Role of IL-27 in Epstein-Barr virus infection revealed by IL-27RA deficiency.

Epstein-Barr virus (EBV) infection can engender severe B cell lymphoproliferative diseases1,2. The primary infection is often asymptomatic or causes infectious mononucleosis (IM), a self-limiting lymphoproliferative disorder3. Selective vulnerability to EBV has been reported in association with inherited mutations impairing T cell immunity to EBV4. Here we report biallelic loss-of-function variants in IL27RA that underlie an acute and severe primary EBV infection with a nevertheless favourable outcome requiring a minimal treatment. One mutant allele (rs201107107) was enriched in the Finnish population (minor allele frequency = 0.0068) and carried a high risk of severe infectious mononucleosis when homozygous. IL27RA encodes the IL-27 receptor alpha subunit5,6. In the absence of IL-27RA, phosphorylation of STAT1 and STAT3 by IL-27 is abolished in T cells. In in vitro studies, IL-27 exerts a synergistic effect on T-cell-receptor-dependent T cell proliferation7 that is deficient in cells from the patients, leading to impaired expansion of potent anti-EBV effector cytotoxic CD8+ T cells. IL-27 is produced by EBV-infected B lymphocytes and an IL-27RA-IL-27 autocrine loop is required for the maintenance of EBV-transformed B cells. This potentially explains the eventual favourable outcome of the EBV-induced viral disease in patients with IL-27RA deficiency. Furthermore, we identified neutralizing anti-IL-27 autoantibodies in most individuals who developed sporadic infectious mononucleosis and chronic EBV infection. These results demonstrate the critical role of IL-27RA-IL-27 in immunity to EBV, but also the hijacking of this defence by EBV to promote the expansion of infected transformed B cells.

[Hematopoietic cell transplantation for monogenic inflammatory bowel disease].

The pathogenesis of inflammatory bowel disease (IBD) may include immune dysregulation. About 20% of inborn errors of immunity (IEIs) are related to IBD, and more than 60 IEIs are known to present IBD. Monogenic IBDs include those that are refractory to traditional treatment and can be cured by allogeneic hematopoietic cell transplantation (HCT), making early diagnosis and treatment essential. In this report, we present a series of monogenic IBDs that are relatively frequently found in Japan, such as interleukin (IL)-10/IL-10 receptor deficiency, chronic granulomatous disease, XIAP deficiency, immunodysregulation, polyendocrinopathy, enteropathy, and X-linked (IPEX) syndrome, NEMO deficiency, and A20 haploinsufficiency and will describe the features of each IEI and the indications for HCT.

Early diagnosis of partial interferon-γ receptor 1 deficiency prevents the development of Bacille de Calmette et Guérin osteomyelitis.

We encountered two patients with partial interferon γ receptor 1 (IFN-γR1) deficiency in whom early diagnosis enhanced disease management. Patient 1 was a 44-year-old woman with enlarged lymph nodes diagnosed in a pre-pregnancy checkup, and pathological examination revealed a Mycobacterium avium infection. Based on her history of unknown multiple osteomyelitis during early childhood, mendelian susceptibility to mycobacterial disease was suspected. Genetic analysis revealed a novel heterozygous variant in IFNGR1. Genetic counseling was administered to the patient and her husband before they had their baby. Patient 2 was a 4-month-old boy whose father was previously diagnosed with autosomal dominant IFN-γR1 deficiency owing to Bacille de Calmette et Guérin (BCG) osteomyelitis. Genetic analysis showed that he had the same INFGR1 variant. He avoided BCG vaccination and has been disease-free since then. Early diagnosis is considered to be useful for genetic counseling and essential for preventing BCG osteomyelitis.

Recurrent tandem duplication of UNC13D in familial hemophagocytic lymphohistiocytosis type 3.

Familial hemophagocytic lymphohistiocytosis type 3 is a fatal inborn error of immunity due to abnormal cytotoxic activity of T and NK cells and is caused by variants in UNC13D, which encodes Munc13-4. One published case was reported to carry a tandem duplication of UNC13D exons 7-12, and we here present another case with the exact same duplication breakpoints. The patient carried the tandem duplication from maternal origin, and a c.2346_2349 variant on the paternal allele. Single nucleotide polymorphism analysis around UNC13D revealed that the allele with tandem duplication was most likely a founder allele. Transposable element analysis showed that the breakpoints occurred within Alu elements in introns 12 and 6. Multiple sequence alignment revealed that Alu elements containing the truncated points are highly homologous. Sequence homology was thought to be a factor predisposing to the tandem duplication variant.

Identification of Germline Non-coding Deletions in XIAP Gene Causing XIAP Deficiency Reveals a Key Promoter Sequence.

PURPOSE: X-linked inhibitor of apoptosis protein (XIAP) deficiency, also known as the X-linked lymphoproliferative syndrome of type 2 (XLP-2), is a rare immunodeficiency characterized by recurrent hemophagocytic lymphohistiocytosis, splenomegaly, and inflammatory bowel disease. Variants in XIAP including missense, non-sense, frameshift, and deletions of coding exons have been reported to cause XIAP deficiency. We studied three young boys with immunodeficiency displaying XLP-2-like clinical features. No genetic variation in the coding exons of XIAP was identified by whole-exome sequencing (WES), although the patients exhibited a complete loss of XIAP expression. METHODS: Targeted next-generation sequencing (NGS) of the entire locus of XIAP was performed on DNA samples from the three patients. Molecular investigations were assessed by gene reporter expression assays in HEK cells and CRISPR-Cas9 genome editing in primary T cells. RESULTS: NGS of XIAP identified three distinct non-coding deletions in the patients that were predicted to be driven by repetitive DNA sequences. These deletions share a common region of 839 bp that encompassed the first non-coding exon of XIAP and contained regulatory elements and marks specific of an active promoter. Moreover, we showed that among the 839 bp, the exon was transcriptionally active. Finally, deletion of the exon by CRISPR-Cas9 in primary cells reduced XIAP protein expression. CONCLUSIONS: These results identify a key promoter sequence contained in the first non-coding exon of XIAP. Importantly, this study highlights that sequencing of the non-coding exons that are not currently captured by WES should be considered in the genetic diagnosis when no variation is found in coding exons.

Clinical and Immunological Heterogeneity in Japanese Patients with Gain-of-Function Variants in STAT3.

PURPOSE: Germline loss-of-function variants in the signal transducer and activator of transcription 3 (STAT3) gene result in autosomal dominant hyper IgE syndrome, whereas somatic gain-of-function (GOF) variants in STAT3 are associated with some malignancies. In addition, germline GOF variants in STAT3 are linked to disorders involving autoimmunity and lymphoproliferation. In this study, we describe five Japanese families with germline GOF variants in STAT3, including three novel variants. We also present the clinical and immunological characteristics of these patients. METHODS: Eight patients from five families were enrolled in this study. We performed genetic and immunological analyses, and collected the associated clinical information. RESULTS: We identified five heterozygous variants in STAT3 using whole-exome sequencing and target gene sequencing. Two of these (E286G and T716M) were previously reported and three (K348E, E415G, and G618A) were novel. A STAT3 reporter assay revealed that all of the variants were GOF. However, the immunological and clinical characteristics among the patients were highly variable. CONCLUSION: Patients with STAT3 GOF variants exhibited clinical and immunological heterogeneity with incomplete penetrance.

APRIL-dependent lifelong plasmacyte maintenance and immunoglobulin production in humans.

BACKGROUND: Interactions between the tumor necrosis factor (TNF) ligand superfamily and TNF receptor superfamily play critical roles in B-cell development and maturation. A proliferation-inducing ligand (APRIL), a member of the TNF ligand superfamily, is secreted from myeloid cells and known to induce the differentiation of memory B cells to plasmacytes. OBJECTIVE: We sought to elucidate the role of APRIL in B-cell differentiation and immunoglobulin production through the analysis of complete APRIL deficiency in human. METHODS: We performed whole exome sequencing in a patient with adult common variable immunodeficiency. His parents were in a consanguineous marriage. TNFSF13 mRNA and protein expression were analyzed in the primary cells and plasma from the patient and in cDNA-transfected cells and supernatants of the cultures in vitro. Immunologic analysis was performed by using flow cytometry and next-generation sequencing. Monocyte-derived dendritic cells differentiated from induced pluripotent stem cells (iPSC-moDCs) were cocultured with memory B cells from healthy controls to examine in vitro plasmacyte differentiation. RESULTS: We identified a homozygous frameshift mutation in TNFSF13, the gene encoding APRIL, in the patient. APRIL mRNA and protein were completely absent in the monocytes and iPSC-moDCs of the patient. In contrast to the results of previous animal model studies, the patient showed hypogammaglobulinemia with a markedly reduced level of plasmacytes in peripheral blood and a clearly increased level of circulating marginal zone B cells. Although iPSC-moDC-induced in vitro plasmacyte differentiation was reduced in the patient, recombinant APRIL supplementation corrected this abnormality. CONCLUSION: The first APRIL deficiency in an adult patient with common variable immunodeficiency revealed the role of APRIL in lifelong maintenance of plasmacytes and immunoglobulin production in humans.

Whole-Exome Sequencing-Based Approach for Germline Mutations in Patients with Inborn Errors of Immunity.

PURPOSE: Owing to recent technological advancements, using next-generation sequencing (NGS) and the accumulation of clinical experiences worldwide, more than 420 genes associated with inborn errors of immunity (IEI) have been identified, which exhibit large genotypic and phenotypic variations. Consequently, NGS-based comprehensive genetic analysis, including whole-exome sequencing (WES), have become more valuable in the clinical setting and have contributed to earlier diagnosis, improved treatment, and prognosis. However, these approaches have the following disadvantages that need to be considered: a relatively low diagnostic rate, high cost, difficulties in the interpretation of each variant, and the risk of incidental findings. Thus, the objective of this study is to review our WES results of a large number of patients with IEI and to elucidate patient characteristics, which are related to the positive WES result. METHODS: We performed WES for 136 IEI patients with negative conventional screening results for candidate genes and classified these variants depending on validity of their pathogenicity. RESULTS: We identified disease-causing pathogenic mutations in 36 (26.5%) of the patients which were found in known IEI-causing genes. Although the overall diagnostic rate was not high and was not apparently correlated with the clinical subcategories and severity, we revealed that earlier onset with longer duration of diseases were associated with positive WES results, especially in pediatric cases. CONCLUSIONS: Most of the disease-causing germline mutations were located in the known IEI genes which could be predicted using patients' clinical characteristics. These results may be useful when considering appropriate genetic approaches in the clinical setting.

Inherited chromosomally integrated human herpesvirus-6 in a patient with XIAP deficiency.

Human herpesvirus-6 (HHV-6) is a common pathogen affecting the human population. Primary HHV-6 infection generally occurs during infancy and causes exanthema subitum. Moreover, HHV-6 may exhibit inherited chromosomally integrated HHV-6 (iciHHV-6) in certain individuals. Although iciHHV-6 is generally known to be nonpathogenic, it may cause reactivation in patients with primary immunodeficiency disease (PID). XIAP deficiency is a rare PID characterized by recurrent hemophagocytic lymphohistiocytosis (HLH). It has been reported that the Epstein-Barr virus primarily causes HLH; however, the other pathogens, including HHV-6, can also cause this complication. We encountered a case of XIAP deficiency accompanied by iciHHV-6. He suffered from recurrent HLH, for which allogeneic bone marrow transplantation (BMT) was performed as a curative therapy. During the course of BMT, the patient experienced HLH three times, but there was no reactivation of endogenous HHV-6 from iciHHV-6. Finally, the patient achieved complete donor chimerism and a decline in HHV-6 DNA copy number in whole blood. This case report demonstrates no evidence of reactivation of iciHHV-6 during BMT in a patient with XIAP deficiency.

Mechanisms of genotype-phenotype correlation in autosomal dominant anhidrotic ectodermal dysplasia with immune deficiency.

BACKGROUND: Autosomal dominant anhidrotic ectodermal dysplasia with immune deficiency (AD EDA-ID) is caused by heterozygous point mutations at or close to serine 32 and serine 36 or N-terminal truncations in IκBα that impair its phosphorylation and degradation and thus activation of the canonical nuclear factor κ light chain enhancer of activated B cells (NF-κB) pathway. The outcome of hematopoietic stem cell transplantation is poor in patients with AD EDA-ID despite achievement of chimerism. Mice heterozygous for the serine 32I mutation in IκBα have impaired noncanonical NF-κB activity and defective lymphorganogenesis. OBJECTIVE: We sought to establish genotype-phenotype correlation in patients with AD EDA-ID. METHODS: A disease severity scoring system was devised. Stability of IκBα mutants was examined in transfected cells. Immunologic, biochemical, and gene expression analyses were performed to evaluate canonical and noncanonical NF-κB signaling in skin-derived fibroblasts. RESULTS: Disease severity was greater in patients with IκBα point mutations than in those with truncation mutations. IκBα point mutants were expressed at significantly higher levels in transfectants compared with truncation mutants. Canonical NF-κB-dependent IL-6 secretion and upregulation of the NF-κB subunit 2/p100 and RELB proto-oncogene, NF-κB subunit (RelB) components of the noncanonical NF-κB pathway were diminished significantly more in patients with point mutations compared with those with truncations. Noncanonical NF-κB-driven generation of the transcriptionally active p100 cleavage product p52 and upregulation of CCL20, intercellular adhesion molecule 1 (ICAM1), and vascular cell adhesion molecule 1 (VCAM1), which are important for lymphorganogenesis, were diminished significantly more in LPS plus α-lymphotoxin ß receptor-stimulated fibroblasts from patients with point mutations compared with those with truncations. CONCLUSIONS: IκBα point mutants accumulate at higher levels compared with truncation mutants and are associated with more severe disease and greater impairment of canonical and noncanonical NF-κB activity in patients with AD EDA-ID.

High frequencies of asymptomatic Epstein-Barr virus viremia in affected and unaffected individuals with CTLA4 mutations.

Patients with CTLA4 mutations present with autoimmune diseases, lymphoproliferation, and hypogammaglobulinemia, and a subset of patients developed Epstein-Barr virus (EBV)-associated malignancies, suggesting an impaired immune function against EBV. Here we investigated EBV infection in individuals with CTLA4 mutations. We measured EBV viral DNA in healthy individuals, individuals with autoimmune diseases, and individuals with CTLA4 mutations. In addition, we evaluated the numbers and function of EBV-specific T cells, invariant NKT cells, and NK cells. More than half of individuals with CTLA4 mutations including asymptomatic ones had detectable EBV DNA, which is a significantly higher frequency with higher viral loads compared with healthy and disease controls. However, individuals with CTLA4 mutations had almost normal immunity against EBV. Individuals with CTLA4 mutations have an increased susceptibility to Epstein-Barr virus infections. Asymptomatic viremia occurs at high frequencies, which can be persistent and can occur in unaffected individuals.

Droplet Digital PCR-Based Chimerism Analysis for Primary Immunodeficiency Diseases.

OBJECTIVE: In the current study, we aimed to accurately evaluate donor/recipient or male/female chimerism in samples from patients who underwent hematopoietic stem cell transplantation (HSCT). METHODS: We designed the droplet digital polymerase chain reaction (ddPCR) for SRY and RPP30 to detect the male/female chimerism. We also developed mutation-specific ddPCR for four primary immunodeficiency diseases. RESULTS: The accuracy of the male/female chimerism analysis using ddPCR was confirmed by comparing the results with those of conventional methods (fluorescence in situ hybridization and short tandem repeat-PCR) and evaluating dilution assays. In particular, we found that this method was useful for analyzing small samples. Thus, this method could be used with patient samples, especially to sorted leukocyte subpopulations, during the early post-transplant period. Four mutation-specific ddPCR accurately detected post-transplant chimerism. CONCLUSION: ddPCR-based male/female chimerism analysis and mutation-specific ddPCR were useful for all HSCT, and these simple methods contribute to following the post-transplant chimerism, especially in disease-specific small leukocyte fractions.

Highly sensitive detection of Epstein-Barr virus-infected cells by EBER flow FISH.

When Epstein-Barr virus (EBV) infection is suspected, identification of infected cells is important to understand the pathogenesis, determinine the treatment strategy, and predict the prognosis. We used the PrimeFlow™ RNA Assay Kit with a probe to detect EBV-encoded small RNAs (EBERs) and multiple surface markers, to identify EBV-infected cells by flow cytometry. We analyzed a total of 24 patients [11 with chronic active EBV disease (CAEBV), 3 with hydroa vacciniforme lymphoproliferative disorder, 2 with X-linked lymphoproliferative disease type 1 (XLP1), 2 with EBV-associated hemophagocytic lymphohistiocytosis, and 6 with posttransplant lymphoproliferative disorder (PTLD)]. We compared infected cells using conventional quantitative PCR methods and confirmed that infected cell types were identical in most patients. Patients with CAEBV had widespread infection in T and NK cells, but a small amount of B cells were also infected, and infection in patients with XLP1 and PTLD was not limited to B cells. EBV-associated diseases are believed to be complex pathologies caused by EBV infecting a variety of cells other than B cells. We also demonstrated that infected cells were positive for HLA-DR in patients with CAEBV. EBER flow FISH can identify EBV-infected cells with high sensitivity and is useful for elucidating the pathogenesis.

Janus kinase inhibitors ameliorate clinical symptoms in patients with STAT3 gain-of-function.

Germline gain-of-function (GOF) variants in the signal transducer and activator of transcription 3 (STAT3) gene is an inborn error of immunity presenting with autoimmunity and lymphoproliferation. Symptoms can vary widely, and no effective treatment has been established. This study investigated the efficacy of Janus kinase (JAK) inhibitors (JAKi) in patients with STAT3-GOF. Four patients were enrolled and their clinical symptoms before and after the initiation of treatment with JAKi were described. A cell stimulation assay was performed using Epstein-Barr virus transformed lymphoid cell lines (EBV-LCLs) that were derived from the patients with STAT3-GOF. The patients presented with various symptoms, and these symptoms mostly improved after the initiation of JAKi treatment. Upon interleukin-6 stimulation, the EBV-LCLs of patients showed enhanced STAT3 phosphorylation compared with those of the EBV-LCLs of healthy controls. In conclusion, four Japanese patients with STAT3-GOF were successfully treated with JAKi. JAKi ameliorated various symptoms and therefore, the use of JAKi could be an effective treatment option for patients with STAT3-GOF.

A complementary approach for genetic diagnosis of inborn errors of immunity using proteogenomic analysis.

Advances in next-generation sequencing technology have identified many genes responsible for inborn errors of immunity (IEI). However, there is still room for improvement in the efficiency of genetic diagnosis. Recently, RNA sequencing and proteomics using peripheral blood mononuclear cells (PBMCs) have gained attention, but only some studies have integrated these analyses in IEI. Moreover, previous proteomic studies for PBMCs have achieved limited coverage (approximately 3000 proteins). More comprehensive data are needed to gain valuable insights into the molecular mechanisms underlying IEI. Here, we propose a state-of-the-art method for diagnosing IEI using PBMCs proteomics integrated with targeted RNA sequencing (T-RNA-seq), providing unique insights into the pathogenesis of IEI. This study analyzed 70 IEI patients whose genetic etiology had not been identified by genetic analysis. In-depth proteomics identified 6498 proteins, which covered 63% of 527 genes identified in T-RNA-seq, allowing us to examine the molecular cause of IEI and immune cell defects. This integrated analysis identified the disease-causing genes in four cases undiagnosed in previous genetic studies. Three of them could be diagnosed by T-RNA-seq, while the other could only be diagnosed by proteomics. Moreover, this integrated analysis showed high protein-mRNA correlations in B- and T-cell-specific genes, and their expression profiles identified patients with immune cell dysfunction. These results indicate that integrated analysis improves the efficiency of genetic diagnosis and provides a deep understanding of the immune cell dysfunction underlying the etiology of IEI. Our novel approach demonstrates the complementary role of proteogenomic analysis in the genetic diagnosis and characterization of IEI.

Case Report: Rotavirus Vaccination and Severe Combined Immunodeficiency in Japan.

Severe combined immunodeficiency (SCID) is an inborn error of immunity that occurs in approximately 1 in 50,000 births, mainly due to impaired lymphocyte differentiation. Without curative treatment, such as hematopoietic cell transplantation (HCT) or gene therapy, severe infection in the first year of life could make this condition fatal. The results of HCT are poor when patients have active infections, thus requiring early diagnosis before onset of infection. In five cases of SCID diagnosed in Japan, the oral rotavirus vaccine had been administered before diagnosis. In this study, we demonstrated that the rotavirus from their stools was a vaccine-derived strain. In some cases, severe gastroenteritis triggered the diagnosis of SCID. However, newborn screening for SCID is available before the first rotavirus vaccination using assays for the detection of T-cell receptor excision circles (TRECs). Therefore, to improve the prognosis of patients with SCID in Japan, we should establish a screening system of TRECs for newborns throughout Japan.

Novel AP3B1 compound heterozygous mutations in a Japanese patient with Hermansky-Pudlak syndrome type 2.

Hermansky-Pudlak syndrome type 2 (HPS2) is an extremely rare autosomal recessive inherited disease characterized by partial oculocutaneous albinism (OCA), bleeding diathesis due to a storage pool deficiency and immunodeficiency. The disorder is caused by disruption of the adapter protein 3 complex, which is involved in impaired intracellular vesicle transport. Here, we report the first case of a 1-year-old girl with HPS2 in Asia. She had no specific symptoms other than OCA and neutropenia. We analyzed her platelet function using transmission electron microscopy and a platelet aggregation test, cytotoxic degranulation assay of her natural killer (NK) cells and bleeding time, the results of which led to the diagnosis of HPS2. Although her NK-cell cytotoxic degranulation was impaired, she had not developed signs of hemophagocytic lymphohistiocytosis (HLH) or fibrosing lung disease. Molecular genetic analyses showed novel heterozygous mutations (c.188T>A [p.M63K] and c.2546>A [p.L849X]) in AP3B1. When examining patients with OCA, blood tests should be performed to confirm neutrophil count, bleeding time and platelet agglutination. When HPS2 is suspected, detailed immunological tests should be considered, and attention should be paid to HLH and pulmonary lesions immediately and over the long term.