Neonatal innate TLR-mediated responses are distinct from those of adults.

The human neonate and infant are unduly susceptible to infection with a wide variety of microbes. This susceptibility is thought to reflect differences from adults in innate and adaptive immunity, but the nature of these differences is incompletely characterized. The innate immune response directs the subsequent adaptive immune response after integrating information from TLRs and other environmental sensors. We set out to provide a comprehensive analysis defining differences in response to TLR ligation between human neonates and adults. In response to most TLR ligands, neonatal innate immune cells, including monocytes and conventional and plasmacytoid dendritic cells produced less IL-12p70 and IFN-alpha (and consequently induced less IFN-gamma), moderately less TNF-alpha, but as much or even more IL-1beta, IL-6, IL-23, and IL-10 than adult cells. At the single-cell level, neonatal innate cells generally were less capable of producing multiple cytokines simultaneously, i.e., were less polyfunctional. Overall, our data suggest a robust if not enhanced capacity of the neonate vs the adult white-blood cell TLR-mediated response to support Th17- and Th2-type immunity, which promotes defense against extracellular pathogens, but a reduced capacity to support Th1-type responses, which promote defense against intracellular pathogens.

Correlation analysis of intracellular and secreted cytokines via the generalized integrated mean fluorescence intensity.

The immune response in humans is usually assessed using immunogenicity assays to provide biomarkers as correlates of protection (CoP). Flow cytometry is the assay of choice to measure intracellular cytokine staining (ICS) of cell-mediated immune (CMI) biomarkers. For CMI analysis, the integrated mean fluorescence intensity (iMFI) was introduced as a metric to represent the total functional CMI response as a CoP. iMFI is computed by multiplying the relative frequency (percent positive) of cells expressing a particular cytokine with the MFI of that population, and correlates better with protection in challenge models than either the percentage or the MFI of the cytokine-positive population. While determination of the iMFI as a CoP can readily be accomplished in animal models that allow challenge/protection experiments, this is not feasible in humans for ethical reasons. As a first step toward extending the iMFI concept to humans, we investigated the correlation of the iMFI derived from a human innate immune response ICS assay with functional cytokine release into the culture supernatant, as innate cytokines need to be released to have a functional impact. Next, we developed a quantitatively more correlative mathematical approach for calculating the functional response of cytokine-producing cells by incorporating the assignment of different weights to the magnitude (frequency of cytokine-positive cells) and the quality (the MFI) of the observed innate immune response. We refer to this model as generalized iMFI.

Identification of B cells through negative gating-An example of the MIFlowCyt standard applied.

Polychromatic flow cytometric analysis takes advantage of the increasing number of available fluorophores to positively identify and simultaneously assess multiple parameters in the same cell (1). Additional parameters may be analyzed through negative identification (i.e., through exclusion of particular stains or antibodies employed). In this report, we tested whether such negative-gating strategy would identify human B lymphocytes in innate immune phenotyping studies. To this end, B cells were identified as the negatively-stained subpopulation from the CD123 vs. CD11c plot of the CD14(neg-low), MHC II(high) human peripheral blood mononuclear cells. To test the specificity of this negative gating approach, we confirmed that negatively gated B cells indeed expressed CD19, the bona fide marker for human B cells. However, a small number of unidentified cells were contained in the negatively-gated B cells. Furthermore, a small percentage cells expressing markers used to identify monocytes and myeloid dendritic cells (mDC) coexpressed CD19. This identifies such negative B-cell gating approach as potentially problematic. When applied to the analysis of Toll-like receptors (TLR) stimulation experiments, we were however able to interpret the results, as B-cells respond to TLR stimulation in a qualitative different pattern as compared to monocytes and DC. This report is presented in a manner that is fully compliant with the Minimum Information about a Flow Cytometry Experiment (MIFlowCyt) standard, which was recently adopted by the International Society for Advancement of Cytometry (ISAC) (2) and incorporated in the publishing policies of Cytometry and other journals. We demonstrate how a MIFlowCyt-compliant report can be prepared with minimal effort, and yet provide the reader with a much clearer picture of the portrayed FCM experiment and data.

Polychromatic flow cytometric high-throughput assay to analyze the innate immune response to Toll-like receptor stimulation.

Polychromatic flow cytometry allows the capture of multidimensional data, providing the technical tool to assess complex immune responses. Interrogation of the adaptive T cell response to infection or vaccination already has benefited greatly from standardized protocols for polychromatic flow cytometric analysis. The innate immune system plays an important role in health and disease, and presents potentially important therapeutic and diagnostic modalities. We describe here a high-throughput polychromatic flow cytometry-based platform that enables the rapid interrogation and large scale screening of human blood antigen presenting cell responses to Toll-like receptor (TLR) ligands and other innate immune modulators. Using this assay, we found that for certain stimuli (e.g., TLR9 and TLR3 ligands), the general protocol for intracellular cytokine cytometry had to be significantly modified to allow response detection. Furthermore, high concentrations of TLR7/8 and TLR4 stimuli caused substantial changes in lineage markers, potentially confounding analysis if one were to use a conventional "lineage-negative" cocktail. The assay we developed is reproducible and has been used to show that a given individual's TLR response pattern is relatively stable over at least several months. This protocol is in strict compliance with published guidelines for polychromatic flow cytometry, provides a common platform for scientists to compare their results directly, and may be applicable to the diagnostic evaluation of Toll-like receptor function and the rapid screening of promising therapeutic innate immune modulators.

Variables to be controlled in the assessment of blood innate immune responses to Toll-like receptor stimulation.

Variability in TLR function influences susceptibility to infectious as well as immune-mediated diseases. Given the outbred nature of humans, identifying functional Toll-like receptor variability and its role in clinical disease requires such analysis to be conducted in large, often multi-centered cohorts. Yet the technically complex measurements involved in innate immune analysis benefit from centralized processing of samples. Centralization requires shipping of samples or prolonged storage, possibly even cryopreservation. Deviation from standard operating procedures (SOP) for sample procurement, storage and processing may alter the final innate immune read out. We here set out to define the impact of variables most likely to be encountered during large, multi-site studies: (i) the source of the sample, (ii) time between sample procurement to processing, and (iii) processing of fresh vs. cryopreserved samples. We found that all of these variables exert a profound impact on the final innate response to TLR stimulation. Specific innate responses appeared to be affected in response to specific TLR stimuli by a particular variable under study, proving it impossible to provide global generalizations. Based on our studies and other published work on this topic, we propose a minimal list of variables that have to be met for samples to be comparable within and across studies: a) timing between procurement and processing cannot vary by more than 10%; b) all samples have to be stored the same; and c) the source of samples needs to be the same. In summary, for innate immune analysis scrupulous adherence to standard operating procedures is paramount.

A single immunization near birth elicits immediate and lifelong protective immunity.

Most existing vaccines do not induce protective immunity immediately following birth, nor do they retain protective efficacy in the latter years of life without booster doses. Using a mouse model, we present evidence that a live-replicating vaccine administered only once shortly after birth was able to induce both immediate and lifelong protection. Newborn mice immunized with a safe, highly attenuated strain of Listeria monocytogenes (Lm) were already protected by day 7 post-vaccination when challenged with a virulent strain of Lm. Furthermore, all mice remained fully protected for 2 years after only a single immunization. Vaccine-specific T cell immune responses were still detectable 2 years later, indicating long-lived immune memory even in neonatal vaccine recipients. Analysis of memory precursor subsets, specific for antigens homologous to Lm or a model vaccine (Ova), demonstrated remarkable similarity between adult and neonatal vaccine recipient effector and central memory CD8 T cell development. The magnitude of expansion of antigen specific memory T cells post-infectious challenge correlated with protection in both groups. This is the first direct evidence that vaccination--even in the absence of a booster dose--is capable of inducing immediate and lifelong protective immune memory regardless of age at the time of initial vaccination.

Ontogeny of Toll-like receptor mediated cytokine responses of human blood mononuclear cells.

Newborns and young infants suffer increased infectious morbidity and mortality as compared to older children and adults. Morbidity and mortality due to infection are highest during the first weeks of life, decreasing over several years. Furthermore, most vaccines are not administered around birth, but over the first few years of life. A more complete understanding of the ontogeny of the immune system over the first years of life is thus urgently needed. Here, we applied the most comprehensive analysis focused on the innate immune response following TLR stimulation over the first 2 years of life in the largest such longitudinal cohort studied to-date (35 subjects). We found that innate TLR responses (i) known to support Th17 adaptive immune responses (IL-23, IL-6) peaked around birth and declined over the following 2 years only to increase again by adulthood; (ii) potentially supporting antiviral defense (IFN-α) reached adult level function by 1 year of age; (iii) known to support Th1 type immunity (IL-12p70, IFN-γ) slowly rose from a low at birth but remained far below adult responses even at 2 years of age; (iv) inducing IL-10 production steadily declined from a high around birth to adult levels by 1 or 2 years of age, and; (v) leading to production of TNF-α or IL-1ß varied by stimuli. Our data contradict the notion of a linear progression from an 'immature' neonatal to a 'mature' adult pattern, but instead indicate the existence of qualitative and quantitative age-specific changes in innate immune reactivity in response to TLR stimulation.

Systems biology evaluation of immune responses induced by human host defence peptide LL-37 in mononuclear cells.

The immune system is very complex, it involves the integrated regulation and expression of hundreds of proteins. To understand in greater detail how the human host defence immunomodulatory peptide LL-37 interacts with innate immunity, a systems approach was pursued. Polychromatic flow cytometry was employed to demonstrate that within human peripheral blood mononuclear cells, CD14+ monocytes, myeloid and plasmocytoid dendritic cells and T- and B-lymphocytes, all responded to LL-37, with the differential production of intracellular cytokines. Microarray analyses with CD14+ monocytes indicated the differential expression of 475 genes in response to stimulation with LL-37. To understand this complex response, bioinformatic interrogation, using InnateDB, of the gene ontology, signalling pathways and transcription factor binding sites was undertaken. Activation of the IkappaBalpha/NFkappaB, mitogen-activated protein kinases p38, ERK1/2 and JNK, and PI3K signalling pathways in response to LL-37 was demonstrated by pathway and ontology over-representation analyses, and confirmed experimentally by inhibitor studies. Computational analysis of the predicted transcription factor binding sites upstream of the genes that were regulated by LL-37 predicted the involvement of several transcription factors including NFkappaB and five novel factors, AP-1, AP-2, SP-1, E2F1, and EGR, which were experimentally confirmed to respond to LL-37 by performing transcription factor array studies on nuclear extracts from LL-37 treated mononuclear cells. These data are discussed as reflecting the integration of several responsive signalling pathways through the involvement of transcription factor complexes in gene expression activated by LL-37 in human mononuclear cells.

Induction of protective immunity to Listeria monocytogenes in neonates.

Neonates suffer unduly from infections and also respond suboptimally to most commonly used vaccines. However, a CD8 T cell response can be elicited in neonates if the Ag is introduced into the cytoplasm of APCs. Listeria monocytogenes (Lm) targets the cytoplasm of APC and is a strong CD8 and CD4 Th1-promoting vaccine vehicle in adult mice. We hypothesized that an attenuated strain of Lm would be safe and induce long-lasting protective immunity, even in neonates. We found that neonatal mice immunized only once with the attenuated strain DeltaactA-Lm developed robust primary and secondary CD8 and CD4 Th1 responses and were fully protected from lethal challenge with virulent wild-type Lm without the need for a booster immunization. Furthermore, DeltaactA-Lm expressing a heterologous recombinant Ag induced a strong CD8 and Th1 memory response to that Ag. Based on these data, we propose that DeltaactA-Lm or derivatives thereof might serve as a vaccine vehicle for neonatal immunization.