology, USA). After washing, membranes were incubated with an HRP-conjugated anti-rabbit IgG secondary antibody (Abmart, China) for 1 h at room temperature. Protein bands were visualized using enhanced chemiluminescence and imaged with a digital detection system.

Flow Cytometry Analysis of Lymphocyte Subsets and T Cell Function

PBMCs from the patient and HCs were stained with fluorochrome-conjugated antibodies against surface markers including CD3, CD4, CD8, CD45RA, CD27, CD19, CD24, CD38, IgD, IgG, and IgM (Biolegend; BD, USA) to analyze lymphocyte subsets. For intracellular staining, cells were fixed and permeabilized using eBioscience™ Fixation/Permeabilization Concentrate (Thermo Fisher, USA). Foxp3 was stained using a directly conjugated antibody, whereas MSN protein was detected by sequential incubation with a primary anti-human MSN antibody (Cell Signaling Technology, USA) and a fluorochrome-conjugated secondary antibody.

To determine Th1 and Th17 cell proportions, PBMCs were stimulated with Cell Activation Cocktail with Brefeldin A (Biolegend, USA) for 6 h, followed by fixation and permeabilization using the Cytofix/Cytoperm™ Kit (BD, USA) and intracellular staining with IFN-γ and IL-17A antibodies.

For T cell activation, proliferation, and apoptosis assays, PBMCs were stimulated with 5 µg/mL Concanavalin A (ConA). Activation was assessed at 36 h by surface expression of CD69 and CD25, proliferation at 72 h by intracellular Ki-67 staining, and apoptosis at 72 h using Annexin V staining. All samples were acquired on a FACSCalibur flow cytometer (BD, USA), and data were analyzed using FlowJo software.

Statistical Analysis

Flow cytometry data were analyzed using FlowJo v10.8.1 (BD Biosciences). Data visualization and descriptive analyses were performed using GraphPad Prism version 10 (GraphPad Software, USA). For antigen microarray data, Z-scores were calculated for each antigen by comparing the patient’s normalized NFI with the mean and standard deviation derived from three HCs. A Z-score > 0 was considered elevated relative to controls, and Z-score > 3 was defined as significantly increased autoantibody reactivity.

Results

Clinical Manifestations

The patient was born at 33 + 2 weeks of gestation via vaginal delivery due to premature rupture of membranes, with a birth weight of 1720 g (P10–25). The Apgar score was 9 at 1 min, and the blood glucose level at birth was 4.8 mmol/L. At 1 week of age, echocardiography demonstrated a small patent ductus arteriosus (1.1 mm) and a patent foramen ovale (2.3 mm). By 1 month of age, the ductus arteriosus had closed spontaneously, while the foramen ovale persisted; cranial MRI showed only mild prematurity-related changes. The infant was clinically stable, tolerated full oral feeding with adequate weight gain, and was discharged in good condition.

From 6 months of age, the patient developed recurrent upper respiratory tract infections and bacterial enteritis, for which he received multiple courses of symptomatic and antibiotic therapy. At 18 months of age, the patient contracted varicella, which initially presented with rash on the legs and subsequently spread to the back, retroauricular areas, and face, without accompanying fever. He was treated with acyclovir for 5 days, after which the rash resolved.

At 2 years 4 months, the patient was admitted with fever, generalized rash, mucocutaneous symptoms, and cervical lymphadenopathy. Laboratory tests showed elevated inflammatory markers (CRP, IL-6, SAA, and ESR), and chest X-ray suggested bronchopneumonia. Respiratory pathogen panel and blood metagenomic sequencing results were negative, and antibiotics were ineffective. A diagnosis of Kawasaki disease was made. Echocardiography during the acute phase revealed no coronary artery abnormalities but identified a small pericardial effusion and trivial mitral regurgitation. The patient received intravenous immunoglobulin and high-dose aspirin. After 7 days of treatment, his symptoms improved, and he was then discharged. On follow-up echocardiography, the pericardial effusion had resolved and no coronary artery involvement was detected. Aspirin was subsequently tapered to 33.3 mg once daily for antiplatelet therapy.

Conventional Immunological Features

White blood cell, lymphocyte, and neutrophil counts remained within normal ranges. Analysis of lymphocyte subsets revealed a reduced proportion of CD16⁺CD56⁺ NK cells, an increased proportion of CD19⁺ B cells, elevated CD4⁺ T cells, and decreased CD8⁺ T cells. Hypogammaglobulinemia was detected at 2 months of age but resolved by 1 year and 5 months. During the episode of Kawasaki disease, serum IL-5, IL-6, IL-8, and IL-12p70 levels were markedly elevated (Table 1).

Table 1.

Immunological profile of the patient

Year of age	2m10d	1y5m	2y3m
Lymphocyte subpopulation
CD16 + CD56+%	2.26↓	2.59↓	6.25↓
CD16 + CD56+(cells/uL)	139.79↓	88.43↓	229.17↓
CD3+%	68.82	68.08	70.17
CD3+(cells/uL)	4248.8↑	2324.2	2572.6
CD19+%	26.91↑	27.32↑	22.11↑
CD19+(cells/uL)	1661.41↑	932.81↑	810.48
CD4+%	52.9↑	50.24↑	47.22↑
CD4+(cells/uL)	3265.57↑	1715.14	1731.18
CD8+%	14.48↓	13.47↓	17.38↓
CD8+(cells/uL)	893.89	459.99↓	637.1
CD45+(cells/uL)	6173.574	3413.878	3665.995
CD4+/CD8+	3.65	3.73	2.72
Immunoglobulin
IgG	2.2↓	6	6.6
IgA	0.05	0.16	0.2
IgM	0.25	0.84	0.69
IgE	16.23	30.42	43.06
Inflammatory cytokine
Il1 beta	-	-	< 1.5
Il2	-	-	1.5
Il4	-	-	< 1.5
Il5	-	-	4.5↑
Il6	-	-	41.9↑
Il8	-	-	84.4↑
Il10	-	-	< 1.5
Il12p70	-	-	39.1↑
Il17	-	-	17.3
ifn-alpha	-	-	< 1.5
ifn-gamma	-	-	3.3
tnf-alpha	-	-	2.2
Autoantibody
ANA 1:80	negative	-	+
Anti-Ro52	negative	-	weakly +

Autoantibody testing performed as part of routine clinical evaluation revealed positivity for antinuclear antibodies (ANA) and anti-Ro52 antibodies by immunofluorescence (Fig. 1a). To further characterize the patient’s autoantibody landscape, we performed a comprehensive antigen microarray profiling covering 120 IgG autoantigens. A total of 40 antigens showed elevated reactivity (Z-score > 0) (Fig. 1b), among which 13 were markedly increased (Z-score > 3). These highly enriched autoantibodies spanned multiple antigen categories, including nuclear antigens (Jo-1, Ro/SSA 60 kDa, ssDNA), cytoplasmic or membrane-associated proteins (α-actinine, CD4), cell matrix proteins (Collagen VI), phospholipid-related antigens (phosphatidyl-serine), and several circulating proteins (Calprotectin, Complement C1q, C-reactive protein, Factor H, Insulin, Troponin-I). This broad autoreactive signature highlights a dysregulated humoral immune response in the patient.

Fig. 1.

Autoantibody assessments in the patient. (a) Immunofluorescence results showing positive (red) and negative (gray) signals; (b) IgG autoantibody profiling of antigen microarray, displaying antigens with Z-score > 0 as a heatmap

Genetic Analysis Reveals p.I115T MSN Mutation

WES identified a hemizygous MSN c.344T > C (p.I115T) variant in the patient. Sanger sequencing confirmed this variant and showed that it was maternally inherited, with the mother being a heterozygous carrier (Fig. 2a-b). This missense variant is not recorded in major population databases and therefore appears to be novel. Multiple in silico prediction tools suggested a damaging effect, supporting its potential pathogenicity (Fig. 2c).

Fig. 2.

Identification of the MSN c.344T > C (p.I115T) mutant. (a) Pedigree illustrating the inheritance pattern of the mutant; (b) Sanger sequencing confirming the c.344T > C (p.I115T) substitution in the patient; (c) Summary of in silico predictions for the functional impact of the mutant

p.I115T MSN Variant Exhibits Reduced Protein Stability

To determine whether the p.I115T substitution affects MSN protein stability, wild-type and p.I115T MSN plasmids were generated and transiently introduced into HEK293T cells. Protein turnover was evaluated using a CHX assay. Quantitative immunoblot analysis demonstrated that the p.I115T mutant exhibited markedly reduced stability, with accelerated degradation relative to the wild-type MSN protein (Fig. 3).

Fig. 3.

HEK293T cells expressing WT or p.I115T MSN were treated with cycloheximide, and MSN protein stability was assessed by Western blot. GAPDH served as a loading control. Representative results of three independent experiments are shown

Reduced MSN Protein Levels in Immune Cells

At baseline, the proportion of MSN + T cells in the patient was 65.6%, which was lower than approximately 80% observed in HCs. After 72 h of ConA stimulation, the proportion of MSN + T cells in the patient decreased to 53.4%, whereas it was slightly increased in HCs, ranging from 80% to 90% (Fig. 4a). These results indicate that T cell activation leads to a more pronounced reduction of MSN expression in the patient compared with HCs, consistent with our HEK293T CHX assay showing reduced stability of the I115T MSN protein. In addition, MSN expression in the patient’s monocytes was lower than in HCs, while expression in B cells was comparable, indicating that the I115T mutation affects MSN protein levels in a cell type-specific manner (Fig. 4b).

Fig. 4.

MSN expression in the patient and HCs. (a) Proportion of MSN + T cells at baseline and after 72 h ConA stimulation; (b) MSN expression in B cells and monocytes

Altered T cell Subsets and Th17-skewed Differentiation

The patient exhibited an increased percentage of CD4⁺ terminal effector memory T cells (TEMRA) at the expense of CD4⁺ naive and central memory cells (TCM). In the CD8⁺ compartment, both TCM and TEMRA subsets were markedly elevated compared with HCs, whereas naive CD8⁺ T cells were reduced (Fig. 5a). The proportion of regulatory T cells (Tregs) and follicular helper T (Tfh) cells in the patient was comparable to that in the controls. However, both the proportion of follicular regulatory T (Tfr) cells and the Tfr/Tfh ratio were decreased, suggesting dysregulation of balance between Tfh and Tfr (Fig. 5b). The proportion of Th1 cells within the patient’s CD4⁺ T cell population was markedly lower than that in the controls, whereas the proportion of Th17 cells was increased, indicating a shift toward a pro-inflammatory Th17-biased phenotype. CD8⁺ T cells showed reduced IFN-γ production, suggesting impaired cytotoxic effector function (Fig. 5c).

Fig. 5.

Altered T cell subsets in the patient. (a) Proportions of naïve, central memory, effector memory, and TEMRA subsets in CD4⁺ and CD8⁺ T cells; (b) Proportions of Treg, Tfh, and Tfr cells; (c) Proportions of Th1 and Th17 in CD4⁺ T cells, and IFN-γ producing CD8 + T cells

Impaired B cell Maturation and Memory Differentiation

The proportions of memory B cells and marginal zone (MZ) B cells were markedly lower in the patient compared with those in HCs, whereas the proportion of naïve B cells was slightly higher, suggesting that MSN deficiency may preferentially impair B-cell maturation or differentiation toward memory and MZ compartments (Fig. 6a). The proportion of atypical memory CD21^−/low B cells was also modestly increased (Fig. 6b). This discordant pattern indicates an abnormal differentiation trajectory of antigen-experienced B cells, potentially limiting the establishment of functionally competent effector and memory B cell pools.

Fig. 6.

Altered B cell subsets in the patient. (a) Proportions of naïve, marginal, memory, transitional B cells and plasmablasts in the patient and HCs; (b) Proportion of atypical CD21^−/low memory B cells

Hyperactivation and Reduced Apoptosis of Patient CD4⁺ T Cells

After 36 h of ConA stimulation, the proportion of CD25⁺CD69⁺ CD4⁺ T cells was higher in the patient than in HCs, indicating enhanced CD4⁺ T cell activation (Fig. 7a). In contrast, CD8⁺ T cell activation was comparable between the patient and controls (Fig. 7d). After 72 h of stimulation, CD4⁺ T cell proliferation was slightly increased in the patient (Fig. 7b), whereas CD8⁺ T cell proliferation remained similar to that of HCs (Fig. 7d). The proportion of Annexin V⁺ CD4⁺ T cells was lower in the patient (Fig. 7c), indicating reduced apoptosis, with a slight decrease also observed in CD8⁺ T cells (Fig. 7d).

Fig. 7.

T cell activation, proliferation, and apoptosis in the patient. (a) Proportion of CD25⁺CD69⁺ CD4⁺ T cells after 36 h of ConA stimulation; (b) Proliferation of CD4⁺ T cells after 72 h of stimulation. (c) Proportion of Annexin V⁺ apoptotic CD4⁺ T cells. (d) Corresponding activation, proliferation, and apoptosis of CD8⁺ T cells

Discussion

In this study, we identified a patient carrying a novel hemizygous MSN c.344T > C (p.I115T) mutant who underwent routine neonatal screening and was followed up at our hospital. The patient exhibited transient hypogammaglobulinemia at 2 months of age and developed recurrent infections from 6 months, including an episode of VZV at 18 months, consistent with the reported viral susceptibility in MSN deficiency [10].

While the clinical spectrum of MSN deficiency has been associated with recurrent infections and susceptibility to tumor and autoimmune diseases [11], our findings add further insight into the cellular mechanisms underlying disease progression. Notably, we observed that MSN protein levels decrease further upon T cell activation, which may exacerbate cytoskeletal dysfunction and impair immune regulation. This activation-induced reduction in MSN indicates that MSN deficiency may become more pronounced during immune activation, emphasizing the importance of early genetic diagnosis, careful longitudinal monitoring, and timely clinical intervention in X-MAID patients.

Autoimmune manifestations are increasingly recognized in MSN deficiency, with established features including eczema, autoimmune cytopenias, antiphospholipid syndrome, and thyroid autoimmunity [14–16]. More recently, the clinical spectrum has been further expanded to include atypical autoimmune manifestations such as dermatomyositis-like symptoms [11], inflammatory bowel disease [17], lupus-like nephritis [18], and inflammatory ocular disease with retinal detachment [19], suggesting a broader range of immune-mediated phenotypes associated with MSN deficiency than previously appreciated. In our patient, a broad spectrum of autoantibodies was detected despite the absence of overt autoimmune symptoms. The patient exhibited extremely high levels of anti-α-actinin reactivity, suggesting pronounced immune dysregulation potentially linked to his cytoskeletal defect, consistent with the known role of MSN in cytoskeleton-associated processes [20]. The appearance of autoantibodies at such a young age is also uncommon, as autoimmune diseases typically arise during adolescence [21], making the early development of autoantibodies in this case particularly noteworthy. In addition, the occurrence of Kawasaki disease in this patient provides further evidence of underlying immune dysregulation. Taken together, these findings support a broader role for MSN in maintaining immune tolerance and suggest that MSN deficiency may predispose individuals to autoimmune diseases.

Evidence from both animal models and previously reported patients has highlighted the role of Tfh and Tfr cells in MSN-associated autoimmunity. Tfh cells are a subset of CD4⁺ T cells that provide help to B cells in germinal centers, promoting antibody production and affinity maturation [22], whereas Tfr cells are regulatory counterparts that restrain Tfh and B cell responses to limit autoreactivity [23]. In aged MSN-deficient mice, moesin deficiency leads to an SLE-like autoimmune disease, accompanied by accumulation of Tfh cells and germinal center B cells in secondary lymphoid organs [24]. In previously reported X-MAID patients, peripheral blood analysis revealed a relative increase in Tfh cells and a decrease in Tfr cells, indicating that autoimmunity associated with MSN mutations may arise from an imbalance in the Tfr/Tfh ratio [11]. In contrast, in our patient, Tfr cells were selectively decreased without a corresponding increase in Tfh cells, indicating that Tfr deficiency may represent an early or primary mechanism by which MSN deficiency contributes to immune dysregulation. This finding highlights the central role of Tfr cells in restraining autoreactive B cell responses and provides new insight into the pathogenesis of autoimmunity in X-MAID.

MSN deficiency may also influence CD4⁺ T cell subset differentiation. Th1 cells are critical for effective antiviral and cytotoxic responses, whereas Th17 cells have been implicated in tissue inflammation and autoimmunity [25]. In our patient, we observed a marked reduction in Th1 cells alongside an increase in Th17 cells, indicating a shift toward a pro-inflammatory Th17-biased phenotype. This Th1/Th17 imbalance may therefore contribute to both impaired host defense and heightened susceptibility to autoimmune diseases in X-MAID. Dedicator of Cytokinesis 8 (DOCK8) deficiency, another cytoskeleton-associated immunodeficiency, has been shown to skew CD4⁺ T cells toward a Th2 effector fate at the expense of Th1 and Th17 differentiation, highlighting a broader role for cytoskeleton-associated proteins in shaping T cell subset commitment [26, 27]. However, additional cases and mechanistic studies are needed to fully understand how MSN regulates Th1/Th17 balance in X-MAID. In addition to the Th1/Th17 skewing, MSN deficiency also affected CD4⁺ T cell activation and survival. In our patient, CD4⁺ T cells showed enhanced activation and proliferation with reduced apoptosis upon ConA stimulation, contrasting with previous reports of impaired activation and normal apoptosis in MSN-deficient patients [10, 11]. Notably, studies in Msn knockout mice have shown that CD4⁺ T cells can proliferate more than wild-type controls in vitro after anti-CD3/CD28 stimulation [28], suggesting that the effects of MSN deficiency on T cell proliferation and survival may depend on the type of stimulus, the cellular context, or the specific mutation. These findings underscore the complex role of MSN in modulating both T cell subset differentiation and T cell functions.

However, this study is based on a single patient with a novel MSN mutation, which represents an inherent limitation of the present work. Therefore, the findings may not be fully generalizable to all patients with X-MAID. Larger cohorts and further functional studies are needed to validate these observations and better define the immunological spectrum associated with MSN deficiency.

In conclusion, we report a patient carrying a novel hemizygous MSN c.344T > C (p.I115T) mutant who presented with recurrent infections, early-onset broad-spectrum autoantibody production, and Kawasaki disease. The patient displayed a reduction in Tfr cells, a Th1/Th17 imbalance, and enhanced CD4⁺ T cell activation and proliferation, highlighting the multifaceted impact of MSN deficiency on immune regulation. These findings expand the clinical and immunological spectrum of X-MAID and emphasize the importance of early recognition and longitudinal monitoring to guide timely interventions.

Author Contributions

K.Z. and B.S. designed the study. K.Z. collected samples, performed experiments, analyzed data, and drafted the manuscript. B.S., W.Z., L.L., W.W., C.W., F.X., and Q.Z. collected clinical information. X.W. and J.S. reviewed and revised the manuscript. All authors have read and approved the final version of the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (92574107) and Shanghai Municipal Health Commission Talent Program (2022XD024).

Data Availability

No datasets were generated or analysed during the current study.

Declarations

Competing Interests

The authors declare no competing interests.