Variants in IGLL1 cause a broad phenotype from agammaglobulinemia to transient hypogammaglobulinemia.

BACKGROUND: Agammaglobulinemia due to variants in IGLL1 has traditionally been considered an exceedingly rare form of severe B-cell deficiency, with only 8 documented cases in the literature. Surprisingly, the first agammaglobulinemic patient identified by newborn screening (NBS) through quantification of kappa-deleting recombination excision circles harbored variants in IGLL1. OBJECTIVE: We comprehensively reviewed clinical and immunologic findings of patients with B-cell deficiency attributed to variants in IGLL1. METHODS: NBS programs reporting the use of kappa-deleting recombination excision circle assays, the European Society for Immunodeficiencies Registry, and authors of published reports featuring patients with B-cell deficiency linked to IGLL1 variants were contacted. Only patients with (likely) pathogenic variants, reduced CD19+ counts, and no alternative diagnosis were included. RESULTS: The study included 13 patients identified through NBS, 2 clinically diagnosed patients, and 2 asymptomatic siblings. All had severely reduced CD19+ B cells (< 0.1 × 109/L) at first evaluation, yet subsequent follow-up assessments indicated residual immunoglobulin production. Specific antibody responses to vaccine antigens varied, with a predominant reduction observed during infancy. Clinical outcomes were favorable with IgG substitution. Two patients successfully discontinued substitution therapy without developing susceptibility to infections and while maintaining immunoglobulin levels. The pooled incidence of homozygous or compound heterozygous pathogenic IGLL1 variants identified by NBS in Austria, Czechia, and Switzerland was 1.3:100,000, almost double of X-linked agammaglobulinemia. CONCLUSION: B-cell deficiency resulting from IGLL1 variants appears to be more prevalent than initially believed. Despite markedly low B-cell counts, the clinical course in some patients may be milder than reported in the literature so far.

Endothelial cells of extremely premature infants display impaired immune response after proinflammatory stimulation.

BackgroundEndothelial cells (ECs) exert immunological functions such as production of proinflammatory cytokines/chemokines as well as facilitation of extravasation of immune cells into infected tissue. Limited data are available on the functionality of ECs from extremely preterm neonates during infection. Accordingly, the aim of our study was to investigate the immune response of premature ECs after proinflammatory stimulation.MethodsCell adhesion receptors' expression and function, nuclear factor 'kappa-light-chain-enhancer' of activated B-cells (NFκB) signaling, and chemokine production were analyzed in umbilical cord ECs from extremely preterm and term neonates after proinflammatory stimulation.ResultsP-selectin and E-selectin surface expression as well as NFκB signaling were lower after lipopolysaccharide (LPS) stimulation in premature ECs. Preterm ECs exhibited lower, but significant, cell-adhesive functions after LPS stimulation compared with term ECs. CCL2/CXCL8 chemokine secretion was significantly upregulated after proinflammatory stimulation in both groups. CXCL10 production was significantly increased in term but not in preterm ECs upon stimulation with tumor necrosis factor compared with unstimulated ECs.ConclusionExtremely premature ECs showed partly reduced expression levels and function of cell adhesion molecules. Both NFκB signaling and chemokine/cytokine production were reduced in premature ECs. The diminished endothelial proinflammatory immune response might result in impaired infection control of preterm newborns rendering them prone to severe infection.

Pentoxifylline modulates LPS-induced hyperinflammation in monocytes of preterm infants in vitro.

BackgroundPentoxifylline (PTX), a methylxanthine derivate with immunomodulating properties, has been used as adjunctive treatment in severe neonatal sepsis. The aim of the study was to investigate the anti-inflammatory effects of PTX on Lipopolysaccharides (LPS)-stimulated monocytes of preterm neonates in vitro compared with monocytes of term infants and adult controls.MethodsWhole cord blood samples and control adult blood samples were incubated with LPS and PTX. The expression of surface markers, phagocytosis, cytokine secretion, and Toll-like receptor (TLR)4 signaling of monocytes were assessed by flow cytometry. Changes of TLR4-messenger RNA (mRNA) levels were confirmed by reverse-transcriptase PCR.ResultsThe expression of CD14, CD11b, CD64, CD71, and CD80 was downregulated by PTX in a dose-dependent manner; the greatest effect was observed on CD14 and CD11b in preterm infants. PTX markedly downregulated LPS-induced tumor necrosis factor-α, interleukin (IL)-1ß, and IL-6 levels in all age groups. Early IL-10 production was significantly downregulated by PTX in term and preterm neonates, while remaining unchanged in adults. Moreover, PTX downregulated TLR4 expression of monocytes on cellular and mRNA level, decreased signaling, and suppressed phagocytosis.ConclusionPTX downregulated TLR4 expression and signaling, thereby leading to strong anti-inflammatory properties in monocytes. Age-dependent differences were identified for CD14 and CD11b expression and IL-10 production.

NF-κB1 Haploinsufficiency Causing Immunodeficiency and EBV-Driven Lymphoproliferation.

PURPOSE: NF-κB signaling is critically important for regulation of both innate and adaptive immune responses. While activation of NF-κB has been implicated in malignancies such as leukemia and lymphoma, loss-of-function mutations affecting different NF-κB pathway components have been shown to cause primary immunodeficiency disorders. Recently, haploinsufficiency of NF-κB1 has been described in three families with common variable immunodeficiency (CVID). METHODS AND RESULTS: We studied a patient with recurrent respiratory infections and bacterial parapharyngeal abscess. Immunological investigations revealed normal total B- cell numbers, but hypogammaglobulinemia, decreased frequencies of class-switched B cells and impaired T-cell proliferation. Targeted next-generation sequencing using a custom-designed panel comprising all known PID genes (IUIS 2014 classification) and novel candidate genes identified a novel heterozygous frameshift mutation in the NFKB1 gene leading to a premature stop codon (c.491delG; p.G165A*31). We could show that the mutation leads to reduced phosphorylation of p105 upon stimulation, resulting in decreased protein levels of p50. The further disease course was mainly characterized by two episodes of severe EBV-associated lymphoproliferative disease responsive to rituximab treatment. Due to disease severity, the patient is considered for allogeneic hematopoietic stem cell transplantation. Interestingly, the father carries the same heterozygous NFKB1 mutation and also shows decreased frequencies of memory B cells but has a much milder clinical phenotype, in line with a considerable phenotypic disease heterogeneity. CONCLUSIONS: Deficiency of NF-κB1 leads to immunodeficiency with a wider phenotypic spectrum of disease manifestation than previously appreciated, including EBV lymphoproliferative diseases as a hitherto unrecognized feature of the disease.

The TLR-specific adjuvants R-848 and CpG-B endorse the immunological reaction of neonatal antigen-presenting cells.

BACKGROUND: Preterm neonates display an impaired vaccine response. Neonatal antigen-presenting cells (APCs) are less effective to induce an adaptive immune response and to promote the development of immunological memory. Efficient adjuvantal toll-like receptor (TLR)-triggering may overcome the neonatal immunological impairment. Accordingly, the aim of this study was to investigate the immunostimulatory action of R-848 and CpG-B on neonatal APCs. METHODS: Surface marker and cytokine secretion of APCs were evaluated after incubation of cord blood and peripheral blood mononuclear cells with the indicated adjuvants and were analyzed using flow cytometry. RESULTS: TLR-specific stimulation resulted in a significant induction of costimulatory molecules on neonatal APCs. Stimulation with R-848 resulted in significant higher secretion of TNFα, IL-6, IL-10, IL-12/IL-23p40, IL-12p70, and IFN-γ. Interestingly, CpG-B resulted in significant higher secretion of TNFα and IL-6. CONCLUSION: In summary, the incubation of TLR-agonists induced activation and maturation of neonatal APCs. These data show that modern TLR-specific adjuvants achieve a direct effect and potent upregulation of activation and maturation markers and cytokines in preterm neonates. We thus conclude that agents triggering TLRs might possibly overcome neonatal lack of vaccine responses.

B-cell deficiency and severe autoimmunity caused by deficiency of protein kinase C δ.

Primary B-cell disorders comprise a heterogeneous group of inherited immunodeficiencies, often associated with autoimmunity causing significant morbidity. The underlying genetic etiology remains elusive in the majority of patients. In this study, we investigated a patient from a consanguineous family suffering from recurrent infections and severe lupuslike autoimmunity. Immunophenotyping revealed progressive decrease of CD19(+) B cells, a defective class switch indicated by low numbers of IgM- and IgG-memory B cells, as well as increased numbers of CD21(low) B cells. Combined homozygosity mapping and exome sequencing identified a biallelic splice-site mutation in protein C kinase δ (PRKCD), causing the absence of the corresponding protein product. Consequently, phosphorylation of myristoylated alanine-rich C kinase substrate was decreased, and mRNA levels of nuclear factor interleukin (IL)-6 and IL-6 were increased. Our study uncovers human PRKCD deficiency as a novel cause of common variable immunodeficiency-like B-cell deficiency with severe autoimmunity.

A novel immunodeficiency syndrome associated with partial trisomy 19p13.

BACKGROUND: Subtelomeric deletions and duplications may cause syndromic disorders that include features of immunodeficiency. To date, no phenotype of immunological pathology has been linked to partial trisomy 19. We report here on two unrelated male patients showing clinical and laboratory signs of immunodeficiency exhibiting a duplication involving Chromosome 19p13. METHODS: Both patients underwent a detailed clinical examination. Extended laboratory investigations for immune function, FISH and array comparative genome hybridization (CGH) analyses were performed. RESULTS: The reported patients were born prematurely with intrauterine growth retardation and share clinical features including neurological impairment, facial dysmorphy and urogenital malformations. Array CGH analyses of both patients showed a largely overlapping terminal duplication affecting Chromosome 19p13. In both affected individuals, the clinical course was marked by recurrent severe infections. Signs of humoral immunodeficiency were detected, including selective antibody deficiency against polysaccharide antigens in patient 1 and reduced IgG1, IgG3 subclass levels and IgM deficiency in patient 2. Class-switched B memory cells were almost absent in both patients. Normal numbers of T cells, B cells and natural killer cells were observed in both boys. Lymphocytic proliferation showed no consistent functional pathology, however, function of granulocytes and monocytes as assessed by oxidative burst test was moderately reduced. Moreover, natural killer cytotoxicity was reduced in both patients. Immunoglobulin substitution resulted in a decreased number and severity of infections and improved thriving in both patients. CONCLUSIONS: Partial trisomy 19p13 represents a syndromic disorder associating organ malformation and hitherto unrecognised immunodeficiency.

Combined immunodeficiency with life-threatening EBV-associated lymphoproliferative disorder in patients lacking functional CD27.

CD27, a tumor necrosis factor receptor family member, interacts with CD70 and influences T-, B- and NK-cell functions. Disturbance of this axis impairs immunity and memory generation against viruses including Epstein Barr virus (EBV), influenza, and others. CD27 is commonly used as marker of memory B cells for the classification of B-cell deficiencies including common variable immune deficiency. Flow cytometric immunophenotyping including expression analysis of CD27 on lymphoid cells was followed by capillary sequencing of CD27 in index patients, their parents, and non-affected siblings. More comprehensive genetic analysis employed single nucleotide polymorphism-based homozygosity mapping and whole exome sequencing. Analysis of exome sequencing data was performed at two centers using slightly different data analysis pipelines, each based on the Genome Analysis ToolKit Best Practice version 3 recommendations. A comprehensive clinical characterization was correlated to genotype. We report the simultaneous confirmation of human CD27 deficiency in 3 independent families (8 patients) due to a homozygous mutation (p. Cys53Tyr) revealed by whole exome sequencing, leading to disruption of an evolutionarily conserved cystein knot motif of the transmembrane receptor. Phenotypes varied from asymptomatic memory B-cell deficiency (n=3) to EBV-associated hemophagocytosis and lymphoproliferative disorder (LPD; n=3) and malignant lymphoma (n=2; +1 after LPD). Following EBV infection, hypogammaglobulinemia developed in at least 3 of the affected individuals, while specific anti-viral and anti-polysaccharide antibodies and EBV-specific T-cell responses were detectable. In severely affected patients, numbers of iNKT cells and NK-cell function were reduced. Two of 8 patients died, 2 others underwent allogeneic hematopoietic stem cell transplantation successfully, and one received anti-CD20 (rituximab) therapy repeatedly. Since homozygosity mapping and exome sequencing did not reveal additional modifying factors, our findings suggest that lack of functional CD27 predisposes towards a combined immunodeficiency associated with potentially fatal EBV-driven hemo-phagocytosis, lymphoproliferation, and lymphoma development.

Transcriptomic analysis identifies lactoferrin-induced quiescent circuits in neonatal macrophages.

Introduction: Upon birth, a hitherto naïve immune system is confronted with a plethora of microbial antigens due to intestinal bacterial colonization. To prevent excessive inflammation and disruption of the epithelial barrier, physiological mechanisms must promote immune-anergy within the neonatal gut. As high concentrations of human lactoferrin (hLF), a transferrin glycoprotein shown to modulate macrophage function, are frequently encountered in colostrum, its direct interaction with intestinal macrophages may satisfy this physiological need. Thus, the primary objective of this study was to investigate transcriptional changes induced by human lactoferrin in neonatal monocyte-derived macrophages. Methods: Cord blood-derived monocytes were differentiated with M-CSF in presence or absence of 500 µg/mL hLF for 7 days and afterwards stimulated with 1 ng/mL LPS or left untreated. RNA was then isolated and subjected to microarray analysis. Results: Differentiation of cord blood-derived monocytes in presence of hLF induced a distinct transcriptional program defined by cell cycle arrest in the G2/M phase, induction of IL-4/IL-13-like signaling, altered extracellular matrix interaction, and enhanced propensity for cell-cell interaction. Moreover, near-complete abrogation of transcriptional changes induced by TLR4 engagement with LPS was observed in hLF-treated samples. Discussion: The global transition towards an M2-like homeostatic phenotype and the acquisition of quiescence elegantly demonstrate the ontogenetical relevance of hLF in attenuating pro-inflammatory signaling within the developing neonatal intestine. The marked anergy towards proinflammatory stimuli such as LPS further underlines the glycoprotein's potential therapeutic relevance.

Immunological characterization of an Italian PANDAS cohort.

This cross-sectional study aimed to contribute to the definition of Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal Infections (PANDAS) pathophysiology. An extensive immunological assessment has been conducted to investigate both immune defects, potentially leading to recurrent Group A ß-hemolytic Streptococcus (GABHS) infections, and immune dysregulation responsible for a systemic inflammatory state. Twenty-six PANDAS patients with relapsing-remitting course of disease and 11 controls with recurrent pharyngotonsillitis were enrolled. Each subject underwent a detailed phenotypic and immunological assessment including cytokine profile. A possible correlation of immunological parameters with clinical-anamnestic data was analyzed. No inborn errors of immunity were detected in either group, using first level immunological assessments. However, a trend toward higher TNF-alpha and IL-17 levels, and lower C3 levels, was detected in the PANDAS patients compared to the control group. Maternal autoimmune diseases were described in 53.3% of PANDAS patients and neuropsychiatric symptoms other than OCD and tics were detected in 76.9% patients. ASO titer did not differ significantly between the two groups. A possible correlation between enduring inflammation (elevated serum TNF-α and IL-17) and the persistence of neuropsychiatric symptoms in PANDAS patients beyond infectious episodes needs to be addressed. Further studies with larger cohorts would be pivotal to better define the role of TNF-α and IL-17 in PANDAS pathophysiology.

Mannan-binding lectin deficiency - Good news, bad news, doesn't matter?

Mannan-binding lectin (MBL) deficiency has been classified as a commonly occurring immune disorder, affecting approximately 30% of the human population. MBL, being part of the innate immune system, supports the recognition of infectious pathogens by binding to carbohydrate moieties expressed on microorganisms and activates the lectin pathway of the complement system. MBL2 gene polymorphisms are associated with quantitative and qualitative MBL abnormalities in the serum. The clinical impact of MBL deficiency and its association to a wide variety of diseases has been extensively studied. The picture is puzzling as the studies suggest a detrimental or beneficial or no impact of low or high MBL serum levels on disease susceptibility. In this review we attempt to extract what is relevant from the literature and address controversial issues. We finally suggest that a comprehensive understanding of the role of MBL in human diseases requires considering its context-dependency.

Perspectives on immunomodulation early in life.

The immune system early in life is characterized by immature activation and function of immune cells and a preponderance of Th2 cytokines. Together with other factors such as genetics and epigenetics, these immature immune responses might prone newborns susceptible to severe infections as well as allergic diseases. Immunomodulation therapy may have potential as therapeutic strategy against those disorders and might have implication in early-life interventions in the future. In this review, we will focus on two immunomodulatory substance classes, Toll-like receptor (TLR) ligands and sphingolipids, which are the focus of extensive research to date. Both TLRs and sphingolipid receptors have a very distinct distribution pattern and function on immune cells. Therefore, they can potentially modulate and balance immune responses, which might be in particular beneficial for the immaturity of the immune response early in life.

Reactogenicity and immunogenicity of the second COVID-19 vaccination in patients with inborn errors of immunity or mannan-binding lectin deficiency.

Background: Patients with inborn errors of immunity (IEI) are at increased risk for severe courses of SARS-CoV-2 infection. COVID-19 vaccination provides effective protection in healthy individuals. However, it remains unclear whether vaccination is efficient and safe in patients with constitutional dysfunctions of the immune system. Thus, we analyzed the humoral response, adverse reactions and assessed the disease activity of the underlying disease after COVID-19 vaccination in a cohort of patients suffering from IEIs or mannan-binding lectin deficiency (MBLdef). Methods: Vaccination response was assessed after basic immunization using the Elecsys anti-SARS-CoV-2 S immunoassay and via Vero E6 cell based assay to detect neutralization capabilities. Phenotyping of lymphocytes was performed by flow cytometry. Patient charts were reviewed for disease activity, autoimmune phenomena as well as immunization status and reactogenicity of the vaccination. Activity of the underlying disease was assessed using a patient global numeric rating scale (NRS). Results: Our cohort included 11 individuals with common variable immunodeficiency (CVID), one patient with warts hypogammaglobulinemia immunodeficiency myelokathexis (WHIM) syndrome, two patients with X-linked agammaglobulinemia (XLA), one patient with Muckle Wells syndrome, two patients with cryopyrin-associated periodic syndrome, one patient with Interferon-gamma (IFN-gamma) receptor defect, one patient with selective deficiency in pneumococcal antibody response combined with a low MBL level and seven patients with severe MBL deficiency. COVID-19 vaccination was generally well tolerated with little to no triggering of autoimmune phenomena. 20 out of 26 patients developed an adequate humoral vaccine response. 9 out of 11 patients developed a T cell response comparable to healthy control subjects. Tested immunoglobulin replacement therapy (IgRT) preparations contained Anti-SARS-CoV-2 S antibodies implicating additional protection through IgRT. Summary: In summary the data support the efficacy and safety of a COVID-19 vaccination in patients with IEIs/MBLdef. We recommend evaluation of the humoral immune response and testing for virus neutralization after vaccination in this cohort.

Immunologically relevant aspects of the new COVID-19 vaccines-an ÖGAI (Austrian Society for Allergology and Immunology) and AeDA (German Society for Applied Allergology) position paper.

BACKGROUND: The vaccines against the coronavirus disease 2019 (COVID-19) approved in the European Union represent a decisive step in the fight against the pandemic. The application of these available vaccines to patients with pre-existing immunological conditions leads to a multitude of questions regarding efficacy, side effects and the necessary patient information. RESULTS: This review article provides insight into mechanisms of action of the currently available severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines and summarises the current state of science as well as expert recommendations regarding tolerability of the vaccines. In addition, the potential to develop protective immune responses is determined. A special focus is given on patients under immunosuppression or in treatment with immunomodulatory drugs. Special groups of the population such as children, pregnant women and the elderly are also considered. CONCLUSION: Despite the need for a patient-specific risk-benefit assessment, the consensus among experts is that patients with immunological diseases in particular benefit from the induced immune protection after COVID-19 vaccination and do not have an increased risk of side effects.

Vaccines against Toxoplasma gondii: challenges and opportunities.

Development of vaccines against Toxoplasma gondii infection in humans is of high priority, given the high burden of disease in some areas of the world like South America, and the lack of effective drugs with few adverse effects. Rodent models have been used in research on vaccines against T. gondii over the past decades. However, regardless of the vaccine construct, the vaccines have not been able to induce protective immunity when the organism is challenged with T. gondii, either directly or via a vector. Only a few live, attenuated T. gondii strains used for immunization have been able to confer protective immunity, which is measured by a lack of tissue cysts after challenge. Furthermore, challenge with low virulence strains, especially strains with genotype II, will probably be insufficient to provide protection against the more virulent T. gondii strains, such as those with genotypes I or II, or those genotypes from South America not belonging to genotype I, II or III. Future studies should use animal models besides rodents, and challenges should be performed with at least one genotype II T. gondii and one of the more virulent genotypes. Endpoints like maternal-foetal transmission and prevention of eye disease are important in addition to the traditional endpoint of survival or reduction in numbers of brain cysts after challenge.