Deciphering B cell Maturation Dynamics in Hyper IgM Syndromes

Deciphering B cell Maturation Dynamics in Hyper IgM Syndromes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Deciphering B cell Maturation Dynamics in Hyper IgM Syndromes Hande Üçler Çınar, Murat Cansever, Şerife Erdem, Abdullah Arık, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8894623/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Purpose: Hyper-IgM syndromes (HIGM) are primary immunodeficiencies characterized by defective class-switch recombination (CSR) and impaired humoral immunity. While genetic causes such as CD40L and AICDA mutations are well-established, a detailed comparison of B cell maturation dynamics across different HIGM subtypes remains limited. This study aims to comprehensively characterize B cell immunophenotypes and functional responses in HIGM patients and to delineate mutation-specific differences in B cell maturation and proliferation. Methods: Four patients with genetically confirmed HIGM (one CD40L and three homozygous AICDA mutations, c.70C>T; p.R24W) and age- and sex-matched healthy controls were studied. Peripheral blood mononuclear cells were analyzed via multiparameter flow cytometry to define B cell subsets based on CD19, CD20, CD24, CD27, CD38, IgD, and IgM expression. Additionally, B cell proliferation was assessed following CpG stimulation. Results: All patients exhibited a marked reduction in class-switched memory B cells (CD27 + IgD - ),and an accumulation of naive B cells (CD27 − IgD + ), consistent with defective CSR. The CD40L-deficient patient demonstrated profound depletion of plasmablasts and precursor skewing, reflecting a failure in germinal center formation. In contrast, AICDA patients showed preserved CD27 expression with variable expansion of transitional and plasmablast populations, suggesting intact T cell–dependent activation but an intrinsic failure of CSR. Functional assays revealed heterogeneous proliferative responses, with inter-individual variability observed particularly among AICDA-deficient patients. Conclusion: Detailed immunophenotyping reveals distinct B cell maturation arrest points in CD40L- versus AICDA-associated HIGM. Flow cytometric analysis provides valuable insights into disease mechanisms, supports differential diagnosis, and informs clinical monitoring and therapeutic decision-making in HIGM Hyper-IgM syndrome B cell maturation CD40L AICDA class-switch recombination Figures Figure 1 Figure 2 1. Introduction Hyper IgM Syndrome (HIGM), first described by Rosen et al. in 1961, is a primary immunodeficiency characterized by normal or elevated serum IgM and markedly low serum IgA, IgG, and IgE levels [ 1 ]. This aberrant immunoglobulin profile results from a fundamental defect in isotype switching, a critical process for the development of effective humoral immunity. Defects occur during in the class switch recombination (CSR) and somatic hypermutation steps of mature B cell development due to various genetic mutations [ 2 ] Since the initial identification of mutations in the CD40L-encoding gene in 1992, genetic heterogeneity has been increasingly recognized in HIGM cases [ 3 , 4 ]. While the most common form of HIGM is X-linked (HIGM type 1), arising from mutations in the CD40 ligand ( CD40L ) gene, which disrupts T cell-dependent B cell activation and germinal center formation, autosomal recessive forms, such as those caused by mutations in the activation-induced cytidine deaminase ( AID ) or CD40 genes[ 5 ], are more prevalent in populations with high rates of consanguineous marriages, such as in Turkiye, and also contribute to the clinical spectrum of HIGM. The development and maturation of B lymphocytes are meticulously orchestrated processes, characterized by the sequential and precise modulation of a plethora of cell surface markers. These markers serve not only as identifiers for distinct B cell subsets but also as pivotal mediators of signal transduction and intercellular communication. In HIGM, disruptions in isotype switching profoundly impact B cell maturation, leading to alterations in the expression patterns of key surface markers. This study aims to comprehensively characterize the B cell compartment in patients with HIGM, focusing on the expression profiles of CD19, CD20, CD24, CD27, CD38, and the immunoglobulin isotypes IgD and IgM. By employing flow cytometry analysis, we seek to delineate the quantitative and qualitative alterations in B cell subpopulations in HIGM patients, providing a deeper understanding of the immunopathogenesis of this complex disorder. This study will contribute to a more refined characterization of the B cell immunophenotype in HIGM, potentially leading to improved diagnostic and therapeutic strategies. 2. Materials and Methods Patients followed with a diagnosis of HIGM at Department of Kayseri City Hospital Pediatric Allergy and Clinical Immunology were retrospectively screened using their ICD codes. The study cohort consisted of four patients diagnosed with Hyper-IgM (HIGM) syndrome who presented to our center for scheduled routine follow-up and clinical management. To establish a comparative baseline, blood samples were also obtained from age- and sex-matched healthy controls. Comprehensive information regarding the study objectives was provided to all participants and their legal guardians; subsequently, written informed consent was obtained from the parents of the patients and from the control subjects prior to their enrollment in the study. The study was conducted in accordance with the 2013 revision of the Declaration of Helsinki and was implemented by the Kayseri City Education and Research Hospital Local Ethics Committee with the approval decision numbered 235 dated 05.11.2024 2.1. PBMC Isolation Peripheral blood mononuclear cells (PBMCs) were isolated from venous blood samples of patients and age/sex-matched healthy individuals using Ficoll-Paque density gradient centrifugation. Briefly, blood samples were diluted 1:1 with PBS and carefully layered onto Ficoll-Paque (GE Healthcare). Samples were centrifuged at 400 g for 30 minutes, and the mononuclear cell layer was carefully collected. Cells were washed twice with PBS and suspended in RPMI-1640 culture medium supplemented with 10% FBS. Cell concentration was determined using a hemocytometer and used for phenotyping and proliferation assays. 2.2. B Cell Surface Staining The gating strategy followed a strict hierarchical approach: lymphocytes were first identified by FSC-A/SSC-A characteristics, debris excluded, followed by singlet selection to exclude doublets, and finally gated on CD19 + to define the total B cell population. For the identification of B cell subpopulations, isolated PBMCs were stained with surface markers after Fc receptor blocking: CD19-FITC, CD20-BV510, CD24-PE, CD27-APC, CD38-PE-Cy7, IgD-PE-Dazzle, CD138-APC-Cy7. After staining, cells were incubated at 4°C for 30 minutes, washed with PBS, and prepared for flow cytometry analysis. Analyses were performed using a BD FACSARIA III device, and data were evaluated using FlowJo software. 2.3. B Cell Proliferation Assay To evaluate B cell proliferation, isolated PBMCs were seeded in 96-well plates at 2x10⁵ cells/well and cultured in RPMI-1640 + 10% FBS medium. Cells were stimulated with CpG (20 µg/mL) and incubated for 5 days. Proliferation analysis was performed using: Control group (unstimulated, no CpG), and Experimental group (stimulated with CpG, 20 µg/mL CpG added). At the end of the incubation period, cells were stained with PE-conjugated CD19 antibody and incubated at 4°C for 30 minutes. After washing with PBS, cells were analyzed by flow cytometry. Proliferation rates were statistically compared. 2.4. Statistical Analysis Methods Data analysis was performed by first testing the assumption of normal distribution. The normality of the groups was assessed using the Shapiro-Wilk test. Parametric tests were used for normally distributed data, and non-parametric tests were used for non-normally distributed data. One-way analysis of variance (ANOVA) was used to compare multiple groups. Tukey's multiple comparison test was performed to determine the source of differences between groups after ANOVA. Statistical Significance was indicated as follows: p < 0.05 values were considered statistically significant. p values were expressed with asterisk symbols: p < 0.05 → * p < 0.01 → ** p < 0.001 → *** p < 0.0001 → ****All analyses were performed using GraphPad Prism 9 software. 3. Result 3.1. Demographic and Clinical Characteristics The study included 4 patients with a mean age of 8.75 years (range: 4–17), comprising 2 males and 2 females. All patients in the study group shared a common clinical history of recurrent respiratory tract infections (RTIs). Patient 2 (Pt 2), who underwent an adenotonsillectomy at the age of three due to chronic infections, was also followed by the pediatric oncology department for persistent recurrent cervical lymphadenopathy. Following her clinical evaluation, her sister (Pt 3) was screened due to a history of recurrent upper RTIs, leading to her subsequent diagnosis. The family history was notable for third-degree consanguinity between the parents and a maternal relative receiving intravenous immunoglobulin (IVIG) therapy for an unknown immunodeficiency. All patients exhibited a laboratory profile consistent with Hyper IgM Syndrome (HIGM), characterized by significantly low serum IgG and IgA levels. Genetic analysis confirmed a hemizygous mutation in the CD40L gene for Pt 1, whereas Pt 2, Pt3, and Pt4 were identified as having a homozygous mutation in the AICDA gene (c.70C>T, p.R24W). Clinical, demographic and laboratory characteristics of patients are shown in Table 1. Table 1. Clinical, demographic and laboratory characteristics of patients Pt1 Pt 2 Pt 3 Pt 4 Age/Sex 4, Male 6, Female 8, Female 17, Male CBC Hemoglobin (g/dl) WBC (/mm 3 ) ANS (/mm 3 ) Lymphocyte (/mm 3 ) 11.3 8390 2290 5280 11.2 7510 3940 2729 13.8 6830 3130 2930 10.8 8470 3730 3790 Lymphocytes subsets CD3 CD4 CD8 CD16+56 CD19 %70 (56-75) %51 (28-47) %14 (16-30) %7 (4-17) %22 (14-33) %72.2 (56-75) %39.8 (28-47) %24.9 (16-30) %7.2 (4-17) %17.8 (14-33) %71 (60-76) %33 (31-47) %31 (18-35) %11 (4-17) %16 (12-27) %70 (56-84) %42 (31-52) %25 (18-35) %8 (3-22) %20 (6-23) IgG (mg/dl) IgA (mg/dl) IgM (mg/dl) IgE (mg/dl) 18 (457-1120) 1 (35.7-192) 240 (58.7-198) 0.2 18 (483-1580) 15 (44.8-276) 2453 (50.3-242) 0.2 T Homozygous AICDA mutation c.70C>T (p.R24W) Complication Lymphadenopathy,tonsillectomy WBC: White blood count ANS: absolute neutrophil count. Serum Ig age-specific reference values are shown in parentheses [6] 3.2. B Cell Subsets Distribution and CSR Defects The immunophenotypic analysis of patient B cell subsets revealed distinct profiles compared to healthy controls (HC). Figure 1 provides a comprehensive immunophenotypic map comparing the patient group with age- and sex-matched healthy controls (Figure 1 A, gate strategy), detailing B cell development from the total reservoir and naive-to-memory distribution (Figure 1 B), through class-switch recombination (CSR) efficiency at the isotype level (Figure 1C), to the terminal differentiation of transitional cells into antibody-secreting plasmablasts (Figure 1 D). In terms of total CD19⁺ B cells, Pt1 showed a significant increase, whereas Pt2, Pt3, and Pt4 exhibited a decrease. Specifically, an analysis of the CD19 + B cell compartment revealed a significant expansion of the CD19 + CD20 + population in Patients 1 and 4. Analysis of B cell maturation revealed that while the frequency of CD27 + memory B cells was decreased across all patients, this reduction achieved statistical significance in Pt1 and Pt3 (p<0.001 and p<0.05, respectively), although CD27 − naive B cells were expanded in the entire cohort, the increase reached statistical significance exclusively in Pt1, Pt2, and Pt3 (p<0.001; p<0.05 and p<0.01, respectively). Further characterization of the CD19⁺CD20⁺ compartment using CD27 and IgD markers indicated that CD27⁺IgD⁺ cells were significantly reduced in Pt1 and Pt2 (p<0.05 and p<0.01, respectively). Similarly, switched memory cells (CD27⁺IgD⁻) were decreased in Pt1, Pt3, and Pt4, and double-negative cells (CD27⁻IgD⁻) were lower in Pt1 and Pt3. Conversely, CD27⁻IgD⁺ cells were significantly increased across all patients (Pt1–Pt4). Analysis of B cell differentiation stages revealed a significant depletion of CD20⁻CD38⁺⁺ plasmablasts in Pt1 (p<0.01), whereas a marked expansion of this population was observed in Pt2 (p<0.001). Sub-analysis within the CD38⁺CD20⁻ compartment showed a significant increase in the frequency of CD27⁻ precursor B cells in Pt1 (p<0.05), contrasted by a downward trend in CD27⁺ plasma B cells (p<0.05). Furthermore, regarding transitional B cells (CD24⁺CD38⁺), Pt1 exhibited a significant reduction compared to the healthy control group (p<0.01), while Pt2 showed a statistically significant elevation (p<0.05) 3.3. B Cell Functional Analysis: CpG Stimulation and Proliferation Functional assays focused on B cell proliferation following CpG stimulation revealed that Pt1 and Pt4 had significantly higher proliferation rates compared to healthy controls (p<0.05 and p<0.0001, respectively), whereas Pt3 showed a significant reduction. Regarding general lymphocyte proliferation, Pt1 again showed a significant increase, while the responses in Pt3 and Pt4 remained stable. Overall lymphocyte and CD19⁺ B cell proliferation profiles are presented in Figure 2. 4. Discussion B cell maturation involves dynamic surface marker changes, enabling the identification of distinct peripheral blood subpopulations via multicolor flow cytometry. Since the impaired formation of these subsets is a hallmark of various primary immunodeficiencies (PIDs), analyzing these stages provides critical insights into immune dysfunction. In this context, the most characteristic finding of our study is the uniform accumulation of B cells at the naive (CD27 − IgD + ) stage and the dramatic loss of class-switched memory B cells (CD27 + IgD − ) across all Hyper-IgM (HIGM) patients, regardless of the underlying genetic mutation ( CD40L or AICDA )[ 7 ]. This cellular profile provides definitive evidence of defective class-switch recombination (CSR) at the systemic level. However, a granular analysis of plasmablast and precursor distributions reveals critical differences in the biological mechanisms underlying these mutations, serving as a pivotal tool for differential diagnosis. In Pt 1, diagnosed with Type 1 HIGM (CD40L deficiency), the proportion of CD19 + B cells within total lymphocytes remains elevated, yet the qualitative integrity of these cells is severely compromised. Memory B cells are categorized into two distinct subsets based on the expression of CD27 and IgD. CD27⁺IgD⁺ memory B cells are thought to develop independently of the germinal center (GC) reaction and represent circulating marginal zone B cells that respond to T-independent antigens. In contrast, CD27⁺IgD⁻ (switched) memory B cells are products of the post-germinal center reaction. Consequently, a marked reduction or absence of these switched memory B cells is highly indicative of a disrupted GC reaction[ 8 ]. Due to the lack of CD40L-mediated T-cell help, these B cells are unable to initiate GC formation, which essentially halts antigen-specific memory development[ 9 ]. The absence of CD27 + and IgD − CD27 + (switched memory) cells confirms a maturation arrest at the naive stage[ 10 ]. Furthermore, the total lack of plasmablast-like cells (CD20 − CD38 ++ ) underscores that terminal differentiation cannot proceed without functional CD40L signaling. The landscape in Type 2 HIGM ( AID deficiency), observed in Pt2, Pt3, and Pt4, is more complex. In these individuals, the CD40/CD40L interaction remains intact, enabling B cells to form GCs and acquire limited CD27 expression. However, the absence of the AID enzyme renders CSR and somatic hypermutation biochemically impossible. While these cells may initiate GC reactions, the marked decrease of IgD − CD27 + cells highlights their inability to successfully switch antibody classes[ 11 ]. When the double-negative (DN; CD27 − IgD − ) B cell subset was analyzed, a significant reduction was observed in Pt1 and Pt3, whereas this decrease did not reach statistical significance in Pt2 and Pt4. According to the literature, the DN1 subpopulation is considered a product of an aborted GC reaction and a precursor to switched memory (CD27 + ) B cells. In contrast, DN2 and DN3 cells are known to originate via GC-independent, extrafollicular pathways. The non-significant results in Pt2 and Pt4 suggest that the extrafollicular pathway might remain partially active or allow for a low level of DN cell production despite the underlying genetic defects. Furthermore, it is established that DN B cells tend to expand with aging and chronic infections[ 12 ]. Therefore, in Pt4—the oldest patient in our cohort—years of cumulative antigenic exposure may have driven the expansion of this subpopulation despite the genetic constraint, potentially through these alternative developmental routes The distinct precursor distributions further delineate the two HIGM types. In Pt1, the increase in CD38 + CD27− precursors alongside a plasmablast deficit reflects the absolute failure of GC formation due to CD40L deficiency. Conversely, the expansion of CD24 + CD38 + populations in Pt2 suggests that AID-deficient B cells are driven toward maturation by antigenic stimulation but remain sequestered in a "pre-switched" or late-naive state. The increased plasmablast levels and high transitional cell (CD24 + CD38 + ) proportions in Pt2 reflect a state where GCs can form but become hyperplastic. In alignment with our findings, a study in the literature involving 18 patients with Type 2 HIGM reported 13 lymphoid hyperplasia leading to tonsillectomy in 5 of these cases [ 13 ]. In a previous study, lymphoid hyperplasia was observed in 20 out of 29 patients prior to IVIG replacement therapy, with peripheral lymph nodes and tonsils being the most commonly affected sites. Similarly, Pt2 in our group presented with recurrent cervical lymphadenopathy and a history of adenotonsillectomy at age 3, suggesting an early-onset lymphoproliferative phenotype. While data in the literature often point to follicular hyperplasia with giant GCs, the clinical progression in our case—including follow-up by pediatric oncology—highlights the importance of monitoring these patients for lymphoproliferative complications[ 14 ]. This hyperplasia is a result of chronic inflammatory responses in GCs that are constantly stimulated but unable to complete CSR. This environment pushes B cells toward a plasmablast phenotype, yet they remain incapable of producing functional, switched antibody classes. Peripheral B cell ontogeny progresses along a linear maturation spectrum defined by the dynamic shifts in CD38 and CD24 expression density. According to the model by Buffa et al., newly emigrated CD38 high CD24 high (Transitional) cells gradually reduce these markers to become CD38 int CD24 int (Mature Naive) and eventually CD38 − memory cells. The marked expansion of the CD24 + CD38 + population in Pt2 indicates a "maturational stasis"[ 15 ]. Viewed through the functional compartmentalization theory of Sanz et al., this finding confirms that while these cells receive antigenic signals, genetic constraints (AID deficiency) prevent the completion of the GC reaction, causing them to accumulate in a "pre-memory" phase [ 16 ]. The analysis of B cell maturation markers in patients with homozygous AICDA mutations (Pt2, Pt3, and Pt4) reveals a distinct biological pattern where the percentages of CD38⁺ CD27⁻ and CD38⁺ CD27⁺ populations remain comparable to healthy controls. This observation confirms that, unlike in CD40L deficiency (Pt1), the T-cell-dependent activation signaling is functionally preserved in AICDA-deficient patients. Since the molecular defect in these individuals is intrinsic to the nuclear enzymatic machinery rather than the surface receptor signaling, their B lymphocytes successfully process activation cues and proceed to express the CD27 surface marker. Consequently, these cells are capable of entering the plasma cell differentiation pathway and phenotypically acquiring a "mature" profile. However, this represents a pseudo-maturation state; while the cells can harbor the CD27 marker on their surface, the underlying genetic defect in AID prevents the execution of CSR within the nucleus, leading to a functional failure in producing switched antibody isotypes. Despite sharing the same homozygous AICDA mutation (c.70C > T, p.R24W) and a common family history of third-degree consanguinity, Pt2, Pt3, and Pt4 exhibit significant phenotypic inconsistencies. This clinical variation is most evident when comparing the two sisters (Pt2 and Pt3); while both share a history of recurrent respiratory tract infections (RTI), Pt2’s clinical course was marked by more severe lymphadenopathy and a history of adenotonsillectomy. These clinical disparities are further mirrored in the patients' cellular profiles and proliferative capacities. Contrary to what might be expected from her peripheral B cell count, Pt4—the oldest patient in the cohort—exhibited a significantly reduced CD19 + B cell frequency (Fig. 1 ) alongside the highest serum IgG levels and a notably robust proliferative response to CpG stimulation (Fig. 2 ). This suggests that while the absolute B cell pool is contracted in Pt4, the remaining cells retain a high functional capacity for activation. In contrast, the cellular profiles of Pt2 and Pt3 show a different correlation. Pt3 exhibited both diminished peripheral B cell counts and a significantly lower proliferative response, indicating a more profound exhaustion or defect in her B cell compartment compared to Pt4. These variations highlight that peripheral B cell frequency alone does not always predict functional competency in Hyper-IgM patients, as compensatory proliferative mechanisms or disease chronicity may influence the immunophenotypic profile differently across individuals. The proliferation data obtained in this study reveal the heterogeneous functional impact of different genetic mutations on B cell activation in HIGM. While CD40L deficiency is characterized by a lack of T-cell-dependent stimulation, it is well-established that these B cells can remain responsive to T-cell-independent signals, such as the TLR9 ligand CpG. The increased proliferation observed in Pt1 suggests that the TLR9 activation pathway is not only preserved but may also exhibit a compensatory hyper-responsiveness in the absence of CD40L-mediated signaling. Imai et al. demonstrated that B cells from both HIGM2 and HIGM4 patients are capable of achieving normal proliferation when stimulated in vitro with sCD40L and IL-4[ 17 ]. Our results using CpG stimulation are consistent with this observation, as Pt1 (CD40L deficiency) and Pt4 (AICDA mutation) exhibited significantly increased proliferation rates compared to healthy controls (p < 0.05 and p < 0.0001, respectively). This supports the thesis that HIGM B cells can remain highly responsive to activation stimuli, even when the downstream terminal differentiation and class-switching machinery is defective. Interestingly, a notable divergence was observed between Pt3 and Pt4 despite both sharing a homozygous AICDA mutation. While Pt4 showed robust proliferation, the significant suppression of B cell proliferation in Pt3 suggests that the impact of AID deficiency extends beyond immunoglobulin class switching and may involve broader signaling or survival defects that vary among individuals. These findings suggesting that compensatory immune mechanisms or environmental exposures may partially mitigate the AICDA defect over time. The clinical presentation of our study group is highly consistent with the established literature on primary B cell immunodeficiencies. As highlighted in the previous study, recurrent RTIs represent the most frequent clinical manifestation in these patients[ 14 , 18 ]. In total alignment with these reports, recurrent upper RTIs were the primary clinical finding across our entire patient group[ 14 ]. De la Morena et al. argue that even patients with Type 1 HIGM who appear stable on immunoglobulin replacement therapy remain at high risk for life-threatening complications, and therefore stem cell transplantation should be considered as the definitive treatment[ 19 ]. For Type 2 HIGM patients, the indications for stem cell transplantation are less standardized compared to Type 1. Specifically, our first patient ( CD40L deficiency), who is continuing IVIG therapy and antimicrobial prophylaxis, is stable with the current treatment; not performing a transplant in this patient is consistent with a more conservative management approach. Several limitations of this study should be acknowledged. First, the sample size is relatively small, which is a common challenge in research involving rare primary immunodeficiencies. While this limited cohort size may constrain the generalizability of our findings to all HIGM subtypes, it reflects the clinical reality of managing such rare conditions. Second, our immunophenotypic analysis was restricted to peripheral blood mononuclear cells (PBMCs), which may not fully represent the complex B-cell dynamics and germinal center reactions occurring within secondary lymphoid organs. Third, we observed significant inter-individual phenotypic and functional variability, particularly among patients with the same homozygous AICDA mutation. A key limitation is the lack of additional parameters, such as detailed environmental exposure history or epigenetic profiling, which might have better elucidated the underlying causes of this clinical and proliferative heterogeneity. Despite these limitations, this study has yielded significant findings in the field of B-cell immunopathology. Specifically, through high-resolution, multi-parameter flow cytometric analysis, we compared X-linked CD40L deficiency with autosomal recessive homozygous AICDA mutations (c.70C > T; p.R24W). By extending our characterization beyond basic subsets to include detailed differentiation stages such as transitional cells and plasmablasts, we have successfully delineated mutation-specific "pseudo-maturation" states and maturation arrest points that are critical for differential diagnosis. Furthermore, the inclusion of functional B cell proliferation assays highlights the heterogeneous effect of different genetic defects on B cell activation, offering valuable insights into disease mechanisms and potential diagnostic strategies for HIGM patients. However, further studies with larger sample sizes are recommended to confirm these results. 5. Conclusions Detailed analysis of B cell subsets elucidates the fundamental biological disparities between HIGM subtypes. The significant loss of CD27 − IgD − cells and the mutation-specific shifts in maturation stages reaffirm the critical importance of flow cytometry in the diagnosis and clinical monitoring of HIGM. Declarations Acknowledgements None Conflict of Interest The authors declare that there are no conflicts of interest regarding the publication of this manuscript. Authorship Contributions All authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by H.Ü.Ç., M.C., and A.A. Methodology and formal analysis were conducted by S.E. and A.E. All authors read and approved the final manuscript.. Funding Disclosure This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Ethical Approval This study was conducted in strict accordance with the ethical principles for medical research involving human subjects as outlined in the Declaration of Helsinki (2013 revision). 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DOI: 10.1016/j.clim.2003.10.007. https://www.ncbi.nlm.nih.gov/pubmed/14962793 https://www.sciencedirect.com/science/article/abs/pii/S1521661603002857?via%3Dihub Buffa S, Pellicano M, Bulati M, Martorana A, Goldeck D, Caruso C, et al. A novel B cell population revealed by a CD38/CD24 gating strategy: CD38(-)CD24 (-) B cells in centenarian offspring and elderly people. Age (Dordr) 2013;35(5):2009-24. (In eng). DOI: 10.1007/s11357-012-9488-5. https://www.ncbi.nlm.nih.gov/pubmed/23129025 https://pmc.ncbi.nlm.nih.gov/articles/PMC3776115/ Sanz I, Wei C, Lee FE, Anolik J. Phenotypic and functional heterogeneity of human memory B cells. Seminars in Immunology 2008;20(1):67-82. (In eng). DOI: 10.1016/j.smim.2007.12.006. https://www.ncbi.nlm.nih.gov/pubmed/18258454 https://pmc.ncbi.nlm.nih.gov/articles/PMC2440717/ Imai K, Catalan N, Plebani A, Marodi L, Sanal O, Kumaki S, et al. Hyper-IgM syndrome type 4 with a B lymphocyte-intrinsic selective deficiency in Ig class-switch recombination. The Journal of Clinical Investigation 2003;112(1):136-42. (In eng). DOI: 10.1172/JCI18161. https://www.ncbi.nlm.nih.gov/pubmed/12840068 https://dm5migu4zj3pb.cloudfront.net/manuscripts/18000/18161/JCI0318161.pdf Moazzami B, Yazdani R, Azizi G, Kiaei F, Tafakori M, Modaresi M, et al. Respiratory Complications in Patients with Hyper IgM Syndrome. J Clin Immunol 2019;39(6):557-568. (In eng). DOI: 10.1007/s10875-019-00650-3. https://www.ncbi.nlm.nih.gov/pubmed/31183658 https://link.springer.com/article/10.1007/s10875-019-00650-3 de la Morena MT, Leonard D, Torgerson TR, Cabral-Marques O, Slatter M, Aghamohammadi A, et al. Long-term outcomes of 176 patients with X-linked hyper-IgM syndrome treated with or without hematopoietic cell transplantation. Journal of Allergy and Clinical Immunology 2017;139(4):1282-1292. (In eng). DOI: 10.1016/j.jaci.2016.07.039. https://www.ncbi.nlm.nih.gov/pubmed/27697500 https://www.jacionline.org/article/S0091-6749(16)30964-2/pdf Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 31 Mar, 2026 Reviews received at journal 26 Mar, 2026 Reviewers agreed at journal 16 Mar, 2026 Reviews received at journal 12 Mar, 2026 Reviewers agreed at journal 05 Mar, 2026 Reviewers invited by journal 03 Mar, 2026 Editor assigned by journal 19 Feb, 2026 Submission checks completed at journal 19 Feb, 2026 First submitted to journal 16 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8894623","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":601418375,"identity":"b02e9980-6bbe-470d-a2cf-05eaeda4b9a1","order_by":0,"name":"Hande Üçler Çınar","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3UlEQVRIiWNgGAWjYFAC5gYGxgYgfYD5AJCUkCFCCyNMC1sCSAsPKVp4DEBcwloMjh9sfFy5wy6P73jP51c3aix4GNgPH92AV8uZxGbDs2eSiyXPnN1mnXMM6DCetLQbeLXcYGyTbGxjTtxwI3ebcQ4bUIsEjxkhLe0/G9vqEzfcf/PMOOcfcVraGBvbDgNt4WF+nNtGhBZJoF+ADjueOPNMmhlzbp8EDxshv/AdP3zwY2NbdWLf8cOPP+d8q5PjZz98DK8WZMAmASaJVQ4CzB9IUT0KRsEoGAUjBwAA7qZP1QQQBSYAAAAASUVORK5CYII=","orcid":"","institution":"Kayseri City Hospital","correspondingAuthor":true,"prefix":"","firstName":"Hande","middleName":"Üçler","lastName":"Çınar","suffix":""},{"id":601418376,"identity":"ceee0bee-776a-4feb-9749-709e59731c27","order_by":1,"name":"Murat Cansever","email":"","orcid":"","institution":"Kayseri City Hospital","correspondingAuthor":false,"prefix":"","firstName":"Murat","middleName":"","lastName":"Cansever","suffix":""},{"id":601418377,"identity":"4f1f7f39-4349-473c-8521-6e2612845e04","order_by":2,"name":"Şerife Erdem","email":"","orcid":"","institution":"Ahi Evran University","correspondingAuthor":false,"prefix":"","firstName":"Şerife","middleName":"","lastName":"Erdem","suffix":""},{"id":601418379,"identity":"87afcb3d-f8de-41ae-bc4f-fbea254e9011","order_by":3,"name":"Abdullah Arık","email":"","orcid":"","institution":"Kayseri City Hospital","correspondingAuthor":false,"prefix":"","firstName":"Abdullah","middleName":"","lastName":"Arık","suffix":""},{"id":601418380,"identity":"91a3b356-f0d2-4171-bfd7-29f085ce7d68","order_by":4,"name":"Ahmet Eken","email":"","orcid":"","institution":"Erciyes University","correspondingAuthor":false,"prefix":"","firstName":"Ahmet","middleName":"","lastName":"Eken","suffix":""}],"badges":[],"createdAt":"2026-02-16 16:09:52","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8894623/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8894623/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104204375,"identity":"58412dd1-8039-49bb-a58e-6e844589e658","added_by":"auto","created_at":"2026-03-09 06:33:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":317004,"visible":true,"origin":"","legend":"\u003cp\u003eImmunophenotypic analysis of B cell subsets across patients (Pt1–Pt4) and \u0026nbsp;\u0026nbsp;healthy controls (HC). Each circle represents an individual subject within \u0026nbsp;\u0026nbsp;the healthy control group (HC 1,2,3,4) or technical replicates for patients. \u0026nbsp;\u0026nbsp;Panel (A) shows gate strategy for flow cytometry. Panel (B) displays total \u0026nbsp;\u0026nbsp;(CD19+), CD20+, memory (CD27+) and naïve (CD27-) B cell frequencies. Panel \u0026nbsp;\u0026nbsp;(C) shows memory and naive B cell distributions based on IgD and CD27 \u0026nbsp;\u0026nbsp;expression. Panel (D) illustrates B cell differentiation stages: \u0026nbsp;\u0026nbsp;CD19+CD20-CD38++( Antibody-Secreting Cells (ASCs), include plasmablasts (PBs) \u0026nbsp;\u0026nbsp;and plasma cells (PCs)); CD19+CD20-CD38+CD27- B cells primarily represent \u0026nbsp;\u0026nbsp;plasmablasts (early antibody-secreting cells) and some activated naive B \u0026nbsp;\u0026nbsp;cells or transitional cells; CD19+CD20-CD38+CD27+ B cells primarily represent \u0026nbsp;\u0026nbsp;Plasma Cells (PCs) or their precursors the Plasmablasts (PBs); \u0026nbsp;\u0026nbsp;CD19+CD24-CD38+ B cells are generally identified as immature or transitional \u0026nbsp;\u0026nbsp;B cells; (CD24-CD38+) represent plasmablasts, and (CD19+CD24+CD38+) rpresent \u0026nbsp;\u0026nbsp;immature transitional B cells. Statistical significance was determined using \u0026nbsp;\u0026nbsp;ANOVA and indicated as: *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026nbsp;\u0026nbsp;\u0026lt; 0.0001; ns: not significant.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8894623/v1/3995de33db32bfb098e11120.png"},{"id":104204376,"identity":"9d3b5964-61fb-4457-b14f-7860d3c321f1","added_by":"auto","created_at":"2026-03-09 06:33:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":216938,"visible":true,"origin":"","legend":"\u003cp\u003eProliferative capacity of lymphocytes and CD19⁺ B cells following CpG stimulation. Representative flow cytometry plots and cumulative data showing the proliferative response in healthy controls (CTRL1, 3, 4) and patients (Pt1, Pt3, Pt4). Representative pseudocolor plots and CFSE dilution histograms for unstimulated (Unstim.) and CpG-stimulated conditions are shown in left. The numbers indicate the percentage of gated cells. Bar graphs represent the percentage of proliferating cells among total lymphocytes and the CD19⁺ B cell population. Statistical analysis was performed using ANOVA followed by Tukey’s multiple comparison test to compare stimulated patients against the stimulated healthy control (HC1,3,4) group. Significance is indicated as: *p \u0026lt; 0.05, ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8894623/v1/a76302f37d2325b89f4475c9.png"},{"id":104204382,"identity":"8d9e95ba-050a-4e40-94d2-bb2cd14fa0cf","added_by":"auto","created_at":"2026-03-09 06:33:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1023126,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8894623/v1/8dd690ed-6a97-4718-8634-e0d97ba49ef9.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Deciphering B cell Maturation Dynamics in Hyper IgM Syndromes","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eHyper IgM Syndrome (HIGM), first described by Rosen et al. in 1961, is a primary immunodeficiency characterized by normal or elevated serum IgM and markedly low serum IgA, IgG, and IgE levels [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. This aberrant immunoglobulin profile results from a fundamental defect in isotype switching, a critical process for the development of effective humoral immunity. Defects occur during in the class switch recombination (CSR) and somatic hypermutation steps of mature B cell development due to various genetic mutations [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] Since the initial identification of mutations in the CD40L-encoding gene in 1992, genetic heterogeneity has been increasingly recognized in HIGM cases [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. While the most common form of HIGM is X-linked (HIGM type 1), arising from mutations in the CD40 ligand (\u003cem\u003eCD40L\u003c/em\u003e) gene, which disrupts T cell-dependent B cell activation and germinal center formation, autosomal recessive forms, such as those caused by mutations in the activation-induced cytidine deaminase (\u003cem\u003eAID\u003c/em\u003e) or \u003cem\u003eCD40\u003c/em\u003e genes[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], are more prevalent in populations with high rates of consanguineous marriages, such as in Turkiye, and also contribute to the clinical spectrum of HIGM.\u003c/p\u003e \u003cp\u003eThe development and maturation of B lymphocytes are meticulously orchestrated processes, characterized by the sequential and precise modulation of a plethora of cell surface markers. These markers serve not only as identifiers for distinct B cell subsets but also as pivotal mediators of signal transduction and intercellular communication. In HIGM, disruptions in isotype switching profoundly impact B cell maturation, leading to alterations in the expression patterns of key surface markers.\u003c/p\u003e \u003cp\u003eThis study aims to comprehensively characterize the B cell compartment in patients with HIGM, focusing on the expression profiles of CD19, CD20, CD24, CD27, CD38, and the immunoglobulin isotypes IgD and IgM. By employing flow cytometry analysis, we seek to delineate the quantitative and qualitative alterations in B cell subpopulations in HIGM patients, providing a deeper understanding of the immunopathogenesis of this complex disorder. This study will contribute to a more refined characterization of the B cell immunophenotype in HIGM, potentially leading to improved diagnostic and therapeutic strategies.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003ePatients followed with a diagnosis of HIGM at Department of Kayseri City Hospital Pediatric Allergy and Clinical Immunology were retrospectively screened using their ICD codes. The study cohort consisted of four patients diagnosed with Hyper-IgM (HIGM) syndrome who presented to our center for scheduled routine follow-up and clinical management. To establish a comparative baseline, blood samples were also obtained from age- and sex-matched healthy controls. Comprehensive information regarding the study objectives was provided to all participants and their legal guardians; subsequently, written informed consent was obtained from the parents of the patients and from the control subjects prior to their enrollment in the study. The study was conducted in accordance with the 2013 revision of the Declaration of Helsinki and was implemented by the Kayseri City Education and Research Hospital Local Ethics Committee with the approval decision numbered 235 dated 05.11.2024\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. PBMC Isolation\u003c/h2\u003e \u003cp\u003ePeripheral blood mononuclear cells (PBMCs) were isolated from venous blood samples of patients and age/sex-matched healthy individuals using Ficoll-Paque density gradient centrifugation. Briefly, blood samples were diluted 1:1 with PBS and carefully layered onto Ficoll-Paque (GE Healthcare). Samples were centrifuged at 400 g for 30 minutes, and the mononuclear cell layer was carefully collected. Cells were washed twice with PBS and suspended in RPMI-1640 culture medium supplemented with 10% FBS. Cell concentration was determined using a hemocytometer and used for phenotyping and proliferation assays.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. B Cell Surface Staining\u003c/h2\u003e \u003cp\u003eThe gating strategy followed a strict hierarchical approach: lymphocytes were first identified by FSC-A/SSC-A characteristics, debris excluded, followed by singlet selection to exclude doublets, and finally gated on CD19\u0026thinsp;+\u0026thinsp;to define the total B cell population. For the identification of B cell subpopulations, isolated PBMCs were stained with surface markers after Fc receptor blocking: CD19-FITC, CD20-BV510, CD24-PE, CD27-APC, CD38-PE-Cy7, IgD-PE-Dazzle, CD138-APC-Cy7. After staining, cells were incubated at 4\u0026deg;C for 30 minutes, washed with PBS, and prepared for flow cytometry analysis. Analyses were performed using a BD FACSARIA III device, and data were evaluated using FlowJo software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. B Cell Proliferation Assay\u003c/h2\u003e \u003cp\u003eTo evaluate B cell proliferation, isolated PBMCs were seeded in 96-well plates at 2x10⁵ cells/well and cultured in RPMI-1640\u0026thinsp;+\u0026thinsp;10% FBS medium. Cells were stimulated with CpG (20 \u0026micro;g/mL) and incubated for 5 days. Proliferation analysis was performed using: Control group (unstimulated, no CpG), and Experimental group (stimulated with CpG, 20 \u0026micro;g/mL CpG added). At the end of the incubation period, cells were stained with PE-conjugated CD19 antibody and incubated at 4\u0026deg;C for 30 minutes. After washing with PBS, cells were analyzed by flow cytometry. Proliferation rates were statistically compared.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Statistical Analysis Methods\u003c/h2\u003e \u003cp\u003eData analysis was performed by first testing the assumption of normal distribution. The normality of the groups was assessed using the Shapiro-Wilk test. Parametric tests were used for normally distributed data, and non-parametric tests were used for non-normally distributed data. One-way analysis of variance (ANOVA) was used to compare multiple groups. Tukey's multiple comparison test was performed to determine the source of differences between groups after ANOVA. Statistical Significance was indicated as follows: p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 values were considered statistically significant. p values were expressed with asterisk symbols: p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 \u0026rarr; * p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 \u0026rarr; ** p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 \u0026rarr; *** p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 \u0026rarr; ****All analyses were performed using GraphPad Prism 9 software.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Result","content":"\u003cp\u003e\u003cstrong\u003e3.1. Demographic and Clinical Characteristics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study included 4 patients with a mean age of 8.75 years (range: 4\u0026ndash;17), comprising 2 males and 2 females. All patients in the study group shared a common clinical history of recurrent respiratory tract infections (RTIs). Patient 2 (Pt 2), who underwent an adenotonsillectomy at the age of three due to chronic infections, was also followed by the pediatric oncology department for persistent recurrent cervical lymphadenopathy. Following her clinical evaluation, her sister (Pt 3) was screened due to a history of recurrent upper RTIs, leading to her subsequent diagnosis. The family history was notable for third-degree consanguinity between the parents and a maternal relative receiving intravenous immunoglobulin (IVIG) therapy for an unknown immunodeficiency. All patients exhibited a laboratory profile consistent with Hyper IgM Syndrome (HIGM), characterized by significantly low serum IgG and IgA levels. Genetic analysis confirmed a hemizygous mutation in the \u003cem\u003eCD40L\u003c/em\u003e gene for Pt 1, whereas Pt 2, Pt3, and Pt4 were identified as having a homozygous mutation in the \u003cem\u003eAICDA\u003c/em\u003e gene (c.70C\u0026gt;T, p.R24W). Clinical, demographic and laboratory characteristics of patients are shown in Table 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003eClinical, demographic and laboratory characteristics of patients\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"586\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 130px;\"\u003e\n \u003cp\u003ePt1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003ePt 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003ePt 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003ePt 4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003eAge/Sex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 130px;\"\u003e\n \u003cp\u003e4, Male\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e6, Female\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e8, Female\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e17, Male\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003eCBC\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;Hemoglobin (g/dl)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;WBC (/mm\u003csup\u003e3\u003c/sup\u003e)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;ANS (/mm\u003csup\u003e3\u003c/sup\u003e)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;Lymphocyte (/mm\u003csup\u003e3\u003c/sup\u003e)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 130px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e11.3\u003c/p\u003e\n \u003cp\u003e8390\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e2290\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e5280\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e11.2\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e7510\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e3940\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e2729\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e13.8\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e6830\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e3130\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e2930\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e10.8\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e8470\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e3730\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e3790\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003eLymphocytes subsets\u003c/p\u003e\n \u003cp\u003eCD3\u003c/p\u003e\n \u003cp\u003eCD4\u003c/p\u003e\n \u003cp\u003eCD8\u003c/p\u003e\n \u003cp\u003eCD16+56\u003c/p\u003e\n \u003cp\u003eCD19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 130px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e%70 (56-75)\u003c/p\u003e\n \u003cp\u003e%51 (28-47)\u003c/p\u003e\n \u003cp\u003e%14 (16-30)\u003c/p\u003e\n \u003cp\u003e%7 (4-17)\u003c/p\u003e\n \u003cp\u003e%22 (14-33)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e%72.2 (56-75)\u003c/p\u003e\n \u003cp\u003e%39.8 (28-47)\u003c/p\u003e\n \u003cp\u003e%24.9 (16-30)\u003c/p\u003e\n \u003cp\u003e%7.2 (4-17)\u003c/p\u003e\n \u003cp\u003e%17.8 (14-33)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e%71 (60-76)\u003c/p\u003e\n \u003cp\u003e%33 (31-47)\u003c/p\u003e\n \u003cp\u003e%31 (18-35)\u003c/p\u003e\n \u003cp\u003e%11 (4-17)\u003c/p\u003e\n \u003cp\u003e%16 (12-27)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e%70 \u0026nbsp; \u0026nbsp; (56-84)\u003c/p\u003e\n \u003cp\u003e%42 (31-52)\u003c/p\u003e\n \u003cp\u003e%25 (18-35)\u003c/p\u003e\n \u003cp\u003e%8 (3-22)\u003c/p\u003e\n \u003cp\u003e%20 (6-23)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003eIgG \u0026nbsp;(mg/dl)\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eIgA \u0026nbsp;(mg/dl)\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eIgM (mg/dl)\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eIgE \u0026nbsp;(mg/dl)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 130px;\"\u003e\n \u003cp\u003e18 (457-1120)\u003c/p\u003e\n \u003cp\u003e1 (35.7-192)\u003c/p\u003e\n \u003cp\u003e240 (58.7-198)\u003c/p\u003e\n \u003cp\u003e0.2\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e18 (483-1580)\u003c/p\u003e\n \u003cp\u003e15 (44.8-276)\u003c/p\u003e\n \u003cp\u003e2453 (50.3-242)\u003c/p\u003e\n \u003cp\u003e0.2\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u0026lt;0.3 (483-1580)\u003c/p\u003e\n \u003cp\u003e1 (44.8-276)\u003c/p\u003e\n \u003cp\u003e904 (50.3-242)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e454 (688-2430)\u003c/p\u003e\n \u003cp\u003e8 (46.3-385)\u003c/p\u003e\n \u003cp\u003e661 (60.7-323)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003eGene, zygosity, mutation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 130px;\"\u003e\n \u003cp\u003e\u003cem\u003eCD40L\u003c/em\u003e, Hemizygous c.346+1G\u0026gt;T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 322px;\"\u003e\n \u003cp\u003eHomozygous \u003cem\u003eAICDA\u003c/em\u003e mutation\u0026nbsp;\u003c/p\u003e\n \u003cp\u003ec.70C\u0026gt;T (p.R24W)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003eComplication\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 130px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003eLymphadenopathy,tonsillectomy\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" valign=\"top\" style=\"width: 586px;\"\u003e\n \u003cp\u003eWBC: White blood count \u0026nbsp; \u0026nbsp; ANS: absolute neutrophil count. \u0026nbsp;\u003c/p\u003e\n \u003cp\u003eSerum Ig age-specific reference values are shown in parentheses [6]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e3.2. B Cell Subsets Distribution and CSR Defects\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe immunophenotypic analysis of patient B cell subsets revealed distinct profiles compared to healthy controls (HC). \u003cstrong\u003eFigure 1\u003c/strong\u003e provides a comprehensive immunophenotypic map comparing the patient group with age- and sex-matched healthy controls (Figure 1 A, gate strategy), detailing B cell development from the total reservoir and naive-to-memory distribution (Figure 1 B), through class-switch recombination (CSR) efficiency at the isotype level (Figure 1C), to the terminal differentiation of transitional cells into antibody-secreting plasmablasts (Figure 1 D). In terms of total CD19⁺ B cells, Pt1 showed a significant increase, whereas Pt2, Pt3, and Pt4 exhibited a decrease. Specifically, an analysis of the CD19\u003csup\u003e+\u003c/sup\u003e B cell compartment revealed a significant expansion of the CD19\u003csup\u003e+\u003c/sup\u003e CD20\u003csup\u003e+\u003c/sup\u003e population in Patients 1 and 4. Analysis of B cell maturation revealed that while the frequency of CD27\u003csup\u003e+\u003c/sup\u003e memory B cells was decreased across all patients, this reduction achieved statistical significance in Pt1 and Pt3 (p\u0026lt;0.001 and p\u0026lt;0.05, respectively), although CD27\u003csup\u003e\u0026minus;\u003c/sup\u003e naive B cells were expanded in the entire cohort, the increase reached statistical significance exclusively in Pt1, Pt2, and Pt3 (p\u0026lt;0.001; p\u0026lt;0.05 and p\u0026lt;0.01, respectively).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Further characterization of the CD19⁺CD20⁺ compartment using CD27 and IgD markers indicated that CD27⁺IgD⁺ cells were significantly reduced in Pt1 and Pt2 (p\u0026lt;0.05 and p\u0026lt;0.01, respectively). Similarly, switched memory cells (CD27⁺IgD⁻) were decreased in Pt1, Pt3, and Pt4, and double-negative cells (CD27⁻IgD⁻) were lower in Pt1 and Pt3. Conversely, CD27⁻IgD⁺ cells were significantly increased across all patients (Pt1\u0026ndash;Pt4). Analysis of B cell differentiation stages revealed a significant depletion of CD20⁻CD38⁺⁺ plasmablasts in Pt1 (p\u0026lt;0.01), whereas a marked expansion of this population was observed in Pt2 (p\u0026lt;0.001). Sub-analysis within the CD38⁺CD20⁻ compartment showed a significant increase in the frequency of CD27⁻ precursor B cells in Pt1 (p\u0026lt;0.05), contrasted by a downward trend in CD27⁺ plasma B cells (p\u0026lt;0.05). Furthermore, regarding transitional B cells (CD24⁺CD38⁺), Pt1 exhibited a significant reduction compared to the healthy control group (p\u0026lt;0.01), while Pt2 showed a statistically significant elevation (p\u0026lt;0.05) \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3. B Cell Functional Analysis: CpG Stimulation and Proliferation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFunctional assays focused on B cell proliferation following CpG stimulation revealed that Pt1 and Pt4 had significantly higher proliferation rates compared to healthy controls (p\u0026lt;0.05 and p\u0026lt;0.0001, respectively), whereas Pt3 showed a significant reduction. Regarding general lymphocyte proliferation, Pt1 again showed a significant increase, while the responses in Pt3 and Pt4 remained stable. Overall lymphocyte and CD19⁺ B cell proliferation profiles are presented in Figure 2.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eB cell maturation involves dynamic surface marker changes, enabling the identification of distinct peripheral blood subpopulations via multicolor flow cytometry. Since the impaired formation of these subsets is a hallmark of various primary immunodeficiencies (PIDs), analyzing these stages provides critical insights into immune dysfunction. In this context, the most characteristic finding of our study is the uniform accumulation of B cells at the naive (CD27\u003csup\u003e\u0026minus;\u003c/sup\u003eIgD\u003csup\u003e+\u003c/sup\u003e) stage and the dramatic loss of class-switched memory B cells (CD27\u003csup\u003e+\u003c/sup\u003eIgD\u003csup\u003e\u0026minus;\u003c/sup\u003e) across all Hyper-IgM (HIGM) patients, regardless of the underlying genetic mutation (\u003cem\u003eCD40L\u003c/em\u003e or \u003cem\u003eAICDA\u003c/em\u003e)[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. This cellular profile provides definitive evidence of defective class-switch recombination (CSR) at the systemic level. However, a granular analysis of plasmablast and precursor distributions reveals critical differences in the biological mechanisms underlying these mutations, serving as a pivotal tool for differential diagnosis.\u003c/p\u003e \u003cp\u003eIn Pt 1, diagnosed with Type 1 HIGM (CD40L deficiency), the proportion of CD19\u003csup\u003e+\u003c/sup\u003e B cells within total lymphocytes remains elevated, yet the qualitative integrity of these cells is severely compromised. Memory B cells are categorized into two distinct subsets based on the expression of CD27 and IgD. CD27⁺IgD⁺ memory B cells are thought to develop independently of the germinal center (GC) reaction and represent circulating marginal zone B cells that respond to T-independent antigens. In contrast, CD27⁺IgD⁻ (switched) memory B cells are products of the post-germinal center reaction. Consequently, a marked reduction or absence of these switched memory B cells is highly indicative of a disrupted GC reaction[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Due to the lack of CD40L-mediated T-cell help, these B cells are unable to initiate GC formation, which essentially halts antigen-specific memory development[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The absence of CD27\u003csup\u003e+\u003c/sup\u003e and IgD\u003csup\u003e\u0026minus;\u003c/sup\u003eCD27\u003csup\u003e+\u003c/sup\u003e (switched memory) cells confirms a maturation arrest at the naive stage[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Furthermore, the total lack of plasmablast-like cells (CD20\u003csup\u003e\u0026minus;\u003c/sup\u003eCD38\u003csup\u003e++\u003c/sup\u003e) underscores that terminal differentiation cannot proceed without functional CD40L signaling.\u003c/p\u003e \u003cp\u003eThe landscape in Type 2 HIGM (\u003cem\u003eAID\u003c/em\u003e deficiency), observed in Pt2, Pt3, and Pt4, is more complex. In these individuals, the CD40/CD40L interaction remains intact, enabling B cells to form GCs and acquire limited CD27 expression. However, the absence of the AID enzyme renders CSR and somatic hypermutation biochemically impossible. While these cells may initiate GC reactions, the marked decrease of IgD\u003csup\u003e\u0026minus;\u003c/sup\u003e CD27\u003csup\u003e+\u003c/sup\u003e cells highlights their inability to successfully switch antibody classes[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWhen the double-negative (DN; CD27\u003csup\u003e\u0026minus;\u003c/sup\u003eIgD\u003csup\u003e\u0026minus;\u003c/sup\u003e) B cell subset was analyzed, a significant reduction was observed in Pt1 and Pt3, whereas this decrease did not reach statistical significance in Pt2 and Pt4. According to the literature, the DN1 subpopulation is considered a product of an aborted GC reaction and a precursor to switched memory (CD27\u003csup\u003e+\u003c/sup\u003e) B cells. In contrast, DN2 and DN3 cells are known to originate via GC-independent, extrafollicular pathways. The non-significant results in Pt2 and Pt4 suggest that the extrafollicular pathway might remain partially active or allow for a low level of DN cell production despite the underlying genetic defects. Furthermore, it is established that DN B cells tend to expand with aging and chronic infections[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Therefore, in Pt4\u0026mdash;the oldest patient in our cohort\u0026mdash;years of cumulative antigenic exposure may have driven the expansion of this subpopulation despite the genetic constraint, potentially through these alternative developmental routes\u003c/p\u003e \u003cp\u003eThe distinct precursor distributions further delineate the two HIGM types. In Pt1, the increase in CD38\u0026thinsp;+\u0026thinsp;CD27\u0026minus; precursors alongside a plasmablast deficit reflects the absolute failure of GC formation due to CD40L deficiency. Conversely, the expansion of CD24\u003csup\u003e+\u003c/sup\u003eCD38\u003csup\u003e+\u003c/sup\u003e populations in Pt2 suggests that AID-deficient B cells are driven toward maturation by antigenic stimulation but remain sequestered in a \"pre-switched\" or late-naive state. The increased plasmablast levels and high transitional cell (CD24\u003csup\u003e+\u003c/sup\u003eCD38\u003csup\u003e+\u003c/sup\u003e ) proportions in Pt2 reflect a state where GCs can form but become hyperplastic. In alignment with our findings, a study in the literature involving 18 patients with Type 2 HIGM reported 13 lymphoid hyperplasia leading to tonsillectomy in 5 of these cases [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In a previous study, lymphoid hyperplasia was observed in 20 out of 29 patients prior to IVIG replacement therapy, with peripheral lymph nodes and tonsils being the most commonly affected sites. Similarly, Pt2 in our group presented with recurrent cervical lymphadenopathy and a history of adenotonsillectomy at age 3, suggesting an early-onset lymphoproliferative phenotype. While data in the literature often point to follicular hyperplasia with giant GCs, the clinical progression in our case\u0026mdash;including follow-up by pediatric oncology\u0026mdash;highlights the importance of monitoring these patients for lymphoproliferative complications[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. This hyperplasia is a result of chronic inflammatory responses in GCs that are constantly stimulated but unable to complete CSR. This environment pushes B cells toward a plasmablast phenotype, yet they remain incapable of producing functional, switched antibody classes.\u003c/p\u003e \u003cp\u003ePeripheral B cell ontogeny progresses along a linear maturation spectrum defined by the dynamic shifts in CD38 and CD24 expression density. According to the model by Buffa et al., newly emigrated CD38\u003csup\u003ehigh\u003c/sup\u003eCD24\u003csup\u003ehigh\u003c/sup\u003e (Transitional) cells gradually reduce these markers to become CD38\u003csup\u003eint\u003c/sup\u003eCD24\u003csup\u003eint\u003c/sup\u003e (Mature Naive) and eventually CD38\u003csup\u003e\u0026minus;\u003c/sup\u003e memory cells. The marked expansion of the CD24\u003csup\u003e+\u003c/sup\u003eCD38\u003csup\u003e+\u003c/sup\u003epopulation in Pt2 indicates a \"maturational stasis\"[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Viewed through the functional compartmentalization theory of Sanz et al., this finding confirms that while these cells receive antigenic signals, genetic constraints (AID deficiency) prevent the completion of the GC reaction, causing them to accumulate in a \"pre-memory\" phase [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The analysis of B cell maturation markers in patients with homozygous AICDA mutations (Pt2, Pt3, and Pt4) reveals a distinct biological pattern where the percentages of CD38⁺ CD27⁻ and CD38⁺ CD27⁺ populations remain comparable to healthy controls. This observation confirms that, unlike in CD40L deficiency (Pt1), the T-cell-dependent activation signaling is functionally preserved in AICDA-deficient patients. Since the molecular defect in these individuals is intrinsic to the nuclear enzymatic machinery rather than the surface receptor signaling, their B lymphocytes successfully process activation cues and proceed to express the CD27 surface marker. Consequently, these cells are capable of entering the plasma cell differentiation pathway and phenotypically acquiring a \"mature\" profile. However, this represents a pseudo-maturation state; while the cells can harbor the CD27 marker on their surface, the underlying genetic defect in AID prevents the execution of CSR within the nucleus, leading to a functional failure in producing switched antibody isotypes.\u003c/p\u003e \u003cp\u003eDespite sharing the same homozygous AICDA mutation (c.70C\u0026thinsp;\u0026gt;\u0026thinsp;T, p.R24W) and a common family history of third-degree consanguinity, Pt2, Pt3, and Pt4 exhibit significant phenotypic inconsistencies. This clinical variation is most evident when comparing the two sisters (Pt2 and Pt3); while both share a history of recurrent respiratory tract infections (RTI), Pt2\u0026rsquo;s clinical course was marked by more severe lymphadenopathy and a history of adenotonsillectomy.\u003c/p\u003e \u003cp\u003eThese clinical disparities are further mirrored in the patients' cellular profiles and proliferative capacities. Contrary to what might be expected from her peripheral B cell count, Pt4\u0026mdash;the oldest patient in the cohort\u0026mdash;exhibited a significantly reduced CD19\u0026thinsp;+\u0026thinsp;B cell frequency (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) alongside the highest serum IgG levels and a notably robust proliferative response to CpG stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This suggests that while the absolute B cell pool is contracted in Pt4, the remaining cells retain a high functional capacity for activation. In contrast, the cellular profiles of Pt2 and Pt3 show a different correlation. Pt3 exhibited both diminished peripheral B cell counts and a significantly lower proliferative response, indicating a more profound exhaustion or defect in her B cell compartment compared to Pt4. These variations highlight that peripheral B cell frequency alone does not always predict functional competency in Hyper-IgM patients, as compensatory proliferative mechanisms or disease chronicity may influence the immunophenotypic profile differently across individuals.\u003c/p\u003e \u003cp\u003eThe proliferation data obtained in this study reveal the heterogeneous functional impact of different genetic mutations on B cell activation in HIGM. While CD40L deficiency is characterized by a lack of T-cell-dependent stimulation, it is well-established that these B cells can remain responsive to T-cell-independent signals, such as the TLR9 ligand CpG. The increased proliferation observed in Pt1 suggests that the TLR9 activation pathway is not only preserved but may also exhibit a compensatory hyper-responsiveness in the absence of CD40L-mediated signaling. Imai et al. demonstrated that B cells from both HIGM2 and HIGM4 patients are capable of achieving normal proliferation when stimulated in vitro with sCD40L and IL-4[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Our results using CpG stimulation are consistent with this observation, as Pt1 (CD40L deficiency) and Pt4 (AICDA mutation) exhibited significantly increased proliferation rates compared to healthy controls (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, respectively). This supports the thesis that HIGM B cells can remain highly responsive to activation stimuli, even when the downstream terminal differentiation and class-switching machinery is defective. Interestingly, a notable divergence was observed between Pt3 and Pt4 despite both sharing a homozygous \u003cem\u003eAICDA\u003c/em\u003e mutation. While Pt4 showed robust proliferation, the significant suppression of B cell proliferation in Pt3 suggests that the impact of AID deficiency extends beyond immunoglobulin class switching and may involve broader signaling or survival defects that vary among individuals. These findings suggesting that compensatory immune mechanisms or environmental exposures may partially mitigate the AICDA defect over time.\u003c/p\u003e \u003cp\u003eThe clinical presentation of our study group is highly consistent with the established literature on primary B cell immunodeficiencies. As highlighted in the previous study, recurrent RTIs represent the most frequent clinical manifestation in these patients[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In total alignment with these reports, recurrent upper RTIs were the primary clinical finding across our entire patient group[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. De la Morena et al. argue that even patients with Type 1 HIGM who appear stable on immunoglobulin replacement therapy remain at high risk for life-threatening complications, and therefore stem cell transplantation should be considered as the definitive treatment[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. For Type 2 HIGM patients, the indications for stem cell transplantation are less standardized compared to Type 1. Specifically, our first patient (\u003cem\u003eCD40L\u003c/em\u003e deficiency), who is continuing IVIG therapy and antimicrobial prophylaxis, is stable with the current treatment; not performing a transplant in this patient is consistent with a more conservative management approach.\u003c/p\u003e \u003cp\u003eSeveral limitations of this study should be acknowledged. First, the sample size is relatively small, which is a common challenge in research involving rare primary immunodeficiencies. While this limited cohort size may constrain the generalizability of our findings to all HIGM subtypes, it reflects the clinical reality of managing such rare conditions. Second, our immunophenotypic analysis was restricted to peripheral blood mononuclear cells (PBMCs), which may not fully represent the complex B-cell dynamics and germinal center reactions occurring within secondary lymphoid organs. Third, we observed significant inter-individual phenotypic and functional variability, particularly among patients with the same homozygous \u003cem\u003eAICDA\u003c/em\u003e mutation. A key limitation is the lack of additional parameters, such as detailed environmental exposure history or epigenetic profiling, which might have better elucidated the underlying causes of this clinical and proliferative heterogeneity. Despite these limitations, this study has yielded significant findings in the field of B-cell immunopathology. Specifically, through high-resolution, multi-parameter flow cytometric analysis, we compared X-linked CD40L deficiency with autosomal recessive homozygous AICDA mutations (c.70C\u0026thinsp;\u0026gt;\u0026thinsp;T; p.R24W). By extending our characterization beyond basic subsets to include detailed differentiation stages such as transitional cells and plasmablasts, we have successfully delineated mutation-specific \"pseudo-maturation\" states and maturation arrest points that are critical for differential diagnosis. Furthermore, the inclusion of functional B cell proliferation assays highlights the heterogeneous effect of different genetic defects on B cell activation, offering valuable insights into disease mechanisms and potential diagnostic strategies for HIGM patients. However, further studies with larger sample sizes are recommended to confirm these results.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eDetailed analysis of B cell subsets elucidates the fundamental biological disparities between HIGM subtypes. The significant loss of CD27\u003csup\u003e\u0026minus;\u003c/sup\u003eIgD\u003csup\u003e\u0026minus;\u003c/sup\u003e cells and the mutation-specific shifts in maturation stages reaffirm the critical importance of flow cytometry in the diagnosis and clinical monitoring of HIGM.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no conflicts of interest regarding the publication of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthorship Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by H.\u0026Uuml;.\u0026Ccedil;., M.C., and A.A. Methodology and formal analysis were conducted by S.E. and A.E. All authors read and approved the final manuscript..\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Disclosure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was conducted in strict accordance with the ethical principles for medical research involving human subjects as outlined in the Declaration of Helsinki (2013 revision). The study protocol was reviewed and approved by the Local Ethics Committee (Approval Date: November 5, 2024; Decision Number: 235). Written informed consent was obtained from the parents or legal guardians of all participants prior to their inclusion in the study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eNotarangelo LD, Duse M, Ugazio AG. Immunodeficiency with hyper-IgM (HIM). Immunodeficiency Reviews 1992;3(2):101-21. (In eng) (https://www.ncbi.nlm.nih.gov/pubmed/1554497). https://www.ncbi.nlm.nih.gov/pubmed/1554497\u003c/li\u003e\n \u003cli\u003eYazdani R, Fekrvand S, Shahkarami S, Azizi G, Moazzami B, Abolhassani H, et al. The hyper IgM syndromes: Epidemiology, pathogenesis, clinical manifestations, diagnosis and management. Clinical Immunology 2019;198:19-30. DOI: 10.1016/j.clim.2018.11.007. https://www.ncbi.nlm.nih.gov/pubmed/30439505 https://www.sciencedirect.com/science/article/abs/pii/S1521661618306399?via%3Dihub\u003c/li\u003e\n \u003cli\u003eKeerthikumar S, Raju R, Kandasamy K, Hijikata A, Ramabadran S, Balakrishnan L, et al. 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DOI: 10.3389/fimmu.2011.00081. https://www.ncbi.nlm.nih.gov/pubmed/22566870 https://public-pages-files-2025.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2011.00081/pdf\u003c/li\u003e\n \u003cli\u003eVale AM, Schroeder HW, Jr. Clinical consequences of defects in B-cell development. J Allergy Clin Immunol 2010;125(4):778-87. DOI: 10.1016/j.jaci.2010.02.018. https://www.ncbi.nlm.nih.gov/pubmed/20371392 https://www.jacionline.org/article/S0091-6749(10)00361-1/pdf\u003c/li\u003e\n \u003cli\u003eBeckers L, Somers V, Fraussen J. IgD(-)CD27(-) double negative (DN) B cells: Origins and functions in health and disease. Immunology Letters 2023;255:67-76. DOI: 10.1016/j.imlet.2023.03.003. https://www.ncbi.nlm.nih.gov/pubmed/36906182 https://www.sciencedirect.com/science/article/abs/pii/S016524782300038X?via%3Dihub\u003c/li\u003e\n \u003cli\u003eRevy P, Muto T, Levy Y, Geissmann F, Plebani A, Sanal O, et al. Activation-induced cytidine deaminase (AID) deficiency causes the autosomal recessive form of the Hyper-IgM syndrome (HIGM2). Cell 2000;102(5):565-75. DOI: 10.1016/s0092-8674(00)00079-9. https://www.ncbi.nlm.nih.gov/pubmed/11007475 https://www.cell.com/cell/pdf/S0092-8674(00)00079-9.pdf\u003c/li\u003e\n \u003cli\u003eQuartier P, Bustamante J, Sanal O, Plebani A, Debre M, Deville A, et al. Clinical, immunologic and genetic analysis of 29 patients with autosomal recessive hyper-IgM syndrome due to Activation-Induced Cytidine Deaminase deficiency. Clin Immunol 2004;110(1):22-9. DOI: 10.1016/j.clim.2003.10.007. https://www.ncbi.nlm.nih.gov/pubmed/14962793 https://www.sciencedirect.com/science/article/abs/pii/S1521661603002857?via%3Dihub\u003c/li\u003e\n \u003cli\u003eBuffa S, Pellicano M, Bulati M, Martorana A, Goldeck D, Caruso C, et al. A novel B cell population revealed by a CD38/CD24 gating strategy: CD38(-)CD24 (-) B cells in centenarian offspring and elderly people. Age (Dordr) 2013;35(5):2009-24. (In eng). DOI: 10.1007/s11357-012-9488-5. https://www.ncbi.nlm.nih.gov/pubmed/23129025 https://pmc.ncbi.nlm.nih.gov/articles/PMC3776115/\u003c/li\u003e\n \u003cli\u003eSanz I, Wei C, Lee FE, Anolik J. Phenotypic and functional heterogeneity of human memory B cells. Seminars in Immunology 2008;20(1):67-82. (In eng). DOI: 10.1016/j.smim.2007.12.006. https://www.ncbi.nlm.nih.gov/pubmed/18258454 https://pmc.ncbi.nlm.nih.gov/articles/PMC2440717/\u003c/li\u003e\n \u003cli\u003eImai K, Catalan N, Plebani A, Marodi L, Sanal O, Kumaki S, et al. Hyper-IgM syndrome type 4 with a B lymphocyte-intrinsic selective deficiency in Ig class-switch recombination. The Journal of Clinical Investigation 2003;112(1):136-42. (In eng). DOI: 10.1172/JCI18161. https://www.ncbi.nlm.nih.gov/pubmed/12840068 https://dm5migu4zj3pb.cloudfront.net/manuscripts/18000/18161/JCI0318161.pdf\u003c/li\u003e\n \u003cli\u003eMoazzami B, Yazdani R, Azizi G, Kiaei F, Tafakori M, Modaresi M, et al. Respiratory Complications in Patients with Hyper IgM Syndrome. J Clin Immunol 2019;39(6):557-568. (In eng). DOI: 10.1007/s10875-019-00650-3. https://www.ncbi.nlm.nih.gov/pubmed/31183658 https://link.springer.com/article/10.1007/s10875-019-00650-3\u003c/li\u003e\n \u003cli\u003ede la Morena MT, Leonard D, Torgerson TR, Cabral-Marques O, Slatter M, Aghamohammadi A, et al. Long-term outcomes of 176 patients with X-linked hyper-IgM syndrome treated with or without hematopoietic cell transplantation. Journal of Allergy and Clinical Immunology 2017;139(4):1282-1292. (In eng). DOI: 10.1016/j.jaci.2016.07.039. https://www.ncbi.nlm.nih.gov/pubmed/27697500 https://www.jacionline.org/article/S0091-6749(16)30964-2/pdf\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-clinical-immunology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"joci","sideBox":"Learn more about [Journal of Clinical Immunology](https://www.springer.com/journal/10875)","snPcode":"10875","submissionUrl":"https://submission.nature.com/new-submission/10875/3","title":"Journal of Clinical Immunology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Hyper-IgM syndrome, B cell maturation, CD40L, AICDA, class-switch recombination","lastPublishedDoi":"10.21203/rs.3.rs-8894623/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8894623/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003ePurpose:\u003c/strong\u003e Hyper-IgM syndromes (HIGM) are primary immunodeficiencies characterized by defective class-switch recombination (CSR) and impaired humoral immunity. While genetic causes such as CD40L and AICDA mutations are well-established, a detailed comparison of B cell maturation dynamics across different HIGM subtypes remains limited. This study aims to comprehensively characterize B cell immunophenotypes and functional responses in HIGM patients and to delineate mutation-specific differences in B cell maturation and proliferation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e Four patients with genetically confirmed HIGM (one CD40L and three homozygous AICDA mutations, c.70C\u0026gt;T; p.R24W) and age- and sex-matched healthy controls were studied. Peripheral blood mononuclear cells were analyzed via multiparameter flow cytometry to define B cell subsets based on CD19, CD20, CD24, CD27, CD38, IgD, and IgM expression. Additionally, B cell proliferation was assessed following CpG stimulation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e All patients exhibited a marked reduction in class-switched memory B cells (CD27\u003csup\u003e+\u003c/sup\u003eIgD\u003csup\u003e-\u003c/sup\u003e),and an accumulation of naive B cells (CD27\u003csup\u003e−\u003c/sup\u003eIgD\u003csup\u003e+\u003c/sup\u003e), consistent with defective CSR. The CD40L-deficient patient demonstrated profound depletion of plasmablasts and precursor skewing, reflecting a failure in germinal center formation. In contrast, AICDA patients showed preserved CD27 expression with variable expansion of transitional and plasmablast populations, suggesting intact T cell–dependent activation but an intrinsic failure of CSR. Functional assays revealed heterogeneous proliferative responses, with inter-individual variability observed particularly among AICDA-deficient patients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e Detailed immunophenotyping reveals distinct B cell maturation arrest points in CD40L- versus AICDA-associated HIGM. Flow cytometric analysis provides valuable insights into disease mechanisms, supports differential diagnosis, and informs clinical monitoring and therapeutic decision-making in HIGM\u003c/p\u003e","manuscriptTitle":"Deciphering B cell Maturation Dynamics in Hyper IgM Syndromes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-09 06:33:04","doi":"10.21203/rs.3.rs-8894623/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-31T12:23:22+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-26T17:46:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"313570287781832264752634146775149903154","date":"2026-03-16T10:14:10+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-12T20:23:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"289894387016284943961374052348254124052","date":"2026-03-05T17:23:41+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-03T12:30:29+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-19T06:36:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-19T06:33:51+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Clinical Immunology","date":"2026-02-16T15:47:24+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-clinical-immunology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"joci","sideBox":"Learn more about [Journal of Clinical Immunology](https://www.springer.com/journal/10875)","snPcode":"10875","submissionUrl":"https://submission.nature.com/new-submission/10875/3","title":"Journal of Clinical Immunology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"bce726b9-7a32-409f-a1f2-0f01e4d7d9ec","owner":[],"postedDate":"March 9th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-11T23:53:30+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-09 06:33:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8894623","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8894623","identity":"rs-8894623","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}