Clinical and functional spectrum of RAC2-related immunodeficiency.

ABSTRACT: Mutations in the small Rho-family guanosine triphosphate hydrolase RAC2, critical for actin cytoskeleton remodeling and intracellular signal transduction, are associated with neonatal severe combined immunodeficiency (SCID), infantile neutrophilic disorder resembling leukocyte adhesion deficiency (LAD), and later-onset combined immune deficiency (CID). We investigated 54 patients (23 previously reported) from 37 families yielding 15 novel RAC2 missense mutations, including one present only in homozygosity. Data were collected from referring physicians and literature reports with updated clinical information. Patients were grouped by presentation: neonatal SCID (n = 5), infantile LAD-like disease (n = 5), or CID (n = 44). Disease correlated to RAC2 activity: constitutively active RAS-like mutations caused neonatal SCID, dominant-negative mutations caused LAD-like disease, whereas dominant-activating mutations caused CID. Significant T- and B-lymphopenia with low immunoglobulins were seen in most patients; myeloid abnormalities included neutropenia, altered oxidative burst, impaired neutrophil migration, and visible neutrophil macropinosomes. Among 42 patients with CID with clinical data, upper and lower respiratory infections and viral infections were common. Twenty-three distinct RAC2 mutations, including 15 novel variants, were identified. Using heterologous expression systems, we assessed downstream effector functions including superoxide production, p21-activated kinase 1 binding, AKT activation, and protein stability. Confocal microscopy showed altered actin assembly evidenced by membrane ruffling and macropinosomes. Altered protein localization and aggregation were observed. All tested RAC2 mutant proteins exhibited aberrant function; no single assay was sufficient to determine functional consequence. Most mutants produced elevated superoxide; mutations unable to support superoxide formation were associated with bacterial infections. RAC2 mutations cause a spectrum of immune dysfunction, ranging from early onset SCID to later-onset combined immunodeficiencies depending on RAC2 activity. This trial was registered at www.clinicaltrials.gov as #NCT00001355 and #NCT00001467.

Heterozygous FOXN1 Variants Cause Low TRECs and Severe T Cell Lymphopenia, Revealing a Crucial Role of FOXN1 in Supporting Early Thymopoiesis.

FOXN1 is the master regulatory gene of thymic epithelium development. FOXN1 deficiency leads to thymic aplasia, alopecia, and nail dystrophy, accounting for the nude/severe combined immunodeficiency (nu/SCID) phenotype in humans and mice. We identified several newborns with low levels of T cell receptor excision circles (TRECs) and T cell lymphopenia at birth, who carried heterozygous loss-of-function FOXN1 variants. Longitudinal analysis showed persistent T cell lymphopenia during infancy, often associated with nail dystrophy. Adult individuals with heterozygous FOXN1 variants had in most cases normal CD4+ but lower than normal CD8+ cell counts. We hypothesized a FOXN1 gene dosage effect on the function of thymic epithelial cells (TECs) and thymopoiesis and postulated that these effects would be more prominent early in life. To test this hypothesis, we analyzed TEC subset frequency and phenotype, early thymic progenitor (ETP) cell count, and expression of FOXN1 target genes (Ccl25, Cxcl12, Dll4, Scf, Psmb11, Prss16, and Cd83) in Foxn1nu/+ (nu/+) mice and age-matched wild-type (+/+) littermate controls. Both the frequency and the absolute count of ETP were significantly reduced in nu/+ mice up to 3 weeks of age. Analysis of the TEC compartment showed reduced expression of FOXN1 target genes and delayed maturation of the medullary TEC compartment in nu/+ mice. These observations establish a FOXN1 gene dosage effect on thymic function and identify FOXN1 haploinsufficiency as an important genetic determinant of T cell lymphopenia at birth.

Dominant-negative loss of function arises from a second, more frequent variant within the SAND domain of autoimmune regulator (AIRE).

A genetic variant in the SAND domain of autoimmune regulator (AIRE), R247C, was identified in a patient with type I diabetes mellitus (T1DM) and his mother with rheumatoid arthritis. In vitro, the variant dominantly inhibited AIRE; however, typical features of Autoimmune Polyendocrinopathy Candidiasis and Ectodermal Dysplasia (APECED) were not seen in the subjects. Rather, early manifestation of autoimmunity appeared to be dependent on additional genetic factors. On a population level, diverse variants were identified in this region. Surprisingly, many likely pathogenic variants were seen disproportionately in Africans when compared to Europeans, reinforcing the importance of these variants in altering the immune repertoire. In light of these findings, we propose that R247C and other variants within the SAND-domain alter protein function in a dominant fashion and hold potential as drivers of autoimmunity.

Inducible and naturally occurring regulatory T cells enhance lung allergic responses through divergent transcriptional pathways.

BACKGROUND: Regulatory T cells attenuate development of asthma in wild-type (WT) mice, with both naturally occurring regulatory T (nTreg) cells and inducible regulatory T (iTreg) cells exhibiting suppressive activity. When transferred into CD8-deficient (CD8-/-) recipients, both cell types enhanced development of allergen-induced airway hyperresponsiveness. OBJECTIVE: We sought to determine whether the pathways leading to enhancement of lung allergic responses by transferred nTreg and iTreg cells differed. METHODS: nTreg cells (CD4+CD25+) were isolated from WT mice and iTreg cells were generated from WT CD4+CD25- T cells after activation in the presence of TGF-ß and transferred into sensitized CD8-/- recipients before challenge. Development of airway hyperresponsiveness, cytokine levels, and airway inflammation were monitored. RESULTS: Transfer of nTreg cells enhanced lung allergic responses, as did transfer of iTreg cells. Although anti-IL-13 reduced nTreg cell-mediated enhancement, it was ineffective in iTreg cell-mediated enhancement; conversely, anti-IL-17, but not anti-IL-13, attenuated the enhancement by iTreg cells. Recovered iTreg cells from the lungs of CD8-/- recipients were capable of IL-17 production and expressed high levels of signature genes of the TH17 pathway, RORγt and Il17, whereas reduced expression of the Treg cell key transcription factor forkhead box p3 (Foxp3) was observed. In vitro exogenous IL-6-induced IL-17 production in iTreg cells, and in vivo conversion of transferred iTreg cells was dependent on recipient IL-6. CONCLUSIONS: iTreg cells, similar to nTreg cells, exhibit functional plasticity and can be converted from suppressor cells to pathogenic effector cells, enhancing lung allergic responses, but these effects were mediated through different pathways.

Long-term outcomes of 176 patients with X-linked hyper-IgM syndrome treated with or without hematopoietic cell transplantation.

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.

DOCK8 deficiency: clinical and immunological phenotype and treatment options - a review of 136 patients.

Mutations in DOCK8 result in autosomal recessive Hyper-IgE syndrome with combined immunodeficiency (CID). However, the natural course of disease, long-term prognosis, and optimal therapeutic management have not yet been clearly defined. In an international retrospective survey of patients with DOCK8 mutations, focused on clinical presentation and therapeutic measures, a total of 136 patients with a median follow-up of 11.3 years (1.3-47.7) spanning 1693 patient years, were enrolled. Eczema, recurrent respiratory tract infections, allergies, abscesses, viral infections and mucocutaneous candidiasis were the most frequent clinical manifestations. Overall survival probability in this cohort [censored for hematopoietic stem cell transplantation (HSCT)] was 87 % at 10, 47 % at 20, and 33 % at 30 years of age, respectively. Event free survival was 44, 18 and 4 % at the same time points if events were defined as death, life-threatening infections, malignancy or cerebral complications such as CNS vasculitis or stroke. Malignancy was diagnosed in 23/136 (17 %) patients (11 hematological and 9 epithelial cancers, 5 other malignancies) at a median age of 12 years. Eight of these patients died from cancer. Severe, life-threatening infections were observed in 79/136 (58 %); severe non-infectious cerebral events occurred in 14/136 (10 %). Therapeutic measures included antiviral and antibacterial prophylaxis, immunoglobulin replacement and HSCT. This study provides a comprehensive evaluation of the clinical phenotype of DOCK8 deficiency in the largest cohort reported so far and demonstrates the severity of the disease with relatively poor prognosis. Early HSCT should be strongly considered as a potential curative measure.

Newborn screening for severe combined immunodeficiency in 11 screening programs in the United States.

IMPORTANCE: Newborn screening for severe combined immunodeficiency (SCID) using assays to detect T-cell receptor excision circles (TRECs) began in Wisconsin in 2008, and SCID was added to the national recommended uniform panel for newborn screened disorders in 2010. Currently 23 states, the District of Columbia, and the Navajo Nation conduct population-wide newborn screening for SCID. The incidence of SCID is estimated at 1 in 100,000 births. OBJECTIVES: To present data from a spectrum of SCID newborn screening programs, establish population-based incidence for SCID and other conditions with T-cell lymphopenia, and document early institution of effective treatments. DESIGN: Epidemiological and retrospective observational study. SETTING: Representatives in states conducting SCID newborn screening were invited to submit their SCID screening algorithms, test performance data, and deidentified clinical and laboratory information regarding infants screened and cases with nonnormal results. Infants born from the start of each participating program from January 2008 through the most recent evaluable date prior to July 2013 were included. Representatives from 10 states plus the Navajo Area Indian Health Service contributed data from 3,030,083 newborns screened with a TREC test. MAIN OUTCOMES AND MEASURES: Infants with SCID and other diagnoses of T-cell lymphopenia were classified. Incidence and, where possible, etiologies were determined. Interventions and survival were tracked. RESULTS: Screening detected 52 cases of typical SCID, leaky SCID, and Omenn syndrome, affecting 1 in 58,000 infants (95% CI, 1/46,000-1/80,000). Survival of SCID-affected infants through their diagnosis and immune reconstitution was 87% (45/52), 92% (45/49) for infants who received transplantation, enzyme replacement, and/or gene therapy. Additional interventions for SCID and non-SCID T-cell lymphopenia included immunoglobulin infusions, preventive antibiotics, and avoidance of live vaccines. Variations in definitions and follow-up practices influenced the rates of detection of non-SCID T-cell lymphopenia. CONCLUSIONS AND RELEVANCE: Newborn screening in 11 programs in the United States identified SCID in 1 in 58,000 infants, with high survival. The usefulness of detection of non-SCID T-cell lymphopenias by the same screening remains to be determined.

Composition of the CD27+ Memory-B-Cell Compartment Delineates Immunoglobulin Deficiency Endotypes.

Abstract Purpose: The finding of reduced numbers of class-switched memory B cells (CSM) in peripheral blood is widely used to assist the diagnosis and subclassification of CVID. Limited data exists on this finding in relation to the entire class of PADs. In this study, consecutive 8-marker comprehensive B-cell panel results were analyzed to determine how reduced CSM quantities might inform the pathophysiology of CVID and other humoral immunodeficiencies. Methods: Subpopulations of CD27+ memory B cells from 64 consecutive subjects with or without humoral immunodeficiency were examined to identify associations with diagnosis and serum immunoglobulin level. Results: CD27+IgM-IgD- percentage (CSM%) was correlated with IgG level in a discontinuous manner with an estimated change point of 9.7% (95% CI: 4.7, 12.4). All subjects with a CSM% below 9.7% had substantially lower serum IgG and IgA levels compared with those above 9.7. CSM% below 9.7% is not associated with serum IgM level. Rather, the proportion of CD27+IgMonly B cells (IgMonly or IgMonly%) is correlated with serum IgM. Conclusion: Low CSM% may mark an endotype of humoral immune dysfunction defined by either loss of class switching or critical failure of the coordinated production of both memory cells and long-lived plasma cells responsible for adequate immunoglobulin levels in humans. In patients with low CSM%, maintenance or expansion of IgMonly cells and IgM production suggests the former explanation, while concomitant loss of IgMonly cells suggests the latter. These findings provide a simple endotypic stratification method for future studies on the failed coordinated B cell response in humans with PAD.

Autophagy-associated immune dysregulation and hyperplasia in a patient with compound heterozygous mutations in ATG9A.

ABBREVIATIONS: BCL2: BCL2 apoptosis regulator; BCL10: BCL10 immune signaling adaptor; CARD11: caspase recruitment domain family member 11; CBM: CARD11-BCL10-MALT1; CR2: complement C3d receptor 2; EBNA: Epstein Barr nuclear antigen; EBV: Epstein-Barr virus; FCGR3A; Fc gamma receptor IIIa; GLILD: granulomatous-lymphocytic interstitial lung disease; HV: healthy volunteer; IKBKB/IKB kinase: inhibitor of nuclear factor kappa B kinase subunit beta; IL2RA: interleukin 2 receptor subunit alpha; MALT1: MALT1 paracaspase; MS4A1: membrane spanning 4-domain A1; MTOR: mechanistic target of rapamycin kinase; MYC: MYC proto-oncogene, bHLH: transcription factor; NCAM1: neural cell adhesion molecule 1; NFKB: nuclear factor kappa B; NIAID: National Institute of Allergy and Infectious Diseases; NK: natural killer; PTPRC: protein tyrosine phosphatase receptor type C; SELL: selectin L; PBMCs: peripheral blood mononuclear cells; TR: T cell receptor; Tregs: regulatory T cells; WT: wild-type.

"Common variable immunodeficiency: Challenges for diagnosis".

Common variable immunodeficiency is a heterogeneous condition characterized by B cell dysfunction with reduced serum immunoglobulin levels and a highly variable spectrum of clinical manifestations ranging from recurrent infections to autoimmune disease. The diagnosis of CVID is often challenging due to the diverse clinical presentation of patients and the existence of multiple diagnostic criteria without a universally adopted consensus. Laboratory evaluation to assist with diagnosis currently includes serum immunoglobulin testing, immunophenotyping, assessment of vaccine response, and genetic testing. Additional emerging techniques include investigation of the B cell repertoire and the use of machine learning algorithms. Advances in our understanding of common variable immunodeficiency will ultimately contribute to earlier diagnosis and novel interventions with the goal of improving prognosis for these patients.

Fluctuations in quality of life and immune responses during intravenous immunoglobulin infusion cycles.

Despite adequate infection prophylaxis, variation in self-reported quality of life (QOL) throughout the intravenous immunoglobulin (IVIG) infusion cycle is a widely reported but infrequently studied phenomenon. To better understand this phenomenon, subjects with humoral immunodeficiency receiving replacement doses of IVIG were studied over 3 infusion cycles. Questionnaire data from 6 time points spread over 3 IVIG infusions cycles (infusion day and 7 days after each infusion) were collected in conjunction with monitoring the blood for number of regulatory T-cells (Treg) and levels of 40 secreted analytes: primarily cytokines, chemokines, and growth factors. At day 7, self-reported well-being increased, and self-reported fatigue decreased, reflecting an overall improvement in QOL 7 days after infusion. Over the same period, percentage of Treg cells in the blood increased (p<0.01). Multiple inflammatory chemokine and cytokine levels increased in the blood by 1 hour after infusion (CCL4 (MIP-1b), CCL3 (MIP-1a), CCL2 (MCP-1), TNF-α, granzyme B, IL-10, IL-1RA, IL-8, IL-6, GM-CSF, and IFN- γ). The largest changes in analytes occurred in subjects initiated on IVIG during the study. A significant decrease in IL-25 (IL-17E) following infusion was seen in most intervals among subjects already receiving regular infusions prior to study entry. These findings reveal several short-term effects of IVIG given in replacement doses to patients with humoral immunodeficiency: QOL consistently improves in the first week of infusion, levels of a collection of monocyte-associated cytokines increase immediately after infusion whereas IL-25 levels decrease, and Treg levels increase. Moreover, patients that are new to IVIG experience more significant fluctuations in cytokine levels than those receiving it regularly.

Expression and activation of the steroidogenic enzyme CYP11A1 is associated with IL-13 production in T cells from peanut allergic children.

Activation of the steroidogenic enzyme CYP11A1 was shown to be necessary for the development of peanut-induced intestinal anaphylaxis and IL-13 production in allergic mice. We determined if levels of CYP11A1 in peripheral blood T cells from peanut-allergic (PA) children compared to non-allergic controls were increased and if levels correlated to IL-13 production and oral challenge outcomes to peanut. CYP11A1 mRNA and protein levels were significantly increased in activated CD4+ T cells from PA patients. In parallel, IL-13 production was significantly increased; IFNγ levels were not different between groups. There were significant correlations between expression levels of CYP11A1 mRNA and levels of IL13 mRNA and protein, levels of serum IgE anti-Ara h 2 and to outcomes of peanut challenge. The importance of CYP11A1 on cytokine production was tested using a CYP11A1 CRISPR/Cas9 KO plasmid or an inhibitor of enzymatic CYP11A1 activity. Inhibition of CYP11A1 activation in patient cells treated with the inhibitor, aminoglutethimide, or CD4+ T cell line transfected with the CYP11A1 KO plasmid resulted in reduced IL-13 production. These data suggest that the CYP11A1-CD4+Tcell-IL-13 axis in activated CD4+ T cells from PA children is associated with development of PA reactions. CYP11A1 may represent a novel target for therapeutic intervention in PA children.