Neonates, particularly those born prematurely, are among the most vulnerable age group for morbidity and mortality due to infections. Immaturity of the innate immune system and a high need for invasive medical procedures in the context of a preterm birth make these infants highly susceptible to common neonatal pathogens. Preterm infants who survive may also suffer permanent disabilities due to organ damage resulting from either the infection itself or from the inflammatory response generated under an oxidative stress. Infections in preterm infants continue to pose important healthcare challenges. Yet, developmental maturation events in the innate immune system that underlie their excessively high vulnerability to infection remain largely understudied. In this review article, we identify pertinent knowledge gaps that must be filled in order to orient future translational research.

Newborn infants, including those born at term without congenital disorders, are at high risk of severe disease from respiratory syncytial virus (RSV) infection. Indeed, our current local surveillance data demonstrate that approximately half of children hospitalized with RSV were ≤3 mo old, and 74% were born at term. Informed by this clinical epidemiology, we investigated antiviral innate immune responses in early life, with the goal of identifying immunological factors underlying the susceptibility of infants and young children to severe viral lower respiratory tract infections. We compared RSV-induced innate cytokine production in blood mononuclear cells from neonates, young children aged 12–59 mo, and healthy adults. RSV-induced IFN-α production was primarily mediated by plasmacytoid dendritic cells (pDCs), and was significantly lower in term infants and young children < 5 y of age than in adults (p < 0.01). RSV-induced IFN-α production in human pDCs proceeded independently of endosomal TLRs, and human pDCs from healthy adult donors produced IFN-α in a retinoic acid–inducible gene I protein (RIG-I)–dependent manner. Of interest, young age and premature birth were independently associated with attenuated RIG-I–dependent IFN-α responses (p < 0.01). In contrast to IFN-α production, proinflammatory IL-6 responses to RSV were mediated by monocytes, appeared less dependent on RIG-I, and were significantly impaired only among preterm infants, not in term infants and young children. Our results suggest that human pDCs are less functional in early life, which may contribute to the increased susceptibility of infants and young children to severe RSV disease.

Interleukin-1β (IL-1β) production is impaired in cord blood monocytes. However, the mechanism underlying this developmental attenuation remains unclear. Here, we analyzed the extent of variability within the Toll-like receptor (TLR)/NLRP3 inflammasome pathways in human neonates. We show that immature low CD14 expressing/CD16 pos monocytes predominate before 33 weeks of gestation, and that these cells lack production of the pro-IL-1β precursor protein upon LPS stimulation. In contrast, high levels of pro-IL-1β are produced within high CD14 expressing monocytes, although these cells are unable to secrete mature IL-1β. The lack of secreted IL-1β in these monocytes parallels a reduction of NLRP3 induction following TLR stimulation resulting in a lack of caspase-1 activity before 29 weeks of gestation, whereas expression of the apoptosisassociated speck-like protein containing a CARD and function of the P2×7 receptor are preserved. Our analyses also reveal a strong inhibitory effect of placental infection on LPS/ATP-induced caspase-1 activity in cord blood monocytes. Lastly, secretion of IL-1β in preterm neonates is restored to adult levels during the neonatal period, indicating rapid maturation of these responses after birth. Collectively, our data highlight important developmental mechanisms regulating IL-1β responses early in gestation, in part due to a downregulation of TLR-mediated NLRP3 expression. Such mechanisms may serve to limit potentially damaging inflammatory responses in a developing fetus.Keywords: Human r Inflammasome r Interleukin-1 beta (IL-1β) r Neonate r Toll-like receptor Additional supporting information may be found in the online version of this article at the publisher's web-site Correspondence: Dr. Pascal M. Lavoie e-mail: plavoie@cw.bc.ca * These authors contributed equally to this work.C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu Eur. J. Immunol. 2015. 45: 238-249 Innate immunity 239 IntroductionNewborns are at high risk of infections, in part due to attenuated innate immune defenses [1]. The cytokine interleukin-1β (IL-1β) is an important inflammatory mediator in response to infections [2]. Mice lacking IL-1β display impaired acute phase and pyrogenic responses [3], and increased susceptibility to pathogens commonly encountered in the neonatal period [4,5]. In contrast, high levels of IL-1β in a fetus can result in autoimmune organ damage [2], as well as lethal metabolic disturbances including severe weight loss and hypoglycemia [6]. Together, these data illustrate the evolutionary importance of a tight regulation of IL-1β in order to avoid inflammation-mediated organ damage, neurological injury [7], or even premature birth in a developing human [2,8].Monocytes are primarily responsible for the production of IL-1β in circulating blood [9,10]. Three subsets predominate in humans: "Classical" monocytes express CD14, but lack expression of the immunoglobulin receptor CD16. These CD14 high /CD16 neg monocytes make up the majority of monocytes in peripheral adult bloo...

NK cell adoptive therapy is a promising cancer therapeutic approach, but there are significant challenges that limiting its feasibility and clinical efficacy. One difficulty is the paucity of clinical grade manufacturing platforms to support the large scale expansion of highly active NK cells. We created an NK cell feeder cell line termed ‘NKF’ through overexpressing membrane bound IL-21 that is capable of inducing robust and sustained proliferation (>10,000-fold expansion at 5 weeks) of highly cytotoxic NK cells. The expanded NK cells exhibit increased cytotoxic function against a panel of blood cancer and solid tumor cells as compared to IL-2-activated non-expanded NK cells. The NKF-expanded NK cells also demonstrate efficacy in mouse models of human sarcoma and T cell leukemia. Mechanistic studies revealed that membrane-bound IL-21 leads to an activation of a STAT3/c-Myc pathway and increased NK cell metabolism with a shift towards aerobic glycolysis. The NKF feeder cell line is a promising new platform that enables the large scale proliferation of highly active NK cells in support of large scale third party NK cell clinical studies that have been recently intiatied. These results also provide mechanistic insights into how membrane-bound IL-21 regulates NK cell expansion.

BackgroundInflammation and infection are major determinants of disease severity and consequently, the quality of life and outcome for patients with cystic fibrosis (CF). Interleukin-1 beta (IL-1β) is a key inflammatory mediator. Secretion of biologically active IL-1β involves inflammasome-mediated processing. Little is known about the contribution of IL-1β and the inflammasomes in CF inflammatory disease. This study examines inflammasome-mediated IL-1β production in CF bronchial epithelial cell lines and human patients with CF.ResultsBronchial epithelial cell lines were found to produce negligible amounts of basal or stimulated IL-1β compared to hematopoeitic cells and they did not significantly upregulate caspase-1 activity upon inflammasome stimulation. In contrast, peripheral blood mononuclear cells (PBMCs) from both CF and healthy control subjects produced large amounts of IL-1β and strongly upregulated caspase-1 activity upon inflammasome stimulation. PBMCs from CF patients and controls displayed similar levels of caspase-1 activation and IL-1β production when stimulated with inflammasome activators. This IL-1β production was dependent on NF-κB activity and could be enhanced by priming with LPS. Finally, chemical inhibition of CFTR activity in control PBMCs and THP-1 cells did not significantly alter IL-1β or IL-8 production in response to P. aeruginosa.ConclusionHematopoeitic cells appear to be the predominant source of inflammasome-induced pro-inflammatory IL-1β in CF. PBMCs derived from CF subjects display preserved inflammasome activation and IL-1β secretion in response to the major CF pathogen Pseudomonas aeruginosa. However, our data do not support the hypothesis that increased IL-1β production in CF subjects is due to an intrinsic increase in NF-κB activity through loss of CFTR function.

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Chimeric antigen receptor T cells (CAR-T cell) targeting CD19 are effective against several subtypes of CD19-expressing hematologic malignancies. Centralized manufacturing has allowed rapid expansion of this cellular therapy, but it may be associated with treatment delays due to the required logistics. We hypothesized that point of care manufacturing of CART cells on the automated CliniMACS Prodigy R device allows reproducible and fast delivery of cells for the treatment of patients with non-Hodgkin lymphoma. Here we describe cell manufacturing results and characterize the phenotype and effector function of CART cells used in a phase I/II study. We utilized a lentiviral vector delivering a second-generation CD19 CAR construct with 4-1BB costimulatory domain and TNFRSF19 transmembrane domain. Our data highlight the successful generation of CART cells at numbers sufficient for all patients treated, a shortened duration of production from 12 to 8 days followed by fresh infusion into patients, and the detection of CART cells in patient circulation up to 1-year post-infusion.

Background: Antimicrobial responses have been shown to be profoundly attenuated in very preterm neonates when examined on cord blood. However, we lack data on these responses at the time these neonates are most vulnerable to infections. Methods: Multiple cytokine responses to two prototypic Toll-like receptor (TLR) agonists: lipopolysaccharide (LPS) (TLR4) and R848 (TLR7/8) were prospectively measured in preterm neonates born ≤30 wk of gestation (n = 50) during the first 28 d of age using whole blood and single-cell multiparameter flow cytometry assays. Results were compared to term neonates (n = 30) and adult controls (n = 25). results: In preterm neonates, LPS and R848 responses remained attenuated in both cord blood and in the first 28 d of age. These responses showed significant maturation over time after adjusting for gestational age and were confirmed in monocytes and dendritic cells on a per-cell basis. We detected no major contribution of chorioamnionitis, maternal antenatal corticosteroids or magnesium sulfate treatment, labor, or mode of delivery to the maturation of cytokine responses. conclusion: Innate immune antimicrobial defenses are profoundly attenuated developmentally in very preterm neonates during the neonatal period, suggesting that exogenous factors drive the sustained systemic inflammation that has been linked to increased morbidities in these infants.d espite improvements in neonatal health care, mortality from infections has continued to increase in very preterm neonates over the last two decades, owing to the improved survival of smaller and more vulnerable infants (1). However, the underlying immunological basis of their high morbidities and high vulnerability to sepsis remains poorly understood (2). To defend themselves against infection, neonates rely on first-line, innate immune defense mechanisms. These mechanisms include the detection of specific microbial molecular structures called pathogenassociated molecular patterns through specialized receptors termed pattern recognition receptors (PRR), but also other defense mechanisms such as phagocytosis and antigen presentation to the adaptive immune system.It is widely stated that the high risk of neonatal sepsis observed in these infants is due to immature immune defense mechanisms. However, data are lacking on the development of immune functions beyond measures performed on umbilical cord blood. Recent studies have reported reduced antigen-presenting receptor expression during the first week of age in infants born below 33 wk of gestation (3,4). In contrast, phagocytic functions in these infants are more comparable to term infants (5). Toll-like receptors (TLRs) play a predominant role as a major family of PRR that act as essential "detectors" in the recognition of microbial pathogens by the innate immune system. We and others have shown that TLR-induced cytokine immune reactivity are profoundly attenuated in cord blood of very preterm neonates and develops asynchronously over the last trimester of gestation (6-10). These infants als...