
==== Front
J Clin Immunol
J Clin Immunol
Journal of Clinical Immunology
0271-9142
1573-2592
Springer US New York

39305354
1805
10.1007/s10875-024-01805-7
Research
Levels of Natural Antibodies Before and After Immunoglobulin Replacement Treatment Affect the Clinical Phenotype in Common Variable Immunodeficiency
Sarrigeorgiou Ioannis 1
Tsinti Gerasimina 12
Kalala Fani 2
Germenis Anastasios 2
Speletas Matthaios 2
Lymberi Peggy plymberi@pasteur.gr

1
1 https://ror.org/035cy3r13 grid.418497.7 Laboratory of Immunology, Department of Immunology, Hellenic Pasteur Institute, Athens, Greece
2 https://ror.org/04v4g9h31 grid.410558.d 0000 0001 0035 6670 Department of Immunology & Histocompatibility, Faculty of Medicine, University of Thessaly, Larissa, Greece
21 9 2024
21 9 2024
2025
45 1 1322 5 2024
9 9 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Natural antibodies (NAbs) occurring in individuals without prior exposure to specific antigens, provide direct first barrier protection against pathogens, and exert immunoregulation thus actively contributing to the maintenance of immune homeostasis, controlling inflammatory processes and preventing autoimmunity. Common variable immunodeficiency (CVID) is a heterogeneous group of disorders characterized by a compromised immune function that brings into focus the role of NAbs. Our aim was to explore whether NAb levels could serve as potential key indicators in CVID for monitoring disease progression and predicting outcomes. In this study, we analyzed a Hellenic cohort of 56 patients with CVID (31 newly diagnosed and 25 under immunoglobulin replacement therapy-IgRT) and 33 healthy controls, for total Ig levels and serum IgM and IgG NAb levels against five informative target-antigens of NAbs, namely, actin, DNA, carbonic anhydrase, F(ab΄)2 fragments of human IgG and TriNitroPhenyl. In addition, follow-up pre- and post- IgRT samples were analyzed in ten (10) patients of our cohort. Results showed that Ig-treated patients exhibited significantly lower IgM NAb levels than untreated patients and healthy controls against all panel antigens. In the follow-up samples, pre-treatment IgM NAb levels negatively correlated with total serum IgM. This imbalance was only partially restored after IgRT, with a significant decrease in IgM NAb levels observed in nine out of ten patients. Moreover, post-treatment patients with recurrent infections presented significantly lower IgM NAb levels, a reduction also observed in patients with bronchiectasis independently of treatment status. On the contrary, post-treatment patients with enteropathy had significantly higher IgM NAb levels against all panel antigens, an increase also noted in patients with autoimmune diseases. Regarding IgG NAbs, replacement therapy restored levels to those of healthy controls. In conclusion, impaired NAb levels are found in CVID patients, particularly related to certain phenotypes. Moreover, the significant decrease in IgM NAb levels after IgRT suggests a potential association with disease course and complications. The results suggest that administration of human IgM NAbs may be an effective combinatorial treatment in selected patients. Further research is needed to understand the functional roles of NAbs in CVID and its complex clinical phenotypes.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10875-024-01805-7.

Keywords

CVID
Clinical phenotype
Immunoglobulin Replacement Treatment (IgRT)
Natural Antibodies (NAbs)
IgM
IgG
issue-copyright-statement© Springer Science+Business Media, LLC, part of Springer Nature 2025
==== Body
pmcIntroduction

Common variable immunodeficiency (CVID) is a heterogeneous group of disorders characterized by permanent and sustained hypogammaglobulinemia, an inadequate or lacking response to immunization, and a wide spectrum of clinical manifestations, including recurrent infections, autoimmunity, granulomas formation, benign lymphoproliferation and an increased incidence of lymphomas and cancer [1, 2]. The golden standard of treatment in CVID is immunoglobulin replacement therapy (IgRT), intravenously (IVIG) or subcutaneously (SCIG), along with antibiotics for the treatment of infections. The management of non-infectious manifestations of CVID remains a challenge for clinicians [3]. Moreover, even following appropriate IgRT and the successful trough IgG levels in the bloodstream, there are still patients who are unable to eradicate infection or who display severe complications, including bronchiectasis [4, 5].

Natural antibodies (NAbs) are germline encoded antibodies present in individuals without prior exposure to specific antigens [6]. NAbs are mainly produced by B1 cells and are mostly of IgM isotype but also of IgG, IgA and IgE isotypes [7]. NAbs are polyreactive, targeting a variety of self and foreign antigens and this property is directly linked to their numerous biological functions [8]. NAbs are considered part of the innate immune system. They play crucial roles in first line host defense by providing immediate protection against pathogens, as well as in clearance of apoptotic cells and immune complexes, and thereby have a major role in maintaining tissue homeostasis and preventing excessive inflammation [9]. Moreover, NAbs serve as important components of the immune system that contribute to immunoregulation and shape the development and function of B and T cells [10, 11]. Despite their potential significance in the pathogenesis of primary antibody deficiency (PAD), the involvement of NAbs in CVID patients is understudied. Lower IgG NAb levels to isohemagglutinins have been previously documented in CVID patients [12], and significant alterations in marginal zone B cells and B1 cells have been reported, yet with contradictory results [13].

To determine whether fluctuations or discrepancies in NAb levels might contribute to the pathogenesis and/or the varied clinical phenotype of CVID, here we investigated NAb levels in CVID patients, before and after treatment initiation (i.e., IVIg replacement) and correlated them with clinical manifestations. We analyzed 56 CVID patients (31 newly-diagnosed, 25 under IgRT), and 33 healthy controls for total Ig levels, and IgM and IgG NAb levels against five target antigens which share informative features associated with NAb recognition and their regulatory functions. Specifically, the target antigens were G-actin, a cytoskeletal component and common target of NAbs implicated in various human diseases [14, 15], DNA, a known target of autoantibodies-markers of systemic autoimmune diseases [16], carbonic anhydrase (CA), an evolutionarily conserved autoantigen associated with red blood cells which frequently mediates autoimmune manifestations [17], F(ab')2 fragments of human IgG, which are major components of NAb-mediated immunoregulatory network via Fab-Fab interactions [18], and TriNitroPhenyl (TNP), a surrogate measure to assess polyreactivity of NAbs [19]. Follow-up analysis of pre- and post-IgRT samples was also performed in ten patients. Data of CVID clinical manifestations for each patient were analyzed together with NAb levels in univariate and multivariate models to reveal potential associations.

Materials and Methods

Patients and Samples

Subjects enrolled in the study are shown in Table 1. Among them, 31 CVID patients were evaluated before the initiation of IgRT, 25 CVID patients were already under IgRT, and 33 were healthy matched controls. All subjects were analyzed in parallel. In addition, 10 newly diagnosed patients were evaluated in follow-up pre- and post IgRT samples. The diagnosis of the disease was based on standard criteria including (a) low serum levels of IgG, IgA and/or IgM, more than two standard deviations below the normal mean for the age; (b) absence of isohemagglutinins and poor responses to vaccines (especially the polysaccharide ones); and (c) an exclusion of other defined causes of hypogammaglobulinemia and/or other types of Inborn Errors of Immunity [20]. The study was approved by the Ethics Committee of the General University Hospital of Larissa in Thessaly (6/18.3.2015), and an informed consent was obtained from all patients. Table 1 Patients and controls enrolled in the study

Subjects	n	Age
(yrs±SD)	Sex
(Male/Female)	IgRT Duration (mo±SD)	
Total	56	43±16	24/32	40±77	
Newly diagnosed	31	43±18	12/19		
Follow-up:	10		4/8		
 Pre-treatment		40±14			
 Post-treatment		44±14		29±25	
Under long term IgRT	25	42±15	12/13	94±107	
Healthy Controls	33	38±10	10/23		

CVID complications such as frequent recurrent infections (3-4 attacks per year), autoimmunity, bronchiectasis, lymphoproliferation, enteropathy, atopy, cancer and granulomatosis were identified and recorded by the clinician at the time of diagnosis (Table 2). Enteropathy includes chronic diarrhea, malabsorption, and inflammatory bowel disease-like symptoms. Table 2 CVID related patients’ characteristics

Characteristics	Patients (n,%)	
Recurrent Infections	52 (85.7%)	
Autoimmunity	39 (69.6%)	
Bronchiectasis	15 (26.8%)	
Lymphoproliferation	34 (60.7%)	
Atopy and/or drug allergy	14 (25%)	
Enteropathy	11 (19.6%)	
Cancer	10 (19.7%)	
Granulomatosis	8 (14.3%)	

ELISA Measurements

IgM and IgG NAb levels in patient sera were measured by indirect in-house ELISA: i.e., NAbs against DNA from calf thymus (D1501, Sigma Aldrich), CA from human erythrocytes (C3934, Sigma Aldrich), as well as TNP-BSA, human G-actin and IgG-F(ab΄)2 fragments prepared as previously described [21, 22]. Antigens were immobilized on high-binding, flat-bottomed 96-well MaxiSorp plates (Nunc, Denmark) at 10μg/ml for actin, DNA and TNP-BSA, 5 μg/ml for CA and 2 μg/ml for F(ab΄)2 fragments.

Briefly, plates were coated overnight at 4 °C in carbonate-bicarbonate buffer 0,1M pH 9,6. Plates were thoroughly washed with PBS, and then saturated for 1 h at 37 °C with PBS containing 1% BSA. Patient sera diluted 1/50 in PBS containing 0.1% Tween and 1% BSA (PBS-T-BSA) were incubated independently with each panel antigen overnight at 4 °C. Plates were washed and incubated with secondary antibodies (goat anti-human -μ, and -γ chain) conjugated with alkaline phosphatase diluted at optimum predefined concentration in PBS-T-BSA for 2 h at 37°C. After thorough wash, antibody binding to immobilized antigen was assessed with the addition of the enzyme’s soluble chromogenic substrate p-nitrophenyl phosphate disodium hexahydrate (pNPP) (Sigma-Aldrich) and optical density (OD) of colored reaction product was measured at 405 nm (620 nm reference) using a spectrophotometer (TECAN Spark Control Magellan V2.2, Grödig/Salzburg, Austria). Values were converted to arbitrary units by the equation AU=OD*1000. All experiments were run in duplicate and three positive control sera (previously defined as reference samples) were always added in each plate for inter-assay normalization (<15%).

Statistical Analysis

The d’Agostino-Pearson omnibus normality test was used to assess the normality of the data distribution. For non-parametric two independent group comparisons the Mann–Whitney U test was used. The non-parametric (ANOVA) Friedman test was used to detect differences between groups. The non-parametric Spearman correlation matrix was employed to assess the strength and direction of monotonic relationships between total serum IgM and IgM NAb levels. In all cases, the significance level was set at 5%, the tests were two sided and a result was considered significant if the estimated p-value was less than the significance level (p<0.05). Statistical analysis was performed and graphs were made in GraphPad Prism version 9.0.0 (GraphPad Software, San Diego, California USA).

Results

IgM NAb Levels among CVID Patients and Healthy Controls

Among the 56 patient sera, five newly diagnosed patients and six patients under IgRT had undetectable serum IgM and were excluded from this analysis. Therefore, levels of serum IgM NAbs against G-actin, DNA, CA, F(ab΄)2 and TNP were estimated in 26 newly diagnosed patients and 19 patients under treatment, as well as in 33 healthy individuals, as shown in Fig. 1. We found significantly lower NAb levels in Ig-treated patients compared to newly diagnosed patients (p<0.01). Moreover, for all antigens tested, NAb levels were significantly lower in both newly diagnosed and treated patients compared to healthy controls, reaffirming the deficiency of antibody production within these patients.Fig. 1 IgM NAb levels in serum of CVID patients and healthy controls. IgM NAb levels against actin, DNA, CA, F(ab΄)2 fragments and TNP were measured for CVID patients without any treatment (n=26), CVID patients on IgRT (n=19) and healthy controls (n=33). *p<0.05. **p<0.01, ***p<0.001

Association of Serum IgM NAb Levels with Total IgM in Follow-Up Samples

Lower total serum IgM was found in IgRT-patients when compared to newly diagnosed patients (p<0.01) and healthy controls (p<0.001). In order to evaluate the relationship of total serum IgM with NAb levels, we analyzed ten (10) patients of our cohort with paired samples (pre- and post- treatment) and with detectable levels of total IgM. Figure 2 illustrates IgM NAb levels of the panel antigens for pre- and post- IgRT patient samples. Eight (8) out of the ten patients exhibited a significant decrease in IgM NAb levels for all panel antigens (p<0.05). The study revealed two outliers, one 47 year-old male, with significantly increased IgM NAb levels for all panel antigens, and one 49 year-old female, with increased anti-CA and anti-TNP IgM NAb levels post-treatment. Both were autoimmune patients, with co-existing clinical manifestations such as lymphoproliferation and enteropathy in the first case, and recurrent infections and brochiectasis in the second, while neither of them had cancer. Interestingly, these two patients had no significant variations in total serum IgM in the pre- and post- treatment samples.Fig. 2 IgM NAb levels among CVID patients and healthy controls. Levels of IgM NAbs against actin, DNA, CA, F(ab΄)2 fragments and TNP were measured in ten (10) CVID patients with follow-up pre- and post- IgRT samples. Every dot in the plot represents an individual patient in the left column and a line is connecting the dot of the same individual in the right column

To evaluate the relationship of total serum IgM with IgM NAb levels, we performed a correlation analysis and Fig. 3 represents a correlation matrix graph with given spearman values (r) (see also supplementary material – Table S1). Levels of IgM NAbs against all studied antigens were negatively correlated with total serum IgM in pre-treatment samples (Fig. 3A), whereas this imbalance was only partially restored post- treatment (Fig. 3B). After IVIg therapy, levels of anti-TNP IgM NAbs were positively correlated with total serum IgM (r=0.49), anti-G-actin, anti-CA, and anti-F(ab΄)2 NAbs showed intermediate correlation, while anti-DNA IgM NAbs remained negatively correlated with total serum IgM (r=-0.36).Fig. 3 Spearman correlation matrix for total IgM concentration and IgM NAb levels. A: Total serum IgM of CVID patients pre-treatment correlated to the respective values of NAb levels. B: Total serum IgM of CVID patients post- IgRT correlated to the respective values of NAb levels. The same patients were analyzed (n=10) before and after the initiation of the IVIg replacement therapy

Association of IgM NAb Levels with CVID-Related Clinical Complications

We analyzed the levels of IgM NAbs in relationship to CVID clinical complications such as the existence of frequent infections, bronchiectasis, autoimmunity, enteropathy, atopy, granulomatosis and cancer. Table 3 presents the significant differences in NAb levels targeting G-actin, DNA, CA, F(ab΄)2 and TNP among patients with different CVID related complications. We found that after the initiation of IgRT, patients with low incidence of infections had significantly higher levels of NAbs against all antigens tested, when compared to those patients with high incidence of infections. Interestingly, we found significantly decreased levels of NAbs in patients with bronchiectasis when compared to those without, independently of treatment status. For CVID patients with enteropathy, we found increased levels of NAbs against all panel antigens after treatment initiation, when compared to those without enteropathy. For CVID patients with autoimmunity, we found significantly increased levels of NAbs against actin, CA and TNP after treatment initiation, when compared to patients without any autoimmune disorder. No significant differences in NAb levels were found in patients exhibiting lymphoproliferation, granulomatosis or cancer. When demographic data (e.g. age and sex) and CVID related phenotypes were analyzed together in multivariate linear regression models (see also supplementary material – Table S2), a correlation between anti-CA IgM NAb levels and brochiectasis and granulomatosis was found in patients at diagnosis. Additionally, for patients on IgRT, the correlation of IgM NAb levels with enteropathy was confirmed and evident for all panel antigens while anti-G-actin and anti-CA IgM NAb levels were correlated with recurrent infections. Table 3 Analysis of IgM NAb levels in relation to different CVID-related clinical complications

Table 3	Infections	Brochiectasis	Enteropathy	Autoimmunity	
Ag	All patients	Patients at Diagnosis	Patients on Treatment	All patients	Patients at Diagnosis	Patients on Treatment	All patients	Patients at Diagnosis	Patients on Treatment	All patients	Patients at Diagnosis	Patients on Treatment	
Actin	↓ (p=0,048)	↓ NS	↓ (p<0.001)	↓ (p=0,011)	↓ NS	↓ NS	↑ (p=0,030)	↑ NS	↑ (p=0.001)	↑ NS	↑ NS	↑ (p=0,011)	
DNA	↓ NS	↓ NS	↓ (p=0.008)	↓ (p=0,001)	↓ (p=0,002)	↓ NS	↑ (p=0,042)	↑ NS	↑ (p=0.033)	↑ NS	↑ NS	↑ NS	
CA	↓ NS	↓ NS	↓ (p<0.001)	↓ NS	↓ NS	↓ (p<0.010)	↑ (p=0,047)	↑ NS	↑ (p=0.005)	↑ NS	↑ NS	↑ (p=0,005)	
F(ab')2	↓ NS	↓ NS	↓ (p=0.001)	↓ (p=0,002)	↓ NS	↓ (p<0.034)	↑ (p=0,012)	↑ NS	↑ (p<0.001)	↑ NS	↑ NS	↑ NS	
TNP	↓ p=0,029	↓ NS	↓ (p=0.002)	↓p<0,001	↓ NS	↓ NS	↑ NS	↑ NS	↑ (p=0.010)	↑ NS	↑ NS	↑ (p=0,006)	
Levels of NAbs against G-actin, DNA, CA, F(ab΄)2, and TNP were compared among patients with or without common CVID complications such as incidence of high infections, brochiectasis, enteropathy and autoimmunity. Arrows show the increase (↑) or decrease (↓) in NAb levels among patients with different CVID complications compared to those without, and p values are given in the cases of significant differences

IgG NAb Levels in CVID Patients Compared to Healthy Controls

Levels of serum IgG NAbs against G-actin, DNA, CA, F(ab΄)2 and TNP were estimated in 24 newly diagnosed patients with detectable serum IgG, 25 patients under IgRT and 33 healthy individuals, as shown in Fig. 4. We found higher NAb levels in treated patients compared to newly diagnosed patients. Anti-F(ab’)2 NAb levels were significantly higher in treated patients compared to healthy controls (p<0.01), reflecting the polyvalency of IVIg administration.Fig. 4 IgG NAb levels in CVID patients and healthy controls. IgG NAb levels against G-actin, DNA, CA, F(ab΄)2 fragments and TNP were measured for CVID patients without any treatment (n=24), CVID patients on IgRT (n=25) and healthy controls (n=33). *p<0.05. **p<0.01, ***p<0.001

Discussion

Underlying immune mechanisms in CVID are yet not fully understood, despite considerable progress in understanding other primary immunodeficiency diseases at the molecular level [23]. The diverse clinical manifestations of CVID suggest that various defects in immune regulation may converge upon a common pathway, resulting in reduced levels of immunoglobulins [24]. The term CVID, currently used to describe this condition, is likely to encompass a range of disorders that share the common feature of deficient B cell differentiation, impaired immunoglobulin production and dysregulated immune responses [25, 26]. Failure of the immune system in PADs is associated with recurrent infections, autoimmunity, and cancer.

Insufficient responses to glycan-based vaccines or low titers of isohemagglutinins, i.e. NAbs to polysaccharide blood group antigens, are characteristic and diagnostic features of CVID [10]. Unfortunately, there are few studies highlighting the importance of identifying other NAbs as informative markers for CVID patients, and clinical assessments are often based on reactivity to a limited antigen panel. Previously, an altered human carbohydrate-specific IgG NAb repertoire was described in CVID patients involving significant qualitative glycan-recognition defect profiles for Gala- and GalNAc-reactivity [27]. Additionally, impaired recognition of microbial, self-antigens, and tumor-associated carbohydrate antigens may also occur in these patients. Therefore, NAb repertoire analysis could serve as a valuable tool to understand the extent and clinical implications of immune system failure in individual patients, and provide valuable insights for clinical decision-making. Here, we selected representative target antigens to assess natural autoimmunity in CVID patients and normal individuals, as previously described [28]. We select proteins like actin and CA, nucleic acids like DNA, and haptens like TNP, to comprehensively analyze the measurement and variability of NAbs in serum. NAbs that bind to actin, CA and DNA are considered to be part of the autoreactive NAb repertoire, and can be used as a measure of internal immune regulation [9]. On the other hand, subjects are not normally exposed to TNP, and it can be used as a measure of polyreactivity and exogenous recognition. Moreover, in vitro measurements of autoreactive NAbs to F(ab')2 fragments of human IgG, known to participate in immunoregulatory mechanisms in vivo, enabled us to detect changes in the dynamics of the antibody (Fab-Fab) mediated network [29]. Even so, given the myriad potential targets of NAbs, defining a minimal set of panel antigens that reflect natural autoimmunity in humans is challenging.

Disturbances in NAb levels may compromise immune responses to pathogens, increasing susceptibility to autoimmune manifestations and promoting cancer development [30]. This study along with other observations [12], suggest that the severity and course of CVID in patients may be impacted by serum levels of NAbs, with lower levels being associated with more severe disease progression, increased susceptibility to infections, and potential complications. Acute lung infections can cause pneumonia, and long-term lung infections can cause a chronic form of bronchitis known as bronchiectasis, which is characterized by thickened airway walls colonized by bacteria. IgM NAbs targeting surface carbohydrates on pathogens are conserved across many species, including fish and mammals [31, 32]. Moreover, Rita Carsetti et al., have previously described an inverse correlation between the frequency of IgM memory B cells and susceptibility to bacterial lower respiratory tract infections, and together with anti-pneumococcal polysaccharide (anti-PnPS) IgM NAb levels can effectively discriminate low or high-risk patients for recurrent infections caused by trapped bacteria and low or high risk of bronchiectasis [33]. Considering the importance of IgM NAbs as key players in first-line defense and pathogen elimination, our results are consistent with the hypothesis that lower IgM NAb levels lead to loss of protection and susceptibility to infection. Most importantly we found that following IVIg replacement therapy, patients who still experienced recurrent infections had significantly lower IgM NAb levels compared to those with lower incidence of infection, a finding confirmed by linear regression analysis for NAbs targeting G-actin and CA. Further, patients with bronchiectasis generally exhibited lower IgM NAb levels regardless of treatment status.

Another possible complication associated with CVID is enteropathy, which may result in symptoms such as chronic diarrhea, malabsorption, weight loss, and abdominal pain. It is known that enteropathy in CVID patients is a result of dysregulated immune responses in the gastrointestinal tract. The specific mechanisms linking NAbs to CVID-associated enteropathy have not been fully elucidated. Previous studies show that levels of secretory IgM in IgA PAD patients are increased as a possible regulatory feedback mechanism involved in mucosal homeostasis and host-microbial interaction [34]. In this study, patients with enteropathy had significantly increased levels of IgM NAb against all panel antigens, and a strong correlation of IgM NAb levels and enteropathy was confirmed for patients on treatment by regression analysis. Considering that enteropathy may emerge independently of infection, IgRT is expected to have limited effect on treated patients although improved clinical outcomes have been observed in some cases [35]. Given the importance of secretory IgM in gut immune homeostasis, our preliminary data makes it challenging to determine whether NAb levels have a direct effect on the enteropathy phenotype and this important finding should be further explored including a larger number of patients [36].

Autoimmune and chronic inflammatory disorders result from a complex interplay of genetic and environmental factors, making it difficult to distinguish specific defects that cause dysregulation of apoptotic cell clearance. Furthermore, despite the observed protective role of higher levels of certain IgM antibodies against various disease manifestations, individuals with autoimmune diseases (autoimmune patients) may still exhibit an overall increase in these beneficial autoantibodies compared to healthy controls [37]. It has been suggested that certain specificities within the pool of circulating IgM might undergo an increase as part of a positive feedback mechanism, indicative of a compensatory effort to resolve inflammation and enhance the clearance of apoptotic cells. This is in line with our results that autoimmune patients had increased levels of NAbs against all panel antigens following IVIg replacement therapy, suggesting that this positive feedback loop was activated by or after initiation of therapy.

It is well known that polyvalent IVIg preparations consisting of pooled IgG antibodies derived from thousands of donors, contain a wide range of specificities, making them very effective in immune replacement therapies [38]. In PAD patients, different IVIg preparations are used at different doses for diverse clinical phenotype and patient profiles. In general, high-dose IVIg elicits a more potent immunosuppressive and anti-inflammatory response compared to lower doses [39]. Although research has shed light on various mechanisms of IVIg’s mode of action, these effects are difficult to generalize and must be considered on a case-by-case basis for each specific disease. A decrease in B-1 cells and IgM immunoglobulins after IVIg replacement treatment in selected CVID patients has been previously described, and to our knowledge the current study is one of the few studies to report a decrease in IgM NAb levels in these patients after IgRT [40]. Considering that in healthy state the vast majority of circulating IgM antibodies are NAbs, the negative correlation of IgM NAb levels with total IgM concentration before IgRT in our follow up analysis of CVID patients is indicative of immune dysregulation in these patients [41]. Furthermore, IgM NAb levels were still not correlated with total IgM concentration after IgRT, indicating a potential treatment inefficiency in terms of restoring IgM NAb levels. Additionally, two out of ten patients showed increased NAb levels after IgRT, without profound changes in total IgM concentration. This could be attributed either to the onset of an infection occurring at the time of sampling or as an outcome of IgRT, emphasizing once more the variability of CVID patients as a group of disorders and the multivalent mode of IgRT effects. Taking all together, decreased IgM NAb levels may affect the clinical phenotype and the course of CVID. This is already a matter of intensive research in order to achieve an effective treatment for all patients with PAD [42].

The dysregulated production of NAbs in CVID implicates these antibodies in several aspects of immune regulation, development of adaptive immune system and the continuous regulation of the adult B cell repertoire. Our study highlights the importance of NAbs in patients with CVID, where serum levels vary among the spectrum of CVID clinical phenotypes and have potential utility in diagnosis and treatment. The significant decrease in IgM NAb levels after IgRT, suggests a potential association with disease course and complications, and that administration of IgM-enriched preparations might enhance the effectiveness of IgRT in selected patients, as has been proposed also by others [43]. Further research is needed to understand the functional roles of NAbs in CVID and its complexity with respect to clinical phenotypes.

Supplementary Information

ESM 1 (XLSX 13 kb)

ESM 2 (XLSX 38 kb)

We express our deepest gratitude to the late Apostolos Balafas, our experienced technician at the Hellenic Pasteur Institute and a victim of COVID-19, for his valuable contributions and dedicated work over the years. We also thank Lesley Probert (Laboratory of Molecular Genetics, Hellenic Pasteur Institute) for proofreading the manuscript and for her useful comments.

Institutional Review Board Statement

The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of the University General Hospital of Larissa (29268/16-07-2019).

Author Contribution

Conceptualization, P.L. and A.G.; methodology, I.S., P.L.; software, I.S., G.T.; validation, M.S and A.G.; formal analysis, I.S, G.T.; investigation, I.S.; resources, F.K., M.S., P.L.; data curation, I.S., G.T., F.K.; writing original draft preparation, I.S., G.T.; writing—review and editing, P.L., M.S., A.G.; visualization, I.S., G.T.; supervision, P.L.; project administration, All authors reviewed the manuscript and have agreed to the published version of the manuscript.

Funding

This research received no external funding

Data Availability

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Declarations

Informed Consent Statement

Informed consent was obtained from all subjects involved in the study.

Competing Interests

The authors declare no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Peggy Lymberi and Matthaios Speletas contributed equally to this work.
==== Refs
References

1. Yazdani R Habibi S Sharifi L Azizi G Abolhassani H Olbrich P Aghamohammadi A Common Variable Immunodeficiency: Epidemiology, Pathogenesis, Clinical Manifestations, Diagnosis, Classification, and Management J Investig Allergol Clin Immunol 2020 30 1 14 34 10.18176/jiaci.0388 30741636
Yazdani R, Habibi S, Sharifi L, Azizi G, Abolhassani H, Olbrich P, Aghamohammadi A. Common Variable Immunodeficiency: Epidemiology, Pathogenesis, Clinical Manifestations, Diagnosis, Classification, and Management. J Investig Allergol Clin Immunol. 2020;30(1):14–34. 10.18176/jiaci.0388.30741636
2. Ameratunga R Assessing Disease Severity in Common Variable Immunodeficiency Disorders (CVID) and CVID-Like Disorders Front Immunol 2018 9 2130 10.3389/fimmu.2018.02130 30323807
Ameratunga R. Assessing Disease Severity in Common Variable Immunodeficiency Disorders (CVID) and CVID-Like Disorders. Front Immunol. 2018;9:2130. 10.3389/fimmu.2018.02130.30323807
3. Gouilleux-Gruart V Chapel H Chevret S Lucas M Malphettes M Fieschi C Patel S Boutboul D Marson MN Gérard L Lee M Watier H Oksenhendler E DEFI study group Efficiency of immunoglobulin G replacement therapy in common variable immunodeficiency: correlations with clinical phenotype and polymorphism of the neonatal Fc receptor Clin Exp Immunol 2013 171 2 186 194 10.1111/cei.12002 23286945
Gouilleux-Gruart V, Chapel H, Chevret S, Lucas M, Malphettes M, Fieschi C, Patel S, Boutboul D, Marson MN, Gérard L, Lee M, Watier H, Oksenhendler E, DEFI study group. Efficiency of immunoglobulin G replacement therapy in common variable immunodeficiency: correlations with clinical phenotype and polymorphism of the neonatal Fc receptor. Clin Exp Immunol. 2013;171(2):186–94. 10.1111/cei.12002.23286945
4. Resnick ES Moshier EL Godbold JH Cunningham-Rundles C Morbidity and mortality in common variable immune deficiency over 4 decades Blood 2012 119 7 1650 1657 10.1182/blood-2011-09-377945 22180439
Resnick ES, Moshier EL, Godbold JH, Cunningham-Rundles C. Morbidity and mortality in common variable immune deficiency over 4 decades. Blood. 2012;119(7):1650–7. 10.1182/blood-2011-09-377945.22180439
5. Salehzadeh M Aghamohammadi A Rezaei N Evaluation of immunoglobulin levels and infection rate in patients with common variable immunodeficiency after immunoglobulin replacement therapy J Microbiol Immunol Infect 2010 43 1 11 17 10.1016/S1684-1182(10)60002-3 20434118
Salehzadeh M, Aghamohammadi A, Rezaei N. Evaluation of immunoglobulin levels and infection rate in patients with common variable immunodeficiency after immunoglobulin replacement therapy. J Microbiol Immunol Infect. 2010;43(1):11–7. 10.1016/S1684-1182(10)60002-3.20434118
6. Avrameas S Natural autoantibodies: from 'horror autotoxicus' to 'gnothi seauton' Immunol Today 1991 12 5 154 159 10.1016/S0167-5699(05)80045-3 1715166
Avrameas S. Natural autoantibodies: from 'horror autotoxicus' to 'gnothi seauton'. Immunol Today. 1991 May;12(5):154–9. 10.1016/S0167-5699(05)80045-3.1715166
7. Holodick NE Rodríguez-Zhurbenko N Hernández AM Defining Natural Antibodies Front Immunol 2017 8 872 10.3389/fimmu.2017.00872 28798747
Holodick NE, Rodríguez-Zhurbenko N, Hernández AM. Defining Natural Antibodies. Front Immunol. 2017;8:872. 10.3389/fimmu.2017.00872.28798747
8. Jarossay A Hadzhieva M Kaveri SV Lacroix-Desmazes S Dimitrov JD Natural and Induced Antibody Polyreactivity Anticancer Agents Med Chem 2015 15 10 1230 1241 10.2174/1871520615666150716104935 26179269
Jarossay A, Hadzhieva M, Kaveri SV, Lacroix-Desmazes S, Dimitrov JD. Natural and Induced Antibody Polyreactivity. Anticancer Agents Med Chem. 2015;15(10):1230–41. 10.2174/1871520615666150716104935.26179269
9. Lymberi P Zannikou M Hatzioannou A Natural Autoantibodies in Health and Disease Comprehensive Pharmacology 2022 Elsevier 342 368
Lymberi P, Zannikou M, Hatzioannou A. Natural Autoantibodies in Health and Disease. In: Comprehensive Pharmacology, vol. 5. Elsevier; 2022. p. 342–68. 10.1016/B978-0-12-820472-6.00197-3.
10. Nguyen TT Baumgarth N Natural IgM and the Development of B Cell-Mediated Autoimmune Diseases Crit Rev Immunol 2016 36 2 163 177 10.1615/CritRevImmunol.2016018175 27910766
Nguyen TT, Baumgarth N. Natural IgM and the Development of B Cell-Mediated Autoimmune Diseases. Crit Rev Immunol. 2016;36(2):163–77. 10.1615/CritRevImmunol.2016018175.27910766
11. Lobo PI Role of Natural IgM Autoantibodies (IgM-NAA) and IgM Anti-Leukocyte Antibodies (IgM-ALA) in Regulating Inflammation Curr Top Microbiol Immunol 2017 408 89 117 10.1007/82_2017_37 28698955
Lobo PI. Role of Natural IgM Autoantibodies (IgM-NAA) and IgM Anti-Leukocyte Antibodies (IgM-ALA) in Regulating Inflammation. Curr Top Microbiol Immunol. 2017;408:89–117. 10.1007/82_2017_37.28698955
12. Bernth Jensen JM Hansen AT Söderström A Jørgensen CS Larsen CS Skov Sørensen UB Thiel S Petersen MS A low level of naturally occurring antibodies associates with functional antibody deficiency Clin Immunol 2022 241 109070 10.1016/j.clim.2022.109070 35779828
Bernth Jensen JM, Hansen AT, Söderström A, Jørgensen CS, Larsen CS, Skov Sørensen UB, Thiel S, Petersen MS. A low level of naturally occurring antibodies associates with functional antibody deficiency. Clin Immunol. 2022 Aug;241:109070. 10.1016/j.clim.2022.109070.35779828
13. Suchanek O Sadler R Bateman EA Patel SY Ferry BL Immunophenotyping of putative human B1 B cells in healthy controls and common variable immunodeficiency (CVID) patients Clin Exp Immunol 2012 170 3 333 341 10.1111/j.1365-2249.2012.04656.x 23121674
Suchanek O, Sadler R, Bateman EA, Patel SY, Ferry BL. Immunophenotyping of putative human B1 B cells in healthy controls and common variable immunodeficiency (CVID) patients. Clin Exp Immunol. 2012 Dec;170(3):333–41. 10.1111/j.1365-2249.2012.04656.x.23121674
14. Dighiero G Guilbert B Fermand JP Lymberi P Danon F Avrameas S Thirty-six human monoclonal immunoglobulins with antibody activity against cytoskeleton proteins, thyroglobulin, and native DNA: immunologic studies and clinical correlations Blood 1983 62 2 264 270 10.1182/blood.V62.2.264.264 6409187
Dighiero G, Guilbert B, Fermand JP, Lymberi P, Danon F, Avrameas S. Thirty-six human monoclonal immunoglobulins with antibody activity against cytoskeleton proteins, thyroglobulin, and native DNA: immunologic studies and clinical correlations. Blood. 1983;62(2):264–70. 10.1182/blood.V62.2.264.264.6409187
15. Zamanou A Samiotaki M Panayotou G Margaritis L Lymberi P Fine specificity and subclasses of IgG anti-actin autoantibodies differ in health and disease J Autoimmun 2003 20 4 333 344 10.1016/s0896-8411(03)00036-2 12791319
Zamanou A, Samiotaki M, Panayotou G, Margaritis L, Lymberi P. Fine specificity and subclasses of IgG anti-actin autoantibodies differ in health and disease. J Autoimmun. 2003;20(4):333–44. 10.1016/s0896-8411(03)00036-2.12791319
16. Rekvig OP The Anti-DNA Antibodies: Their Specificities for Unique DNA Structures and Their Unresolved Clinical Impact-A System Criticism and a Hypothesis Front Immunol 2022 12 808008 10.3389/fimmu.2021.808008 35087528
Rekvig OP. The Anti-DNA Antibodies: Their Specificities for Unique DNA Structures and Their Unresolved Clinical Impact-A System Criticism and a Hypothesis. Front Immunol. 2022;12:808008. 10.3389/fimmu.2021.808008.35087528
17. Alver A Menteşe A Karahan SC Erem C Keha EE Arikan MK Eminağaoğlu MS Deger O Increased serum anti-carbonic anhydrase II antibodies in patients with Graves' disease Exp Clin Endocrinol Diabetes 2007 115 5 287 291 10.1055/s-2007-960498 17516290
Alver A, Menteşe A, Karahan SC, Erem C, Keha EE, Arikan MK, Eminağaoğlu MS, Deger O. Increased serum anti-carbonic anhydrase II antibodies in patients with Graves' disease. Exp Clin Endocrinol Diabetes. 2007;115(5):287–91. 10.1055/s-2007-960498.17516290
18. Brezski RJ Knight DM Jordan RE The origins, specificity, and potential biological relevance of human anti-IgG hinge autoantibodies ScientificWorldJournal 2011 11 1153 1167 10.1100/tsw.2011.107 21623461
Brezski RJ, Knight DM, Jordan RE. The origins, specificity, and potential biological relevance of human anti-IgG hinge autoantibodies. ScientificWorldJournal. 2011;11:1153–67. 10.1100/tsw.2011.107.21623461
19. Gunti S Notkins AL Polyreactive Antibodies: Function and Quantification J Infect Dis 2015 212 Suppl 1 S42 S46 10.1093/infdis/jiu512 26116731
Gunti S, Notkins AL. Polyreactive Antibodies: Function and Quantification. J Infect Dis. 2015;212(Suppl 1):S42–6. 10.1093/infdis/jiu512.26116731
20. ESID - European Society for Immunodeficiencies. https://esid.org/Working-Parties/Registry-Working-Party/Diagnosis-criteria (accessed 2024-02-15)
21. Dighiero G Lymberi P Holmberg D Lundquist I Coutinho A Avrameas S High frequency of natural autoantibodies in normal newborn mice J Immunol 1985 134 765 771 10.4049/jimmunol.134.2.765 4038410
Dighiero G, Lymberi P, Holmberg D, Lundquist I, Coutinho A, Avrameas S. High frequency of natural autoantibodies in normal newborn mice. J Immunol. 1985;134:765–71.4038410
22. Sali AD Karakasiliotis I Evangelidou M Avrameas S Lymberi P Immunological evidence and regulatory potential for cell-penetrating antibodies in intravenous immunoglobulin Clin Transl Immunology 2015 4 10 e42 10.1038/cti.2015.18 26682050
Sali AD, Karakasiliotis I, Evangelidou M, Avrameas S, Lymberi P. Immunological evidence and regulatory potential for cell-penetrating antibodies in intravenous immunoglobulin. Clin Transl Immunology. 2015;4(10):e42. 10.1038/cti.2015.18.26682050
23. Wehr C Kivioja T Schmitt C Ferry B Witte T Eren E Vlkova M Hernandez M Detkova D Bos PR Poerksen G von Bernuth H Baumann U Goldacker S Gutenberger S Schlesier M Bergeron-van der Cruyssen F Le Garff M Debré P Jacobs R Jones J Bateman E Litzman J van Hagen PM Plebani A Schmidt RE Thon V Quinti I Espanol T Webster AD Chapel H Vihinen M Oksenhendler E Peter HH Warnatz K The EUROclass trial: defining subgroups in common variable immunodeficiency Blood 2008 111 1 77 85 10.1182/blood-2007-06-091744 17898316
Wehr C, Kivioja T, Schmitt C, Ferry B, Witte T, Eren E, Vlkova M, Hernandez M, Detkova D, Bos PR, Poerksen G, von Bernuth H, Baumann U, Goldacker S, Gutenberger S, Schlesier M, Bergeron-van der Cruyssen F, Le Garff M, Debré P, et al. The EUROclass trial: defining subgroups in common variable immunodeficiency. Blood. 2008;111(1):77–85. 10.1182/blood-2007-06-091744.17898316
24. Bonilla FA Barlan I Chapel H Costa-Carvalho BT Cunningham-Rundles C de la Morena MT Espinosa-Rosales FJ Hammarström L Nonoyama S Quinti I Routes JM Tang ML Warnatz K International Consensus Document (ICON): Common Variable Immunodeficiency Disorders J Allergy Clin Immunol Pract 2016 4 1 38 59 10.1016/j.jaip.2015.07.025 26563668
Bonilla FA, Barlan I, Chapel H, Costa-Carvalho BT, Cunningham-Rundles C, de la Morena MT, Espinosa-Rosales FJ, Hammarström L, Nonoyama S, Quinti I, Routes JM, Tang ML, Warnatz K. International Consensus Document (ICON): Common Variable Immunodeficiency Disorders. J Allergy Clin Immunol Pract. 2016;4(1):38–59. 10.1016/j.jaip.2015.07.025.26563668
25. Warnatz K Denz A Dräger R Braun M Groth C Wolff-Vorbeck G Eibel H Schlesier M Peter HH Severe deficiency of switched memory B cells (CD27(+)IgM(-)IgD(-)) in subgroups of patients with common variable immunodeficiency: a new approach to classify a heterogeneous disease Blood 2002 99 5 1544 1551 10.1182/blood.v99.5.1544 11861266
Warnatz K, Denz A, Dräger R, Braun M, Groth C, Wolff-Vorbeck G, Eibel H, Schlesier M, Peter HH. Severe deficiency of switched memory B cells (CD27(+)IgM(-)IgD(-)) in subgroups of patients with common variable immunodeficiency: a new approach to classify a heterogeneous disease. Blood. 2002;99(5):1544–51. 10.1182/blood.v99.5.1544.11861266
26. Roskin KM Simchoni N Liu Y Lee JY Seo K Hoh RA Pham T Park JH Furman D Dekker CL Davis MM James JA Nadeau KC Cunningham-Rundles C Boyd SD IgH sequences in common variable immune deficiency reveal altered B cell development and selection Sci Transl Med 2015 7 302 302ra135 10.1126/scitranslmed.aab1216 26311730
Roskin KM, Simchoni N, Liu Y, Lee JY, Seo K, Hoh RA, Pham T, Park JH, Furman D, Dekker CL, Davis MM, James JA, Nadeau KC, Cunningham-Rundles C, Boyd SD. IgH sequences in common variable immune deficiency reveal altered B cell development and selection. Sci Transl Med. 2015;7(302):302ra135. 10.1126/scitranslmed.aab1216.26311730
27. Jandus P Boligan KF Smith DF de Graauw E Grimbacher B Jandus C Abdelhafez MM Despont A Bovin N Simon D Rieben R Simon HU Cummings RD von Gunten S The architecture of the IgG anti-carbohydrate repertoire in primary antibody deficiencies Blood 2019 134 22 1941 1950 10.1182/blood.2019001705 31537530
Jandus P, Boligan KF, Smith DF, de Graauw E, Grimbacher B, Jandus C, Abdelhafez MM, Despont A, Bovin N, Simon D, Rieben R, Simon HU, Cummings RD, von Gunten S. The architecture of the IgG anti-carbohydrate repertoire in primary antibody deficiencies. Blood. 2019;134(22):1941–50. 10.1182/blood.2019001705.31537530
28. Tsitsami E Sarrigeorgiou I Tsinti M Rouka EC Zarogiannis SG Lymberi P Natural autoimmunity in oligoarticular juvenile idiopathic arthritis Pediatr Rheumatol Online J 2023 21 1 44 10.1186/s12969-023-00823-w 37138302
Tsitsami E, Sarrigeorgiou I, Tsinti M, Rouka EC, Zarogiannis SG, Lymberi P. Natural autoimmunity in oligoarticular juvenile idiopathic arthritis. Pediatr Rheumatol Online J. 2023;21(1):44. 10.1186/s12969-023-00823-w.37138302
29. Terness PI Navolan D Dufter C Welschof M Opelz G Immunosuppressive anti-immunoglobulin autoantibodies: specificity, gene structure and function in health and disease Cell Mol Biol (Noisy-le-grand) 2002 48 3 271 278 12030431
Terness PI, Navolan D, Dufter C, Welschof M, Opelz G. Immunosuppressive anti-immunoglobulin autoantibodies: specificity, gene structure and function in health and disease. Cell Mol Biol (Noisy-le-grand). 2002;48(3):271–8.12030431
30. Rothstein TL Natural Antibodies as Rheostats for Susceptibility to Chronic Diseases in the Aged Front Immunol 2016 7 127 10.3389/fimmu.2016.00127 27092140
Rothstein TL. Natural Antibodies as Rheostats for Susceptibility to Chronic Diseases in the Aged. Front Immunol. 2016;7:127. 10.3389/fimmu.2016.00127.27092140
31. Zhou ZH Zhang Y Hu YF Wahl LM Cisar JO Notkins AL The broad antibacterial activity of the natural antibody repertoire is due to polyreactive antibodies Cell Host Microbe 2007 1 1 51 61 10.1016/j.chom.2007.01.002 18005681
Zhou ZH, Zhang Y, Hu YF, Wahl LM, Cisar JO, Notkins AL. The broad antibacterial activity of the natural antibody repertoire is due to polyreactive antibodies. Cell Host Microbe. 2007;1(1):51–61. 10.1016/j.chom.2007.01.002.18005681
32. Rapaka RR Ricks DM Alcorn JF Chen K Khader SA Zheng M Plevy S Bengtén E Kolls JK Conserved natural IgM antibodies mediate innate and adaptive immunity against the opportunistic fungus Pneumocystis murina J Exp Med 2010 207 13 2907 2919 10.1084/jem.20100034 21149550
Rapaka RR, Ricks DM, Alcorn JF, Chen K, Khader SA, Zheng M, Plevy S, Bengtén E, Kolls JK. Conserved natural IgM antibodies mediate innate and adaptive immunity against the opportunistic fungus Pneumocystis murina. J Exp Med. 2010;207(13):2907–19. 10.1084/jem.20100034.21149550
33. Carsetti R Rosado MM Donnanno S Guazzi V Soresina A Meini A Plebani A Aiuti F Quinti I The loss of IgM memory B cells correlates with clinical disease in common variable immunodeficiency J Allergy Clin Immunol 2005 115 2 412 417 10.1016/j.jaci.2004.10.048 15696104
Carsetti R, Rosado MM, Donnanno S, Guazzi V, Soresina A, Meini A, Plebani A, Aiuti F, Quinti I. The loss of IgM memory B cells correlates with clinical disease in common variable immunodeficiency. J Allergy Clin Immunol. 2005;115(2):412–7. 10.1016/j.jaci.2004.10.048.15696104
34. Mella MA Lavrinienko A Akhi R Hindström R Nissinen AE Wang C Kullaa A Salo T Auvinen J Koskimäki JJ Hörkkö S Compensatory IgM to the Rescue: Patients with Selective IgA Deficiency Have Increased Natural IgM Antibodies to MAA-LDL and No Changes in Oral Microbiota Immunohorizons 2021 5 4 170 181 10.4049/immunohorizons.2100014 33893180
Mella MA, Lavrinienko A, Akhi R, Hindström R, Nissinen AE, Wang C, Kullaa A, Salo T, Auvinen J, Koskimäki JJ, Hörkkö S. Compensatory IgM to the Rescue: Patients with Selective IgA Deficiency Have Increased Natural IgM Antibodies to MAA-LDL and No Changes in Oral Microbiota. Immunohorizons. 2021;5(4):170–81. 10.4049/immunohorizons.2100014.33893180
35. Shah SN Todoric K Tarrant TK Improved outcomes on subcutaneous IgG in patients with humoral immunodeficiency and co-morbid bowel disease Clin Case Rep Rev 2015 1 7 151 152 10.15761/CCRR.1000149 27042340
Shah SN, Todoric K, Tarrant TK. Improved outcomes on subcutaneous IgG in patients with humoral immunodeficiency and co-morbid bowel disease. Clin Case Rep Rev. 2015;1(7):151–2. 10.15761/CCRR.1000149.27042340
36. Ding Y, Fern Ndez-Montero A, Mani A, Casadei E, Shibasaki Y, Takizawa F, Miyazawa R, Salinas I, Sunyer JO. Secretory IgM (sIgM) is an ancient master regulator of microbiota homeostasis and metabolism. bioRxiv. 2023; 10.1101/2023.02.26.530119.
37. Grönwall C Vas J Silverman GJ Protective Roles of Natural IgM Antibodies Front Immunol 2012 3 66 10.3389/fimmu.2012.00066 22566947
Grönwall C, Vas J, Silverman GJ. Protective Roles of Natural IgM Antibodies. Front Immunol. 2012;3:66. 10.3389/fimmu.2012.00066.22566947
38. Kaveri SV Intravenous immunoglobulin: exploiting the potential of natural antibodies Autoimmun Rev 2012 11 11 792 794 10.1016/j.autrev.2012.02.006 22349620
Kaveri SV. Intravenous immunoglobulin: exploiting the potential of natural antibodies. Autoimmun Rev. 2012;11(11):792–4. 10.1016/j.autrev.2012.02.006.22349620
39. Matucci A Maggi E Vultaggio A Mechanisms of action of Ig preparations: immunomodulatory and anti-inflammatory effects Front Immunol 2015 5 690 10.3389/fimmu.2014.00690 25628625
Matucci A, Maggi E, Vultaggio A. Mechanisms of action of Ig preparations: immunomodulatory and anti-inflammatory effects. Front Immunol. 2015;5:690. 10.3389/fimmu.2014.00690.25628625
40. Kraljevic K Wong S Fulcher DA Circulating phenotypic B-1 cells are decreased in common variable immunodeficiency and correlate with immunoglobulin M levels Clin Exp Immunol 2013 171 3 278 282 10.1111/cei.12008 23379434
Kraljevic K, Wong S, Fulcher DA. Circulating phenotypic B-1 cells are decreased in common variable immunodeficiency and correlate with immunoglobulin M levels. Clin Exp Immunol. 2013;171(3):278–82. 10.1111/cei.12008.23379434
41. Mosaed M Pourfathollah AA Moghadam M Jazayeri MH Safdarian AR Evaluation of serum natural autoantibodies reaction in different hematological disorders with prospective view to their probable utilization in predictive medicine Asian J Transfus Sci 2020 14 2 167 171 10.4103/ajts.AJTS_15_17 33767544
Mosaed M, Pourfathollah AA, Moghadam M, Jazayeri MH, Safdarian AR. Evaluation of serum natural autoantibodies reaction in different hematological disorders with prospective view to their probable utilization in predictive medicine. Asian J Transfus Sci. 2020;14(2):167–71. 10.4103/ajts.AJTS_15_17.33767544
42. Langereis JD van der Flier M de Jonge MI Limited Innovations After More Than 65 Years of Immunoglobulin Replacement Therapy: Potential of IgA- and IgM-Enriched Formulations to Prevent Bacterial Respiratory Tract Infections Front Immunol 2018 9 1925 10.3389/fimmu.2018.01925 30190722
Langereis JD, van der Flier M, de Jonge MI. Limited Innovations After More Than 65 Years of Immunoglobulin Replacement Therapy: Potential of IgA- and IgM-Enriched Formulations to Prevent Bacterial Respiratory Tract Infections. Front Immunol. 2018;9:1925. 10.3389/fimmu.2018.01925.30190722
43. Späth PJ Schneider C von Gunten S Clinical Use and Therapeutic Potential of IVIG/SCIG, Plasma-Derived IgA or IgM, and Other Alternative Immunoglobulin Preparations Arch Immunol Ther Exp (Warsz) 2017 65 3 215 231 10.1007/s00005-016-0422-x 27638480
Späth PJ, Schneider C, von Gunten S. Clinical Use and Therapeutic Potential of IVIG/SCIG, Plasma-Derived IgA or IgM, and Other Alternative Immunoglobulin Preparations. Arch Immunol Ther Exp (Warsz). 2017;65(3):215–31. 10.1007/s00005-016-0422-x.27638480
