The impaired infection control related to the functional immaturity of the neonatal immune system is an important cause of infection in preterm newborns. We previously reported that constitutive Toll-like receptor (TLR) 4 expression and cytokine secretion on lipopolysaccharide (LPS) stimulation increases with gestational age. Here, we analyzed constitutive monocyte TLR2 expression and evaluated the expression profiles of the proximal downstream adapter molecule myeloid differentiation factor 88 (MyD88). We further investigated activation of protein kinases p38 and extracellular regulated kinsase (ERK) 1/2 in CD14 monocytes after ex vivo stimulation with bacterial TLR ligands (LPS and lipoteichoic acid [LTA]). The functional outcome of the stimulation was determined by cytokine secretion. Monocytes from 31 preterm newborns (<30 weeks of gestation, n=16; 30-37 weeks of gestation, n=15), 10 term newborns, and 12 adults were investigated. In contrast to TLR4 expression, TLR2 levels did not differ between age groups. However, MyD88 levels were significantly lower in preterm newborns. Activation of p38 and ERK1/2 was impaired in all newborn age groups after stimulation with TLR-specific ligands. Accordingly, after LTA stimulation, the levels of interleukin (IL)-1 beta , IL-6, and IL-8 cytokine production were substantially lower (P<.001) in preterm newborns than in adults. The reduced functional response to bacterial cell wall components appears to be part of the functional immaturity of the neonatal immune system and might predispose premature newborns to bacterial infection.

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Premature newborns are highly susceptible to severe bacterial infections. This is partially due to their immature innate immune system, characterized by decreased neutrophil and monocyte activity as well as by reduced concentrations of complement factors. However, additional mechanisms might be important for innate immunity and are still the subject of considerable debate. The importance of pattern recognition domains such as Toll-like receptors (TLR) has been fully acknowledged within the last few years. Therefore, we investigated age-related monocyte TLR4 expression and lipopolysaccharide-induced cytokine secretion from very low birth weight infants (VLBWI) and from newborns after wk 30 of gestation in comparison to healthy adults. In VLBWI, expression of TLR4 surface protein, detected by flow cytometry, and TLR4-specific mRNA, quantified by real time-PCR, were significantly reduced in comparison to mature infants and to adults. Reduced TLR4 expression was paralleled by significantly diminished ex vivo LPS stimulated IL-1␤, IL-6, and tumor necrosis factor-␣ secretion into whole blood. We conclude that, in VLBWI, the minimized expression of TLR4 contributes to the susceptibility of VLBWI to infections with Gram-negative bacteria due to the lack of cytokines to boost initial immune response. (Pediatr Res 58: 121-124, 2005) Abbreviations CB, cord blood LPS, lipopolysaccharide PAMP, pathogen-associated molecular pattern PB, peripheral blood PBMC, peripheral blood mononuclear cells TLR, Toll-like receptor TNF, tumor necrosis factor VLBWI, very low birth weight infants Bacterial infections are a major cause of neonatal morbidity (1,2), and premature newborns are highly susceptible to infections (3). This increased susceptibility appears to be related to immaturity of the immune system. Maturation of the immune system includes specific humoral and cellular responses as well as nonspecific innate immune mechanisms, such as factors of the complement system and phagocyte activity (3-5).Monocytes and macrophages play a central role in both specific and nonspecific immunity against bacterial, viral, and fungal infection. In VLBWI, a substantial decrease of fully functional phagocytes has recently been reported (6). The Toll protein has been identified as a receptor controlling dorsoventral polarization during embryonic development of Drosophila larvae and serves a defensive function (7). Structures homologous to Toll receptors were found in mammalian cells (8,9) and termed TLR. Within the last few years, 10 human TLR have been described and characterized as important sensors of the innate immune system (9,10). These TLR are phylogenetically conserved and are involved in recognition of microbial PAMP. TLR4 functions as the transmembrane component of the LPS receptor. In addition to LPS, the major glycolipid component of the outer membrane of Gram-negative bacteria, TLR4 recognizes saturated and unsaturated fatty acids such as lipoteichoic acids from Gram-positive bacteria, and heat-shock proteins (8 -10). Binding of these ...

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 NKcell 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 hemophagocytosis, lymphoproliferation, and lymphoma development.

Key Points• PRKCD deficiency causes a novel primary immunodeficiency with B-cell deficiency and severe autoimmunity.• Protein kinase C d may represent a key factor controlling immune homeostasis and autoimmunity.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 1 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 splicesite mutation in protein C kinase d (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. (Blood. 2013;121(16):3112-3116)

PurposeNF-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 ResultsWe 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.ConclusionsDeficiency 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.Electronic supplementary materialThe online version of this article (doi:10.1007/s10875-016-0306-1) contains supplementary material, which is available to authorized users.