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There is increasing evidence that lung-resident memory T and B cells play a critical role in protecting against respiratory reinfection. With a unique transcriptional and phenotypic profile, resident memory lymphocytes are maintained in a quiescent state, constantly surveying the lung for microbial intruders. Upon reactivation with cognate antigen, these cells provide rapid effector function to enhance immunity and prevent infection. Immunization strategies designed to induce their formation, alongside novel techniques enabling their detection, have the potential to accelerate and transform vaccine development. Despite most data originating from murine studies, this review will discuss recent insights into the generation, maintenance and characterisation of pulmonary resident memory lymphocytes in the context of respiratory infection and vaccination using recent findings from human and non-human primate studies.

Small-molecule adjuvants that boost and direct adaptive immunity provide a powerful means to increase the effectiveness of vaccines. Through rational design several novel imidazoquinoline and oxoadenine TLR7/8 agonists, each with unique molecular modifications, were synthesized and assessed for their ability to augment adaptive immunity. All agonists bound human TLR7 and TLR8 and induced maturation of both human mDCs and pDCs. All agonists prompted production of type I interferon and/or proinflammatory cytokines, albeit with varying potencies. In most in vitro assays, the oxoadenine class of agonists proved more potent than the imidazoquinolines. Therefore, an optimized oxoadenine TLR7/8 agonist that demonstrated maximal activity in the in vitro assays was further assessed in a vaccine study with the CRM197 antigen in a porcine model. Antigen-specific antibody production was greatly enhanced in a dose dependent manner, with antibody titers increased 800-fold compared to titers from pigs vaccinated with the non-adjuvanted vaccine. Moreover, pigs vaccinated with antigen containing the highest dose of adjuvant promoted a 13-fold increase in the percentage of antigen-specific CD3(+)/CD8(+) T cells over pigs vaccinated with antigen alone. Together this work demonstrates the promise of these novel TLR7/8 agonists as effective human vaccine adjuvants.

Soluble antigens diffuse out of the brain and can thus stimulate a systemic immune response, whereas particulate antigens (from infectious agents or tumor cells) remain within brain tissue, thus failing to stimulate a systemic immune response. Immune privilege describes how the immune system responds to particulate antigens localized selectively within the brain parenchyma. We believe this immune privilege is caused by the absence of antigen presenting dendritic cells from the brain. We tested the prediction that expression of fms-like tyrosine kinase ligand 3 (Flt3L) in the brain will recruit dendritic cells and induce a systemic immune response against exogenous influenza hemagglutinin in BALB/c mice. Coexpression of Flt3L with HA in the brain parenchyma induced a robust systemic anti-HA immune response, and a small response against myelin basic protein and proteolipid protein epitopes. Depletion of CD4 + CD25+ regulatory T cells (Tregs) enhanced both responses. To investigate the autoimmune impact of these immune responses, we characterized the neuropathological and behavioral consequences of intraparenchymal injections of Flt3L and HA in BALB/c and C57BL/6 mice. T cell infiltration in the forebrain was time and strain dependent, and increased in animals treated with Flt3L and depleted of Tregs; however, we failed to detect widespread defects in myelination throughout the forebrain or spinal cord. Results of behavioral tests were all normal. These results demonstrate that Flt3L overcomes the brain's immune privilege, and supports the clinical development of Flt3L as an adjuvant to stimulate clinically effective immune responses against brain neo-antigens, for example, those associated with brain tumors.

Gene therapy is proposed as a novel therapeutic strategy for treating glioblastoma multiforme (GBM), a devastating brain cancer. In the clinic, antivector immune responses pose formidable challenges. Herein we demonstrate that high-capacity adenovirus vectors (HC-Ads) carrying the conditional cytotoxic gene herpes simplex virus type 1-thymidine kinase (TK) induce tumor regression and long-term survival in an intracranial glioma model, even in the presence of systemic antiadenovirus immunity, as could be encountered in patients. First-generation Ad-TK failed to elicit tumor regression in this model. These results pave the way for implementing HC-Ad-TK-mediated gene therapy as a powerful adjuvant for treating GBM.

Immune responses against vectors or encoded transgenes can impose limitations on gene therapy. We demonstrated that tetracycline-regulated high-capacity adenoviral vectors (HC-Ads) sustain regulated transgene expression in the brain even in the presence of systemic pre-existing immune responses against adenoviruses. In this study we assessed whether systemic pre-existing immune responses against the transgene products, i.e., beta-Gal or the tetracycline-dependent (TetON) regulatory transcription factors (rtTA2(S)M2 and the tTS(Kid)), affect transgene expression levels and the safety profile of HC-Ads in the brain. We pre-immunized mice with plasmids encoding the TetON switch expressing rtTA2(S)M2 and the tTS(Kid) or beta-Gal. HC-Ads expressing beta-Gal under the control of the TetON switch were then injected into the striatum. We assessed levels and distribution of beta-Gal expression, and evaluated local inflammation and neuropathological changes. We found that systemic immunity against beta-Gal, but not against the TetON switch, led to inflammation and reduction of transgene expression in the striatum. Therefore, the regulatory TetON switch appears to be safe to use, and capable of sustaining transgene expression in the brain even in the presence of an immune response against its components. Systemic immunity against the transgene had the effect of curtailing its expression, thereby affecting the efficacy and safety of gene delivery to the brain. This factor should be considered when developing gene therapies for neurological use.

The quaking viable (qk(v)) mice have attracted attention because of their characteristic tremor caused by their dysmyelination. In the central nervous system, qk(v) mice fail to develop mature myelinating oligodendrocytes and display uncompacted myelin. The genetic defect in the qk(v) mice prevents the proper expression of alternatively spliced KH-type QKI RNA binding proteins. Thus qk(v) mice provide a unique animal model linking RNA binding proteins to defects in oligodendrocyte cell fate and myelination. The fact that QKI proteins are modified post-translationally makes them Signal Transduction Activiators of RNA (STAR) proteins. We have used a gain-of-function approach with the ectopic expression of the separate QKI isoforms using adenoviruses and retroviruses to determine their separate roles in cell fate and myelination. Herein, we discuss the recent advances in characterizing the QKI KH-type proteins as glial cell fate and myelin egulators.

The quaking viable mice have myelination defects and develop a characteristic tremor 10 d after birth. The quaking gene encodes at least five alternatively spliced QUAKING (QKI) isoforms that differ in their C-terminal 8–30-amino-acid sequence. The reason for the existence of the different QKI isoforms and their function are unknown. Here we show that only one QKI isoform, QKI-7, can induce apoptosis in fibroblasts and primary rat oligodendrocytes. Heterodimerization of the QKI isoforms results in the nuclear translocation of QKI-7 and the suppression of apoptosis. The unique C-terminal 14 amino acids of QKI-7 confers the ability to induce apoptosis to heterologous proteins such as the green fluorescent protein and a QKI-related protein, Caenorhabditis elegansGLD-1. Thus, the unique C-terminal sequences of QKI-7 may function as a life-or-death ‘sensor’ that monitors the balance between the alternatively spliced QKI isoforms. Moreover, our findings suggest that nuclear translocation is a novel mechanism of inactivating apoptotic inducers.

Alzheimer’s disease (AD) is the most common cause of dementia worldwide. The pathogenesis of this neurodegenerative disease, currently without curative treatment, is associated with the accumulation of amyloid β (Aβ) in brain parenchyma and cerebral vasculature. AD patients are unable to clear this toxic peptide, leading to Aβ accumulation in their brains and, presumably, the pathology associated with this devastating disease. Compounds that stimulate the immune system to clear Aβ may therefore have great therapeutic potential in AD patients. Monophosphoryl lipid A (MPL) is an LPS-derived Toll-like receptor 4 agonist that exhibits unique immunomodulatory properties at doses that are nonpyrogenic. We show here that repeated systemic injections of MPL, but not LPS, significantly improved AD-related pathology in APP swe /PS1 mice. MPL treatment led to a significant reduction in Aβ load in the brain of these mice, as well as enhanced cognitive function. MPL induced a potent phagocytic response by microglia while triggering a moderate inflammatory reaction. Our data suggest that the Toll-like receptor 4 agonist MPL may be a treatment for AD.

We are pleased to announce the publication of this special issue in the journal “Clinical and Developmental Immunology.” We are happy to perceive the growing interest, from a wide range of scientists, regarding the peculiar intercommunication between the immune system and the central nervous system (CNS). We have finally reached a balanced compilation of papers that we collect in this special issue highlighting recent fundamental advances in our understanding of brain immunology with an emphasis on new therapeutic targets covering such emerging topics as chemical suppression of glial activation, inflammation following acute demylination, Notch signalling as a potential therapeutic target in EAE and a link between neuroinflammatory signaling and reproduction. The idea that the CNS is an immune-privileged site is gradually vanishing. However, increasing evidence shows that the relation between the CNS and the immune system is special and in many aspects, different from the rest of the organs and tissues [1]. One of the peculiarities of the CNS is the presence of glial cells, which are the initial responders to brain injuries and degenerative processes [2]. Glial cells, especially microglia, get locally activated in the damaged brain areas and are able to induce the recruitment of scavenger blood cells, such as monocytes and lymphocytes to injured sites, contributing to the inflammatory reaction [3, 4]. In our special issue, R. A. Taylor and L. H. Sansing, describe, in a comprehensive review, the role of microglial cells in ischemic stroke and intracerebral hemorrhage. Importantly, authors highlight the distinct phenotypes of microglia, M1-inflammatory, and M2-repairing microglia, which express different surface molecules and releasing factors. They propose that understanding the mechanism of this switching phenotype will be crucial for future therapeutic purposes. In line with this, E. Assi et al., review the recent literature about the role of microglia in inflammatory signaling cascades in brain pathology, but focusing their paper on the role of sphingolipids in the inflammatory reaction, which is proposed as a potential target to control glial-mediated neuroinflammation. In this context, it is becoming more evident that glial activation is a critical event that should be targeted to avert inflammation in the CNS. B. Rocamonde et al. describe, in an original study, using a rat model of brain injury, that the reduction of glial activation by lipoic acid underlies the restorative effects in the brain. F. Cloutier et al. suggest in an interesting paper that the role of microglia and astrocytes during spinal cord injury and repair may be different depending on the scenario of CNS damage. Authors report, using a rat model of acute demyelination in dorsal funiculus, how immunological using anti GalC demyelination triggers macrophage/microglial cells activation in comparison of a stab injury. Interestingly, in their model of axon regeneration, the participation of astrocytes is limited, whereas microglia and infiltrated macrophages have a prominent role, which emphasize the potential of targeting microglial cells for therapeutic purposes. From this perspective, papers on CNS autoimmunity, MS and MS' experimental models have largely contributed to our special issue. We have compiled extensive reviews and original papers about the initial immune-pathogenesis of MS as well as the immunology and oxidative stress underlying the disease. R. Bassil et al. provide a comprehensive reassessment on the important function of Notch signaling in the activation of T helper cells in experimental autoimmune encephalitis (EAE). They propose targeting Notch signaling as a potential strategy for MS therapy, but they also report the limitations of this approach due to the wide range of functions of Notch signaling, suggesting interesting new research avenues. Hernandez-N. Y. Pedro et al. discuss the innate immune responses occurring during the immune-pathogenesis of MS, which are hypothesized as a critical trigger of the chronic inflammatory response. In line with this, G. G. Ortiz et al. describe that oxidative stress and inflammation are important elements in the self-perpetuation inflammatory/immune cycle of MS. These pathways may contain targets that could be promising to avert the inflammatory responses in MS. Concluding this MS section from a practical point of view, S. F. Goncalves Zorzella-Pezavento et al contribute to settle down the potential controversy regarding the tuberculosis (TB) vaccination protocols, suggesting that TB vaccination does not trigger or worsen EAE pathology. Immunotherapy is also an important topic covered by this special issue. In the CNS, T-lymphocytes infiltrate into the inflamed brain parenchyma and the manipulation of different T-cell subpopulations may have beneficial effect on neurodegenerative disorders and diverse brain injuries. T cell infiltration is observed in many neurodegenerative diseases including Parkinson's and Alzheimer's disease and we are getting to know more aspects on how T-cells may affect neurodegeneration [5, 6]. Importantly, immunotherapy based on targeting T cells (Tregs and Th17 cells) for autoimmunity disorders and cancer is becoming a rising field of investigation [7]. However, the precise lymphocyte function in the damaged brain parenchyma is still poorly understood. S. Chen et al. propose, in a completed review, that the modulation of Tregs may have a favorable outcome for stroke patients. This is based on the fact that Tregs downregulates the excessive brain immune-reaction, which could be favorable for tissue restoration. In line with this, M. S. Zaborowski and Michalak describe how the appearance of paraneoplastic neurological syndromes may be linked with the certain antitumor responses triggered by T cells. Thus, authors suggest that the manipulation of specific subpopulation of T cells, pondering the balance between Tregs and CTLs through immunotherapy, could be beneficial for these neurological disorders. In this context of immunotherapy, the search for antigens that may trigger autoimmune responses in the brain is a crucial field of research. The review by A. Seppanen proposes collagen XVII as a candidate antigen. Autoantibodies against different domains of collagen XVII are found elevated in serum of patients within a wide range of neurological disorders as well as in skin autoimmune disorders, suggesting that an autoimmune response may be the common trigger of those disorders. On the other hand, increasing evidence indicates that immune system dysfunction may also be underlying a number of psychiatric disorders. K. Pathmanandavel et al. hypothesize in their thought-provoking review that some neuronal autoantibodies may be linked with psychiatric symptoms and could open new immunomodulatory approaches for neuropsychiatric disorders in the near future. We scarcely know the factors that trigger CNS diseases. It is thought that genetic and environmental factors, which include the exposure to chemical compounds or particular pathogens, may contribute to the appearance of these disorders. T. T. Win-Shwe et al. discuss an interesting point regarding the immune-related inflammation that environmental volatile compounds may induce. The review hypothesizes how environmental volatile elements may affect respiratory and immune system, having neurological consequences for the population. On the other hand, T. Hautala et al. report in our special issue how viruses, in this case Puumala virus, a northern European type of Hantavirus, are important entities that may seriously affect the CNS. Other pathogen infections, such as Trypanosoma brucei, which causes sleeping sickness, may also cause severe brain damage. Interestingly, D. N. Maranga et al., members of a research group based in Kenya, present in our special issue a research study performed in few monkeys showing that the increase of the proinflammatory cytokine IL-6 in spinal fluid may be a reliable marker to manage human African trypanosomiasis in areas where the diagnostic tools are very limited and derived neurological consequences are frequent. Lastly, A. P. Herman et al. describe how the inflammatory-mediated release of cytokines, such as IL-1β, may have consequences in the hypothalamic neurons affecting the reproductive system. This highlights that it is also important to consider the effect that neuroimmune interactions and neuroinflammation may have in many other systems. We hope this collection of papers is helpful for readers to understand better the peculiar relationship between the immune system and CNS and may be a stimulus to continue the research on this complex field. Carlos Barcia James Curtin Jeffrey Zirger Daniel Larocque

Adjuvant System 04 (AS04) combines the TLR4 agonist MPL (3-O-desacyl-4′-monophosphoryl lipid A) and aluminum salt. It is a new generation TLR-based adjuvant licensed for use in human vaccines. One of these vaccines, the human papillomavirus (HPV) vaccine Cervarix, is used in this study to elucidate the mechanism of action of AS04 in human cells and in mice. The adjuvant activity of AS04 was found to be strictly dependent on AS04 and the HPV Ags being injected at the same i.m. site within 24 h of each other. During this period, AS04 transiently induced local NF-κB activity and cytokine production. This led to an increased number of activated Ag-loaded dendritic cells and monocytes in the lymph node draining the injection site, which further increased the activation of Ag-specific T cells. AS04 was also found to directly stimulate those APCs in vitro but not directly stimulate CD4+ T or B lymphocytes. These AS04-induced innate responses were primarily due to MPL. Aluminum salt appeared not to synergize with or inhibit MPL, but rather it prolonged the cytokine responses to MPL at the injection site. Altogether these results support a model in which the addition of MPL to aluminum salt enhances the vaccine response by rapidly triggering a local cytokine response leading to an optimal activation of APCs. The transient and confined nature of these responses provides further supporting evidence for the favorable safety profile of AS04 adjuvanted vaccines.

BACKGROUND:The quaking viable (qk(v)) mice have uncompacted myelin in their central and peripheral nervous system (CNS, PNS). The qk gene encodes 3 major alternatively spliced isoforms that contain unique sequence at their C-terminus dictating their cellular localization. QKI-5 is a nuclear isoform, whereas QKI-6 and QKI-7 are cytoplasmic isoforms. The qk(v) mice harbor an enhancer/promoter deletion that prevents the expression of isoforms QKI-6 and QKI-7 in myelinating cells resulting in a dysmyelination phenotype. It was shown that QKI regulates the differentiation of oligodendrocytes, the myelinating cells of the CNS, however, little is known about the role of the QKI proteins, or RNA binding proteins in PNS myelination. METHODOLOGY/PRINCIPAL FINDINGS:To define the role of the QKI proteins in PNS myelination, we ectopically expressed QKI-6 and QKI-7 in primary rat Schwann cell/neuron from dorsal root ganglia cocultures. We show that the QKI isoforms blocked proliferation and promoted Schwann cell differentiation and myelination. In addition, these events were coordinated with elevated proteins levels of p27(KIP1) and myelin basic protein (MBP), markers of Schwann cell differentiation. QKI-6 and QKI-7 expressing co-cultures contained myelinated fibers that had directionality and contained significantly thicker myelin, as assessed by electron microscopy. Moreover, QKI-deficient Schwann cells had reduced levels of MBP, p27(KIP1) and Krox-20 mRNAs, as assessed by quantitative RT-PCR. CONCLUSIONS/SIGNIFICANCE:Our findings suggest that the QKI-6 and QKI-7 RNA binding proteins are positive regulators of PNS myelination and show that the QKI RNA binding proteins play a key role in Schwann cell differentiation and myelination.

The main challenge of gene therapy is to provide long-term, efficient transgene expression. Long-term transgene expression from first generation adenoviral vectors (Advs) delivered to the central nervous system (CNS) is elicited in animals not previously exposed to adenovirus (Ad). However, upon systemic immunization against Ad, transgene expression from a first generation Adv is abolished. High-capacity Advs (HC-Advs) provide sustained very long-term transgene expression in the brain, even in animals pre-immunized against Ad. In this study, we tested the hypothesis that a HC-Adv in the brain would allow for long-term transgene expression, for up to 1 year, in the brain of mice immunized against Ad prior to delivery of the vector to the striatum. In naïve animals, the expression of beta-galactosidase from Adv or HC-Adv was sustained for 1 year. In animals immunized prior to vector delivery, expression from a first generation Adv was abolished. These results point to a very long-term HC-Adv-mediated transgene expression in the brain, even in animals that had been immunized systemically against Ad before the delivery of HC-Adv into the brain. This study therefore indicates the utility of HC-Adv as a powerful gene therapy vector for chronic neurological disorders, even in patients who had been pre-exposed to Ad prior to gene therapy.

The breast tumor kinase (BRK) is a growth promoting non-receptor tyrosine kinase overexpressed in the majority of human breast tumors. BRK is known to potentiate the epidermal growth factor (EGF) response in these cells. Although BRK is known to phosphorylate the RNA-binding protein Sam68, the specific tyrosines phosphorylated and the exact role of this phosphorylation remains unknown. Herein, we have generated Sam68 phospho-specific antibodies against C-terminal phosphorylated tyrosine residues within the Sam68 nuclear localization signal. We show that BRK phosphorylates Sam68 on all three tyrosines in the nuclear localization signal. By indirect immunofluorescence we observed that BRK and EGF treatment not only phosphorylates Sam68 but also induces its relocalization. Tyrosine 440 was identified as a principal modulator of Sam68 localization and this site was phosphorylated in response to EGF treatment in human breast tumor cell lines. Moreover, this phosphorylation event was inhibited by BRK small interfering RNA treatment, consistent with Sam68 being a physiological substrate of BRK downstream of the EGF receptor in breast cancer cells. Finally, we observed that Sam68 suppressed BRK-induced cell proliferation, suggesting that Sam68 does indeed contain anti-proliferative properties that may be neutralized in breast cancer cells by phosphorylation.

Antibody-mediated capture of amyloid-beta (Aβ) in peripheral blood was identified as an attractive strategy to eliminate cerebral toxic amyloid in Alzheimer's disease (AD) patients and murine models. Alternatively, defective capacity of peripheral monocytes to engulf Aβ was reported in individuals with AD. In this report, we developed different approaches to investigate cellular uptake and phagocytosis of Aβ, and to examine how two immunological devices – an immunostimulatory Adjuvant System and different amyloid specific antibodies – may affect these biological events. Between one and thirteen months of age, APPswe X PS1.M146V (TASTPM) AD model mice had decreasing concentrations of Aβ in their plasma. In contrast, the proportion of blood monocytes containing Aβ tended to increase with age. Importantly, the TLR-agonist containing Adjuvant System AS01B primed monocytes to promote de novo Aβ uptake capacity, particularly in the presence of anti-Aβ antibodies. Biochemical experiments demonstrated that cells achieved Aβ uptake and internalization followed by Aβ degradation via mechanisms that required effective actin polymerization and proteolytic enzymes such as insulin-degrading enzyme. We further demonstrated that both Aβ-specific monoclonal antibodies and plasma from Aβ-immunized mice enhanced the phagocytosis of 1μm Aβ-coated particles. Together, our data highlight a new biomarker testing to follow amyloid clearance within the blood and a mechanism of Aβ uptake by peripheral monocytes in the context of active or passive immunization, and emphasize on novel approaches to investigate this phenomenon.

Quaking viable ( qk v ) mice fail to properly compact myelin in their central nervous systems. Although the defect in the qk v mice involves a mutation affecting the expression of the alternatively spliced qk gene products, their roles in myelination are unknown. We show that the QKI RNA binding proteins regulate the nuclear export of MBP mRNAs. Disruption of the QKI nucleocytoplasmic equilibrium in oligodendrocytes results in nuclear and perikaryal retention of the MBP mRNAs and lack of export to cytoplasmic processes, as it occurs in qk v mice. MBP mRNA export defect leads to a reduction in the MBP levels and their improper cellular targeting to the periphery. Our findings suggest that QKI participates in myelination by regulating the mRNA export of key protein components.