Major Histocompatibility Complex II Research Articles

Anti-lung cancer effect of myeloid and plasmacytoid dendritic cell combined vaccines loaded with tumor cell lysates in vitro

Objective: To investigate the feasibility of myeloid and plasmacytoid dendritic cell combined vaccines loaded with heat-treated Lewis lung cancer cell lysates for treatment of lung cancer in mice. Methods: Bone marrow cells were induced by the recombinant mouse fms-like tyrosine kinase receptor 3 ligand (rmFlt3-L) in vitro, myeloid dendritic cells (mDC) and plasmacytoid dendritic cells (pDC) were separated by magnetic beads. The mDC, pDC, and mDC∶pDC=1∶1 were stimulated with heat-treated Lewis lung cancer cell lysates, respectively. The effects of each group on stimulating of lymphocyte proliferation and inducing of T cell to kill tumor cells in vitro were compared. The alternations of the immunophenotypes of CD80, CD86, CD40 and major histocompatibility complex Ⅱ (MHC-Ⅱ) were detected by flow cytometry. The secretion of cytokines including interlukin-12 (IL-12), interlukin-6 (IL-6), and tumor necrosis factor α (TNF-α) were detected by enzyme-linked immunosorbent assay (ELISA). Results: The lymphocyte proliferation in mice stimulated with mDC+ pDC group loaded with heat-treated Lewis lung cancer cell lysates was 10.80±0.66, significantly higher than 8.63±0.65 of mDC group and 7.10±0.46 pDC group under the same culture conditions, respectively (P<0.05). When the ratio of effector cells: target cells (E∶T) was 10∶1, the killing rate of the mDC+ pDC group loaded with heat-treated tumor cell lysate was 31.68%±2.93%, significantly higher than 17.44%±0.97% of mDC group and 10.29%±1.33% of pDC group, respectively (P<0.05). When the ratio of E∶T was 20∶1, the killing rate of the mDC+ pDC group loaded with heat-treated tumor cell lysate was 54.77%±3.28%, significantly higher than 35.25%±1.51% of mDC group and 15.52%±0.73% of pDC group, respectively (P<0.05). When the ratio of E∶T was 40∶1, the killing rate of the mDC+ pDC group loaded with heat-treated tumor cell lysate was 73.01%±0.91%, significantly higher than 51.36%±0.58% of mDC group and 22.65%±1.28% of pDC group, respectively (P<0.05). With the rate of E∶T increased, the killing rate also increased. The mean fluorescence intensities of surface molecules including CD80, CD86, CD40 and MHC-Ⅱ of mDC: pDC=1 group pulsed with heat-treated Lewis lung cancer cell lysates were higher than those of mDC group and pDC group. The IL-6 cytokine concentrations of mDC+ pDC group, mDC group and pDC group loaded with heat-treated Lewis lung cancer cell lysates were (586.67±52.52) pg/ml, (323.33±67.14) pg/ml and (166.67±16.07) pg/ml, respectively. The concentrations of IL-12 in each group were (2 568.75±119.24) pg/ml, (2 156.25±120.55) pg/ml and (672.92±31.46) pg/ml, respectively. The concentrations of TNF-α in each group were (789.33±48.08) pg/ml, (584.89±116.49) pg/ml and (291.56±40.73) pg/ml, respectively. The concentrations of IL-6, IL-12 and TNF-α secreted by mDC+ pDC group were much higher than those of mDC group and pDC group under the same culture conditions (P<0.05). Conclusions: The mDCs and pDCs combined vaccines pulsed with heat-treated Lewis lung cancer cell lysates have synergistic effects on inducing of T lymphocyte proliferation and killing tumor cells in vitro. This synergistic anti-tumor effect is related with up-regulation of co-stimulatory molecules and increased secretion of cytokines.

Immunoglobulin-Like Transcript 5 Inhibits Macrophage-Mediated Bacterial Killing and Antigen Presentation During Sepsis.

Immunosuppression contributes to the mortality of sepsis. However, the underlying mechanism remains unclear. In the present study, we investigated the role of inhibitory receptor immunoglobulin-like transcript 5 (ILT5) in sepsis. We first screened the expression of ILT family members, and we found that ILT5 was dramatically up-regulated in the peripheral blood mononuclear cells from sepsis patients versus healthy donors. Knockdown of ILT5 by small interfering ribonucleic acid increased bacterial killing and reactive oxygen species production in THP-1 and RAW264.7 cells. Moreover, ILT5-expressing monocytes/macrophages exhibited lower expression of antigen-presenting molecules including major histocompatibility complex-II and CD80. In the in vitro coculture system with monocytes/macrophages, blockage of ILT5 facilitated Th1 proliferation and differentiation of CD4+ T cells. Furthermore, in vivo experiments demonstrated that pretreatment with ILT5 blocking peptide improved the survival and pulmonary pathology of septic mice. Together, our study identified ILT5 as an immunosuppressive regulator during sepsis, which may provide potential therapeutic strategy for sepsis.

Studying Organelle Dynamics in B Cells During Immune Synapse Formation.

Recognition of surface-tethered antigensby the B cell receptor (BCR)triggers the formation of an immune synapse (IS), where both signalingand antigen uptake are coordinated. IS formation involves dynamic actin remodeling accompanied by the polarized recruitment to the synaptic membrane of the centrosome and associated intracellular organelles such as lysosomes and the Golgi apparatus. Initial stages of actin remodeling allow B cells to increase their cell surface and maximize the quantity of antigen-BCR complexes gathered at the synapse. Under certain conditions, when B cells recognize antigens associated to rigid surfaces, this process is coupled to the local recruitment and secretion of lysosomes, which can facilitate antigen extraction. Uptaken antigensare internalized into specialized endo-lysosome compartments for processing into peptides, which are loaded onto major histocompatibility complex II (MHC-II) molecules for further presentation to T helper cells. Therefore, studying organelle dynamics associated with the formation of an IS is crucial to understanding how B cells are activated. In the present article we will discuss both imaging and a biochemical technique used to study changes in intracellular organelle positioning and cytoskeleton rearrangements that are associated with the formation of an IS in B cells.

Role of PAFR-MHC-II-CD38 interactions in infections

Abstract Infectious diseases are the most prevalent cause of deaths around the world. Chronic contagious ailments will account for seven out of ten deaths in the world by 2020. The most significant problem in the treatment of the infectious disease is the ever-increasing rate of resistance to drugs mounted by pathogens. Our preliminary results suggest that during antigen presentation the major histocompatibility complex II (MHC-II) engages two associate protein molecules CD38 and Platelet Activating Factor Receptor (PAFR). CD38 and PAFR elicit two counteractive signaling pathways in APCs resulting in the release of either proinflammatory cytokine such as IL-12 or anti-inflammatory cytokines such as IL-10 respectively. Pathogens show remarkable adaptability to the drugs by either metabolizing the drug molecules or by changing the surface profile to neutralize binding of drugs to the pathogens. Therefore, there is a requirement to find new treatment modalities that do not target the pathogens but aid the immune system to eliminate the pathogens. Our preliminary data further suggest that the use of associate molecule during antigen presentation decides the fate of pathogens such as Salmonella Typhimurium or Staphylococcus aureus in macrophages. For example, if MHC-II engages CD38 to signal inside the macrophages leading to reduced pathogen survival in macrophages and mice, while MHC-II-signaling via PAFR aid the pathogen survival in macrophages and mice. Our studies also suggest that combinatorial use of PAFR inhibitor (WEB2086) and antibiotics ampicillin reduced the growth of S. Typhimurium or S. aureus in macrophages and mice.

SAT-057 Endoplasmic Reticulum Aminopeptidase-1 Is a Novel Regulator of the Renin-Angiotensin-Aldosterone System and Blood Pressure Homeostasis

Endoplasmic Reticulum Aminopeptidase-1 (ERAP1) has been proposed to metabolize angiotensin II (ANGII), an effector molecule of the renin-ANGII-aldosterone (ALDO) system (RAAS) and key regulator of volume and blood pressure (BP) homeostasis. ERAP1 plays an important role in immune function as it cleaves cytokine cell surface receptors and HLA binding precursors thereby modulating their inclusion into major histocompatibility complex-I (MHC). Indeed, absence of ERAP1 in vivo shows MHC-I peptide presentation defects and decreased T regulatory cells in mice. However, the interaction between ERAP1 and RAAS remains unclear. We hypothesized that ERAP1 mediates the interaction between BP homeostasis and cardiorenal disease. We studied ERAP1 deficient heterozygous (ERAP1+/-) mice; a mouse with a phenotype likely more equivalent to what would be seen in human subjects, i.e. reduced, not absent, ERAP1. We observed that ERAP1+/- mice had 45% lower ERAP1 levels by western blot analyses in heart and kidney as compared to wild-type (WT) littermate control mice maintained on regular rodent chow (n= 10 of each type, p=0.029). We measured ANGII tissue levels by LC/MS and show increased levels in heart, kidney and aortas from ERAP1+/- as compared to WT control mice (P<0.05). On regular rodent chow, urinary 24 hr ALDO levels were increased in ERAP1+/- compared to WT mice (7.11 ±0.82 [WT, n=10] vs 9.92 ±0.88 ng/dL [ERAP1+/-, n=8], p=0.03) while plasma renin activity (PRA) were not significantly different between the two mouse types (65.3±3.2 [WT, n=10] vs 66.16±2.3 ng/ml*h [ERAP1+/-, n=8]). We also evaluated BP by tail cuff on mice maintained on regular rodent chow and showed increased systolic BP in ERAP1+/- as compared to WT mice, (122.8±0.8 [ERAP1, n=9] vs. 110.2±1.6 mmHg [WT, n=12], p=0.003). We then studied the effects of salt intake on BP in these mice. ERAP1+/- mice had increased salt sensitivity of BP, defined as systolic BP on high, 1.6% Na+ diet minus systolic BP on low, 0.03% Na+ diet, when compared to WT mice (SSBP: 8.5±2.3 [ERAP1, n=15] vs -0.2 ±1.6 mmHg [WT, n=15], p=0.0043). Similar results were obtained using continuous intraarterial BP monitoring by telemetry in freely moving mice (SSBP by telemetry: 6.8±1.2 [ERAP1, n=3] vs 0.4±0.7 mmHg [WT, n=2], p<0.001). We then studied renal hemodynamics using 2D and Color-Doppler imaging and showed that ERAP1+/- mice have increased peak-systolic velocity (PSV) and end-diastolic velocity (EDV) compared to WT mice on regular mouse chow (PSV: 412.6±38.6 [WT, n=4] vs 570.2±27.7 mm/s [ERAP1+/-, n=3] p=0.027; EDV: 127.9±9.8 [WT, n=4] vs 191.0±16.3 mm/s [ERAP1+/-, n=6], p=0.02). In conclusion, ERAP1 deficiency is associated with SSBP, likely secondary both to increased ALDO and altered renovascular function.

Characterizing αSyn Pre‐Formed Fibril Animal Models and Exploring Endpoints Beyond αSyn for Parkinson Disease

The second most common neurodegenerative disease, Parkinson disease (PD), has been characterized by conformational mutations in the synaptic protein, alpha‐synuclein (αSyn). In the disease state, the protein becomes increasingly insoluble and aggregates, forming Lewy bodies and impairing cellular function, as seen in PD. To further study this synucleinopathy in animals, a model has been established in which αSyn Lewy bodies are induced through the addition of exogenous αSyn preformed fibrils (PFFs) (Luk et al., 2009). These PFFs allow for the “seeding” of endogenous αSyn, producing the Lewy body neuropathology typical of PD. Our group used this seeding model in‐vivo, by injecting 4 uL and 8 uL doses of 2 ug/ul PFFs directly into the striatum of wild‐type rats, and histologically examining the spread of the pathology 30 days later. Pathological αSyn is correlated with phosphorylation of the protein at serine129 (pSer129), and thus all samples were stained for pSer129 using immunohistochemistry methods at the 30 day time point.Additional endpoints were also explored to better understand the biology of PD. Aside from αSyn accumulation, PD is also characterized by neurodegeneration that could be due to an inflammatory or immune response to the aggregated protein. To explore this relationship, immunohistochemistry staining was performed for glial marker Iba‐1 to visualize microglia and GFAP to visualize astrocytes. Staining was also performed for lymphocyte‐activation gene 3 (LAG3) and major histocompatibility complex II (MHCII) as markers for a potential immune response. These results will enrich understanding of additional inflammation and immune components of this rodent model of PD.Support or Funding InformationMerck Research LaboratoriesThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Biochemical and molecular biomarkers in integument biopsies of free-ranging coastal bottlenose dolphins from southern Brazil

Adverse effects of exposure to persistent organic pollutants (POPs) threaten the maintenance of odontocete populations. In southern Brazil, coastal bottlenose dolphins from the Laguna Estuarine System (LES) and Patos Lagoon Estuary (PLE) were sampled using remote biopsies during the winter and summer months. Levels of bioaccumulated POPs were measured in the blubber. The activities of glutathione S-transferase (GST), glutathione reductase (GR), glutathione peroxidase (GPx), and superoxide dismutase (SOD) were also quantified, as were the mRNA transcript levels of aryl hydrocarbon receptor (AhR), AhR nuclear translocator (ARNT), cytochrome P450 1A1-like (CYP1A1), metallothionein 2A (MT2A), GST-π, GPx-4, GR, interleukin 1 alpha (IL-1α), and major histocompatibility complex II (MHCII) in the skin. In general, levels of POPs were similar among sites, sexes, ages and seasons. For most animals, total polychlorinated biphenyl (ΣPCBs) levels were above the threshold level have physiological effects and pose risks to cetaceans. The best-fitting generalized linear models (GLMs) found significant associations between GR, IL-1α and GPx-4 transcript levels, SOD and GST activities, and total polybrominated diphenyl ether (ΣPBDEs) and pesticide levels. GLMs and Kruskal-Wallis analyses also indicated that there were higher transcript levels for most genes and lower GST activity in the winter. These results reinforce the need to consider the influence of environmental traits on biomarker values in wildlife assessments.

Innate-immune responses of tilapia (Oreochromis mossambicus) exposure to acute cold stress.

In this present study, Oreochromis mossambicus tilapia were transferred to cold water at 12°C for various time intervals (1, 4, 8, 24, and 48 hr) and its innate immune response was analyzed by studying cellular and humoral parameters. In vivo, alternative complement pathway activity in blood plasma was rapidly increased at 1 hr of cold water (12°C) exposure. Lysozyme activity and cortisol levels of plasma were increased at 4 and 1 hr, respectively. Surprisingly, only plasma cortisol levels remained unchanged through 24 hr of cold water transfer. Phagocytic ability, phagocytic capacity, and respiratory burst (RB) activity of head kidney (HK) leukocytes and splenocytes showed no any significant changes. In peripheral blood leukocytes, phagocytic capacity, and RB activity were increased at 24 hr of cold water exposure. The expressions of genes involved innate immunity in splenocytes and HK leukocytes of tilapia cold water exposure were analyzed, messenger RNA (mRNA) expressions of HSP70, HSP90, and immunoglobulin M failed to change upon exposure to cold stress. Major histocompatibility complex-I and II mRNAs were significantly increased in tilapia splenocytes at 1 hr of cold water transferred. Whereas myxovirus (Mx) expression was increased in splenocytes and HK leukocytes of tilapia after 1 hr of cold water exposed. Our result reveals that the exposure of tilapia to acute cold stress condition significantly enhances plasma acid phosphatase activity and both phagocytic capacity and RB activity. Furthermore, cold stress significantly stimulates Mx gene expression in splenocytes and HK leukocytes.

Dendritic cells treated with exogenous indoleamine 2,3-dioxygenase maintain an immature phenotype and suppress antigen-specific T cell proliferation

Indoleamine 2,3-dioxygenase (IDO), an intracellular enzyme responsible for catalyzing the rate limiting step of tryptophan catabolism, plays a critical role in immune cell suppression and tolerance. Indoleamine 2,3-dioxygenase-mediated depletion of the essential amino acid tryptophan increases susceptibility of T cells to apoptosis, while kynurenine and its downstream metabolites, such as 3-hydroxyanthranilic acid and quinolinic acid, have a direct cytotoxic effect on conventional effector T cells. Additionally, IDO-expressing antigen presenting cells (APCs) induce proliferation of regulatory T cells. When expressed by an APC, the immunosuppressive effects of IDO may act directly on the APC as well as indirectly upon local T cells. One approach to elicit immune tolerance or reduce inflammation therefore is to promote expression of IDO. However, this approach is constrained by several factors including the potential for deleterious biologic effects of conventional IDO-inducing agents such as interferon gamma (IFNγ), and the potential limitations of constitutive gene transfection. Alternatively, direct action of recombinant IDO enzyme supplied exogenously as a potential therapeutic in the extracellular space has not been investigated previously, and is the focus of this work. Results indicate exogenous recombinant human IDO supplementation influences murine dendritic cell (DC) maturation and ability to suppress antigen specific T cell proliferation. Following treatment, DCs were refractory to maturation by LPS as defined by co-stimulatory molecule expression (CD80 and CD86) and major histocompatibility complex II (MHC-II) expression. Dendritic cells exhibited skewing toward an anti-inflammatory cytokine release profile, with reduced secretion of IL-12p70 and maintained basal level of secreted IL-10. Notably, IDO-treated DCs suppressed proliferation of ovalbumin (OVA) antigen-specific CD4+ and CD8+ T cells in the presence of cognate antigen presentation in a manner dependent on active enzyme, as introduction of IDO inhibitor 1-methyl-tryptophan, restored T cell proliferation. Defined media experiments indicate a cumulative role for both tryptophan depletion and kynurenine presence, in the suppressive programming of DCs. In sum, we report that exogenously supplied IDO maintains immunoregulatory function on DCs, suggesting that IDO may have potential as a therapeutic protein for suppressive programming with application toward inflammation and tolerance.

Microglial nodules provide the environment for pathogenic T cells in human encephalitis

Microglia nodule formation is a common feature in inflammatory brain diseases mediated by T lymphocytes such as viral and paraneoplastic encephalitis, multiple sclerosis, and Rasmussen encephalitis (RE). However, its role has not been fully understood yet. We hypothesized that, in RE, microglial nodules provide an environment for the initiation of the later dominating T-cell cytotoxicity. In RE stage 0, small primary microglia nodules could be identified in the absence of T cells. These primary nodules showed inflammasome activation and endosomal Toll-like receptor upregulation. In stage 1, T cells migrate into the parenchyma and intermingle with microglial cells, thereby forming secondary nodules in which neurons are destroyed. Whole-genome transcriptome analysis at this point showed upregulation of several inflammatory pathways including interferon signaling and major histocompatibility complex-I signaling. Inflammatory profiles, like the ones observed in RE, could be induced upon TLR3 stimulation in neonatal microglial cell cultures. Taken together, our results point towards activation of endosomal TLRs, resulting in increased interferon signaling, inflammasome activation, and chemokine upregulation as early steps in RE pathogenesis. This activity sets the scene for subsequent infiltration of T cells and destruction of neurons. Similar to RE, this microglial microenvironment might be a crucial step in other T-cell-mediated inflammatory brain diseases.

The GLP-1 analog, liraglutide prevents the increase of proinflammatory mediators in the hippocampus of male rat pups submitted to maternal perinatal food restriction

BackgroundPerinatal maternal malnutrition is related to altered growth of tissues and organs. The nervous system development is very sensitive to environmental insults, being the hippocampus a vulnerable structure, in which altered number of neurons and granular cells has been observed. Moreover, glial cells are also affected, and increased expression of proinflammatory mediators has been observed. We studied the effect of Glucagon-like peptide-1 receptor (GLP-1R) agonists, liraglutide, which have very potent metabolic and neuroprotective effects, in order to ameliorate/prevent the glial alterations present in the hippocampus of the pups from mothers with food restriction during pregnancy and lactation (maternal perinatal food restriction—MPFR).MethodsPregnant Sprague-Dawley rats were randomly assigned to 50% food restriction (FR; n = 12) or ad libitum controls (CT, n = 12) groups at day of pregnancy 12 (GD12). From GD14 to parturition, pregnant FR and CT rats were treated with liraglutide (100 μg/kg) or vehicle. At postnatal day 21 and before weaning, 48 males and 45 females (CT and MPFR) were sacrificed. mRNA expression levels of interleukin-1β (IL1β), interleukin-6 (IL-6), nuclear factor-κβ, major histocompatibility complex-II (MHCII), interleukin 10 (IL10), arginase 1 (Arg1), and transforming growth factor (TGFβ) were assessed in the hippocampus by quantitative real-time polymerase chain reaction. Iba1 and GFAP-immunoreactivity were assessed by immunocytochemistry.ResultsThe mRNA expression IL1β, IL6, NF-κB, and MHCII increased in the hippocampus of male but not in female pups from MPFR. In addition, there was an increase in the percentage of GFAP and Iba1-immupositive cells in the dentate gyrus compared to controls, indicating an inflammatory response in the brain. On the other hand, liraglutide treatment prevented the neuroinflammatory process, promoting the production of anti-inflammatory molecules such as IL10, TGFβ, and arginase 1, and decreasing the number and reactivity of microglial cells and astrocytes in the hippocampus of male pups.ConclusionTherefore, the GLP-1 analog, liraglutide, emerges as neuroprotective drug that minimizes the harmful effects of maternal food restriction, decreasing neuroinflammation in the hippocampus in a very early stage.

Rejection of immunogenic tumor clones is limited by clonal fraction.

Tumors often co-exist with T cells that recognize somatically mutated peptides presented by cancer cells on major histocompatibility complex I (MHC-I). However, it is unknown why the immune system fails to eliminate immune-recognizable neoplasms before they manifest as frank disease. To understand the determinants of MHC-I peptide immunogenicity in nascent tumors, we tested the ability of thousands of MHC-I ligands to cause tumor subclone rejection in immunocompetent mice by use of a new 'PresentER' antigen presentation platform. Surprisingly, we show that immunogenic tumor antigens do not lead to immune-mediated cell rejection when the fraction of cells bearing each antigen ('clonal fraction') is low. Moreover, the clonal fraction necessary to lead to rejection of immunogenic tumor subclones depends on the antigen. These data indicate that tumor neoantigen heterogeneity has an underappreciated impact on immune elimination of cancer cells and has implications for the design of immunotherapeutics such as cancer vaccines.

Tissue-enhanced plasma proteomic analysis for disease stratification in amyotrophic lateral sclerosis

BackgroundIt is unclear to what extent pre-clinical studies in genetically homogeneous animal models of amyotrophic lateral sclerosis (ALS), an invariably fatal neurodegenerative disorder, can be informative of human pathology. The disease modifying effects in animal models of most therapeutic compounds have not been reproduced in patients. To advance therapeutics in ALS, we need easily accessible disease biomarkers which can discriminate across the phenotypic variants observed in ALS patients and can bridge animal and human pathology. Peripheral blood mononuclear cells alterations reflect the rate of progression of the disease representing an ideal biological substrate for biomarkers discovery.MethodsWe have applied TMTcalibrator™, a novel tissue-enhanced bio fluid mass spectrometry technique, to study the plasma proteome in ALS, using peripheral blood mononuclear cells as tissue calibrator. We have tested slow and fast progressing SOD1G93A mouse models of ALS at a pre-symptomatic and symptomatic stage in parallel with fast and slow progressing ALS patients at an early and late stage of the disease. Immunoassays were used to retest the expression of relevant protein candidates.ResultsThe biological features differentiating fast from slow progressing mouse model plasma proteomes were different from those identified in human pathology, with only processes encompassing membrane trafficking with translocation of GLUT4, innate immunity, acute phase response and cytoskeleton organization showing enrichment in both species. Biological processes associated with senescence, RNA processing, cell stress and metabolism, major histocompatibility complex-II linked immune-reactivity and apoptosis (early stage) were enriched specifically in fast progressing ALS patients. Immunodetection confirmed regulation of the immunosenescence markers Galectin-3, Integrin beta 3 and Transforming growth factor beta-1 in plasma from pre-symptomatic and symptomatic transgenic animals while Apolipoprotein E differential plasma expression provided a good separation between fast and slow progressing ALS patients.ConclusionsThese findings implicate immunosenescence and metabolism as novel targets for biomarkers and therapeutic discovery and suggest immunomodulation as an early intervention. The variance observed in the plasma proteomes may depend on different biological patterns of disease progression in human and animal model.

Co-delivery of CpG and antigen using hyaluronic acid bioconjugates-decorated nanoparticles to promote maturation and activation of dendritic cells

Objective To study the maturation and activation effects of hyaluronic acid (HA) modified polymer nanoparticles co-delivering adjuvants and antigens on mouse bone marrow dendritic cells (BMDCs). Methods HA-modified polylactic acid-glycolic acid copolymer (PLGA) and cationic lipid DOTAP were used as nanocarriers (DOTAP-PLGA) to co-deliver adjuvant CpG with model antigen ovalbumin (OVA). In the drug-loaded nanocarriers, CpG was covalently bound to the surface of HA, and OVA was physically blended into DOTAP-PLGA nanocarriers. The nanoparticles were characterized by transmission electron microscopy and dynamic light scattering. The in vitro release of CpG and OVA in the nanoparticles was investigated. The uptake and distribution of nanoparticles in mouse BMDCs were studied by flow cytometry and laser scanning confocal microscopy. The maturation and cytokine expression of mouse BMDCs were evaluated by flow cytometry and enzyme-linked immunosorbent assay, respectively. Results The CpG-HA-OVA-PLGA nanoparticles loading CpG and OVA were prepared. The average particle size was (305.1±2.2) nm and the polydispersity index was 0.203. A core-shell structure of the nanoparticles modified by HA was clearly observed by transmission electron microscopy. Cellular experiment results showed that CpG-HA-OVA-PLGA nanoparticles could be efficiently uptaken by mouse BMDCs, and promote lysosomal release of CpG and cytoplasmic delivery of antigen OVA. Compared with free OVA group and free OVA+CpG group, the CpG-HA-OVA-PLGA nanoparticles significantly up-regulated the expression of co-stimulatory molecules CD86 and CD40 (all P<0.01), major histocompatibility complex I (MHC-I) (P<0.01), and cytokine tumor necrosis factor-α (TNF-α) (P<0.01). Conclusions HA-modified CpG and OVA nanoparticle co-delivery vectors can effectively promote the maturation and activation of dendritic cells, which provides a basis for the development of novel vaccine vectors for the co-delivery of antigens and adjuvants. Key words: Nanoparticles; Co-delivery; CpG; Antigen; Dendritic cells

Age Influences Microglial Activation After Cuprizone-Induced Demyelination.

Multiple sclerosis (MS) is a chronic inflammatory CNS disease, which causes demyelinated lesions and damages white and gray matter regions. Aging is a significant factor in the progression of MS, and microglia, the immune cells of the CNS tissue, play an important role in all disease stages. During aging, microglia are functionally altered. These age-related changes probably already begin early and might influence the progression of CNS pathologies. The aim of the present study was to investigate whether microglia in the middle-aged CNS already react differently to demyelination. For this purpose, several microglia markers (ionized calcium binding adaptor molecule 1 (Iba-1), P2RY12, F4/80, CD68, major histocompatibility complex II (MHCII), macrophage receptor with collagenous structure (Marco), Translocator protein 18 kD (TSPO), CD206, and CD163) were analyzed in the acute cuprizone demyelination model in young (2-month-old) and middle-aged (10-month-old) mice. In addition, microglial proliferation was quantified using double-labeling with proliferating cell nuclear antigen (PCNA) and bromodeoxyuridine (BrdU), which was injected with the onset of remyelination. To compare age-related microglial changes during de- and remyelination in both gray and white matter, the hilus of the dorsal hippocampal dentate gyrus (DG) and the splenium of the corpus callosum (CC) were analyzed in parallel. Age-related changes in microglia of healthy controls were more pronounced in the analyzed gray matter region (higher levels of F4/80 and Marco as well as lower expression of CD68 in middle-aged mice). During de- and remyelination, a stronger increase of the microglial markers Iba-1, CD68 and TSPO was observed in the splenium of the younger groups. There was a significant reduction of P2RY12 during demyelination, however, this was age- and region-dependent. The induction of the anti-inflammatory markers CD206 and CD163 was stronger in the middle-aged group, but also differed between the two analyzed regions. De- and remyelination led to a significant increase in PCNA+ microglia only in young groups within the white matter region. The number of BrdU+ microglia was not changed during de- or remyelination. These results clearly show that microglia are already altered during middle-age and also react differently to CNS demyelination, however, this is highly region-dependent.

Targeting of the class II transactivator attenuates inflammation and neurodegeneration in an alpha-synuclein model of Parkinson\u2019s disease

BackgroundParkinson’s disease (PD) is characterized by intracellular alpha-synuclein (α-syn) inclusions, progressive death of dopaminergic neurons in the substantia nigra pars compacta (SNpc), and activation of the innate and adaptive immune systems. Disruption of immune signaling between the central nervous system (CNS) and periphery, such as through targeting the chemokine receptor type 2 (CCR2) or the major histocompatibility complex II (MHCII), is neuroprotective in rodent models of PD, suggesting a key role for innate and adaptive immunity in disease progression. The purpose of this study was to investigate whether genetic knockout or RNA silencing of the class II transactivator (CIITA), a transcriptional co-activator required for MHCII induction, is effective in reducing the neuroinflammation and neurodegeneration observed in an α-syn mouse model of PD.MethodsIn vitro, we utilized microglia cultures from WT or CIITA −/− mice treated with α-syn fibrils to investigate inflammatory iNOS expression and antigen processing via immunocytochemistry (ICC). In vivo, an adeno-associated virus (AAV) was used to overexpress α-syn in WT and CIITA −/− mice as a model for PD. Concurrently with AAV-mediated overexpression of α-syn, WT mice received CIITA-targeted shRNAs packaged in lentiviral constructs. Immunohistochemistry and flow cytometry were used to assess inflammation and peripheral cell infiltration at 4 weeks post transduction, and unbiased stereology was used 6 months post transduction to assess neurodegeneration.ResultsUsing ICC and DQ-ovalbumin, we show that CIITA −/− microglial cultures failed to upregulate iNOS and MHCII expression, and had decreased antigen processing in response to α-syn fibrils when compared to WT microglia. In vivo, global knock-out of CIITA as well as local knockdown using lentiviral shRNAs targeting CIITA attenuated MHCII expression, peripheral immune cell infiltration, and α-syn-induced neurodegeneration.ConclusionOur data provide evidence that CIITA is required for α-syn-induced MHCII induction and subsequent infiltration of peripheral immune cells in an α-syn mouse model of PD. Additionally, we demonstrate that CIITA in the CNS drives neuroinflammation and neurodegeneration. These data provide further support that the disruption or modulation of antigen processing and presentation via CIITA is a promising target for therapeutic development in preclinical animal models of PD.

Ionizing radiation reduces the capacity of activated macrophages to induce T-cell proliferation, but does not trigger dendritic cell-mediated non-targeted effects

Purpose: Previous investigations revealed influences of irradiation up to 2Gy on the cytokine secretion profile of inflammatory and peritoneal mouse macrophages (pMФ). This raised the question if those alterations impact on dendritic cells and consecutive T-cell responses. Further, the impact of irradiation directly on pMФ capacity to induce T-cell responses was analyzed.Materials and methods: pMФ were LPS-activated, irradiated and the expression of activation markers was assessed. Treated pMФ were co-incubated with T-cells to investigate proliferation. To verify modulating properties of pMФ supernatants isolated 24 h after irradiation, bone marrow-derived dendritic cells (BMDC) were co-incubated with supernatants and activation markers as well as the BMDC-induced proliferation of T-cells were measured.Results: pMФ showed a highly significantly decreased major histocompatibility complexII (MHCII) expression within a dose range from 0.7–2Gy. Further, the proliferation rate of cluster of differentiation 4+ (CD4+) T-cells was decreased after co-incubation particularly with 2 Gy irradiated pMФ. The co-incubation of BMDC with supernatants of activated, irradiated pMФ significantly reduced the CD40 expression, but did not impact on the BMDC-derived induction of T-cell proliferation.Conclusions: Inflammatory macrophages being exposed to irradiation have the potential to modulate consecutive adaptive immune reactions. But supernatants of irradiated macrophages do not influence the dendritic cells (DC)-mediated induction of T cell proliferation.

Counteracting roles of MHCI and CD8+ T cells in the peripheral and central nervous system of ALS SOD1G93A mice

BackgroundThe major histocompatibility complex I (MHCI) is a key molecule for the interaction of mononucleated cells with CD8+T lymphocytes. We previously showed that MHCI is upregulated in the spinal cord microglia and motor axons of transgenic SOD1G93A mice.MethodsTo assess the role of MHCI in the disease, we examined transgenic SOD1G93A mice crossbred with β2 microglobulin-deficient mice, which express little if any MHCI on the cell surface and are defective for CD8+ T cells.ResultsThe lack of MHCI and CD8+ T cells in the sciatic nerve affects the motor axon stability, anticipating the muscle atrophy and the disease onset. In contrast, MHCI depletion in resident microglia and the lack of CD8+ T cell infiltration in the spinal cord protect the cervical motor neurons delaying the paralysis of forelimbs and prolonging the survival of SOD1G93A mice.ConclusionsWe provided straightforward evidence for a dual role of MHCI in the peripheral nervous system (PNS) compared to the CNS, pointing out regional and temporal differences in the clinical responses of ALS mice. These findings offer a possible explanation for the failure of systemic immunomodulatory treatments and suggest new potential strategies to prevent the progression of ALS.

Abstract 317: Nlrc5 Positively Regulates Intimal Formation via Interaction With PPARã in Vascular Smooth Muscle Cells

Background: Innate immunity and vascular smooth muscle cells play crucial role in the development of vascular remodeling and intimal hyperplasia. NLR family CARD domain containing 5 (Nlrc5), known as a pattern recognition receptor, has been noted for its implication in immune disease in recent studies. However, whether Nlrc5 regulates intimal hyperplasia is still unclear. Aim: We aimed to determine the role of Nlrc5 in intimal hyperplasia and its underlying molecular mechanism. Methods and Results: We showed that an elevated expression of Nlrc5 in neointima induced by carotid ligation compared normal carotid artery. In human aortic smooth muscle cells (HASMCs), PDGF induced Nlrc5 expression and nuclear translocation. By utilizing Nlrc5 knockout mice, we found Nlrc5 deficiency prominently aggravated neointimal formation compared to wild type (WT) mice. On the contrary, gain of function of Nlrc5 based on local adenovirus injection attenuated intimal hyperplasia. The neointimal area was comparable in WT mice receiving either WT or Nlrc5-/- bone marrow transplantation, demonstrating that hematopoietic cell-derived Nlrc5 did not affect vascular hyperplasia. In vitro experiments unveiled that knockdown of Nlrc5 resulted in excessive proliferation, migration and dedifferentiation of HASMCs. Nevertheless, depletion of Nlrc5 in HASMCs did not influence proinflammatory cytokine excretion and immune activation reflected by major histocompatibility complex-I expression. Mechanistic study revealed that Nlrc5 bound to nuclear transcription factor PPARγ and positively regulated PPARγ luciferase activity, ensuing contributing to KLF4 ubiquitination and degradation. Furthermore, we generated five deletion constructs of Nlrc5 and identified that the functional domain containing DD and NACHT constructs was responsible for the interaction with PPARγ. Conclusions: Our study reveals that Nlrc5 alleviates intimal formation through interaction with PPARγ and inhibition of smooth muscle cell dedifferentiation, suggesting that Nlrc5 constitutes a novel molecular target for vascular remodeling.

Comprehensive Analysis of Alternative Splicing Across Tumors from 8,705 Patients

Our comprehensive analysis of alternative splicing across 32 The Cancer Genome Atlas cancer types from 8,705 patients detects alternative splicing events and tumor variants by reanalyzing RNA and whole-exome sequencing data. Tumors have up to 30% more alternative splicing events than normal samples. Association analysis of somatic variants with alternative splicing events confirmed known trans associations with variants in SF3B1 and U2AF1 and identified additional trans-acting variants (e.g., TADA1, PPP2R1A). Many tumors have thousands of alternative splicing events not detectable in normal samples; on average, we identified ≈930 exon-exon junctions ("neojunctions") in tumors not typically found in GTEx normals. From ClinicalProteomic Tumor Analysis Consortium data available for breast and ovarian tumor samples, we confirmed≈1.7 neojunction- and ≈0.6 single nucleotide variant-derived peptides per tumor sample that are also predicted major histocompatibility complex-I binders ("putative neoantigens").