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X-linked agammaglobulinemia (XLA) is a primary immunodeficiency (PID) caused by a defect in the gene encoding for Bruton tyrosine kinase (BTK). In the absence of a functional BTK, patients have low or absent circulating B cells and low or absent serum immunoglobulin. Despite gammaglobulin replacement and prompt use of antimicrobial agents, patients with XLA continue to experience infectious and non-infectious complications throughout their lifetime. The purpose of this study was to understand self-perceived health status of US-based patients with XLA, and examine the associations amongst clinical characteristics, treatment experience, and quality of life (QoL).A 46 and 68 question survey, developed by the Immune Deficiency Foundation (IDF) and a Short Form-12item v2® (SF-12v2®) for adults and SF-10™ for children to assess QoL, were mailed by IDF to patients in 2017 and 2018. Those that self-identified as having XLA or males with agammaglobulinemia were selected for analysis. Mean physical and mental composite scores (PCS and MCS) from SF-12v2® and mean physical health component (PHS) and psychological health summary (PSS) from SF-10™ scores were compared to the US normative data.Ninety-one patients completed the surveys: 58 (63.7%) adults and 33 (36.3%) children. For the combined surveys, the overall median age at time of the survey was 28.5 years (yrs); Inter-Quartile-Range (IQR) 13-49.5 yrs; the median age at diagnosis was 2 yrs (IQR = 0-4 yrs) and the median number of years with XLA diagnosis was 23 (IQR 10.75-40yrs). Amongst adult patients, physical scores were noted to be below the general adult population but did not reach statistical significance. In contrast, 2 or more chronic conditions impacted both physical and mental QoL (p .001) and hospitalization was associated with significantly decreased physical health QoL (p .001); three or more infections in the past 12 months exhibited impact on physical health although was not found to be statistically significant. Adult patients with public insurance fared worse in mental health domains compared to those with combined public and private or those with private alone (p = 0.001). Employment status did not impact QoL. None of these variables met statistical significance nor demonstrated impact within the pediatric population in either physical or mental domains of health.Our study provides further insight into what factors impact both physical and mental domains of health amongst patients with XLA. Early detection to prevent the development of associated morbidity, as well as vigilant care to prevent hospitalizations and infections, can limit the impact this disease may have on the overall well-being of XLA patients.

Purpose To understand the natural history and clinical outcomes for patients with X-linked agammaglobulinemia (XLA) in the United States utilizing the United States Immunodeficiency Network (USIDNET) patient registry. Methods The USIDNET registry was queried for data from XLA patients collected from 1981 to 2019. Data fields included demographics, clinical features before and after diagnosis of XLA, family history, genetic mutation in Bruton’s tyrosine kinase (BTK), laboratory findings, treatment modalities, and mortality. Results Data compiled through the USIDNET registry on 240 patients were analyzed. Patient year of birth ranged from 1945 to 2017. Living status was available for 178 patients; 158/178 (88.8%) were alive. Race was reported for 204 patients as follows: White, 148 (72.5%); Black/African American, 23 (11.2%); Hispanic, 20 (9.8%); Asian or Pacific Islander, 6 (2.9%), and other or more than one race, 7 (3.4%). The median age at last entry, age at disease onset, age at diagnosis, and length of time with XLA diagnosis was 15 [range (r) = 1–52 years], 0.8 [r = birth–22.3 years], 2 [r = birth–29 years], and 10 [r = 1–56 years] years respectively. One hundred and forty-one patients (58.7%) were Conclusions Current therapies for XLA patients reduce early mortality, but patients continue to experience complications that impact organ function. With improved life expectancy, more efforts will be required to improve post-diagnosis organ dysfunction and quality of life. Neurologic manifestations are an important co-morbidity associated with mortality and not yet clearly fully understood.

Purpose The human antibody repertoire forms in response to infections, the microbiome, vaccinations, and environmental exposures. The specificity of such antibody responses was compared among a cohort of toddlers to identify differences between seropositive versus seronegative responses. Methods An assessment of the serum IgM and IgG antibody reactivities in 197 toddlers of 1- and 2-years of age was performed with a microfluidic array containing 110 distinct antigens. Longitudinal profiling was done from years 1 to 2. Seropositivity to RNA and DNA viruses; bacteria; live attenuated, inactive, and subunit vaccines; and autoantigens was compared. A stratification was developed based on quantitative variations in the IgG responses. Clinical presentations and previously known genetic risk alleles for various immune system conditions were investigated in relation to IgG responses. Results IgG reactivities stratified toddlers into low, moderate, and high responder groups. The high group (17%) had elevated IgG responses to multiple RNA and DNA viruses (e.g., respiratory syncytial virus, Epstein-Barr virus, adenovirus, Coxsackievirus) and this correlated with increased responses to live attenuated viral vaccines and certain autoantigens. This high group was more likely to be associated with gestational diabetes and an older age. Genetic analyses identified polymorphisms in the IL2RB, TNFSF4, and INS genes in two high responder individuals that were associated with their elevated cytokine levels and clinical history of eczema and asthma. Conclusion Serum IgG profiling of toddlers reveals correlations between the magnitude of the antibody responses towards viruses, live attenuated vaccines, and certain autoantigens. A low responder group had much weaker responses overall, including against vaccines. The serum antibody screen also identifies individuals with IgG responses to less common infections (West Nile virus, parvovirus, tuberculosis). The characterization of the antibody responses in combination with the identification of genetic risk alleles provides an opportunity to identify children with increased risk of clinical disease. Supplementary Information The online version contains supplementary material available at 10.1007/s10875-021-00993-w.

The SKIV2L RNA exosome is an evolutionarily conserved RNA degradation complex in the eukaryotes. Mutations in theSKIV2Lgene are associated with a severe inherited disorder, trichohepatoenteric syndrome (THES), with multisystem involvement but unknown disease mechanism. Here, we reported a THES patient withSKIV2Lmutations showing severe primary B cell immunodeficiency, hypogammaglobulinemia, and kappa-restricted plasma cell dyscrasia but normal T cell and NK cell function. To corroborate these findings, we made B cell–specificSkiv2lknockout mice (Skiv2lfl/flCd79a-Cre), which lacked both conventional B-2 and innate-like B-1 B cells in the periphery and secondary lymphoid organs. This was linked to a requirement of SKIV2L RNA exosome activity in the bone marrow during early B cell development at the pro–B cell to large pre–B cell transition. Mechanistically,Skiv2l-deficient pro–B cells exhibited cell cycle arrest and DNA damage. Furthermore, loss of Skiv2l led to substantial out-of-frame V(D)J rearrangement of immunoglobulin heavy chain and severely reduced surface expression of μH, both of which are crucial for pre-BCR signaling and proliferative burst during early B cell development. Together, our data demonstrated a crucial role for SKIV2L RNA exosome in early B cell development in both human and mice by ensuring proper V(D)J recombination andIghexpression, which serves as the molecular basis for immunodeficiency associated with THES.

Type I interferons (IFN-I) play a critical role in human antiviral immunity, as demonstrated by the exceptionally rare deleterious variants of IFNAR1 or IFNAR2. We investigated five children from Greenland, Canada, and Alaska presenting with viral diseases, including life-threatening COVID-19 or influenza, in addition to meningoencephalitis and/or hemophagocytic lymphohistiocytosis following live-attenuated viral vaccination. The affected individuals bore the same homozygous IFNAR2 c.157T>C, p.Ser53Pro missense variant. Although absent from reference databases, p.Ser53Pro occurred with a minor allele frequency of 0.034 in their Inuit ancestry. The serine to proline substitution prevented cell surface expression of IFNAR2 protein, small amounts of which persisted intracellularly in an aberrantly glycosylated state. Cells exclusively expressing the p.Ser53Pro variant lacked responses to recombinant IFN-I and displayed heightened vulnerability to multiple viruses in vitro—a phenotype rescued by wild-type IFNAR2 complementation. This novel form of autosomal recessive IFNAR2 deficiency reinforces the essential role of IFN-I in viral immunity. Further studies are warranted to assess the need for population screening.

Thymus hypoplasia is reported for individuals with autosomal recessive, compound heterozygous and single allelic FOXN1 mutations. FOXN1 is the master transcriptional regulator of thymus epithelial cell development. Autosomal recessive mutations in FOXN1 lead to a Nude/SCID phenotype due to the ensuing T cell lymphopenia and impaired hair follicle extrusion. Targeted exome and whole genome sequencing for patients with low TRECs (measure of T cell output) has increased the number of diverse FOXN1 mutations to over 40. The consequence of these FOXN1 mutations on patients is varied and somewhat complicated by some individuals having only a transient delay in T cell development that corrects over time. We compared the functions of the FOXN1 mutants with luciferase reporter assays and nuclear localization experiments. For selected FOXN1 mutations, mice were developed to genocopy these to assess impacts on thymopoiesis. We identify partial and complete loss-of-function mutations along with gain-of-function and dominant negatives. Comparative analyses of murine thymopoiesis reveal some compound het Foxn1 mutations cause a transient thymus hypoplasia while others cause a permanent small thymus. Taken together, our findings establish FOXN1 genotype-phenotype relationships and suggest rapid functional screening approaches can be used to define the impact of different mutations of clinical relevance. Supported by grants from NIH (R01AI114523, R21AI144140) and the Jeffrey Modell Foundation (MdlM, CAW)

Thymus hypoplasia occurs in several clinical conditions including 22q11.2 deletion syndrome (22q11.2DS). 22q11.2DS is the most common human microdeletion disorder, affecting 1/4000. Thymuses from 60–70% 22q11.2DS patients are small and produce fewer T cells than normal. For individuals with a severe hypoplasia, an allogenic thymus tissue graft is needed to restore thymopoiesis. To determine the molecular mechanisms contributing to a small thymus in 22q11.2DS, we compared the development of the thymus in embryos from various 22q11.DS mouse models, normal controls and Foxn1 mutant mice. Reaggregate fetal thymic organ culture assays reveal that replacing mesenchymal cells from 22q11.2del hypoplastic lobes with normal ones restores tissue expansion and thymopoiesis. Thymic epithelial cells used as substitutes cannot. This is distinct from the Foxn1 mutant mice, wherein defective thymic epithelial cell functions lead to thymus hypoplasia/aplasia. Single cell RNA sequencing of normal and hypoplastic thymus lobes revealed differential expression of transcripts that primarily impacted 5 distinct mesenchymal cell subsets in 22q11.2DS. These transcripts are involved in cell-cell interactions, collagen deposition and growth. Elevated levels of collagen are present in the hypoplastic thymus tissues, suggesting a structural restriction. Mesenchymal and epithelial cell differentiation/expansion assays reveal a selective reduction in mesenchymal tissue expansion due to 22q11.2DS Supported by grants from the National Institutes of Health (R01AI114523, R21AI144140) and the Jeffrey Modell Foundation (MdlM, CAW)

Purpose of review Overview of neuroendocrine neoplasms in the context of their associations with primary and secondary immunodeficiency states. Recent findings Malignancies of neuroendocrine origin are well known to be associated with hereditary syndromes, including multiple endocrine neoplasia type 1, von Hippel-Lindau syndrome, neurofibromatosis type 1, and tuberous sclerosis. This review includes the X-linked form of hyper-IgM syndrome (XHIGM), due to mutations in the CD40Ligand gene (CD40LG), as an additional inherited disorder with susceptibility to such malignancies, and discusses neuroendocrine tumors (NETs) arising in other immunocompromised states. Of all primary immune deficiency diseases, NETs appear to be unique to XHIGM patients. Outcomes for XHIGM patients with NETs is poor, and the mechanism behind this association remains unclear. In secondary immune deficiency states, NET occurrences were primarily in patients with HIV or AIDS, the autoimmune disease systemic lupus erythematosus and solid organ transplant recipients. Gastroenteropancreatic NETs were most frequent in XHIGM patients, whereas nongastroenteropancreatic-NETs, like Merkel cell carcinoma and small-cell lung carcinoma, affected HIV/AIDS patients. Possible mechanisms as to the nature of these associations are discussed, including chronic infections and inflammation, and CD40-CD40L interactions. Many questions remain, and further studies are needed to clarify the predisposition of patients with XHIGM to the development of NETs. Given that many of these patients present late in their disease state and have poor outcomes, it is imperative to keep a high index of suspicion at the advent of early signs and symptoms. Regular monitoring with laboratory or imaging studies, including tumor markers, may be warranted, for which further studies are needed. Summary Of all primary immunodeficiency diseases, NETs appear to be unique to XHIGM, and the mechanism behind this association remains unclear. Outcome for XHIGM patients with NETs is poor, and it is imperative to keep a high index of suspicion at the advent of early signs and symptoms.

NF-κB signaling through its NFKB1-dependent canonical and NFKB2-dependent noncanonical pathways plays distinctive roles in a diverse range of immune processes. Recently, mutations in these 2 genes have been associated with common variable immunodeficiency (CVID). While studying patients with genetically uncharacterized primary immunodeficiencies, we detected 2 novel nonsense gain-of-function (GOF) NFKB2 mutations (E418X and R635X) in 3 patients from 2 families, and a novel missense change (S866R) in another patient. Their immunophenotype was assessed by flow cytometry and protein expression; activation of canonical and noncanonical pathways was examined in peripheral blood mononuclear cells and transfected HEK293T cells through immunoblotting, immunohistochemistry, luciferase activity, real-time polymerase chain reaction, and multiplex assays. The S866R change disrupted a C-terminal NF-κΒ2 critical site affecting protein phosphorylation and nuclear translocation, resulting in CVID with adrenocorticotropic hormone deficiency, growth hormone deficiency, and mild ectodermal dysplasia as previously described. In contrast, the nonsense mutations E418X and R635X observed in 3 patients led to constitutive nuclear localization and activation of both canonical and noncanonical NF-κΒ pathways, resulting in a combined immunodeficiency (CID) without endocrine or ectodermal manifestations. These changes were also found in 2 asymptomatic relatives. Thus, these novel NFKB2 GOF mutations produce a nonfully penetrant CID phenotype through a different pathophysiologic mechanism than previously described for mutations in NFKB2.

IntroductionInflammatory bowel disease (IBD) affects approximately 1/3 of patients with chronic granulomatous disease (CGD). Comprehensive investigation of the effect of allogeneic hematopoietic cell transplantation (HCT) on CGD IBD and the impact of IBD on transplant outcomes is lacking.MethodsWe collected data retrospectively from 145 patients with CGD who had received allogeneic HCT at 26 Primary Immune Deficiency Treatment Consortium (PIDTC) centers between January 1, 2005 and June 30, 2016.ResultsForty-nine CGD patients with IBD and 96 patients without IBD underwent allogeneic HCT. Eighty-nine percent of patients with IBD and 93% of patients without IBD engrafted (p = 0.476). Upper gastrointestinal acute GVHD occurred in 8.5% of patients with IBD and 3.5% of patients without IBD (p = 0.246). Lower gastrointestinal acute GVHD occurred in 10.6% of patients with IBD and 11.8% of patients without IBD (p = 0.845). The cumulative incidence of acute GVHD grades II-IV was 30% (CI 17-43%) in patients with IBD and 20% (CI 12-29%) in patients without IBD (p = 0.09). Five-year overall survival was equivalent for patients with and without IBD: 80% [CI 66-89%] and 83% [CI 72-90%], respectively (p = 0.689). All 33 surviving evaluable patients with a history of IBD experienced resolution of IBD by 2years following allogeneic HCT.ConclusionsIn this cohort, allogeneic HCT was curative for CGD-associated IBD. IBD should not contraindicate HCT, as it does not lead to an increased risk of mortality. This study is registered at clinicaltrials.gov NCT02082353.

Chromosome 22q11.2 deletion syndrome (22q11.2del) is a complex, multi-organ disorder noted for its varying severity and penetrance among those affected. The clinical problems comprise congenital malformations; cardiac problems including outflow tract defects, hypoplasia of the thymus, hypoparathyroidism, and/or dysmorphic facial features. Additional clinical issues that can appear over time are autoimmunity, renal insufficiency, developmental delay, malignancy and neurological manifestations such as schizophrenia. The majority of individuals with 22q11.2del have a 3 Mb deletion of DNA on chromosome 22, leading to a haploinsufficiency of ~106 genes, which comprise coding RNAs, noncoding RNAs, and pseudogenes. The consequent haploinsufficiency of many of the coding genes are well described, including the key roles of T-box Transcription Factor 1 (TBX1) and DiGeorge Critical Region 8 (DGCR8) in the clinical phenotypes. However, the haploinsufficiency of these genes alone cannot account for the tremendous variation in the severity and penetrance of the clinical complications among those affected. Recent RNA and DNA sequencing approaches are uncovering novel genetic and epigenetic differences among 22q11.2del patients that can influence disease severity. In this review, the role of coding and non-coding genes, including microRNAs (miRNA) and long noncoding RNAs (lncRNAs), will be discussed in relation to their bearing on 22q11.2del with an emphasis on TBX1.

Liver disease in X-linked hyper IgM syndrome (XHIGM) is an important predictor of mortality. In case liver transplantation (LT) is required, a survival benefit is observed when LT is combined with HSCT. ispartof: JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY vol:143 issue:5 pages:1952-+ ispartof: location:United States status: published

The primary immunodeficiency (PID) diseases comprise a heterogeneous group of inherited disorders of immune function. Technical advancements in whole-genome, whole-exome, and RNA-sequencing have seen the explosion of genetic discoveries in the field of PIDs. The present review aims to focus on a group of immunodeficiency disorders associated with elevated levels of IgM (hyper IgM; HIGM) and provides a clinical differential diagnosis. Most patients present for evaluation of immunodeficiency due to recurrent infections, and laboratory studies show either a clear isolated elevation of serum immunoglobulin M (IgM) with low or absent IgG, IgA, and IgE. Alternatively, IgM levels may be normal or moderately elevated while other serum immunoglobulins are reported below the norms for age but not absent. Mechanistically, these disorders are recognized as defects in immunoglobulin (Ig) class switch recombination (CSR). Importantly, to safeguard genetic stability, CSR utilizes elements of the DNA repair machinery including multi-protein complexes involved in mismatch repair (MMR). Therefore, it is not uncommon for defects in the DNA repair machinery, to present with laboratory findings of HIGM. This review will discuss clinical phenotypes associated with congenital defects associated with HIGM. Clinical manifestations, relevant immunologic testing, inheritance pattern, molecular diagnosis, presumed pathogenesis, and OMIM number, when annotated are compiled. Accepted therapeutic options, when available, are reviewed for each condition discussed.

Despite the known importance of zinc for human immunity, molecular insights into its roles have remained limited. Here we report a novel autosomal recessive disease characterized by absent B cells, agammaglobulinemia and early onset infections in five unrelated families. The immunodeficiency results from hypomorphic mutations of SLC39A7, which encodes the endoplasmic reticulum-to-cytoplasm zinc transporter ZIP7. Using CRISPR-Cas9 mutagenesis we have precisely modeled ZIP7 deficiency in mice. Homozygosity for a null allele caused embryonic death, but hypomorphic alleles reproduced the block in B cell development seen in patients. B cells from mutant mice exhibited a diminished concentration of cytoplasmic free zinc, increased phosphatase activity and decreased phosphorylation of signaling molecules downstream of the pre-B cell and B cell receptors. Our findings highlight a specific role for cytosolic Zn2+ in modulating B cell receptor signal strength and positive selection.

Background The antibody specificities of an infant progressively form in response to infections, environmental exposures, and vaccinations. While many adults develop antibodies to self-antigens, it remains unknown if these are present in infants and toddlers. Methods Serum, peripheral blood, and clinical data have been collected from 102 healthy children (1–2 year of age). ANA titers were measured by QuantiaLite ELISA (Inova) and reactivity to 125 diverse autoantigens tested by autoantibody array. Targeted sequencing was performed in 60 children to capture HLA alleles and potentially pathogenic genetic variations in 100 plus loci that are implicated in various autoimmune, rheumatic and immune system related diseases. Sequencing libraries were made using KAPA Biosystems kits. Custom target oligos were synthesized from Nimblegen. Deep sequencing was performed using Illumina HiSeq 4000 platform. Sequencing reads were aligned to reference genome and variants were called using GATK Pipeline. Secondary data analysis is done using Metlab, GraphPad Prism, Haploview and Golden Helix programs. Results Approximately 28% of very young children have moderate to high-titer autoantibodies, similar to adults, however, no female gender bias was observed. Significant differences between the child and adult immune repertoires were seen. Some samples demonstrated strong signatures of non-nuclear antigens reactivities. Interestingly, the ANA positive group of children exhibited significantly high titers of anti-alpha fodrin IgG (p=0.01), an autoantibody reported in juvenile Sjogren’s syndrome. Analysis of sequencing data identified regulatory polymorphisms in HLA class II and III regions that were associated with ANA positivity. About 8% of the ANA positive children also carried autoimmune disease associated HLA-DR-DQ alleles. HLA alleles and regulatory haplotypes were analyzed in relation to various clinical features in children. Serum C4 level measured in subset of ANA positive children identified few with reduced expression. Conclusions While the immune repertoire of very young children is typically thought of as naive and self-tolerant, a significant fraction of very young children makes autoantibodies as detected by commercial ANA ELISA and autoantigen arrays. This supports the conclusion that ANA and other autoantibodies are consequences of general body development and immune upregulation and not markers of pathology. One of the major genetic determinants of ‘pre-clinical’ autoimmunity as measured by ANA is the human major histocompatibility locus, HLA. Within HLA, several ‘endophenotypes’ emerge, including expression of a complement protein involved in immune complex removal, as well as multiple proteins associated with antigen presentation, including MHC class II. These findings support the idea that there is quantifiable genetic risk for the development of autoimmunity that can be measured in very young individuals.

The International Collaboration in Asthma, Allergy and Immunology initiated an international coalition among the American Academy of Allergy, Asthma & Immunology; the European Academy of Allergy and Clinical Immunology; the World Allergy Organization; and the American College of Allergy, Asthma & Immunology on common variable immunodeficiency. An author group was formed and then divided into individual committees. Within the committee, teams of authors were subgrouped to generate content for specific sections of the document. Content was derived from literature searches, relevant published guidelines, and clinical experience. After a draft of the document was assembled, it was collectively reviewed and revised by the authors. Where evidence was lacking or conflicting, the information presented represents the consensus expert opinion of the group. The full document was then independently reviewed by 5 international experts in the field, none of whom was among the authors of the original. The comments of these reviewers were incorporated before submission for publication.

Despite the known importance of zinc for human immunity, molecular insights into its roles have remained limited. Here we report a novel autosomal recessive disease characterized by absent B cells, agammaglobulinemia and early onset infections in five unrelated families. The immunodeficiency results from hypomorphic mutations of SLC39A7, which encodes the endoplasmic reticulum-to-cytoplasm zinc transporter ZIP7. Using CRISPR-Cas9 mutagenesis we have precisely modeled ZIP7 deficiency in mice. Homozygosity for a null allele caused embryonic death, but hypomorphic alleles reproduced the block in B cell development seen in patients. B cells from mutant mice exhibited a diminished concentration of cytoplasmic free zinc, increased phosphatase activity and decreased phosphorylation of signaling molecules downstream of the pre-B cell and B cell receptors. Our findings highlight a specific role for cytosolic Zn

NFKB2 GOF mutations are associated with CID without endocrine or ectodermal manifestations.As most autosomal-dominant primary immunodeficiencies, NFKB2 GOF changes have incomplete penetrance and variable expressivity.

WOS: 000398771800023, PubMed ID: 27697500, Background: X-linked hyper-IgM syndrome (XHIGM) is a primary immunodeficiency with high morbidity and mortality compared with those seen in healthy subjects. Hematopoietic cell transplantation (HCT) has been considered a curative therapy, but the procedure has inherent complications and might not be available for all patients. Objectives: We sought to collect data on the clinical presentation, treatment, and follow-up of a large sample of patients with XHIGM to (1) compare long-term overall survival and general well-being of patients treated with or without HCT along with clinical factors associated with mortality and (2) summarize clinical practice and risk factors in the subgroup of patients treated with HCT. Methods: Physicians caring for patients with primary immunodeficiency diseases were identified through the Jeffrey Modell Foundation, United States Immunodeficiency Network, Latin American Society for Immunodeficiency, and Primary Immune Deficiency Treatment Consortium. Data were collected with a Research Electronic Data Capture Web application. Survival from time of diagnosis or transplantation was estimated by using the Kaplan-Meier method compared with log-rank tests and modeled by using proportional hazards regression. Results: Twenty-eight clinical sites provided data on 189 patients given a diagnosis of XHIGM between 1964 and 2013; 176 had valid follow-up and vital status information. Sixty-seven (38%) patients received HCT. The average follow-up time was 8.5 +/- 7.2 years (range, 0.1-36.2 years). No difference in overall survival was observed between patients treated with or without HCT (P = .671). However, risk associated with HCT decreased for diagnosis years 1987-1995; the hazard ratio was significantly less than 1 for diagnosis years 1995-1999. Liver disease was a significant predictor of overall survival (hazard ratio, 4.9; 95% confidence limits, 2.2-10.8; P < .001). Among survivors, those treated with HCT had higher median Karnofsky/Lansky scores than those treated without HCT (P < .001). Among patients receiving HCT, 27 (40%) had graft-versus-host disease, and most deaths occurred within 1 year of transplantation. Conclusion: No difference in survival was observed between patients treated with or without HCT across all diagnosis years (1964-2013). However, survivors treated with HCT experienced somewhat greater well-being, and hazards associated with HCT decreased, reaching levels of significantly less risk in the late 1990s. Among patients treated with HCT, treatment at an early age is associated with improved survival. Optimism remains guarded as additional evidence accumulates., Jeffrey Modell Foundation; National Institutes of Health Office of Rare Diseases, National Center for Advancing Translational Sciences and National, Institute of Allergy and Infectious Disease [U54 AI 082973, R13AI094943], Supported by a grant from Jeffrey Modell Foundation (to M.d.l.M.). The Primary Immune Deficiency Treatment Consortium (PIDTC) is supported by the National Institutes of Health Office of Rare Diseases, National Center for Advancing Translational Sciences and National, Institute of Allergy and Infectious Disease grants U54 AI 082973 and R13AI094943.

The thymus, an organ responsible for T cell development, is one of the more stress-sensitive tissues in the body. Stress, in the form of infections, radiation exposure, and steroids, impairs thymic epithelial cell (TEC) functions and induces the programmed cell death of immature thymocytes. MicroRNAs are small noncoding RNAs involved in tissue repair and homeostasis, with several supporting T cell development. We report that miR-205, an epithelial-specific miR, maintains thymopoiesis following inflammatory perturbations. Thus, the activation of diverse pattern recognition receptors in mice causes a more severe thymic hypoplasia and delayed T cell recovery when miR-205 is conditionally ablated in TECs. Gene expression comparisons in the TECs with/without miR-205 revealed a significant differential regulation of chemokine/chemokine receptor pathways, antigen processing components, and changes in the Wnt signaling system. This was partly a consequence of reduced expression of the transcriptional regulator of epithelial cell function, Forkhead Box N1 (Foxn1), and its two regulated targets, stem cell factor and ccl25, following stress. miR-205 mimics supplemented into miR-205-deficient fetal thymic organ cultures restored Foxn1 expression along with ccl25 and stem cell factor. A number of putative targets of miR-205 were up-regulated in TECs lacking miR-205, consistent with an important role for this miR in supporting T cell development in response to stress.

We describe a previously unreported 437 T→G missense mutation producing a V146G substitution in the first coiled-coil (CC1) domain of nuclear factor-κB essential modulator (NEMO) in a 9-month-old boy with ectodermal dysplasia with immunodeficiency who presented with methicillin-resistant Staphylococcus aureus subdural empyema. We performed in vitro experiments to determine if this novel mutation resulted in impaired NF-κB signaling. IκBα phosphorylation experiments were performed using a Jurkat T cell line lacking endogenous NEMO expression that was transfected with vectors containing either the wild type or the patient’s V146G mutation. The cells were stimulated with TNF-α to activate the NF-κB pathway. Phosphorylated IκBα was detected by immunoblotting with anti-phospho-IκBα antibodies. Peripheral blood mononuclear cells from the patient were stimulated with TNF-α or anti-CD3 and anti-CD28. Impaired IκBα degradation was detected using antibodies against the IκBα protein. While TNF-α stimulation resulted in IκBα phosphorylation in NEMO-deficient Jurkat cells reconstituted with wild-type NEMO, cell transfected with the V146G mutant exhibited a 75% reduction in phospho-IκBα. Peripheral blood mononuclear cells from the patient showed impaired degradation of IκBα after stimulation when compared with normal controls. The patient’s V146G mutation results in impaired NF-κB activation in vitro. The mutation extends the known N-terminal boundary within the CC1 domain that produces an ectodermal dysplasia phenotype, and defines an infectious susceptibility previously unappreciated in ectodermal dysplasia with immunodeficiency (methicillin-resistant S. aureus subdural empyema), broadening the clinical spectrum associated with the disease.

Patients with 22q11.2 deletion syndrome have variable, multi-system disorders including a thymic hypoplasia, cardiac anomalies, and hypoparathyroidism. Over 90% have a deletion of 2.5 Mb on chromosome 22q11.2, affecting protein coding genes, microRNAs, long noncoding RNAs, and pseudogenes. 50%~70% have some degree of thymic hypoplasia (DiGeorge syndrome), resulting in a T cell lymphopenia. Successful transplantation of thymic tissue in patients with a thymic aplasia suggests stromal tissue abnormalities. The thymic stroma consists of thymic epithelial cells (TEC), mesenchymal cells, and endothelial populations. We are exploring the TEC-mesenchyme-endothelial interactions during thymus organogenesis in the mouse model of 22q11.2 deletion syndrome (Df1/+). Comparative transcriptome analyses of hypoplastic and normal thymic lobes from embryos revealed a unique mesenchymal cells mRNA expression signature, including reduced levels of the PDGFRa. PDGFRaH2B-EGFP heterozygous mice that have lower level of PDGFRa expression are being crossed with the Df1/+ model to ascertain whether the hypoplasia is influenced by this receptor. Additional mouse models with prominent penetrance of thymic hypoplasia are being used to determine how Tbx1 (encoded on 22q11.2) influences pharyngeal pouch mesenchymal-TEC development. The results from the study will likely lead to novel approaches for thymus reconstitution in various clinical settings.

The antibody repertoire of an infant develops in response to infections, environmental exposures, and vaccinations. By adulthood, many will produce antibodies that react against self-antigens, often causal to autoimmune diseases such as lupus erythematosus. Limited scientific literature exists as to whether such autoreactive antibodies develop early in infancy. For this reason, the antibody specificities in the serum of healthy infants at 1 and 2 years of age was analyzed. Screening in a cohort of 82 infants revealed that 23 (28%) had moderate to high titered antibodies directed to diverse self-antigens. These numbers are consistent with observed ANA positivity in adults. Ongoing custom targeted DNA sequencing analysis will assess the genetic load of known autoimmune risk alleles in the ANA positive infants. Relationships between the antibody specificities and the genetic risk alleles assembled for each infant will be presented. The comparisons will include clinical information; sex, ethnicity, growth records, vaccination status, infectious history, antibiotic and antiviral treatments, disease status, and family history. Genomic analysis of infants with autoantibodies may facilitate implementation of a new wellness screen to identify antibody positive infants that have significant genetic predisposition and therefore are at risk for developing diverse immune system abnormalities including autoimmune disorders.

Patients with mutations in the Forkhead Box N1 (FOXN1) transcription factor are born with a severe T-cell lymphopenia in conjunction with alopecia and nail dystrophy (OMIM # 600838). The T-cell lymphopenia results from the impaired development and/or function of the thymic epithelial cells (TECs). TECs are essential regulators of positive and negative selection of developing thymocytes. We report on 3 independent patients with compound heterozygous mutations in FOXN1. All 3 patients had a severe T cell lymphopenia. However, each had normal hair growth and nail bed formation, suggesting that the compound heterozygous mutations result in clinical presentations distinct from classic cases. To determine how the mutations impact murine Foxn1 function, transcriptional reporter assays and protein expression studies were done. Only one of the mutations affected the transcriptional activity of murine Foxn1, with Western blot analyses indicating that this mutation caused production of a truncated protein. CRISPR/Cas9 technologies were used to create mouse lines with compound heterozygous mutations in Foxn1. The mice are currently being intercrossed. The impact of the compound heterozygous Foxn1 mutations on T cell development in the thymus will be presented. Findings from this study may suggest novel therapeutic strategies at restoring thymopoiesis in different clinical settings.

MicroRNAs (miRNAs) are processed from primary RNA transcripts (pri-miRNAs) encoded in host genes. Several miRNAs including miR-205 are present within long noncoding RNAs (lncRNAs). MiR-205 is an epithelial-specific miRNA that supports thymopoiesis by positively regulating Forkhead Box N1 (Foxn1) expression. We assessed whether the host gene for miR-205, MIR205HG, has independent functions as a lncRNA. The more severe stress-induced thymic atrophy reported in miR-205-deficient mice is also evident in MIR205HG knockout lines. However, MIR205HG knockouts have a small stature phenotype. A new set of miR-205KI mice did not have this phenotype. The smaller mouse size of the MIR205HG null animals is partly a consequence of reduced levels of endocrine hormones produced by the anterior pituitary. In addition, the MIR205HG null animals had abnormal development of the lacrimal and harderian glands that produce eye secretions. Transcriptome analyses revealed that MIR205HG regulates gene expression in the anterior pituitary unlike miR-205. In contrast, transcripts regulated by miR-205 and MIR205HG in the epithelial cells of the skin and thymus overlap significantly. These data indicate that MIR205HG has a specific function as a lncRNA in the anterior pituitary in addition to its primary role as the host gene for miR-205 in thymic epithelial cells.

The last 40 years has seen the emergence of hematopoietic stem cell transplantation as a therapeutic modality for fatal diseases and as a curative option for individuals born with inherited disorders that carry limited life expectancy and poor quality of life. Despite the rarity of many primary immunodeficiency diseases, these disorders have led the way toward innovative therapies and further provide insights into mechanisms of im- munologic reconstitution applicable to all hematopoietic stem cell transplants. This article represents a historical perspective of the early investigators and their contributions. It also reviews the parallel work that oncologists and immunologists have undertaken to treat both primary immunodeficiencies and hematologic malignancies.

Disease caused by these environmental bacteria is almost exclusively limited to patients with this condition., Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by a defect in production of phagocyte-derived reactive oxygen species, which leads to recurrent infections with a characteristic group of pathogens not previously known to include methylotrophs. Methylotrophs are versatile environmental bacteria that can use single-carbon organic compounds as their sole source of energy; they rarely cause disease in immunocompetent persons. We have identified 12 infections with methylotrophs (5 reported here, 7 previously reported) in patients with CGD. Methylotrophs identified were Granulibacter bethesdensis (9 cases), Acidomonas methanolica (2 cases), and Methylobacterium lusitanum (1 case). Two patients in Europe died; the other 10, from North and Central America, recovered after prolonged courses of antimicrobial drug therapy and, for some, surgery. Methylotrophs are emerging as disease-causing organisms in patients with CGD. For all patients, sequencing of the 16S rRNA gene was required for correct diagnosis. Geographic origin of the methylotroph strain may affect clinical management and prognosis.

MiR-205 is an epithelial-specific microRNA (miR) that supports thymopoiesis. This miR positively regulates Forkhead Box N1 (Foxn1) transcription factor expression. MiR-205 is embedded in a long noncoding RNA (lncRNA), termed MIR205HG in humans. Several lncRNAs that contain miRs have independent functional roles in tissue development and/or regeneration. We characterized the transcriptome assembly of the murine locus containing miR-205 and the larger lncRNA. Conditional knockout mice that harbored a targeted deletion of the proximal region of the lncRNA, with miR-205 sequences retained, were developed. The phenotypes in these mice were compared to those with a miR-205 deficiency. The more severe stress-induced thymic atrophy reported in miR-205-deficient mice, compared to littermate controls, is also evident in lncRNA205 knockout lines. In contrast, a lncRNA205 deficiency results in a smaller mouse stature, which may be coupled with a reduced fat but normal lean mass. These phenotypic differences are explained, in part, by the differential regulation of the lncRNA transcript relative to the pre-mature miR-205. Interferon and IL-22, cytokines released following inflammation, transiently reduce miR-205 while increasing the lncRNA. This is partly determined by the DNp63 transcription factor, which controls miR-205 expression. Gene expression comparisons are being undertaken to reveal how the transcriptome is differentially regulated by these two non-coding RNA species. Further, the mechanism by which these noncoding RNAs regulate Foxn1 is being elucidated. Taken together, these findings suggest both overlapping and independent functions for the lncRNA205 and the embedded miR-205.

Forkhead Box N1 (FoxN1) is an epithelial-specific transcription factor essential for the development of the thymus. Patients with mutations in Foxn1 (OMIM # 600838) are born with a severe T-cell lymphopenia that presents in combination with alopecia and nail dystrophy. The nude mouse, which developed from a spontaneous genetic mutation in Foxn1, phenocopies the human disease. We report on 3 independently identified patients that presented with low to absent circulating T cells. Genetic workup of these patients revealed mutations in Foxn1. Each patient had distinct compound heterozygous mutations that were localized in the distal exons of Foxn1. These were predicted to maintain Foxn1 expression while adversely affect its function. Of significance, each patient had normal hair and skin, without any evidence of nail dystrophy, distinct from previously reported phenotypes. To better define the molecular mechanisms leading to this novel clinical presentation, we used CRISPR/Cas techniques to introduce the corresponding mutations in the mouse Foxn1 sequence. We will present data on the phenotypes of these mice, using intercrosses between individual mutant mice. Comparative transcriptome analyses of fetal thymii from these mice will reveal how the Foxn1 mutations impacts thymic epithelial gene expression and function compared to normal Foxn1. Our findings may lead to better understanding of the role of Foxn1 epithelial cell development and function in both the thymus and skin.

Chromosome 22q11.2 deletion syndrome (22q11.2 ΔS) is the most common microdeletion disorder reported (1/4000). Individuals with this deletion have variable, multi-system disorders including thymic hypoplasia, cardiac anomalies, hypoparathyroidism, and/or dysmorphic facial features. Over 90% of patients have a deletion of 2.4 Mb, which comprises 90 genes, 50% protein coding and the remainder microRNAs, long noncoding RNAs and pseudogenes. The principal cause of the development defects is a haploinsufficiency of the T-box 1 transcription factor (Tbx1). Between 40–60% of patients have some degree of thymic hypoplasia, resulting in systemic T cell lymphopenia. Defects in the thymic stromal tissue is the underlying cause of the hypoplasia. We analyzed the thymic tissue in the mouse models of 22q11.2 ΔS (Df1/+). Comparative transcriptome analyses of hypoplastic and normal-sized lobes derived from the same Df1/+ embryo revealed a signature mRNA expression pattern unique to hypoplastic lobes. Ingenuity pathway analysis uncovered selective pathways compromised in the hypoplastic lobes. Fetal thymic organ culture and reaggregate cultures are currently being used to identify the genes essential for the specification and expansion of the thymic stroma. In addition, normal and hypoplastic thymii, including some from 22q11.2 ΔS patients, are being characterized for the expression of the over- and under-represented genes identified in the mouse model. Findings from these studies may lead to better strategies for improving human thymopoiesis in patients with conditions including 22q11.2 and 10p syndromes, those undergoing chemoablative treatments, and other conditions leading to a thymic hypoplasia and ensuing T cell lymphopenia.

Attempts to understand why the fetus is not rejected by the mother have led us to an ongoing and fascinating understanding of both innate and adaptive immune mechanisms of maternal recognition during pregnancy. As gestation evolves, the developing embryo becomes isolated within a semi-permeable environment. The anatomic organization of the maternal–fetal interface, absence of classical MHC molecules on the trophoblast surface, uterine NK cells, the cytokine milieu, macrophages, T and B cell populations, antibody production, indoleamine 2,3-deoxygenase, complement regulatory proteins, and sex hormones allow for the integrity of the maternal–fetal interface and provide an example of cellular recognition. As immunologic changes develop, the balance required for host defense and control of autoimmunity is transformed and the pregnant female becomes susceptible to infections and changes in disease activity. This chapter provides an overview of the fundamental immunologic principles that determine the immunobiology of pregnancy by focusing on some of the cellular and molecular interactions known to be involved in immune function during gestation. How such changes influence the clinical manifestations of autoimmune diseases in the pregnant female and affect susceptibility to infection is discussed briefly.

miR-185 is a microRNA (miR) that targets Bruton's tyrosine kinase in B cells, with reductions in miR-185 linked to B cell autoantibody production. In hippocampal neurons, miR-185 targets both sarcoplasmic/endoplasmic reticulum calcium ATPase 2 and a novel Golgi inhibitor. This miR is haploinsufficient in 90–95% of individuals with chromosome 22q11.2 deletion syndrome, patients who can present with immune, cardiac, and parathyroid problems, learning disorders, and a high incidence of schizophrenia in adults. The reduced levels of miR-185 in neurons cause presynaptic neurotransmitter release. Many of the 22q11.2 deletion syndrome patients have a thymic hypoplasia, which results in a peripheral T cell lymphopenia and unusual T helper cell skewing. The molecular targets of miR-185 in thymocytes are unknown. Using an miR-185 T cell transgenic approach, increasing levels of miR-185 attenuated T cell development at the T cell receptor β (TCRβ) selection checkpoint and during positive selection. This caused a peripheral T cell lymphopenia. Mzb1, Nfatc3, and Camk4 were identified as novel miR-185 targets. Elevations in miR-185 enhanced TCR-dependent intracellular calcium levels, whereas a knockdown of miR-185 diminished these calcium responses. These effects concur with reductions in Mzb1, an endoplasmic reticulum calcium regulator. Consistent with their haploinsufficiency of miR-185, Mzb1 levels were elevated in thymocyte extracts from several 22q11.2 deletion syndrome patients. Our findings indicate that miR-185 regulates T cell development through its targeting of several mRNAs including Mzb1. Background: MicroRNAs have critical roles in T cell development under normal and stress conditions. Results: Increasing miR-185 levels attenuate T cell development via the targeting of Mzb1, Nfatc3, and Camk4. Conclusion: Elevations in miR-185 impair thymopoiesis at several developmental stages. Significance: miR-185 levels are reduced in 22q11.2 deletion syndrome patients and the identification of its gene targets is clinically informative.

Hematopoietic cell transplantation (HCT) is a curative therapeutic option for severe combined immunodeficiency (SCID), a group of diseases which otherwise carry life expectancies that are of limited duration and quality. Survival following HCT for SCID has improved from approximately 23 to 91 % over the last 40 years. Success with SCID prompted efforts to apply HCT to the therapeutic challenge of well over 20 molecularly defined primary immune deficiency diseases (PID). Such success is due to both early recognition of PIDs and advances in the field of transplantation. Such advances include high-resolution HLA DNA donor–recipient matching, expansion of donor sources, better tolerated conditioning, new antibiotics, and wider availability. International collaborative efforts have provided patients and caregivers information that permit better treatment decisions now, and direct clinicians and investigators to ensure progress in the future. Pioneers in screening for SCID have taken steps to correct the fundamental challenge to successful treatment, which is the rapid discovery and characterization of cases and offering the transplant option to an affected child early in life; blood spot testing for T and B cell receptor quantification is now available to a growing fraction of newborns. Organizations including the Primary Immune Deficiency Treatment Consortium in the USA, The European Society for Primary Immunodeficiency, the European Group for Blood and Marrow Transplantation, the Pediatric Blood and Marrow Transplant Consortium, the United States Immunodeficiency Network, the Immune Deficiency Foundation, and the Jeffrey Modell Foundation are contributing mightily to increase awareness and standardize optimal utilization to the benefit of patients. This review will update the allergist–immunologist concerning disease presentations, indications for transplantation, methodologies, conditioning regimens, and clinical outcomes for patients with PID for which timely HCT is critical.

Primary immunodeficiency diseases (PIDDs) are inherited disorders of the immune system. The most severe form, severe combined immunodeficiency (SCID), presents with profound deficiencies of T cells, B cells, or both at birth. If not treated promptly, affected patients usually do not live beyond infancy because of infections. Genetic heterogeneity of SCID frequently delays the diagnosis; a specific diagnosis is crucial for life-saving treatment and optimal management.We developed a next-generation sequencing (NGS)-based multigene-targeted panel for SCID and other severe PIDDs requiring rapid therapeutic actions in a clinical laboratory setting.The target gene capture/NGS assay provides an average read depth of approximately 1000×. The deep coverage facilitates simultaneous detection of single nucleotide variants and exonic copy number variants in one comprehensive assessment. Exons with insufficient coverage (20× read depth) or high sequence homology (pseudogenes) are complemented by amplicon-based sequencing with specific primers to ensure 100% coverage of all targeted regions.Analysis of 20 patient samples with low T-cell receptor excision circle numbers on newborn screening or a positive family history or clinical suspicion of SCID or other severe PIDD identified deleterious mutations in 14 of them. Identified pathogenic variants included both single nucleotide variants and exonic copy number variants, such as hemizygous nonsense, frameshift, and missense changes in IL2RG; compound heterozygous changes in ATM, RAG1, and CIITA; homozygous changes in DCLRE1C and IL7R; and a heterozygous nonsense mutation in CHD7.High-throughput deep sequencing analysis with complete clinical validation greatly increases the diagnostic yield of severe primary immunodeficiency. Establishing a molecular diagnosis enables early immune reconstitution through prompt therapeutic intervention and guides management for improved long-term quality of life.