Oligoclonal IgA Research Articles

Immunogenetics of Kawasaki disease.

Kawasaki disease (KD) is a medium vessel vasculitis that affects young children. Despite extensive research over the last 50years, the etiology of KD remains an enigma. Seasonal change in wind patterns was shown to have correlation with the epidemics of KD in Japan. Occurrence of disease in epidemiological clusters, seasonal variation, and a very low risk of recurrence suggest that KD is triggered by an infectious agent. The identification of oligoclonal IgA response in the affected tissues suggests an antigen-driven inflammation. The recent identification of a viral antigen in the cytoplasm of bronchial ciliated epithelium also favors infection as the main trigger for KD. Pointers that suggest a genetic basis of KD include a high disease prevalence in North-East Asian populations, a high risk among siblings, and familial occurrence of cases. Dysregulated innate and adaptive immune responses are evident in the acute stages of KD. In addition to the coronary wall inflammation, endothelial dysfunction and impaired vascular remodeling contribute to the development of coronary artery abnormalities (CAAs) and thrombosis. Genetic aberrations in certain intracellular signaling pathways involving immune effector functions are found to be associated with increased susceptibility to KD and development of coronary artery abnormalities (CAAs). Several susceptible genes have been identified through genome-wide association studies (GWAS) and linkage studies (GWLS). The genes that are studied in KD can be classified under 4 major groups-enhanced T cell activation (ITPKC, ORAI1, STIM1), dysregulated B cell signaling (CD40, BLK, FCGR2A), decreased apoptosis (CASP3), and altered transforming growth factor beta signaling (TGFB2, TGFBR2, MMP, SMAD). The review aims to highlight the role of several genetic risk factors that are linked with the increased susceptibility to KD.

The Epidemiology and Pathogenesis of Kawasaki Disease.

Epidemiologic and clinical features of Kawasaki Disease (KD) strongly support an infectious etiology. KD is worldwide, most prominently in Japan, Korea, and Taiwan, reflecting increased genetic susceptibility among Asian populations. In Hawaii, KD rates are 20-fold higher in Japanese ethnics than in Caucasians, intermediate in other ethnicities. The age distribution of KD, highest in children < 2 yo, lower in those < 6 months, is compatible with infection by a ubiquitous agent resulting in increasing immunity with age and with transplacental immunity, as with some classic viruses. The primarily winter-spring KD seasonality and well-documented Japanese epidemics with wave-like spread also support an infectious trigger. We hypothesize KD pathogenesis involves an RNA virus that usually causes asymptomatic infection but KD in a subset of genetically predisposed children. CD8 T cells, oligoclonal IgA, and upregulation of cytotoxic T cell and interferon pathway genes in the coronaries in fatal KD also support a viral etiology. Cytoplasmic inclusion bodies in ciliated bronchial epithelium identified by monoclonal antibodies made from oligoclonal IgA heavy chains also supports a viral etiology. Recent availability of “second generation” antibodies from KD peripheral blood plasmablasts may identify a specific viral antigen. Thus, we propose an unidentified (“new”) RNA virus infects bronchial epithelium usually causing asymptomatic infection but KD in a subset of genetically predisposed children. The agent persists in inclusion bodies, with intermittent respiratory shedding, entering the bloodstream via macrophages targeting coronaries. Antigen-specific IgA plasma cells and CD8 T cells respond but coronaries can be damaged. IVIG may include antibody against the agent. Post infection, 97–99% of KD patients are immune to the agent, protected against recurrence. The agent can spread either from those with asymptomatic primary infection in winter-spring or from a previously infected contact who intermittently sheds the agent.

Monoclonal paraprotein influences baseline B-cell repertoire diversity and perturbates influenza vaccination-induced B-cell response

Monoclonal gammopathy of undetermined significance (MGUS) arises from a clonal expansion of plasma cells in the bone marrow, secreting monoclonal (M) paraprotein. It is associated with increased susceptibility to infections, which may reflect altered B-cell repertoire. To investigate this, we examined the immunoglobulin (Ig) M, IgG, and IgA B-cell repertoire diversity in MGUS at baseline and after influenza vaccination (n=16) in comparison with healthy controls (HCs; n=16). The Complementary Determining Region 3 region of the immunoglobulin heavy chain variable region gene was amplified and B-cell spectratypes analyzed by high-resolution electrophoresis. Spectratype Gaussian distribution, kurtosis, and skewness were quantified to measure repertoire shifts. Both HC and MGUS baseline spectratypes show interindividual variability that is more pronounced in the IGHG and IGHA repertoires. Overall, baseline B-cell repertoire is more altered in MGUS, with oligoclonality observed in 50% (p=0.01). Postvaccination, significant differences emerged in MGUS in relation to M-protein levels. High M-protein concentration is associated with a more oligoclonal IgG and IgA response at day 7 postvaccination, and, in contrast to HCs, vaccination also induced significant perturbations in the MGUS IgM repertoire at day 7 (p=0.005). Monoclonal expansion in MGUS thus has an effect on the baseline B-cell repertoire and influences the recruited repertoire upon vaccination.

Re-utilization of germinal centers in multiple Peyer's patches results in highly synchronized, oligoclonal, and affinity-matured gut IgA responses.

Whereas gut IgA responses to the microbiota may be multi-centered and diverse, little is known about IgA responses to T-cell-dependent antigens following oral immunizations. Using a novel approach, gut IgA responses to oral hapten (4-hydroxy-3-nitrophenyl)acetyl-cholera toxin (NP-CT) conjugates were followed at the cellular and molecular level. Surprisingly, these responses were highly synchronized, strongly oligoclonal, and dominated by affinity matured cells. Extensive lineage trees revealed clonal relationships between NP-specific IgA cells in gut inductive and effector sites, suggesting expansion of the same B-cell clone in multiple Peyer's patches (PPs). Adoptive transfer experiments showed that this was achieved through re-utilization of already existing germinal centers (GCs) in multiple PPs by previously activated GC GL7(+) B cells, provided oral NP-CT was given before cell transfer. Taken together, these results explain why repeated oral immunizations are mandatory for an effective oral vaccine.

Kawasaki Disease: Novel Insights into Etiology and Genetic Susceptibility

Kawasaki disease (KD) is a vasculitis of young childhood that particularly affects the coronary arteries. Molecular analysis of the oligoclonal IgA response in acute KD led to production of synthetic KD antibodies. These antibodies identify intracytoplasmic inclusion bodies in acute KD tissues. Light and electron microscopic studies indicate that the inclusion bodies are consistent with aggregates of viral proteins and RNA. Advances in molecular genetic analysis and completion of the Human Genome Project have sparked a worldwide effort to identify genes associated with KD. A polymorphism of one such gene, ITPKC, a negative regulator of T cell activation, confers susceptibility to KD in Japanese populations and increases the risk of developing coronary artery abnormalities in both Japanese and U.S. children. Identification of the etiologic agent and of genes conferring KD susceptibility are the best means of improving diagnosis and therapy and enabling prevention of this important disorder of childhood.

Recent Advances in the Understanding and Management of Kawasaki Disease

Kawasaki disease (KD) is an acute systemic inflammatory illness of childhood that can result in coronary artery aneurysms, myocardial infarction, and sudden death. Clinical and epidemiologic data point to an unknown infectious agent as the cause. We discovered that an oligoclonal IgA immune response is present in arterial tissue in acute KD. Synthetic versions of prevalent IgA antibodies in the KD arterial wall identify cytoplasmic inclusion bodies in acute KD ciliated bronchial epithelium and other inflamed KD tissues. Light and electron microscopic studies show that the inclusion bodies are consistent with aggregates of viral protein and RNA, and are likely formed by the KD etiologic agent. KD susceptibility is likely to be polygenic. Treatment of gammaglobulin nonresponders usually consists of additional intravenous immunoglobulin, methylprednisolone, and/or infliximab. Additional data regarding KD pathogenesis are urgently needed to provide other targets for therapy for those patients at highest risk of developing coronary artery abnormalities.

Cloning the Arterial IgA Antibody Response during Acute Kawasaki Disease

Kawasaki disease (KD) is the most common acquired cardiac disease in children in developed nations. The etiology of KD is unknown but likely to be a ubiquitous microbial agent. Previously, we showed that oligoclonal IgA plasma cells infiltrate coronary arteries and other inflamed tissues in acute KD. We demonstrated that a synthetic Ab made using an alpha H chain sequence prevalent in acute KD arterial tissue detected Ag in acute KD coronary arteries, lung, and other inflamed tissues and that Ag localized to cytoplasmic inclusion bodies in the acute KD ciliated bronchial epithelium. In this study, we synthesized a panel of mAbs from alpha and kappa chain sequences present in the KD arterial wall and tested the Abs for binding to acute KD tissues. We report that all of the synthetic mAbs that bind to acute KD tissues detect Ag in cytoplasmic inclusion bodies in the acute KD ciliated bronchial epithelium. Abs made from alpha sequences that were prevalent in KD arterial tissue show stronger binding to acute KD tissues than Abs made from less prevalent sequences. These findings highlight the likely importance of the inclusion bodies in the etiopathogenesis of acute KD, confirm that the IgA Ab response in acute KD is Ag driven, and demonstrate the usefulness of cloning the Ab response in diseased tissues to identify disease-relevant Ags.

Advances in Kawasaki disease.

Recent studies have increased our understanding of the etiopathogenesis of Kawasaki disease (KD). The inflammatory infiltrate in KD coronary artery aneurysms has been shown to consist of CD8 T lymphocytes, macrophages, and IgA plasma cells, consistent with an immune response to an intracellular pathogen with a mucosal portal of entry. The identification of an oligoclonal IgA response in the vascular wall and the detection of a KD-associated antigen in inflamed KD tissues using a synthetic antibody derived from KD oligoclonal IgA antibodies have provided new approaches to identification of the etiologic agent. Highly effective therapy has evolved for KD, even in the absence of identification of the etiologic agent. The existence of incomplete KD cases remains a significant diagnostic dilemma for the clinician. The development of a diagnostic test, more specific therapy, and ultimate prevention of this potentially fatal illness of childhood are dependent upon continued advances in determining the etiopathogenesis of this fascinating disorder.

Identification of Kawasaki Disease (KD) Antigens Using Synthetic KD Antibodies

We reported the unique finding that IgA plasma cells are prominent in the inflammatory infiltrate in the vascular wall in acute fatal Kawasaki Disease (KD). Additionally, we sequenced IgA heavy chain genes in vascular tissue from three fatal acute KD cases and found evidence of an oligoclonal IgA response in all three KD patients, indicating an antigen-driven process. The presence of oligoclonal IgA-producing cells in the KD vascular wall provides a unique opportunity to identify the specific antigens being targeted by synthesizing the corresponding oligoclonal antibodies in vitro. Recent advances in molecular immunology enable the cloning of specific immunoglobulin heavy and light chain variable region genes into mammalian immunoglobulin expression vectors for the production of specific antibodies in tissue culture. We have prepared four synthetic KD antibodies from oligoclonal IgA genes prevalent in the KD vascular wall. In preliminary experiments, one of the synthetic antibodies binds to tissue sections from the spleen of a child who died of KD on day 13 of illness, but not to control spleen sections. This synthetic KD antibody also binds to upper respiratory tract sections from two other fatal KD cases, but not to control respiratory tract. Identification of the targeted antigen is underway by Western blot and cDNA library screening analysis. The use of synthetic KD antibodies provides a new approach for identifying antigens important in KD pathogenesis, as well for identification of the etiologic agent of KD and the development of a diagnostic test.

Switch recombination in fetal porcine thymus is uncoupled from somatic mutation

Since fetal serum Ig isotype profiles suggested that IgG and IgA could be of de novo origin, we studied their transcription and secretion. IgM transcripts were present at 50 days of gestation in major fetal lymphoid tissues, IgG and IgA transcription was pronounced at 60 days in fetal thymus and both transcription and secretion in this organ increased in late fetal life. The CDR3 spectratype of thymic IgG and IgA transcripts was as polyclonal as that of IgM already at 70 days in utero indicating a broad repertoire of switched B-cells. However, VDJs transcribed with the switched isotypes were not hypermutated as were those from immunized fetuses, indicating that switch recombination and somatic mutation are not coupled in utero in piglets. This finding and the fact that the oligoclonal IgA and IgM repertoires in a non-inductive site of the mucosal immune system (parotid gland) becomes polyclonal in piglets reared germ-free, suggest that initial expansion of switched B-cells in fetal and neonatal piglets is not driven by environmental antigen. Our findings collectively suggest that all IgA and IgM may result from de novo synthesis while some IgG probably results from selective transport. The latter is consistent with the gradual decline in serum IgG concentration in germ-free isolator piglets and the expression of FcRn in the porcine placenta.

Oligoclonal IgA response in the vascular wall in acute Kawasaki disease.

Kawasaki Disease (KD) is a potentially fatal acute vasculitis of childhood. Although KD is the leading cause of acquired heart disease in children in developed nations, its pathogenesis remains unknown. We previously reported the novel observation that IgA plasma cells infiltrate the vascular wall in acute KD. We have now examined the clonality of this IgA response in vascular tissue from three fatal cases of KD to determine whether it is oligoclonal, suggesting an Ag-driven process, or polyclonal, suggesting nonspecific B cell activation or a response to a superantigen. We first sequenced VDJ junctions of 44 alpha genes isolated from a primary, unamplified KD vascular cDNA library. Five sets of clonally related alpha sequences were identified, comprising 34% (15 of 44) of the isolated alpha sequences. Furthermore, point mutations consistent with somatic mutation were detected in the related sequences. Next, using formalin-fixed coronary arteries from two additional fatal KD cases, we sequenced VDJ junctions of alpha genes isolated by RT-PCR, and a restricted pattern of CDR3 usage was observed in both. We conclude that the vascular IgA response in acute KD is oligoclonal. The identification of an oligoclonal IgA response in KD strongly suggests that the immune response to this important childhood illness is Ag-driven.

Molecular analysis of the junctional diversity of IgA heavy-chains in human intestine and peripheral blood: Evidence for oligoclonal IgA B-cell expansions in healthy subjects

Previous studies have demonstrated that the f/~ T cell receptor (TCR) repertoire of intraepithelial and peripheral lymphocytes is oligoclonal in healthy individuals. T/5 T ceils and IgA B cells share many properties. Thus, //5 T cells can recognize antigen without the need of antigen presentation, and the structural features of the antigen binding CDR3 domains of 5 chains are very similar to immunoglobulin heavy chains. IgA secreting B cells and //~ T ceils are predominantly located at mucosal sites and are thought to protect the host from pathogens to which the mucosal surfaces are exposed continually. In the present study we analyzed the junctional diversity of IgA heavy chains and asked whether the intestinal and peripheral IgA receptor repertoires are also oligoclonal. Methods: Endoscopic mucosal biopsies from different colonic sites and two blood samples were obtained from healthy adults. RNA was reverse transcribed into eDNA and the CDR3 domains of all known IgA VH-families (VH 1-VH7) were PCR amplified. PCR products were size separated by denaturing PAGE and visualized by silver staining. Prominent bands were reamplified and directly sequenced. Results: CDR3 length analysis indicated, that parts of the intestinal n.0_q_d_d peripheral IgA receptor repertoire of all subjects were highly restricted. The intestinal VH5, VH6 and VH7 repertoires were as oligoclonal as the intestinal TCR repertoire. Furthermore, CDR3 length analysis of distinct colonic sites resulted in strikingly similar band patterns, and sequencing of dominant bands of the same length from those sites revealed identical CDR3 sequences. Intestinal VH1-VH4 transcripts had a polyclonal CDR3 length profile. However, some signs of clonal expansions could be observed and were reproducible in a second biopsy of the same subject. The VH1, VH2, VH5, VH6 and VH7 IgA receptor repertoire of the peripheral blood was also oligoclonal. Furthermore, the same band patterns and sequences could be obtained from the second blood sample. Conclusions: Large parts of intestinal and peripheral IgA B cells are clonally expanded and in this respect similar to f/~ T cells. Thus, it is likely that also IgA producing B-cells recognize a limited set of antigens.

Cerebrospinal fluid in the diagnosis of multiple sclerosis: a consensus report.

The Committee of the European Concerted Action for Multiple Sclerosis (Charcot Foundation) organised five workshops to discuss CSF analytical standards in the diagnosis of multiple sclerosis. This consensus report from 12 European countries summarises the results of those workshops. It is hoped that neurologists will confer with their colleagues in clinical chemistry to arrange the best possible local practice. The most sensitive method for the detection of oligoclonal immunoglobulin bands is isoelectric focusing. The same amounts of IgG in parallel CSF and serum samples are used and oligoclonal bands are revealed with IgG specific antibody staining. All laboratories performing isoelectric focusing should check their technique at least annually using "blind" standards for the five different CSF and serum patterns. Quantitative measurements of IgG production in the CNS are less sensitive than isoelectric focusing. The preferred method for detection of blood-CSF barrier dysfunction is the albumin quotient. The CSF albumin or total protein concentrations are less satisfactory. These results must be interpreted with reference to the age of the patient and the local method of determination. Cells should be counted. The normal value is no more than 4 cells/microliters. Among evolving optional tests, measurement of the combined local synthesis of antibodies against measles, rubella, and/or varicella zoster could represent a significant advance if it offers higher specificity (not sensitivity) for identifying chronic rather than acute inflammation. Other tests that may have useful correlations with clinical indices include those for oligoclonal free light chains, IgM, IgA, or myelin basic protein concentrations.

Polyclonal and oligoclonal IgA synthesis in the cerebrospinal fluid of neurological patients: An immunoaffinity-mediated capillary blot study

An intrathecal synthesis of IgA has been reported in various neurological disorders. However, the frequency of its occurence and the electrophoretic characteristics of the locally produced IgA remained a matter of controversy. We developed a sensitive immunoaffinity-mediated capillary blot technique for the detection of polyclonal and oligoclonal IgA in the CSF of 115 patients with various neurological disorders. Paired CSF and serum samples containing 50 ng IgA after appropriate dilutions were submitted to isoelectric focusing in agarose gels; IgA was then blotted onto a polyvinylidene difluoride sheet coated by an anti-IgA antiserum or by infectious antigens. The immunoblots were revealed by an alkaline phosphatase-conjugated anti-IgA antiserum. Only five samples displayed CSF-restricted oligoclonal IgA bands, including two out of 33 from MS patients. In herpetic encephalitis ( n = 5) and varicella-zoster meningitis ( n = 2), a strong intrathecal production of virus-specific IgA antibodies was detectable. In such cases, faint oligoclonal IgA antibodies were superimposed on a polyclonal background. A weak local production of anti- Borrelia burgdorferi IgA antibodies was present in two out of four cases of neuroborreliosis.

Detection of oligoclonal IgA in cerebrospinal fluid samples by an isoelectric focusing procedure.

An isoelectric focusing procedure for the detection of oligoclonal IgA in cerebrospinal fluid (CSF) samples is presented. CSF IgA bands were found in 69% of patients with various inflammatory neurological diseases, in 22% of patients with other neurological diseases, but in none of the patients with non-neurological diseases. In patients with inflammatory neurological diseases we examined the relationship between an intrathecal synthesis of oligoclonal IgA and an intrathecal IgA synthesis as proved by a formula recently described by Reiber and Felgenhauer: 83% of patients with an intrathecal IgA synthesis according to the formula of Reiber and Felgenhauer also showed CSF IgA bands whereas 59% of patients without an intrathecal IgA synthesis displayed an intrathecal synthesis of oligoclonal IgA. Seventeen percent of patients with inflammatory neurological diseases were characterized by an intrathecal synthesis of oligoclonal IgA but did not show either an intrathecal IgA synthesis or an intrathecal synthesis of oligoclonal IgG, the detection of oligoclonal IgA therefore being the only evidence of an intrathecal humoral immune response in these patients.

Intrathecal synthesis of IgM and IgA in neurological diseases: comparison of two formulae with isoelectric focusing

The intrathecal IgM and IgA immune response was studied by detecting oligoclonal IgM and IgA bands in CSF and by determining both the IgM/IgA indices as well as the intrathecal production of IgM and IgA by the Reiber formula. A good correlation was found between the demonstration of oligoclonal IgM ( r = 0.890) and IgA bands ( r = 0.927) and the Reiber formula. Compared with the finding of oligoclonal IgM/IgA bands calculation of an intrathecal IgM/IgA synthesis by the Reiber formula was less sensitive (86%). In two out of 22 controls and in 12 of 19 patients with polyradiculitis IgM and IgA indices were elevated while the other evaluation methods were negative. Even though oligoclonal IgM and IgA bands are considered to be a useful diagnostic tool in detecting intrathecal synthesis of IgM and IgA, i.e. in meningoencephalitis or neuroborreliosis for clinical practice, the Reiber formula provides reliable results. Detection sensitivity of assays for CSF-IgM and -IgA should be at least 1.0 mg/l.

Significance of CSF immunoglobulins in monitoring neurologic disease activity in Behçet's disease

We examined the intrathecal production of immunoglobulins (Ig) G, A, and M in 16 patients with Behçet's disease, 13 of whom have CNS involvement, and in 40 neurologic controls. Oligoclonal IgA and IgM bands were mainly detected in CSF samples from patients with active neuro-Behçet's disease and were documented to disappear when neurologic manifestations remit. Oligoclonal IgG bands, however, were not related to disease activity and were also found in some neurologic controls. High immunoglobulin index values were detected in both active and quiescent diseases and were high in some patients with impaired blood-CSF barriers. The study presented here demonstrates that CSF oligoclonal IgA and IgM may be helpful in monitoring CNS disease activity in neuro-Behçet's and could be useful in understanding the pathogenesis of this disease.

Affinity immunoblotting: Rapid and sensitive detection of oligoclonal IgG, IgA and IgM in unconcentrated CSF by agarose isoelectric focusing

Two methods for the investigation of oligoclonal immunoglobulin bands in cerebrospinal fluid are compared. Isoelectric focusing (IEF) in agarose gels combined with a highly sensitive affinity immunoblotting proved to be superior to PAGE-IEF and silver staining regarding detection sensitivity and expenditure of labour. The procedure presented here allows examination of oligoclonal bands not only of IgG, but also of IgA and IgM antibodies in less than 4 h. Oligoclonal IgM bands could be detected in 6 patients with neuroborreliosis or neurosyphilis as well as in one patient with a peripheral neuropathy where other electrophoretic methods, including immunoelectrophoresis, were negative. Oligoclonal IgA bands were demonstrated only in 2 patients with a bacterial infection of the CNS. In patients with multiple sclerosis amounts of IgM and IgA in the CSF were too low to allow investigation of oligoclonal IgM or IgA bands.

Immunoglobulin abnormalities in the cerebrospinal fluid during bacterial meningitis

11 patients with bacterial meningitis, examined during the course of the disease for immunoglobulin (Ig) abnormalities in the cerebrospinal fluid (CSF), all had an increased CSF IgM index equal to (CSF/serum IgM):(CSF/serum albumin), indicating intrathecal IgM production. Seven patients had a slightly increased CSF IgG index, and 7 a slightly increased IgA index. Six of the 11 patients had an increased IgM index in the presence of normal indices for IgG and IgA. Follow-up revealed the return of these values to normal. Four patients had identical oligoclonal IgG bands in the CSF and serum, probably representing a systemic immune response, but in only one case were oligoclonal bands suggestive of intrathecal IgG production found. No oligoclonal IgA response was demonstrable in the 4 patients examined. Antigen-immunofixation or antigen-absorption studies revealed evidence of a specific, intrathecal IgG antibody response in only 2 patients, while a search for IgG antibodies against aetiologically unrelated bacterial and viral antigens was negative. With the exception of IgM production, therefore, a humoral intrathecal immune response is less common in bacterial than in aseptic meningitis.

Oligoclonal IgA bands in multiple sclerosis and subacute sclerosing panencephalitis.

CSF oligoclonal IgG bands are often found in MS or cerebral diseases in which there is chronic antigenic stimulation. Using agarose isoelectric focusing followed by immunoblotting, we found oligoclonal IgA bands in CSF from 16 of 20 randomly selected patients with MS, 7 of 7 with subacute sclerosing panencephalitis (SSPE), and 0 of 10 with noninflammatory neurologic or psychiatric disease. IgA bands did not correlate with the course or stage of MS. Serial samples from two patients with MS and one with SSPE demonstrated only minor changes in IgA banding pattern. One MS patient without oligoclonal IgG bands had oligoclonal IgA bands, indicating that the latter test may be helpful in the diagnosis of MS.