Cellular and Molecular (2018) 15, 610–617 & 2018 CSI and USTC All rights reserved 2042-0226/18 $32.00 www..com/cmi

RESEARCH ARTICLE

Comprehensive review of in patients with hyper-IgM syndrome

Mohamed-Ridha Barbouche1, Qubo Chen2,3, Marco Carbone4, Imen Ben-Mustapha1, Zakera Shums5, Mehdi Trifa6, Federica Malinverno4, Francesca Bernuzzi4, Haiyan Zhang2,4,7, Nourhen Agrebi1, Gary L Norman5, Christopher Chang8, M Eric Gershwin8 and Pietro Invernizzi2,4

Hyper- syndrome is an X-linked primary immunodeficiency disease caused by mutations in the CD40 ligand gene. The CD40 ligand has been recently highlighted as playing a key role in the pathogenesis of primary biliary cholangitis. In the present study, we assessed an extensive set of serum autoantibodies in a series of well-defined patients with hyper-immunoglobulin M syndrome. Serum, liver-related and liver-not-related autoantibodies IgG, IgM and IgA were tested by ELISA and standard indirect immunofluorescence in HEp-2 cells in 13 Tunisian patients (8 males and 5 females, aged 1–12 years) with hyper-immunoglobulin M syndrome during 1995–2012 and, as controls, 21 age- and gender-matched blood donors. The level of IgM against MIT3 was significantly higher in patients than in controls (35.8 vs 10.7, P = 0.002). Half of the hyperimmunoglobulin M syndrome patients were found to be anti-MIT3 IgM positive vs none of the controls (Po0.0001). Twenty-three percent of patients were found to be anti-sp100 antibody positive vs only 0.05% of controls. By immunofluorescence, 92.3% of patients were MIT3 IgM positive vs none of the controls. In conclusion, the IgM class of anti-MIT3 was shown to be present by both ELISA and immunofluorescence in most of the patients with hyper-immunoglobulin M syndrome. The presence of the hallmark of primary biliary cholangitis, a disease where the CD40 ligand is a key player, in an immunodeficiency disease caused by mutations in the CD40 ligand gene is very intriguing and opens new scenarios in understanding the immune pathogenesis of primary biliary cholangitis. Cellular and Molecular Immunology (2018) 15, 610–617; doi:10.1038/cmi.2017.140; published online 5 February 2018

Keywords: immunoglobulin M; autoantibodies; immunodeficiency; hyper-immunoglobulin M syndrome; primary biliary cholangitis

INTRODUCTION subsequent development of immune responses against foreign Immunoglobulin M (IgM) is the first antibody to be produced pathogens and self- by accelerating the production of by mature B cells and plasma cells during an high-affinity IgG. The process controlling B-cell maturation after an initial encounter. It is the predominant and immunoglobulin isotype switching is largely regulated by secreted in T-cell-independent immune responses.1 Although the cross-talk between CD40 and CD40L. the existence of IgM has been known for several decades, its Hyper-immunoglobulin M (HIGM) syndrome is a rare form biological significance in and con- of X-linked primary immunodeficiency disease (PIDD) caused tinues to emerge. IgM plays an important regulatory role in the by mutations in the gene that encodes the CD40 ligand

1Laboratory of Immunology, Institute Pasteur de Tunis and Faculty of , University Tunis El Manar, 1002 Tunis, Tunisia; 2Humanitas Clinical and Research Center, 20089 Rozzano, Italy; 3Clinical Laboratory, Guangdong Provincial Hospital of Chinese medicine, 510000 Guangzhou, China; 4Division of and Center for Autoimmune Liver Diseases, Department of Medicine and , University of Milan-Bicocca, 20900 Monza (MB), Italy; 5Department of Research and Development, Inova Diagnostics, 92131 San Diego, CA, USA; 6Department of Anesthesia and Intensive Care, Children Hospital Bechir Hamza, Tunis and Faculty of Medicine, University Tunis El Manar, 1007 Tunis, Tunisia; 7Division of Gastroenterology and , Key Laboratory of Gastroenterology and Hepatology, Ministry of Health, State, Key Laboratory for Oncogenes and Related Genes, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Institute of Digestive Disease, 200001 Shanghai, China and 8Division of , and Clinical Immunology, University of California at Davis, 95616 Davis, CA, USA Correspondence: Dr Pietro Invernizzi MD PhD, Professor and Chief, Division of Gastroenterology and Hepatology, Center for Autoimmune Liver Diseases, Department of Medicine and Surgery, University of Milan-Bicocca, 20900 Monza (MB), Italy. E-mail: [email protected] Received: 18 October 2017; Accepted: 26 October 2017 Table 1 Clinical features, abnormal biochemistry parameters and serum autoantibodies in patients with HIGM syndrome

Serum autoantibodies and abnormal biochemistry Patient Age Gender Gene defect Clinical manifestations parameters

p1 8 years M ATM Recurrent ENT and pulmonary . His sister with HIGM and died due High IgM level to Hodgkin lymphoma. Later development of ataxia-telangiectasia p2 7 years F ATM Cousin of patient 1. Recurrent pulmonary infections with bronchiectasis, eczema, . High IgM /ALP/GGT level, Low IgG, AMA-IgM, sp100- Later development of Ataxia-telangiectasia IgM, AMA (1:640), nuclear dots (1:320) p3 3 years M Bronchopneumopathies, hepatosplenomegaly, adenopathies, skin rash High IgM level, Low IgG, AMA-IgM, sp100-IgM, AMA (1:1280), nuclear dots (1:40) p4 6 years M AID/ /AID-C ter def Recurrent ENT and pulmonary infections, cervical adenophlegmon AMA (1:40), MPO-IgG p5 12 years M AID /AID-C ter def Brother of patient 4, recurrent pulmonary and ENT infections, cervical adenophlegmon, Low IgG, AMA-IgM, sp100-IgM, AMA (1:1280), nuclear bilateral mastoiditis, lambliasis and coeliac disease dots (1:640), Centromere p6 5 years F Oral candidiasis, adenophlegmon, uveitis, deafness AMA (1:160), p7 1 years M Oral thrush, bronchopneumopathies, ENT infections, diarrhea Low IgG, AMA (1:80), p8 M CD40 ligand Chronic diarrhea, lambliasis with villous atrophy, ulcéronecrotic cutaneous lesions, recurrent High IgM level, Low IgG, AMA-IgM, sp100-IgM, gp210- pneumopathies IgM, AMA/nuclear dots/rim (1:160), ANA, GPA p9 12 years F AID /AID-C ter def Multiple adenopathies, arthralgia with unidentified etiology, nephrotic syndrom due to a High IgM level, Low IgG, AMA-IgM, sp100-IgM, AMA /AID-C ter def membranoproliferative glomerulonephritis at biopsy and type II (1:2560), nuclear dots/Rim (1:640), p10 6 years M AID /AID-C ter def Bronchopneumopathies and ENT infections High IgM level, Low IgG, AMA-IgM, AMA (1:160), p11 3 years F AID /AID-C ter def Recurrent otitis and pneumonia High IgM/ALT level, Low IgG, AMA-IgM, AMA (1:320), p12 4 years M Recurrent otitis, adenopathies, gingival hypertrophy, molluscum contagiosum lesions High IgM level, Low IgG, AMA-IgM, sp100-IgM, AMA eu uonioisi ye-g syndrome Barbouche hyper-IgM M-R in autoantibodies Serum (1:1280), nuclear dots (1:640) p13 3 years F Diarrhea, oral candidiadis, pneumopathy, cervical skin rash, hepatosplenomegaly, erythe- Low IgG, AMA-IgM, sp100-IgM, AMA (1:2560), nuclear matous and necrotic lesions of the anal margin dots (1:640), Rim (1:160)

Abbreviations: AID, activation-induced cytidine deaminase; ATM, the gene responsible for Ataxia-telangiectasia, located on chromosome 11q22-23; ENT, ear, nose and throat; F, female; HIGM, hyper-immunoglobulin M;

IgM, immunoglobulin M; M, male. al et ellradMlclrImmunology Molecular and Cellular 611 Serum autoantibodies in hyper-IgM syndrome M-R Barbouche et al

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(CD40L) in which defective immunoglobulin (Ig) class-switch Although the first description of HIGM syndrome cases recombination (CSR) leads to deficiencies in IgG, IgA and IgE dates to the 1960s, there has been no systematic evaluation of with preserved or elevated levels of IgM. Six main forms of either the prevalence or spectrum of antibodies to common HIGM are known. They can generally be divided into those autoantigens in this disorder. In the present study, we that result from mutations in the genes encoding CD40L and/ performed an extensive assessment of serum autoantibodies fi or CD40 and those resulting from defects in genes encoding in a series of well-de ned patients with HIGM syndrome. intrinsic factors involved in immunoglobulin CSR and somatic MATERIALS AND METHODS hypermutation (SHM) in B cells. HIGM caused by genetic Patient population and study design defects of CD40L or CD40 presents with features of cellular Thirteen patients with HIGM who were referred to the and humoral immunodeficiencies, while that caused by intrin- Children’s Medical Center in Tunis, Tunisia during 1995– sic B-cell defects shows pure humoral immunodeficiencies.2 2012 were enrolled in this study. The criteria for the diagnosis The major defect shared by all forms of HIGM syndromes is a of HIGM were low serum IgG and IgA levels ≤ 2s.d.below failure in the ability of B to switch from IgM to normal values for age, normal or elevated IgM, and recurrent 3 the other immunoglobulin isotypes. HIGM is characterized by or severe infections. Informed consent was obtained from all increased susceptibility to infections and low serum levels of patients and their parents, and blood was collected under IgG, IgA and IgE but normal or elevated levels of IgM and institutional guidelines. The clinical features of the enrolled normal numbers of circulating B cells, with or without defects patients are summarized in Table 1. Eight of the 13 patients of SHM.4 underwent genetic testing for HIGM. At the same time, 21 age- Autoimmunity has been described in all forms of HIGM and gender-matched blood donors served as controls (Table 2). syndrome, with variable presentation and immunological and These control individuals had undergone minor surgery for clinical phenotype.5 In a somewhat paradoxical manner, the orthopedic, non-infectious conditions. The sera were analyzed susceptibility to opportunistic and bacterial infections seen in for basic chemistry and liver panels, including AST, ALT, ALP, HIGM patients is accompanied by a vulnerability to develop GGT, total and direct bilirubin, and baseline IgG, IgA and IgM autoimmune diseases such as immune thrombocytopenia, levels. Coombs-positive and nephritis, suggesting that tolerance is not correctly maintained in these patients.5 It Detection of repertoires by ELISA has been reported that the sera from CD40L-deficient patients Serum autoantibody screening was performed in a blinded fi fashion at a 1:101 dilution using commercially available FDA- contain speci c antibodies against self-antigens (anti-erythro- ® cyte, anti-erythropoietin, anti-platelet, anti-smooth muscle, cleared ELISA kits (QUANTA Lite ; Inova Diagnostics, Inc., San Diego, CA, USA) for IgG antibodies against M2 EP anti-cardiolipin, anti-Ro, anti-RNP, anti-nuclear and anti- (MIT3), gp210, sp100, f-actin, SLA, LKM-1, TPO, ATG, thyroid).6,7 Other autoimmune manifestations seen in HIGM GPA, MPO and anti-centromere. To detect IgA and IgM patients include inflammatory bowel disease, autoimmune antibodies against M2 EP (MIT3), gp210, and sp100, , seronegative , and discoid 8 QUANTA Lite ELISA plates and anti-peroxidase-conjugated erythematosus. It appears that most patients with HIGM goat anti-human immunoglobulin IgA or IgM were used to also develop some form of , particularly perform an in-house research assay. No manufacturer-supplied fi 9 those with HIGM due to intrinsic B-cell de ciencies. controls were used in these assays. Briefly, 100 μlof1:101- diluted serum was added to polystyrene microwells coated with Table 2 Comparison of laboratory features between patients the respective antigen. The samples were run in duplicate, and with HIGM and controls the reactions were allowed to proceed for 30 min at room n Hyper IgM (n = 13) Controls (n = 21) P Value temperature. After washing three times, 100 μlofperoxidase- conjugated goat anti-human immunoglobulin IgG, IgA or IgM Age (years) 6 (3) 4 (4) NS was added to each well, followed by incubation for 30 min. Female/male (n) 5/8 9/12 NS Following washing, 100 μlof3,3,5,5′-tetramethylbenzidine ALP (U/l) 224.62 (178.17) 182.71 (47.30) NS chromogen was added to the wells, which were then incubated GGT (U/l) 95.00 (281.99) 11.48 (3.25) NS for 30 min, followed by the addition of 100 μl of 0.344 M ALT (U/l) 19.00 (23.35) 6.29 (0.64) NS H SO stop solution to terminate the reaction. The absorbance AST (U/l) 7.09 (4.41) 14.57 (4.55) o0.001 2 4 was read at 450/620 nm, and the results, expressed in arbitrary Ig-G (mg/dl) 498.23 (304.18) 736.76 (200.13) 0.007 units, were calculated in reference to a kit-provided calibrator Ig-A (mg/dl) 46.08 (52.89) 63.48 (28.19) 0.001 ’ Ig-M (mg/dl) 408.69 (428.07) 82.57 (26.32) o0.001 for the IgG assays. The manufacturer s cutoff was established at fi TBIL (mg/dl) 0.06 (0.05) 0.14 (0.07) o0.001 25 units depending on the speci c assay. DBIL (mg/dl) 0.04 (0.04) 0.11 (0.02) o0.001 Analysis of AMA reactivity by indirect immunofluorescence Abbreviatoons: IgM, immunoglobulin M, NS, non significant. Continuous variables are expressed as median (STD), P valueso0.05 are Serum AMAs were evaluated by standard indirect immuno- illustrated. fluorescence (IIF) in HEp-2 cells (INOVA Diagnostics, Inc.,

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San Diego, CA, USA). Briefly, reactivities were analyzed by various forms of infections (Table 3), with ENT being the most serial dilutions in phosphate-buffered saline (PBS) on human common (53.85%), followed by recurrent pneumonias and epithelial cell (HEp-2 cell)-fixed slides in a moist chamber at bronchopneumopathies (38.46%). Protracted or recurrent room temperature for 30 min. After washing with PBS, the diarrhea was present in four patients (30.77%). Oral thrush slides were incubated with fluorescent antibody conjugate due to candidiasis was observed in three patients (23.08%). (fluorescein isothiocyanate-labeled goat anti-human immuno- Moreover, three patients (23.08%) developed adenopathy. Two globulin) in a moist chamber at room temperature for 30 min. patients developed episodes of chronic otitis, with one devel- Immunofluorescent images were acquired using fixed exposure oping permanent hearing loss. Of note, two patients (P1 and times and enhancement parameters using a × 40 objective and P2) developed ataxia-telangiectasia during follow-up. an Olympus BX51 fluorescence microscope (Olympus Amer- ica, Inc., NY, USA). Laboratory findings The biochemical and immunological profiles of the HIGM Statistical analysis patients and controls are presented in Table 2. The quantitative An optimized cutoff for the detection of the IgA and IgM levels of serum immunoglobulins were measured at the time of antibodies for the three antigens MIT3, sp100 and gp210 was diagnosis, showing median values (± s.d.) for IgG, IgA and IgM calculated using the formula ‘mean OD of matched controls+3 of 498.23 ( ±304.18), 46.08 (±52.89) and 408.69 (±428.07) mg/ s.d.’. This value was referenced as 25 units, and relative unit dl, respectively. Notably, patients with HIGM had significantly values were calculated based on this cutoff. The data were higher levels of IgM than controls (Po0.001). Conversely, the analyzed by the Kruskal–Wallis H test and χ2-test; the coin- levels of IgG and IgA in the HIGM patient group were cidence of two tests was compared using the Kappa index. significantly lower than those in the control group (P = 0.007 Two-tailed P values o0.05 were considered statistically sig- and P = 0.001, respectively). The levels of AST, total bilirubin nificant. Scattergrams were plotted using GraphPad Prism and direct bilirubin in HIGM patients were statistically lower (GraphPad Software, Inc., San Diego, CA, USA). than those in controls; however, the levels of these parameters in both groups were all within the normal range. Regarding the RESULTS remaining biochemical parameters, there were no significant Patient characteristics differences between patients with HIGM and controls. Thirteen patients (8 male and 5 female, aged 1–12 years) were identified as having HIGM based on a hyper-IgM screen to Autoantibody repertoires detect high levels of IgM and low or absent levels of IgA and The autoantibody profiles of HIGM patients and controls are IgG. The clinical features, abnormal biochemistry parameters presented in Table 4 and Figure 1. When PBC autoantibodies and serum autoantibody repertoires of the enrolled cases are were measured, it was found that the level of IgM antibody summarized in Table 1. Four patients had a family history of against MIT3 was significantly higher in HIGM patients than in immunodeficiency. Patient 1 (P1) and P2 are cousins, while the controls (P = 0.002). By contrast, the levels of both IgG and IgA sister of P1 was also diagnosed with HIGM and died of against MIT3 were significantly lower in HIGM patients than Hodgkin’s lymphoma. In addition, P4 and P5 are brothers. in controls (P = 0.002 and Po0.0001, respectively). In addi- Gene mutations were found in 8 of 13 patients (61.54%), in tion, both IgG and IgA autoantibodies against sp100 or gp210 which 5 patients showed a mutation in the AID or C terminal were also significantly lower in the HIGM cohorts than in the end of the AID Gene, 1 patient had a mutation in the CD40 controls (P = 0.0001 and P = 0.018; P = 0.004 and P = 0.004, ligand gene, and 2 patients had an ATM gene mutation. respectively). Similarly, HIGM patients also had lower IgG autoantibody levels against f-actin than the controls Clinical presentations (P = 0.032). However, there were no statistical differences in All patients experienced chronic and recurrent infections, the IgG levels of other autoimmune liver diseases-related particularly recurrent ear, nose and throat (ENT) infections autoantibodies, including anti-SLA and anti-LKM1. Regarding involving the respiratory and gastrointestinal systems, during extrahepatic autoantibodies, there was no significant difference the course of the disease. These HIGM patients presented with in the levels of ANA, anti-SS-A and SS-B, anti-centromere, anti-Sm, anti-Scl-70, anti-MPO, anti-TPO, anti-Thyroid T, and Table 3 Clinical manifestation of patients with Hyper IgM anti-GPA autoantibodies between the two groups. syndrome Furthermore, we set out to evaluate the prevalence of the fi Condition Number of patients (%) PBC-speci c autoantibody IgM class in HIGM patients and controls. With the cutoff value at 25 units, 7/13 HIGM patients ENT infections 7 (53.85) (53.84%) were found to be anti-MIT3 IgM positive, whereas Recurrent pneumonias 5 (38.46) none of the controls were found to be positive (Po0.00). Bronchopneumopathies 5 (38.46) Regarding anti-sp100 autoantibodies, 3/13(23.08%) HIGM Oral thrush 3 (23.08) patients were found to have anti-sp100 IgM autoantibodies, Adenopathy 3 (23.08) while only 1/21 controls (0.05%) were positive for anti-sp100 Abbreviations: ENT, ear, nose and throat; IgM, immunoglobulin M. IgM autoantibodies (P40.05). Additionally, 1/13 (7.69%)

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Table 4 Comparison of autoantibodies profiles between with a nuclear dot staining pattern (Figures 2a and b). patients with HIGM and controls Additionally, some patients had both the smooth nuclear rim-staining and nuclear dot-staining patterns (Figure 2c). Hyper-IgM Autoantibodies (n = 13) Controls (n = 21) P-value DISCUSSION fi Liver-specific autoantibodies HIGM syndrome is the prototypic disorder of immunode - PBC autoantibodies (mg/dl) ciency caused by genetic defects in the CD40-CD40L pathway MIT3 or enzymes required for immunoglobulin class-switch recom- 10,11 Ig-G 0.93 (0.78) 3.52 (3.55) 0.001 bination and . Thus, patients with Ig-A 0.65 (0.88) 4.48 (6.23) 0.000 HIGM have an increased susceptibility to opportunistic infec- Ig-M 35.84 (24.54) 10.74 (5.06) 0.002 tions, , autoimmune disease and cancer, particu- sp100 larly of the liver or biliary tree.12 Affected patients frequently Ig-G 1.21 (2.65) 1.74 (1.44) 0.000 present with sclerosing cholangitis (due to Cryptosporidium Ig-A 3.34 (2.91) 5.74 (4.32) 0.018 parvum infections), chronic hepatitis (related to Ig-M 16.55 (13.49) 7.89 (6.12) NS , hepatitis C virus and cytomegalovirus), and liver and gp210 biliary tract tumors.9 Interestingly, several studies have reported Ig-G 0.35 (0.34) 1.26 (2.62) 0.004 that HIGM patients are prone to developing autoimmune Ig-A 3.19 (1.33) 4.18 (0.78) 0.004 diseases such as Coombs-positive hemolytic anemia, Crohn’s Ig-M 7.39 (14.86) 2.57 (1.89) disease, mellitus, immune thrombocytopenia AIH autoantibodies and nephritis, suggesting that is not (mg/dl) correctly maintained in HIGM.9,13 Because the liver is a unique f-actin 5.26 (6.52) 5.91 (2.96) NS lymphoid organ that plays a significant role in immune SLA 0.42 (0.16) 0.43 (0.25) NS homeostasis, it is quite possible that autoimmune liver diseases LKM-1 0.45 (0.23) 0.66 (0.45) NS may be a part of the spectrum of HIGM . A previous Extrahepatic autoantibodies (mg/dl) report had described that one HIGM patient developed ANA 16.78 (18.98) 12.16 (4.88) NS with several typical autoantibodies of – SSA 3.323 (2.03) 3.14 (2.96) NS the IgM isotype, including anti-liver kidney-microsome SSB 1.42 (0.69) 1.82 (1.91) NS (LKM), anti-hepatocyte membrane and anti-smooth muscle 13 Sm 2.06 (1.28) 2.14 (1.09) NS antibodies. However, in patients with HIGM syndrome, the Scl-70 4.20 (4.04) 2.57 (1.54) NS prevalence of antibodies against autoantigens that are common Centromere 7.97 (9.65) 4.41 (2.36) NS in other autoimmune disorders, particularly autoimmune liver MPO 6.83 (14.44) 5.18 (7.24) NS diseases, has never been systematically assessed. TPO 21.48 (29.74) 28.53 (26.50) NS In the present study, we first comprehensively analyzed the Thyroid T 7.80 (7.00) 12.76 (16.77) NS antibody repertoires of HIGM patients compared with those of GPA 9.03 (20.37) 3.88 (4.18) NS controls. Second, we dissected in detail the PBC-specific Abbreviations: HIGM, hyper-IgM; Ig, immunoglobulin; NS, non significant. autoantibodies covering IgG, IgA and IgM isotypes using Continuous variables are expressed as median (± s.d.). NS indicates a semi-quantitative ELISA. Strikingly, the level of MIT3 IgM ⩾ P-value 0.05. antibodies in HIGM patients was significantly higher than that in controls. Moreover, our IIF results further confirmed the high incidence of IgM autoantibodies against MIT3 in this HIGM patients were positive for anti-gp210 IgM antibodies, cohort of HIGM patients. Finally, representative IIF patterns while no control was found to be positive for anti-gp210 IgM for reactivity to HEP-2 cells, similar to typical PBC patients, P4 class ( 0.05). were noted in these HIGM children. fi fi To further con rm our nding that HIGM children had Our data demonstrated no differences in the prevalence of higher IgM antibodies against MIT3, we also analyzed auto- autoantibodies for a broad spectrum of autoimmune diseases fl antibodies against AMA by immuno uorescence. Twelve (including systemic lupus erythematosus, Sjögren’ssyndrome, patients (92.31%) were found to be positive for AMA IgM systemic sclerosis, microscopic polyangiitis, Graves’ disease, (titer 1/40, n = 2; 1/80, n = 1; 1/160, n = 3; 1/320, n = 1; 1/640, and Hashimoto’s thyroiditis) between HIGM children and a n = 1; 1/1280, n = 3; 1/2560, n = 3). Conversely, none of the group of matched controls. Moreover, upon probing for liver- controls (0%) were found to be positive by immunofluores- specific autoantibody profiles, HIGM patients demonstrated a cence (Po0.001). In addition, no HIGM or control was found negative AIH-specific autoantibody pattern (anti-LKM-1, anti- to be positive for the AMA IgA and IgG isotypes. Moreover, SLA and anti-f-actin). On the other hand, elevated levels of sera from HIGM patients showed a representative IIF pattern IgM antibodies were observed in our HIGM cohorts compared in HEP-2 cells similar to that seen for typical PBC patients. For with those in contemporary controls by our MIT3-based example, some HIGM patients presented with cytoplasmic and ELISA. The MIT3 ELISA utilizes the immunodominant targets a smooth nuclear rim-staining pattern, while some presented of AMA (PDC-E2, OGDC-E2 and BCOADC-E2) and has been

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Figure 1 Scattergrams depicting the concentration of liver-specific autoantibodies in hyper-immunoglobulin M (HIGM) patients and controls (CTR). (a) The levels of autoantibodies in the serum of HIGM patients (n = 13), mean ± s.e.m. (b) The levels of autoantibodies in the serum of controls (n = 21), mean ± s.e.m.

Figure 2 Representative immunofluorescence pattern of hyper-immunoglobulin M (HIGM) sera in HEP-2 cells. Serum samples from patients with HIGM were diluted at 1:40 and were analyzed for reactivity in HEP-2 cells by immunofluorescence. (a) Typical cytoplasmic and a smooth nuclear rim staining pattern. (b)Nucleardotstainingpattern.(c) Nuclear rim- and nuclear dot-staining pattern. shown to have enhanced performance with well-balanced hallmark of PBC, an autoimmune cholestatic liver disease specificity and sensitivity.14,15 Consistent results were obtained, characterized by immune-mediated destruction of small intra- showing that most of the HIGM children showed positive hepatic bile ducts with portal inflammation.16,17 Notably, the AMA reactivity at high titers (41/40) by IIF. AMA is the prevalence of anti-sp100 IgM antibodies in our HIGM cohort

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was much higher than that in controls. In addition to AMA, of autoreactive T cells and destruction of self-tissue, thus anti-sp100 autoantibody is also an important complementary perpetuating the autoimmune response.24,28 This paradigm biomarker for the diagnosis of PBC, with also a prognostic may be an additional explanation for the susceptibility to PBC role.18 in our HIGM cohorts. Although the prevalence and, possibly, the incidence of PBC One intriguing finding of our study is that patients with are increasing worldwide, middle-aged females are still the HIGM have higher levels of anti-MIT3 IgM antibodies but most susceptible demographic for PBC.19 Unlike other auto- lower levels of IgG and IgA MIT3 antibodies than controls. In a immune liver diseases, such as AIH, few PBC cases had been normal immune response to , IgM class anti-MIT3 20 reported in childhood. However, these HIGM children antibodies may appear initially, with the subsequent appear- fi identi ed with AMA-positive IgM antibodies have not shown ance of IgG class anti-MIT3 antibodies.29 This elevated any manifestation of liver injury or bile duct damage (for presence of IgM class anti-MIT3 antibodies in our HIGM example, abnormal liver function tests). Because the presence patients may result from the failure of IgM to switch to IgG of AMA can precede the development of symptomatic disease, isotypes. Of relevance, a congenic study proposed that IgM some PBC patients tend to have a long latency period before class anti-M2 antibody (specific AMA for PBC) was detected the onset of clinical symptoms or signs such as cholestasis.21 more frequently in autoimmune cholangitis patients, whom PBC is generally a slowly progressive disease. Hence, close they classified as early PBC.30 follow-up of these children with AMA autoantibodies is In conclusion, the IgM class of anti-AMA antibodies was essential to improve our understanding of the relationship shown to be present by both ELISA and IIF in most of our between autoimmunity and HIGM, as is close monitoring so that the disease can be treated appropriately should any of these HIGM patients. The presence of hallmarks of PBC, a disease in fi children develop PBC in the future. which CD40L is a key player, in an immunode ciency disease It is well known that IgM responses play an essential role in caused by mutations in the CD40L gene is very intriguing and innate immunity by providing a first line of host defense may suggest new avenues of investigation for understanding the against microbial infections. Moreover, IgM has been proposed immune pathogenesis of PBC. It is likely that these HIGM as an important link between innate and acquired immunity by patients who are positive for the MIT3 autoimmune marker promoting antigen-driven IgG responses.22 An elevation in the may eventually present with PBC during long-term follow up. total serum IgM has been observed and is an important Longitudinal investigation of these patients will help to diagnostic feature in PBC. However, whether increased levels determine whether this latent vulnerability to developing PBC of IgM exert a pathogenic or a protective role in PBC remains exists. controversial.23,24 Several studies have tried to dissect the possible mechanisms underlying hyper-IgM in PBC. Our previous study demonstrated that the levels of IgM are inversely correlated with the levels of CD40L methylation in 1 Ehrenstein MR, Notley CA. The importance of natural IgM: scavenger, protector and regulator. Nat Rev Immunol 2010; 10:778–786. PBC, suggesting direct involvement of the CD40-CD40L 2 Duarte-Rey C, Bogdanos DP, Leung PS, Anaya JM, Gershwin ME. IgM interaction in the production of high amounts of IgM in predominance in autoimmune disease: genetics and gender. Auto- PBC.25 If this is the case, then patients with HIGM who possess immun Rev 2012; 11:A404–A412. 3 Karaca NE, Durandy A, Gulez N, Aksu G, Kutukculer N. Study of mutations in the gene encoding the CD40-CD40L interaction patients with hyper-IgM type IV phenotype who recovered sponta- may be particularly vulnerable to developing PBC. neously during late childhood and review of the literature. Eur J Pediatr The expression of the CD40 ligand by activated T cells in the 2011; 170:1039–1047. 4 Davies EG, Thrasher AJ. Update on the hyper immunoglobulin M liver stimulates B cells to increase the production of immu- syndromes. Br J Haematol 2010; 149:167–180. noglobulin, activates to produce IL-12, leading to 5 Herve M, Isnardi I, Ng YS, Bussel JB, Ochs HD, Cunningham-Rundles a shift of T-helper cells to the Th1 phenotype and subsequent C et al. CD40 ligand and MHC class II expression are essential for human peripheral tolerance. J Exp Med 2007; 204: IL-2 and IFN-gamma production, and induces cholangiocytes 1583–1593. to undergo apoptosis at an increased rate. Inhibiting the CD40 6 Wolpert KA, Webster AD, Whittaker SJ. Discoid lupus erythematosus pathway in PBC may dampen the autoreactive immune associated with a primary immunodeficiency syndrome showing fea- tures of non-X-linked hyper-IgM syndrome. Br J Dermatol 1998; 138: response, thereby improving liver function and symptoms of 1053–1057. the disease. Indeed, there is a molecule against CD40 currently 7 Gulino AV, Notarangelo LD. Hyper IgM syndromes. 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Cellular and Molecular Immunology