Inherited human RelB deficiency impairs innate and adaptive immunity to infection.

We report two unrelated adults with homozygous (P1) or compound heterozygous (P2) private loss-of-function variants of V-Rel Reticuloendotheliosis Viral Oncogene Homolog B (RELB). The resulting deficiency of functional RelB impairs the induction of NFKB2 mRNA and NF-κB2 (p100/p52) protein by lymphotoxin in the fibroblasts of the patients. These defects are rescued by transduction with wild-type RELB complementary DNA (cDNA). By contrast, the response of RelB-deficient fibroblasts to Tumor Necrosis Factor (TNF) or IL-1ß via the canonical NF-κB pathway remains intact. P1 and P2 have low proportions of naïve CD4+ and CD8+ T cells and of memory B cells. Moreover, their naïve B cells cannot differentiate into immunoglobulin G (IgG)- or immunoglobulin A (IgA)-secreting cells in response to CD40L/IL-21, and the development of IL-17A/F-producing T cells is strongly impaired in vitro. Finally, the patients produce neutralizing autoantibodies against type I interferons (IFNs), even after hematopoietic stem cell transplantation, attesting to a persistent dysfunction of thymic epithelial cells in T cell selection and central tolerance to some autoantigens. Thus, inherited human RelB deficiency disrupts the alternative NF-κB pathway, underlying a T- and B cell immunodeficiency, which, together with neutralizing autoantibodies against type I IFNs, confers a predisposition to viral, bacterial, and fungal infections.

Successful Haematopoietic Stem Cell Transplantation for LRBA Deficiency with Fludarabine, Treosulfan, and Thiotepa-Based Conditioning.

LRBA deficiency is an inborn error of immunity defined by autoimmunity, lymphoproliferation, recurrent infections, cytopenia, and inflammatory bowel disease. Despite recent advances in managing this disease with targeted biologic therapy, haematopoietic stem cell transplant (HSCT) remains the only cure. However, great variability exists between protocols used to transplant patients with LRBA deficiency. We describe a cohort of seven patients with LRBA deficiency who underwent HSCT using a myeloablative, reduced toxicity regime of fludarabine, treosulfan, and thiotepa at two transplantation centres from 2016 to 2019. Data were collected both retrospectively and prospectively, measuring time to engraftment, infectious complications, incidence of graft versus host disease, and post-transplantation chimerism. Six of seven patients survived transplantation, and four of six surviving patients achieving treatment-free survival. We thus recommend that HSCT with fludarabine, treosulfan, and thiotepa-based conditioning be considered in patients with LRBA deficiency.

A Novel Case of IFNAR1 Deficiency Identified a Common Canonical Splice Site Variant in DOCK8 in Western Polynesia: The Importance of Validating Variants of Unknown Significance in Under-Represented Ancestries.

Advanced genomic technologies such as whole exome or whole genome sequencing have improved diagnoses and disease outcomes for individuals with genetic diseases. Yet, variants of unknown significance (VUS) require rigorous validation to establish disease causality or modification, or to exclude them from further analysis. Here, we describe a young individual of Polynesian ancestry who in the first 13 mo of life presented with SARS-CoV-2 pneumonia, severe enterovirus meningitis and adenovirus gastroenteritis, and severe adverse reaction to MMR vaccination. Genomic analysis identified a previously reported pathogenic homozygous variant in IFNAR1 (c.1156G > T, p.Glu386* LOF), which is common in Western Polynesia. Moreover, a new and putatively deleterious canonical splice site variant in DOCK8 was also found in homozygosity (c.3234 + 2T > C). This DOCK8 variant is common in Polynesians and other under-represented ancestries in large genomic databases. Despite in silico bioinformatic predictions, extensive in vitro and ex vivo analysis revealed the DOCK8 variant likely be neutral. Thus, our study reports a novel case of IFNAR1 deficiency, but also highlights the importance of functional validation of VUS, including those predicted to be deleterious, and the pressing need to expand our knowledge of the genomic architecture and landscape of under-represented populations and ancestries.

STAT3 gain of function: Too much of a good thing in the skin!

Germline activating mutations in STAT3 cause a multi-systemic autoimmune and autoinflammatory condition. By studying a mouse model, Toth et al. (https://doi.org/10.1084/jem.20232091) propose a role for dysregulated IL-22 production by Th17 cells in causing some aspects of immune-mediated skin inflammation in human STAT3 GOF syndrome.

Flow Cytometric Identification of Human IgE+ B Lineage Subsets.

The use of flow cytometry for immunophenotyping is contingent on the ability to accurately assign biological relevance to the detected signal. This process has historically been challenging when defining IgE expressing B cells or IgE expressing antibody-secreting cells due to widespread expression of receptors for IgE on various leukocyte subsets, including human B cells. Here we describe our implementation of intracellular staining for human IgE following a blocking step to negate the challenge of surface-bound IgE. We also describe our experience with a human B cell culture system that can be used to robustly validate this approach before application to primary human samples. Orthogonal confirmatory techniques remain essential; these are not described in detail, but several possible strategies are suggested.

Development of EBV Related Diffuse Large B-cell Lymphoma in Deficiency of Adenosine Deaminase 2 with Uncontrolled EBV Infection.

Deficiency of Adenosine Deaminase 2 (DADA2) patients presenting with primary immunodeficiency are at risk of uncontrolled EBV infection and secondary malignancies including EBV-related lymphoproliferative disorders (LPD). This paper describes the first case of EBV related diffuse large B-cell lymphoma in a patient with DADA2 and uncontrolled EBV infection. Consideration should be given to monitoring for EBV viraemia and to preventative EBV specific therapy in DADA2 and patients with at risk primary immunodeficiencies. A type I interferon (IFN) gene signature is associated with DADA2 though its association with immune dysregulation is unclear.

Lack of association between classical HLA genes and asymptomatic SARS-CoV-2 infection.

Human genetic studies of critical COVID-19 pneumonia have revealed the essential role of type I interferon-dependent innate immunity to SARS-CoV-2 infection. Conversely, an association between the HLA-B∗15:01 allele and asymptomatic SARS-CoV-2 infection in unvaccinated individuals was recently reported, suggesting a contribution of pre-existing T cell-dependent adaptive immunity. We report a lack of association of classical HLA alleles, including HLA-B∗15:01, with pre-omicron asymptomatic SARS-CoV-2 infection in unvaccinated participants in a prospective population-based study in the United States (191 asymptomatic vs. 945 symptomatic COVID-19 cases). Moreover, we found no such association in the international COVID Human Genetic Effort cohort (206 asymptomatic vs. 574 mild or moderate COVID-19 cases and 1,625 severe or critical COVID-19 cases). Finally, in the Human Challenge Characterisation study, the three HLA-B∗15:01 individuals infected with SARS-CoV-2 developed symptoms. As with other acute primary infections studied, no classical HLA alleles favoring an asymptomatic course of SARS-CoV-2 infection were identified.

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.

A Novel Heterozygous Variant in AICDA Impairs Ig Class Switching and Somatic Hypermutation in Human B Cells and is Associated with Autosomal Dominant HIGM2 Syndrome.

B cells and their secreted antibodies are fundamental for host-defense against pathogens. The generation of high-affinity class switched antibodies results from both somatic hypermutation (SHM) of the immunoglobulin (Ig) variable region genes of the B-cell receptor and class switch recombination (CSR) which alters the Ig heavy chain constant region. Both of these processes are initiated by the enzyme activation-induced cytidine deaminase (AID), encoded by AICDA. Deleterious variants in AICDA are causal of hyper-IgM syndrome type 2 (HIGM2), a B-cell intrinsic primary immunodeficiency characterised by recurrent infections and low serum IgG and IgA levels. Biallelic variants affecting exons 2, 3 or 4 of AICDA have been identified that impair both CSR and SHM in patients with autosomal recessive HIGM2. Interestingly, B cells from patients with autosomal dominant HIGM2, caused by heterozygous variants (V186X, R190X) located in AICDA exon 5 encoding the nuclear export signal (NES) domain, show abolished CSR but variable SHM. We herein report the immunological and functional phenotype of two related patients presenting with common variable immunodeficiency who were found to have a novel heterozygous variant in AICDA (L189X). This variant led to a truncated AID protein lacking the last 10 amino acids of the NES at the C-terminal domain. Interestingly, patients' B cells carrying the L189X variant exhibited not only greatly impaired CSR but also SHM in vivo, as well as CSR and production of IgG and IgA in vitro. Our findings demonstrate that the NES domain of AID can be essential for SHM, as well as for CSR, thereby refining the correlation between AICDA genotype and SHM phenotype as well as broadening our understanding of the pathophysiology of HIGM disorders.

Filaggrin-Associated Atopic Skin, Eye, Airways, and Gut Disease, Modifying the Presentation of X-Linked Reticular Pigmentary Disorder (XLPDR).

BACKGROUND: X-linked reticular pigmentary disorder (XLPDR) is a rare condition characterized by skin hyperpigmentation, ectodermal features, multiorgan inflammation, and recurrent infections. All probands identified to date share the same intronic hemizygous POLA1 hypomorphic variant (NM_001330360.2(POLA1):c.1393-354A > G) on the X chromosome. Previous studies have supported excessive type 1 interferon (IFN) inflammation and natural killer (NK) cell dysfunction in disease pathogenesis. Common null polymorphisms in filaggrin (FLG) gene underlie ichthyosis vulgaris and atopic predisposition. CASE: A 9-year-old boy born to non-consanguineous parents developed eczema with reticular skin hyperpigmentation in early infancy. He suffered recurrent chest infections with chronic cough, clubbing, and asthma, moderate allergic rhinoconjunctivitis with keratitis, multiple food allergies, and vomiting with growth failure. Imaging demonstrated bronchiectasis, while gastroscopy identified chronic eosinophilic gastroduodenitis. Interestingly, growth failure and bronchiectasis improved over time without specific treatment. METHODS: Whole-genome sequencing (WGS) using Illumina short-read sequencing was followed by both manual and orthogonal automated bioinformatic analyses for single-nucleotide variants, small insertions/deletions (indels), and larger copy number variations. NK cell cytotoxic function was assessed using 51Cr release and degranulation assays. The presence of an interferon signature was investigated using a panel of six interferon-stimulated genes (ISGs) by QPCR. RESULTS: WGS identified a de novo hemizygous intronic variant in POLA1 (NM_001330360.2(POLA1):c.1393-354A > G) giving a diagnosis of XLPDR, as well as a heterozygous nonsense FLG variant (NM_002016.2(FLG):c.441del, NP_0020.1:p.(Arg151Glyfs*43)). Compared to healthy controls, the IFN signature was elevated although the degree moderated over time with the improvement in his chest disease. NK cell functional studies showed normal cytotoxicity and degranulation. CONCLUSION: This patient had multiple atopic manifestations affecting eye, skin, chest, and gut, complicating the presentation of XLPDR. This highlights that common FLG polymorphisms should always be considered when assessing genotype-phenotype correlations of other genetic variation in patients with atopic symptoms. Additionally, while the patient exhibited an enhanced IFN signature, he does not have an NK cell defect, suggesting this may not be a constant feature of XLPDR.

The trajectory of human B-cell function, immune deficiency, and allergy revealed by inborn errors of immunity.

The essential role of B cells is to produce protective immunoglobulins (Ig) that recognize, neutralize, and clear invading pathogens. This results from the integration of signals provided by pathogens or vaccines and the stimulatory microenvironment within sites of immune activation, such as secondary lymphoid tissues, that drive mature B cells to differentiate into memory B cells and antibody (Ab)-secreting plasma cells. In this context, B cells undergo several molecular events including Ig class switching and somatic hypermutation that results in the production of high-affinity Ag-specific Abs of different classes, enabling effective pathogen neutralization and long-lived humoral immunity. However, perturbations to these key signaling pathways underpin immune dyscrasias including immune deficiency and autoimmunity or allergy. Inborn errors of immunity that disrupt critical immune pathways have identified non-redundant requirements for eliciting and maintaining humoral immune memory but concomitantly prevent immune dysregulation. Here, we will discuss our studies on human B cells, and how our investigation of cytokine signaling in B cells have identified fundamental requirements for memory B-cell formation, Ab production as well as regulating Ig class switching in the context of protective versus allergic immune responses.

Isolated Chronic Mucocutaneous Candidiasis due to a Novel Duplication Variant of IL17RC.

PURPOSE: Inborn errors of the IL-17A/F-responsive pathway lead to chronic mucocutaneous candidiasis (CMC) as a predominant clinical phenotype, without other significant clinical manifestations apart from mucocutaneous staphylococcal diseases. Among inborn errors affecting IL-17-dependent immunity, autosomal recessive (AR) IL-17RC deficiency is a rare disease with only three kindreds described to date. The lack of an in vitro functional evaluation system of IL17RC variants renders its diagnosis difficult. We sought to characterize a 7-year-old Japanese girl with CMC carrying a novel homozygous duplication variant of IL17RC and establish a simple in vitro system to evaluate the impact of this variant. METHODS: Flow cytometry, qPCR, RNA-sequencing, and immunoblotting were conducted, and an IL17RC-knockout cell line was established for functional evaluation. RESULTS: The patient presented with oral and mucocutaneous candidiasis without staphylococcal diseases since the age of 3 months. Genetic analysis showed that the novel duplication variant (Chr3: 9,971,476-9,971,606 dup (+131bp)) involving exon 13 of IL17RC results in a premature stop codon (p.D457Afs*16 or p.D457Afs*17). Our functional evaluation system revealed this duplication to be loss-of-function and enabled discrimination between loss-of-function and neutral IL17RC variants. The lack of response to IL-17A by the patient's SV40-immortalized fibroblasts was restored by introducing WT-IL17RC, suggesting that the genotype identified is responsible for her clinical phenotype. CONCLUSIONS: The clinical and cellular phenotype of the current case of AR IL-17RC deficiency supports a previous report on this rare disorder. Our newly established evaluation system will be useful for the diagnosis of AR IL-17RC deficiency, providing accurate validation of unknown IL17RC variants.

For whom the B(c)ell tolls: CXCL4 AIDs human autoimmunity.

In this issue of JEM, Çakan et al. (2023. J. Exp. Med.https://doi.org/10.1084/jem.20230944) explore a CXCL4-mediated mechanism by which TLRs cause autoimmunity in human B cells, breaching bone marrow tolerance.

Isolated chronic mucocutaneous candidiasis due to a novel duplication variant of IL17RC.

Purpose: Inborn errors of the IL-17A/F-responsive pathway lead to chronic mucocutaneous candidiasis (CMC) as a predominant clinical phenotype, without other significant clinical manifestations apart from mucocutaneous staphylococcal diseases. Amongst inborn errors affecting IL-17-dependent immunity, autosomal recessive (AR) IL-17RC deficiency is a rare disease with only three kindreds described to date. The lack of an in vitro functional evaluation system of IL17RC variants renders its diagnosis difficult. We sought to characterize a seven-year-old Japanese girl with CMC carrying a novel homozygous duplication variant of IL17RC and establish a simple in vitro system to evaluate the impact of this variant. Methods: Flow cytometry, qPCR, RNA-sequencing, and immunoblotting were conducted, and an IL17RC-knockout cell line was established for functional evaluation. Results: The patient presented with oral and mucocutaneous candidiasis without staphylococcal diseases since the age of three months. Genetic analysis showed that the novel duplication variant (Chr3: 9,971,476-9,971,606 dup (+ 131bp)) involving exon 13 of IL17RC results in a premature stop codon (p.D457Afs*16 or p.D457Afs*17). Our functional evaluation system revealed this duplication to be loss-of-function and enabled discrimination between loss-of-function and neutral IL17RC variants. The lack of response to IL-17A by the patient's SV40-immortalized fibroblasts was restored by introducing WT-IL17RC, suggesting that the genotype identified is responsible for her clinical phenotype. Conclusions: The clinical and cellular phenotype of the current case of AR IL-17RC deficiency supports a previous report on this rare disorder. Our newly established evaluation system will be useful for diagnosis of AR IL-17RC deficiency, providing accurate validation of unknown IL17RC variants.

Inborn errors of human B cell development, differentiation, and function.

B cells develop from hematopoietic stem cells in the bone marrow. Once generated, they serve multiple roles in immune regulation and host defense. However, their most important function is producing antibodies (Ab) that efficiently clear invading pathogens. This is achieved by generating memory B cells that rapidly respond to subsequent Ag exposure, and plasma cells (PCs) that continually secrete Ab. These B cell subsets maintain humoral immunity and host protection against recurrent infections for extended periods of time. Thus, the generation of antigen (Ag)-specific memory cells and PCs underlies long-lived serological immunity, contributing to the success of most vaccines. Our understanding of immunity is often derived from animal models. However, analysis of individuals with monogenic defects that disrupt immune cell function are unprecedented models to link genotypes to clinical phenotypes, establish mechanisms of disease pathogenesis, and elucidate critical pathways for immune cell development and differentiation. Here, we review fundamental breakthroughs in unraveling the complexities of humoral immunity in humans that have come from the discovery of inborn errors disrupting B cell function.

The Th17/IL-17 Axis and Host Defense Against Fungal Infections.

Chronic mucocutaneous candidiasis (CMC) was recognized as a primary immunodeficiency in the early 1970s. However, for almost 40 years, its genetic etiology remained unknown. The progressive molecular and cellular description of inborn errors of immunity (IEI) with syndromic CMC pointed toward a possible role of IL-17-mediated immunity in protecting against fungal infection and CMC. Since 2011, novel IEI affecting either the response to or production of IL-17A and/or IL-17F (IL-17A/F) in patients with isolated or syndromic CMC provided formal proof of the pivotal role of the IL-17 axis in mucocutaneous immunity to Candida spp, and, to a lesser extent, to Staphylococcus aureus in humans. In contrast, IL-17-mediated immunity seems largely redundant against other common microbes in humans. In this review, we outline the current knowledge of IEI associated with impaired IL-17A/F-mediated immunity, highlighting our current understanding of the role of IL-17A/F in human immunity.

Human PIK3R1 mutations disrupt lymphocyte differentiation to cause activated PI3Kδ syndrome 2.

Heterozygous loss-of-function (LOF) mutations in PIK3R1 (encoding phosphatidylinositol 3-kinase [PI3K] regulatory subunits) cause activated PI3Kδ syndrome 2 (APDS2), which has a similar clinical profile to APDS1, caused by heterozygous gain-of-function (GOF) mutations in PIK3CD (encoding the PI3K p110δ catalytic subunit). While several studies have established how PIK3CD GOF leads to immune dysregulation, less is known about how PIK3R1 LOF mutations alter cellular function. By studying a novel CRISPR/Cas9 mouse model and patients' immune cells, we determined how PIK3R1 LOF alters cellular function. We observed some overlap in cellular defects in APDS1 and APDS2, including decreased intrinsic B cell class switching and defective Tfh cell function. However, we also identified unique APDS2 phenotypes including defective expansion and affinity maturation of Pik3r1 LOF B cells following immunization, and decreased survival of Pik3r1 LOF pups. Further, we observed clear differences in the way Pik3r1 LOF and Pik3cd GOF altered signaling. Together these results demonstrate crucial differences between these two genetic etiologies.