Expression Of Immune Receptors Research Articles

Immunization of recombinant NS3 protein (protease region) of dengue virus induces high levels of CTLA-4 and apoptosis in splenocytes of BALB/c mice.

DENV infection outcomes depend on the host's variable expression of immune receptors and mediators, leading to either resolution or exacerbation. While the NS3 protein is known to induce robust immune responses, the specific impact of its protease region epitopes remains unclear. This study investigated the effect of recombinant NS3 protease region proteins from all four DENV serotypes on splenocyte activation in BALB/c mice (n = 5/group). Mice were immunized with each protein, and their splenocytes were subsequently stimulated with homologous antigens. We measured the expression of costimulatory molecules (CD28, CD80, CD86, CD152) by flow cytometry, along with IL-2 production, CD25 expression, and examined the antigen-specific activation of CD4 + and CD8 + T cells. Additionally, the expression of IL-1, IL-10, and TGF-β1 in splenocytes from immunized animals was assessed. Apoptosis was evaluated using Annexin V/PI staining and DNA fragmentation analysis. Stimulation of splenocytes from immunized mice triggered apoptosis (phosphatidylserine exposure and caspase 3/7 activation) and increased costimulatory molecule expression, particularly CD152. Low IL-2 production and low CD25 expression, as well as sustained expression of the IL-10 gene. These results suggest that these molecules might be involved in mechanisms by which the NS3 protein contributes to viral persistence and disease pathogenesis.

Two adjacent NLR genes conferring quantitative resistance to clubroot disease in Arabidopsis are regulated by a stably inherited epiallelic variation

Clubroot caused by the protist Plasmodiophora brassicae is a major disease affecting cultivated Brassicaceae. Using a combination of quantitative trait locus (QTL) fine mapping, CRISPR-Cas9 validation, and extensive analyses of DNA sequence and methylation patterns, we revealed that the two adjacent neighboring NLR (nucleotide-binding and leucine-rich repeat) genes AT5G47260 and AT5G47280 cooperate in controlling broad-spectrum quantitative partial resistance to the root pathogen P. brassicae in Arabidopsis and that they are epigenetically regulated. The variation in DNA methylation is not associated with any nucleotide variation or any transposable element presence/absence variants and is stably inherited. Variations in DNA methylation at the Pb-At5.2 QTL are widespread across Arabidopsis accessions and correlate negatively with variations in expression of the two genes. Our study demonstrates that natural, stable, and transgenerationally inherited epigenetic variations can play an important role in shaping resistance to plant pathogens by modulating the expression of immune receptors.

Insights into Repeated Renal Injury Using RNA-Seq with Two New RPTEC Cell Lines.

Renal proximal tubule epithelial cells (RPTECs) are a primary site for kidney injury. We created two RPTEC lines from CD-1 mice immortalized with hTERT (human telomerase reverse transcriptase) or SV40 LgT antigen (Simian Virus 40 Large T antigen). Our hypothesis was that low-level, repeated exposure to subcytotoxic levels of 0.25-2.5 μM cisplatin (CisPt) or 12.5-100 μM aflatoxin B1 (AFB1) would activate distinctive genes and pathways in these two differently immortalized cell lines. RNA-seq showed only LgT cells responded to AFB1 with 1139 differentially expressed genes (DEGs) at 72 h. The data suggested that AFB1 had direct nephrotoxic properties on the LgT cells. However, both the cell lines responded to 2.5 μM CisPt from 3 to 96 h expressing 2000-5000 total DEGs. For CisPt, the findings indicated a coordinated transcriptional program of injury signals and repair from the expression of immune receptors with cytokine and chemokine secretion for leukocyte recruitment; robust expression of synaptic and substrate adhesion molecules (SAMs) facilitating the expression of neural and hormonal receptors, ion channels/transporters, and trophic factors; and the expression of nephrogenesis transcription factors. Pathway analysis supported the concept of a renal repair transcriptome. In summary, these cell lines provide in vitro models for the improved understanding of repeated renal injury and repair mechanisms. High-throughput screening against toxicant libraries should provide a wider perspective of their capabilities in nephrotoxicity.

Differential expression of immune receptors in pediatric Acute Lymphoblastic Leukemia (ALL)

Abstract Acute Lymphoblastic Leukemia (ALL) represents the most common pediatric cancer. Significant improvements in chemotherapy regimens caused a leap in the 5-year survival rates to surpass 90%. However, relapse significantly lowers the chances of survival to less than 50% in some cases due to resistance developed by malignant cells to chemotherapy. Natural Killer (NK) cells are a promising immunotherapy alternative for their ability to recognize and kill malignant cells directly through activation or inhibition signals or through cytokine release. We have previously identified 2B4 (SLAMF4), CS1 (SLAMF7) and LLT1 that can activate or inhibit NK cells upon the interaction with their ligands. In this study, the expression of 2B4, CS1, LLT1, NKp30 and NKp46 was determined in pediatric ALL and healthy subjects. Whole blood was collected from 42 pediatric ALL subjects before and after induction chemotherapy and the surface expression of receptors/ligand on T-cells, B-cells, monocytes, and NK-cells was compared with blood samples from 20 healthy subjects. The flow cytometry data showed a significant increase in CS1 and NKp46 expression on monocytes and LLT1 in T cells and monocytes of ALL subjects before treatment. A decrease of LLT1, 2B4, CS1 and NKp46 on T-cells of ALL subjects was also observed after treatment along with elevated expression of LLT1 on ALL subjects’ NK-cells before and after treatment. Furthermore, mRNA data showed altered expression of receptors in ALL subjects pre and post induction chemotherapy treatment. The results indicate that the differential expression of the receptors/ligand may play a role in the T cell and NK-cell mediated immune surveillance of pediatric ALL. Supported by funds from Cancer Research Foundation of North Texas and Team Connor Childhood Cancer Foundation

New participants and pathways in immune receptor trafficking

Expression of immune receptors at the cell surface is a key determinant of immunity. Understanding the molecular mechanisms that govern receptor expression patterns will enable exploitation of receptors in settings of immunotherapy or vaccination. Here, we have aimed to analyse the molecular mechanisms and immune consequences of immune receptor trafficking. One mechanism by which receptor expression is controlled is ubiquitination. Ubiquitination regulates the cell surface display of MHC II (major antigen presenting molecule) and CD86 (co-stimulatory molecule). First, we have identified the ubiquitin codes (ubiquitin chain linkage types) associated with MHC II isolated from primary murine immune cells. Second, we have identified a new role for MHC II ubiquitination in the regulation of dendritic cell homeostasis that involves a surprising intersection between innate and adaptive immunity. Third, we have performed a genome-wide CRISPR/Cas9 screen to identify new molecular participants in ubiquitin-mediated immune trafficking. Finally, we have analysed the trafficking of dendritic cell receptors that are of significant interest for antibody-targeted vaccination, including C-type lectin DEC-205. We have conducted CRISPR/Cas9 screens to identify new genes that regulate both the expression and the internalisation of DEC-205 from the cell surface. In summary, our analysis provides significant advances to understanding how immune receptors are trafficked inside cells with important consequences for immunity.

Concentration-Dependent Decitabine Effects on Primary NK Cells Viability, Phenotype, and Function in the Absence of Obvious NK Cells Proliferation-Original Article.

Acute myeloid leukemia (AML) cells can evade innate immune killing by modulating natural killer (NK) cells receptors and their cognate ligands in tumor cells, thus it may be possible to restore proper expression of immune receptors or ligands with immune sensitive drugs. Decitabine, as a hypomethylation agent, was approved for the treatment of AML and myelodysplastic syndrome. While clinical responses were contributed by epigenetic effects and the induction of cancer cell apoptosis, decitabine also has immune-mediated anti-tumor effects. After exposure to various concentration of decitabine for 24 h, the primary NK cells (AML-NK cells) cytotoxicity and receptor expression (NKG2D and NKp46) displayed parabola-shaped response, while U-shaped response was seen in cytokine release (IFN-γ and IL-10), and these effects were regulated by ERK and STAT3 phosphorylation level. Furthermore, AML-NK cells function displayed different response when the competitive MEK and STAT3 inhibitors applied respectively. Thus, we could conclude that the different dose of decitabine makes various effects on AML-NK cells function and receptors expression.

Characterization of Cellular Heterogeneity and an Immune Subpopulation of Human Megakaryocytes.

Megakaryocytes (MKs) and their progeny platelets function in a variety of biological processes including coagulation, hemostasis, inflammation, angiogenesis, and innate immunity. However, the divergent developmental and cellular landscape of adult MKs remains mysterious. Here, by deriving the single‐cell transcriptomic profiling of MKs from human adult bone marrow (BM), cellular heterogeneity within MKs is unveiled and an MK subpopulation with high enrichment of immune‐associated genes is identified. By performing the dynamic single‐cell transcriptomic landscape of human megakaryopoiesis in vitro, it is found that the immune signatures of MKs can be traced back to the progenitor stage. Furthermore, two surface markers, CD148 and CD48, are identified for mature MKs with immune characteristics. At the functional level, these CD148+CD48+ MKs can respond rapidly to immune stimuli both in vitro and in vivo, exhibit high‐level expression of immune receptors and mediators, and may function as immune‐surveillance cells. The findings uncover the cellular heterogeneity and a novel immune subset of human adult MKs and should greatly facilitate the understanding of the divergent functions of MKs under physiological and pathological conditions.

Physiological substrates and ontogeny-specific expression of the ubiquitin ligases MARCH1 and MARCH8

MARCH1 and MARCH8 are ubiquitin ligases that control the expression and trafficking of critical immunoreceptors. Understanding of their function is hampered by three major knowledge gaps: (i) it is unclear which cell types utilize these ligases; (ii) their level of redundancy is unknown; and (iii) most of their putative substrates have been described in cell lines, often overexpressing MARCH1 or MARCH8, and it is unclear which substrates are regulated by either ligase in vivo. Here we address these questions by systematically analyzing the immune cell repertoire of MARCH1- or MARCH8-deficient mice, and applying unbiased proteomic profiling of the plasma membrane of primary cells to identify MARCH1 and MARCH8 substrates. Only CD86 and MHC II were unequivocally identified as immunoreceptors regulated by MARCH1 and MARCH8, but each ligase carried out its function in different tissues. MARCH1 regulated MHC II and CD86 in professional and “atypical” antigen presenting cells of hematopoietic origin, including neutrophils, eosinophils and monocytes. MARCH8 only operated in non-hematopoietic cells, such as thymic and alveolar epithelial cells. Our results establish the tissue-specific functions of MARCH1 and MARCH8 in regulation of immune receptor expression and reveal that the range of cells constitutively endowed with antigen-presentation capacity is wider than generally appreciated.

Differential expression of immune receptors in two marine sponges upon exposure to microbial-associated molecular patterns

The innate immune system helps animals to navigate the microbial world. The response to microbes relies on the specific recognition of microbial-associated molecular patterns (MAMPs) by immune receptors. Sponges (phylum Porifera), as early-diverging animals, provide insights into conserved mechanisms for animal-microbe crosstalk. However, experimental data is limited. We adopted an experimental approach followed by RNA-Seq and differential gene expression analysis in order to characterise the sponge immune response. Two Mediterranean species, Aplysina aerophoba and Dysidea avara, were exposed to a “cocktail” of MAMPs (lipopolysaccharide and peptidoglycan) or to sterile artificial seawater (control) and sampled 1 h, 3 h, and 5 h post-treatment for RNA-Seq. The response involved, first and foremost, a higher number of differentially-expressed genes in A. aerophoba than D. avara. Secondly, while both species constitutively express a diverse repertoire of immune receptors, they differed in their expression profiles upon MAMP challenge. The response in D. avara was mediated by increased expression of two NLR genes, whereas the response in A. aerophoba involved SRCR and GPCR genes. From the set of annotated genes we infer that both species activated apoptosis in response to MAMPs while in A. aerophoba phagocytosis was additionally stimulated. Our study assessed for the first time the transcriptomic responses of sponges to MAMPs and revealed conserved and species-specific features of poriferan immunity as well as genes potentially relevant to animal-microbe interactions.

Type I IFN signature in childhood-onset systemic lupus erythematosus: a conspiracy of DNA- and RNA-sensing receptors?

BackgroundChildhood-onset systemic lupus erythematosus (cSLE) is an incurable multi-systemic autoimmune disease. Interferon type I (IFN-I) plays a pivotal role in the pathogenesis of SLE. The objective of this study was to assess the prevalence of the IFN-I signature and the contribution of cytosolic nucleic acid receptors to IFN-I activation in a cohort of primarily white cSLE patients.MethodsThe IFN-I score (positive or negative), as a measure of IFN-I activation, was assessed using real-time quantitative PCR (RT-PCR) expression values of IFN-I signature genes (IFI44, IFI44L, IFIT1, Ly6e, MxA, IFITM1) in CD14+ monocytes of cSLE patients and healthy controls (HCs). Innate immune receptor expression was determined by RT-PCR and flow cytometry. To clarify the contribution of RNA-binding RIG-like receptors (RLRs) and DNA-binding receptors (DBRs) to IFN-I activation, peripheral blood mononuclear cells (PBMCs) from patients were treated with BX795, a TANK-binding kinase 1 (TBK1) inhibitor blocking RLR and DBR pathways.ResultsThe IFN-I signature was positive in 57% of cSLE patients and 15% of the HCs. Upregulated gene expression of TLR7, RLRs (IFIH1, DDX58, DDX60, DHX58) and DBRs (ZBP-1, IFI16) was observed in CD14+ monocytes of the IFN-I-positive cSLE patients. Additionally, RIG-I and ZBP-1 protein expression was upregulated in these cells. Spontaneous IFN-I stimulated gene (ISG) expression in PBMCs from cSLE patients was inhibited by a TBK1-blocker.ConclusionsIFN-I activation, assessed as ISG expression, in cSLE is associated with increased expression of TLR7, and RNA and DNA binding receptors, and these receptors contribute to IFN-I activation via TBK1 signaling. TBK1-blockers may therefore be a promising treatment target for SLE.

Distinct and common expression of receptors for inflammatory mediators in vagal nodose versus jugular capsaicin-sensitive/TRPV1-positive neurons detected by low input RNA sequencing.

Capsaicin-sensitive sensory C-fibers derived from vagal ganglia innervate the visceral organs, and respond to inflammatory mediators and noxious stimuli. These neurons play an important role in maintenance of visceral homeostasis, and contribute to the symptoms of visceral inflammatory diseases. Vagal sensory neurons are located in two ganglia, the jugular ganglia (derived from the neural crest), and the nodose ganglia (from the epibranchial placodes). The functional difference, especially in response to immune mediators, between jugular and nodose neurons is not fully understood. In this study, we microscopically isolated murine nodose and jugular capsaicin-sensitive / Trpv1-expressing C-fiber neurons and performed transcriptome profiling using ultra-low input RNA sequencing. RNAseq detected genes with significantly differential expression in jugular and nodose neurons, which were mostly involved in neural functions. Transcriptional regulators, including Cited1, Hoxb5 and Prdm12 showed distinct expression patterns in the two C-fiber neuronal populations. Common and specific expression of immune receptor proteins was characterized in each neuronal type. The expression of immune receptors that have received little or no attention from vagal sensory biologists is highlighted including receptors for certain chemokines (CXCLs), interleukins (IL-4) and interferons (IFNα, IFNγ). Stimulation of immune receptors with their cognate ligands led to activation of the C-fibers in isolated functional assays.

The effects of voluntary wheel running on neuroinflammatory status: Role of monocyte chemoattractant protein-1.

The health benefits of exercise and physical activity (PA) have been well researched and it is widely accepted that PA is crucial for maintaining health. One of the mechanisms by which exercise and PA exert their beneficial effects is through peripheral immune system adaptations. To date, very few studies have looked at the regulation of neuroimmune reactions in response to PA. We studied the effect of voluntary wheel running (VWR) on pro- and anti-inflammatory cytokine levels, patterns of glial cell activation and expression of immune receptors in the brains of female C57BL/6 mice. By using homozygous monocyte chemoattractant protein (MCP)-1 null mice, we investigated the role of this key immunoregulatory cytokine in mediating VWR-induced neuroinflammatory responses. We demonstrated that, compared to their sedentary counterparts, C57BL/6 mice exposed for seven weeks to VWR had increased levels of pro- and anti-inflammatory cytokines, markers of glial cell activation and a trend towards increased expression of toll-like receptor (TLR) 4 in the brain. Measurements of serum cytokines revealed that the alterations in brain cytokine levels could not be explained by the effects of PA on peripheral cytokine levels. We propose that the modified neuroimmune status observed in the VWR group represents an activated immune system, as opposed to a less activated immune system in the sedentary group. Since MCP-1 knockout mice displayed differing patterns of pro- and anti-inflammatory brain cytokine expression and glial activation when compared to their wild-type counterparts, we concluded that the effects of VWR on neuroimmune reactions may be modulated by MCP-1. These identified immunomodulatory effects of PA in the brain could contribute to the observed positive relationship between physically active lifestyles and a reduced risk for a number of neurodegenerative diseases that possess a significant neuroinflammatory component.

Defining the Immune Checkpoint Landscape in Patients (pts) with Acute Myeloid Leukemia (AML)

Introduction: Immune checkpoint therapy has been successful in many solid tumors andHodgkins lymphoma.The expression and clinical implications of immune checkpoints in the tumor microenvironment in patients (pts) with AML remain poorly defined.Identification and targeting of immune checkpoints in AMLwill guide the rational selection of specific antibodies for clinical trials.Methods:Between March, 2015 and April 2016 we performed17-colormulti-parameter flow-cytometry (MFC) on bone marrows (BM) aspirate from 74 pts with AML (36 untreated AML, 38 relapsed AML). We evaluated expression of immune receptors (inhibitory receptors: PD1, CTLA4, LAG3, TIM3, and activating receptors GITR, OX40, 41BB, ICOS) on T-cell subsets: CD4 T effector cells [Teff]: CD3+CD4+CD127lo/+Foxp3, CD4 T regulatory cells [Treg]: CD3+CD4+CD127-Foxp3+, and CD8 T cells. AML blasts were assessed for both immune receptors (as above), and ligands (4-1BBL, B7-1, B7-2, ICOSL, PDL-1, PDL-2, OX40L).Eight healthy human BMs were used as control. These analyses were performed on freshly collected BM aspirate by the M. D. Anderson Cancer Center Immunotherapy Platform.Results: A total of 74 pts were evaluated. Our analyses show that T-cell populations are preserved in BM of untreated and relapsed AML patients. In untreated AML BM, median frequency of live CD3+, CD4+, and CD8+ T cells were 16.5%, 10.4%, and 5.1% respectively. In relapsed AML BM, median frequency of live CD3+, CD4+, and CD8+ T cells were 19.2%, 9.5%, and 4.5% respectively.The expression of the8 costimreceptors and 7 ligands varied significantly between individual AML pts. On analysis of all markers PD-1 and OX40 emerged as potential checkpoint receptors of importance in AML.The baseline expression of OX40 and PD-1 on the CD8, Teff, and Treg cells are shown in Figure 1. All T cell subpopulations had significantly higher PD1 expression in relapsed AML (P<0.006) and untreated AML (P<0.05) compared to healthy controls. There were no significant differences in PD1 expression between relapsed and untreated AML. OX40 expression was significantly higher on all T cell subpopulations in relapsed AML pts (P<0.006) and specifically on Teff cells in untreated AML (P<0.006) when compared to healthy controls. Moreover, Treg cells in relapsed AML pts expressed significantly higher OX40 compared to untreated AML.The expression of costimulatory receptors and ligands differed significantly between BM and PB for the same time-point in the same pt (PB data not shown here).We compared the T-cell repertoire and checkpoint receptor/ligand expression to baseline clinical characteristics (age, cytogenetics, therapy-related status) and outcomes. Pts with untreated AML with adverse karyotype had a higher expression of PDL2 on AML blasts (P=0.07) in the BM. Pts with untreated AML who responded to induction therapy had a trend to higher Teff live cells (P=0.2), and lower OX40 on CD8 cells (P=0.04) in the BM. In relapsed AML higher T-eff live cells (P=0.04) and higher OX40 expression on CD8 (P=0.009) in BM were associated with improved survival.Conclusions: Total T-cell and T-cell subset populations are preserved in the BM of pts with untreated and relapsed AML and may be manipulated by checkpoint antibodies. Clinically targetable checkpoint receptors, specifically PD1 and OX40 are overexpressed in the BM of pts with AML and may be manipulated to unleash T-cells. [Display omitted] DisclosuresDaver:BMS: Research Funding; Kiromic: Research Funding; Karyopharm: Honoraria, Research Funding; Pfizer: Consultancy, Research Funding; Sunesis: Consultancy, Research Funding; Otsuka: Consultancy, Honoraria; Ariad: Research Funding. Cortes:ARIAD: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Research Funding. Konopleva:Calithera: Research Funding; Cellectis: Research Funding.

Defining the Immune Checkpoint Landscape in the Bone Marrow and Peripheral Blood of Patients with Chronic Lymphocytic Leukemia (CLL)

Background: Limited data is available on expression levels of checkpoint receptors, respective ligands, and other immune markers in patients with CLL (Ramsay et al. Blood 2012). Checkpoint blockade has been a successful therapy of many cancers including melanoma, and more recently, Hodgkin's lymphoma. Understanding expression patterns of checkpoint receptors and ligands may help in the clinical development of checkpoint blockade as a therapy for patients with CLL.Methods: Between September 2015 and April 2016, we performed 17-color multi-parameter flow-cytometry (MFC) in paired peripheral blood (PB) and bone marrow (BM) samples from 30 patients with CLL who presented as new patients for evaluation at MDACC. Patients may have received prior CLL therapy. We evaluated expression of immune receptors (inhibitory receptors: PD1, CTLA4, LAG3, TIM3; activating receptors: GITR, OX40, 41BB, ICOS) on T cell subsets: CD4 T effector cells [Teff]: CD3+CD4+CD127lo/+Foxp3-, CD4 T regulatory cells [Treg]: CD3+CD4+CD127-Foxp3+, and CD8 T cells. CLL cells were assessed for both immune receptors (as above), and ligands (4-1BBL, B7-1, B7-2, ICOSL, PDL-1, PDL-2, OX40L). These analyses were performed on freshly collected PB and BM samples by the M. D. Anderson Cancer Center Immunotherapy Platform.Results: A total of 30 patients with CLL were enrolled. The median age was 66 years (range, 35-83). Nine were women. Nineteen were treatment-naive. Prognostic markers included FISH [del(17p) = 6; del(13q) = 9, del(11q) = 4, trisomy 12 = 3, negative = 8]. IGHV mutation status was available for 19 patients (13 unmutated IGHV, 6 mutated IGHV). B2M was ≥3.5 in 11 pts.Baseline expression of costimulatory receptors in CD8 T cells in the marrow, and of the ligands in CLL cells in the marrow is shown in Figure 1. In paired PB and BM sample analysis, there was a high correlation between expression level of PD1 on Treg (Pearson correlation, r = 0.90, p<0.00001), Teff (r = 0.87, p<0.00001), CD8+ cells (r = 0.80, p<0.00001), and CLL cells (r = 0.75, p<0.00001). PD-L1 expression on CLL cells was moderately correlated between PB and BM (r = 0.57, p<0.001).Patients with prior therapy had significantly higher expression of PDL1 on the CLL cells in both PB and BM (p=0.01 and p=0.002, respectively) compared to previously untreated patients. OX40 expression on CD8 cells was significantly higher in both PB and BM in previously treated patients (compared to previously untreated patients). Patients with unmutated IGHV (p = 0.003) and del17p (p = .03) had higher PDL1 expression on CLL cells in the marrow.Conclusions: There is a strong correlation in the expression levels of PD1 on various T cell subsets between PB and BM. Clinically targetable checkpoint receptors including PD1, OX40, CTLA4, and ICOS are consistently expressed across patients with CLL, and present on cells in both PB and BM. [Display omitted] DisclosuresJain:BMS: Research Funding; Abbvie: Research Funding; ADC Therapeutics: Consultancy, Honoraria, Research Funding; Novimmune: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria, Research Funding; Genentech: Research Funding; Infinity: Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding; Incyte: Research Funding; Novartis: Consultancy, Honoraria; Servier: Consultancy, Honoraria; Seattle Genetics: Research Funding; Celgene: Research Funding. Burger:Roche: Other: Travel, Accommodations, Expenses; Pharmacyclics, LLC, an AbbVie Company: Research Funding; Janssen: Consultancy, Other: Travel, Accommodations, Expenses; Portola: Consultancy; Gilead: Research Funding. Thompson:Pharmacyclics: Consultancy, Honoraria. Daver:Otsuka: Consultancy, Honoraria; Ariad: Research Funding; Karyopharm: Honoraria, Research Funding; BMS: Research Funding; Sunesis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Kiromic: Research Funding. Wierda:Acerta: Research Funding; Genentech: Research Funding; Gilead: Research Funding; Novartis: Research Funding; Abbvie: Research Funding.

Reduced Expression of HLA-DR on Monocytes During Severe Respiratory Syncytial Virus Infections.

Respiratory syncytial virus (RSV) is a common cause of bronchiolitis in infants with a wide spectrum of disease severity. Besides environmental and genetic factors, it is thought that the innate immune system plays a pivotal role. The aim of this study was to investigate the expression of immune receptors on monocytes and the in vitro responsiveness from infants with severe RSV infections. Peripheral blood mononuclear cells (PBMCs) from infants with RSV infections were isolated. Classical, intermediate and nonclassical monocytes were immunophenotyped for the expression of CD14, CD16, human leukocyte antigen (HLA)-ABC and HLA-DR. PBMCs were stimulated with lipopolysaccharide to determine the secretion of tumor necrosis factor and interleukin (IL)-10 with enzyme-linked immunosorbent assay. During RSV infection, intermediate monocytes are increased in the peripheral blood, whereas classical and nonclassical monocytes are reduced. The expression of CD14 and HLA-ABC is increased on monocytes, whereas the expression of HLA-DR is suppressed. Low HLA-DR expression is correlated with increased disease severity. PBMCs from infants with severe RSV infections show an impaired IL-10 response in vitro. Phenotyping subpopulations of monocytes combined with in vitro responsiveness reveals significant differences between nonsevere and severe RSV infections. Reduced HLA-DR expression and impaired IL-10 production in vitro during severe RSV infections indicate that an imbalanced innate immune response may play an important role in disease severity.

The Ground State of Innate Immune Responsiveness Is Determined at the Interface of Genetic, Epigenetic, and Environmental Influences

Monocytes and macrophages form the major cellular component of the innate immune system, with roles in tissue development, homeostasis, and host defense against infection. Environmental factors were shown to play a significant part in determining innate immune responsiveness, and this included systemic conditions, such as circulating glucose levels, gut microflora, time of year, and even diurnal rhythm, which had a direct impact on innate immune receptor expression. Although the underlying molecular processes are just beginning to emerge, it is clear that environmental factors may alter epigenetic states of peripheral blood monocytes and resident tissue macrophages. We conclude that some measure of cellular ground state must become an essential part of the analysis of myeloid responsiveness or infectious susceptibility.

Role of intestinal fibroblasts in inflammation across the intestinal epithelium (488.10)

Fibroblasts are mesenchymal cells that produce extracellular matrix (ECM) proteins and play a major role in tissue repair (i.e., resolution of inflammation). Fibroblasts can affect cells in the local environment through the release of cytokines, growth and differentiation factors. CD36 is a membrane‐bound and scavenger receptor that is an indicator of inflammation and interacts with TLR‐2, 4, and 6. We investigated intestinal fibroblasts for expression of immune receptors and for a role of CD36 in intestinal inflammation using fibroblasts exposed to a series of cytokines. Analysis of supernatants revealed the presence of IL‐8 (potent chemoattractant for polymorphonuclear leukocytes, (PMN)) in IL‐6 treated normal and CD36‐deficient fibroblasts. However, results reveal a statistically significant reduction in PMN migration using IL‐6 treated CD36‐deficient cells. Kinase inhibitor experiments reveal that PI3 kinase pathway may be involved in the lL‐6 mediated release of IL‐8 from fibroblasts. Fibroblasts exposed to IL‐8 have been reported to display a myofibroblast‐like phenotype. Differences in cell adhesion and migration were also revealed using CD36‐deficient fibroblasts. Results show that fibroblasts can play a major role in the inflammatory responses in the intestine, PI3 kinase may be involved, and highlight CD36 as a target‐receptor that may modulate the inflammatory responses of fibroblasts.Grant Funding Source: NSF‐RII, NIH/NIDDK 5T35 DK007431

Innate Immune Receptor Expression in Peri‐Implant Tissues of Patients With Different Susceptibility to Periodontal Diseases

Although inflammation mediates the pathogenesis of periodontal diseases, the effects of innate immune responses on implant therapies have not been evaluated. Innate immune receptors, including toll-like-receptors (TLRs) and the receptor for advanced glycated end-products (RAGE), are upregulated within inflamed gingiva and are responsible for initiation of detrimental host responses. The aim of this study is to compare the expression of TLR2, TLR4, and RAGE in gingival tissues from participants susceptible to periodontitis and participants not susceptible to periodontitis before and after implant therapy. Periodontally healthy participants received implant therapy for non-periodontal edentulism. Participants susceptible to periodontitis were diagnosed with chronic periodontitis prior to implant therapy. Gingival biopsies were collected from edentulous ridges before implant installation and from peri-implant mucosa 2 months after treatment. Histology, real-time PCR, and Western blot were used to evaluate levels of inflammatory infiltrate, TLR2, TLR4, and RAGE expression. Before implant therapy, elevated levels of RAGE were detected in gingival tissues from participants susceptible to periodontitis when compared to those from participants with healthy periodontiums, whereas no differences in the expression of TLR2 or TLR4 were detected. After implant therapy, there was an upregulation of RAGE and TLR4 levels that coincided with a downregulation of TLR2 levels in biopsies from participants susceptible to periodontitis. Levels of RAGE and TLR4 remained unchanged in biopsies from participants with healthy periodontiums, whereas TLR2 levels were significantly upregulated. Histologically, post-implant biopsies from participants susceptible to periodontitis displayed higher levels of inflammatory infiltrate. Elevated levels of inflammatory potential were found after implant therapy in participants susceptible to periodontitis.

The Clinical Impact of Vitamin D in Children With Atopic Dermatitis

The Clinical Impact of Vitamin D in Children With Atopic Dermatitis

Comparative evaluation of T11 target structure and its deglycosylated derivative nullifies the importance of glycan moieties in immunotherapeutic efficacy

Sheep red blood cell (SRBC), a non-specific biological response modifier that has long been used as a classical antigen, has been shown to exert an immunomodulatory and anti-tumor activities in experimental animals. The active component of SRBC, which is responsible for such effects, was found to be a cell surface acidic glycoprotein molecule, known as T11 target structure (T11TS). In the present study, T11TS was isolated and purified to homogeneity using a five-step protocol involving isolation of sheep erythrocyte membrane from packed cell volume, 20% ammonium sulfate cut of the crude membrane proteins mixture, immunoaffinity purification using mouse anti-sheep CD58 mAb (L180/1) tagged matrix, preparative gel electrophoresis, and gel electroelution process. Finally, the purity and identity of the proteins were confirmed by the matrix-assisted laser desorption/ionization (MALDI) mass spectrometric analysis. The in silico glycosylation site analysis showed that the extracellular domain contained three N-glycosylation sites (N-12, N-62, and N-111) and one O-glycosylation site (T-107). However, the experimental analysis negated the presence of O-linked glycan moieties on T11TS. To investigate the role of glycan moieties in the current immunotherapeutic regime, T11TS and its deglycosylated form (dT11TS) were administered intraperitoneally (i.p.) in N-ethyl-N-nitrosourea-induced immune-compromised mice at 0.4 mg/kg body weight. It was observed that both the forms of T11TS could activate the compromised immune status of mice by augmenting immune receptor expression (CD2, CD25, CD8, and CD11b), T-helper 1 shift of cytokine network, enhanced cytotoxicity, and phagocytosis activity. Therefore, the results nullify the active involvement of the N-linked glycan moieties in immunotherapeutic efficacy of T11TS.