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investigate the role of miRNA in TEC development and function, we generated
mice (Foxn1-Cre::Dicerflox/flox) with TEC that are deficient for Dicer and
hence miRNA expression. These mice reveal severe morphological changes
in the composition and architecture of the thymic microenvironment initially pertaining to the thymic medulla but later also affecting the cortex. These changes
influence intrathymic T cell differentiation including a reduction in positive thymocyte selection and a selection of a T cell repertoire able to elicit autoimmunity. In
aged Foxn1-Cre::Dicerflox/flox mice, thymopoiesis is almost completely
replaced by in-situ B cell maturation and the thymic microenvironment of
these mutant mice is marked by morphological features typical of secondary
lymphoid tissue. In summary, our data demonstrate the importance of miRNA
expression in TECs for the regular postnatal maintenance of a microenvironment
competent to instruct normal thymopoiesis.

WS/PP-062-04 The role of MicroRNAs in anti-listerial immune
responses of macrophages
A. K. D. Schnitger, N. Papadopoulou. Institute for Medical Microbiology,
Immunology and Hygiene (IMMIH), Cologne, Germany
Macrophages are key players of the innate immune response against Listeria
monocytogenes, a Gram-positive facultative intracellular bacterium.
L. monocytogenes induces host cell transcriptional responses via membranebound/Toll-like receptors (TLR) and cytosolic/nuclear oligomerisation domainlike receptors (NLR), which lead to the production of proinflammatory cytokines
and antimicrobial mediators. However, our knowledge on the posttranscriptional
regulation of gene expression in anti-bacterial immune responses of macrophages is far from comprehensive.
MicroRNAs are small noncoding RNAs that bind to mRNAs resulting in mRNA
degradation or translational inhibition. To investigate the potential role of
miRNAs to posttranscriptionally regulate the response to bacterial infection,
we first determined the expression of 585 murine microRNAs in bone marrow
derived macrophages (BMDMs) infected in vitro with L. monocytogenes, by
TaqMan low density arrays. We identified 14 miRNAs that are significantly upregulated at 6 hours postinfection, including miR-155 and miR-146a, which are
predicted to target components of receptor-induced signalling
cascades.Interestingly, expression of miR-155 and other selected miRNAs
was highly induced upon infection with L. monocytogenes deletion mutant
Dhly, which in macrophages is unable to escape from the phagosome and
induce cytosolic immune response in contrast to infection with wildtype bacteria. Our findings support a role of miRNAs in the early vacuolar-dependent
anti-listerial response of macrophages. Ongoing work focuses on the interaction
of specific TLR signalling components and NFkappaB transcription factor with
listeria-induced microRNAs using macrophages from relevant transgenic
mice. The results of our studies contribute to our understanding on the molecular mechanisms regulating host-pathogen interactions.

WS/PP-062-05 Kinetics of microRNA action in innate immune
cells during early inflammatory response
S. Hedlund1, J. Winnewisser1, Y. Fukuda1, H. Kayo1, M. Tanabe2,
E. Vigorito3, T. Fukao1. 1Max Planck Institute of Immunobiology, Freiburg,
Germany, 2Department of Tropical Medicine and Parasitology, Keio
University School of Medicine, Tokyo, Japan, 3Babraham Institute,
Cambridge, United Kingdom
MicroRNAs (miRNAs) are short non-coding RNAs involved in posttranscriptional gene regulation through base pairing with target mRNAs. It is
well established that microRNAs play important roles in both the innate and
adaptive immune responses. Here, we examined how miRNAs can modulate
gene expression patterns in critical innate immune cells during early inflammatory responses. MicroRNA-mediated transcriptomic regulation is expected to be
quite complex, thus, examining the change of the repertoire of target genes is
crucial for understanding the kinetics of miRNA action during dynamic
immune responses. By utilizing reverse genetics on cells from specific miRNA
knockout mice, it might be possible to look for aberrant target genes of the
miRNA of interest. An example of our proposed study models is miR-155, a
so-called inducible immune miRNA. Adopting miR-155 KO mouse line, we
examined the impact of miR-155 induction on the gene expression profiles in
dendritic cells and/or macrophages during inflammation. Monitoring the gene
expression kinetics in miR-155-deficient DCs and/or Macrophages revealed
that the biological action of miR-155 might be quite broad as the repertoires
of miR-155 targets dramatically change at the different time points of inflammatory reaction. Our results hopefully bring us one step closer to revealing the role
of microRNAs in the immune system.

WS/PP-062-06 MicroRNA gene regulation is associated with
CD8+ T cells immune deviation in Renal Cell Carcinoma patients:
role of JAK3 and MCL-1
M. Gigante1, M. Gigante1, W. Herr2, E. Cavalcanti3, P. Pontrelli1,
G. Zaza3, V. Servidio1, E. Montemurno1, V. Mancini3, M. Battaglia3,
W. J. Storkus4, L. Gesualdo1, E. Ranieri1. 1University of Foggia, Foggia,
Italy, 2University of Mainz, Mainz, Germany, 3University of Bari, Bari, Italy,
4
University of Pittsburgh, Pittsburgh, PA, United States

iii122 Wednesday

Mammalian microRNAs (miRNAs) are important regulators of gene expression
and are pivotal in both adaptive and innate immunity, including controlling the
differentiation of immune cell subsets as well as their immunological functions.
In the present study, we aimed to assess gene expression profiles and their regulatory mechanisms by miRNAs, on CD8+ Tcells from patients with renal cell carcinoma (RCC), a disease where Tcell immune dysfunctions have been reported. We
compared autologous and allogenic CD8+ T-cell responses against RCC line generated from RCC patients and their HLA-matched healthy donors, after mixed lymphocyte/tumor cell stimulation (MLTC). On tumor-specific CD8+ Tcells generated,
we analyzed the gene expression profiles by microarray approach and the molecular mechanisms of gene regulation by miRNAs analysis.
Comparison of gene expression data in allogenic vs autologous RCC-reactive
CD8+ T cells demonstrated differential expression of genes involved in apoptosis
and regulation of cell proliferation. Among these genes, JAK3 and MCL-1 genes
were down-regulated in patient CD8+ Tcells versus their healthy counterpart with evidence for defective suppressor activity of miR-29b and miR-198 in regulating their
gene expression. Transfection experiments on Jurkat cells using Anti-hsa-miR-29b
and Anti-hsa-miR-198 inhibitors revealed a significant up-regulation of both proteins
confirming the role of miR-29b and miR-198 on JAK3 and MCL-1 expression.
Our results indicate that miR-29b and miR-198 play a key role in regulating
immune-mediated mechanisms by interfering in CD8+ T cells gene expression
of JAK3 and MCL-1 and may have important implications for therapeutic strategies in RCC.

WS/PP-062-07 MicroRNA -146a is a significant brake on
autoimmunity, myeloproliferation and cancer in mice
M. P. Boldin1,2, K. D. Taganov1,2, D. S. Rao2, L. Yang2, M. Kalwani2,
Y. Garcia-Flores2, M. Luong2, A. Devrekanli2, J. Xu1, G. Sun1, J. Tay1,
P. S. Linsley1, D. Baltimore2. 1Regulus Therapeutics, Carlsbad, CA,
United States, 2California Institute of Technology, Pasadena, CA, United
States
Excessive or inappropriate activation of the immune system can be deleterious
to the organism, warranting multiple molecular mechanisms to control and properly terminate immune responses. MicroRNAs, 22 nucleotide long noncoding
RNAs, have recently emerged as key post-transcriptional regulators controlling
diverse biological processes, including responses to non-self.
We have examined the biological role of miRNA-146a using genetically engineered mice and showed that mutation of this gene, whose expression is strongly
upregulated following immune cell maturation and/or activation, results in a
number of immune-related phenotypes. First, lack of miRNA-146a expression
results in hyperresponsiveness of macrophages to LPS and leads to an exaggerated inflammatory response in endotoxin-challenged mice.
Later on in life, miR-146a null mice develop a spontaneous autoimmune-like
disorder, characterized by splenomegaly, lymphoadenopathy, and multiorgan
inflammation; as a result many die prematurely. Mechanistically, autoimmunity
in the knockout mice correlated with the loss of peripheral T cell tolerance.
In addition, we found that miRNA-146a seems to play a role in the control of
immune cell proliferation - miR-146a null mice displayed excessive production
of myeloid cells and developed frank tumors suggesting that miR-146a can
function as a tumor suppressor in the context of the immune system.
Using a combination of gain- and loss-of-function approaches, we have confirmed TRAF6 and IRAK1 proteins as bona fide miR-146a targets, whose derepression in miR-146a knockout mice might account for some of the observed
phenotypes. Taken together, our findings suggest that miR-146a plays a key
role as a molecular brake on inflammation, myeloid proliferation and oncogenic
transformation.

PP-062 MicroRNAs in immune development/
responses
PP-062-08 Cellular oncomiR orthologue in EBV oncogenesis
S. G. Babu, S. Saxena. Department of Biotechnology, Babasaheb
Bhimrao Ambedkar University, Lucknow, India
microRNAs (miRNAs) are a class of small, non coding and endogenous RNAs
that regulate gene expression at multiple levels as per the degree of complementarity to the 3′ UTR of targeted mRNAs. They are now known to be key
players in the regulation of genes involved in diverse processes such as development, differentiation, apoptosis and proliferation. The recent discovery of
virally encoded miRNAs (vmiRNAs), mostly in the family of oncogenic herpes
viruses, has attracted immense attention towards their suggested role in viral
replication and pathogenesis. Recently, the kaposi’s-sarcoma-associated
herpes virus (KSHV or HHV8) has been shown to encode a miRNA that functions
as an orthologue of cellular miR-155. Keeping the same view, here, we extend
the analysis of miRNA-homology search to the Epstein-Barr virus (EBV) with
respect to humans. Our In silico analyses shows EBV encoded miR-BART-5
has significant ‘seed’ sequence homology to human cellular miR-18(miR-17-92
polycistron). Further, the computational predictions of human genes involved
in cellular growth that could potentially be targeted by EBV miRNA, i.e.,
vmiR-BART-5, reveal a common set of mRNA transcripts that are also down
regulated by hsa-miR-18. With the above computational results we can speculate that vmirBART-5 may be orthologous to hsa-miR-18.

14th International Congress of Immunology

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14th ICI Abstract book

Macedo1. E. Kasagi2. Munich. Guerau-de-Arellano. Germany. In such condition. a set of self-antigens seem to be Aire independent and their control remains elusive. we examined the expression profile of miRNAs in macrophages under vesicular stomatitis virus (VSV) challenge via microarray technology and identified a range of miRNAs changed significantly. E. Y. K. Racke. a key ribonuclease implicated in maturation of microRNAs. 1 Max-Planck-Institute for Immunobiology. Absence of miRNAs caused a strongly disturbed steady-state homeostasis of LCs by increasing their turnover and apoptosis rate leading to progressive ablation of LCs with age. Ohio State University. 3Institute for Anatomy. C. 1 Molecular Immunogenetics Group. A. the cellular identity of each DC subset should be defined by an unique gene expression pattern linked with their respective role in the immune system. 2 Department of Clinical Pathology and Immunology. (Aire) gene plays a role as a transcription factor in great part of these genes. T. we hypothesized that self-antigens might be under network regulation: 1) Aire might indirectly modulate self-antigen genes in cascade. miR-146a and miR-147 and their potential role in regulation of the observed IFN-b induction triggered by L. Together these data indicate the miR(s) are responsible for controlling cytokine expression and act as negative regulators of inflammation. Germany. Fehling2.and loss-of-function experiments in human TH1-polarized cells. Multiple sclerosis (MS) is an inflammatory disease of the central nervous system mediated by autoreactive CD4+ T cells of a TH1 and TH17 phenotype. H. In this condition Gucy2d established larger number of interactions with selfantigen genes. Nevertheless. 2Institute of Immunology. Recently we have demonstrated that Lactobacillus acidophilus NCFM is capable of inducing IFN-b in murine bone marrow derived dendritic cells. M. Wang. this phenomenon has been termed promiscuous gene expression (PGE). Department of Basic Sciences and Environment. OH. Denmark . Our results identify miRs that are key players in the developmental programming of TH cells and are able to modulate T cell effector function.2. acidophilus NCFM. X. MicroRNAs (miRs) are a class of small non-coding RNAs that negatively regulate post-transcriptional gene expression. Weiss. MiRs have an established role in immune system homeostasis. 1 Institute for Immunology. Sakamoto-Hojo1. S. A. In contrast to the differentiation of epidermal Langerhans cells (LCs) and their seeding into the epidermis. Kawano1. conventional DCs and their common DC progenitor showed subset specific variations in the microRNA signature along differentiation. Frederiksberg C. 3Oswaldo Cruz Institute. Freiburg. both positively and negatively. Indirect participation of Aire in cascade and the effect of microRNAs represent additional mechanisms regulating PGE. W. Schnorfeil1. H. Taken together. migratory pathways and detailed immunological function. Kuipers1. Kobe University Hospital. School of Medicine. T. Ulm. Cao. 1 Department of clinical labolatory. Metzdorff. Rio de Janeiro. M. Ribeira˜o Preto. L. Whitacre. 2Keio University. which generates mature microRNAs (miRNAs). Gucy2d and several other mRNAs were detected in higher levels compared to control mTECs. we have identified miRs that are predicted to target factors that mediate TH1 specification and function. China Symposia Dendritic cells respond to microorganism through Toll-like receptor binding of specific microbial compounds. (FAPESP) Master Lectures PP-062-09 Toll-like receptor stimulation by different bacteria strains and microbial components trigger different expression profiles of miRNAs in murine bone marrow derived dendritic cells S. Germany PP-062-13 Inducible expression of microRNA-155 and microRNA-146a tightly regulate host antiviral immune response P. as expected. In addition. Kayo1. Y. This points to a crucial impact of the dynamic microRNome during subset specification on subsequent DC effector functions. However. Bartels3. Fundamental for DCs to control the immune system is their differentiation from precursors into various DC subsets with distinct functions and locations in lymphoid organs and tissues. Evangelista1.10 line. which are critical hallmarks of functional DC maturation. Dicer-dependent generation of miRNAs affects homeostasis of epidermal LCs. B. PI staining assay for cell cycle. Kobe University Graduate School of Medicine. acidophilus NCFM. Then. Effective recognition of viral infection and subsequent triggering of appropriate antiviral immune response are essential to avoid virus infected disease and over activated immune response. featured Gucy2d gene (an olfactory neuron selfantigen) interacting with downstream genes. A. we identified in DCs several microRNAs that are regulated in a differentiation and subset specific manner. Due to heterogeneity of self-antigen representation. Methods: Fibroblast-like synoviocytes (FLS) from RA patients were compared to those from osteoarthritis (OA) patients for their expression of a panel of 156 miRNAs with quantitative stem-loop RT-PCR. H. We are now investigating three miRNAs. Lunchtime Lectures PP-062-10 Dicer-dependent miRNA deficiency leads to loss of Langerhans cells in vivo F. in which the autoimmune regulator PP-062-15 miR-124a is a regulator of proliferation and MCP1 secretion in fibroblast like synoviocytes from patients with rheumatoid arthritis Y. resulting in the lack of Birbeck granules. Fukao1. Kumagai2. Moreover the expression patterns of the three miRNAs are different for L. Ribeirao Preto. Tokyo. Thus. Frøkiær. and profiling studies have found differential expression of miRs in autoimmune diseases. Using target prediction algorithms. Japan PP-062-14 MicroRNAs: Modulators of CD4+ T cell phenotype in multiple sclerosis K. M. In contrast. Hereby. and IRAK21. We have also profiled the expression of these miRs in naı¨ve CD4+ T cells from MS patients relative to healthy controls. Hence. Tanabe2. To study the impact of microRNAs in defining cellular identities we utilized a comprehensive system biological approach. This allowed demonstration that PGE may be regulated at post-transcriptional level.3. H. Japan Purpose: Micro RNAs (miRNAs) are small (22 nt) endogenous noncoding RNAs that regulate the stability or the translational efficiency of target messenger RNAs. Passos1. E. M. C. S. Dicer-deficient CD4+ T cells that lack miRs produce more cytokine upon stimulation. T. Institute of Immunology. Clinical Seminars PP-062-11 The role of microRNAs in dendritic cells M.14th ICI Abstract book PP-062-12 Promiscuous gene expression in medullary thymic epithelial cells is under network regulation involving Aire and microRNA-mRNA interactions G. Brazil. C. and performed XTT assay for proliferation. its effect on predicted target protein/mRNA was examined by western blot analysis 14th International Congress of Immunology Wednesday iii123 Influenza Arising from previous work it is clear that there are many distinct dendritic cell (DC) subtypes that differ in location. Smith. T. A. To further investigate PGE. Gucy2d was down regulated demonstrating that is Aire-dependent. Lacerda1. Fukuda1. Silencing Aire in mTECs by siRNA. CD40 and CD86 surface molecules upon stimulation. Brazil The expression of self-antigens in the thymus by medullary thymic epithelial cells (mTECs) is essential for the negative selection of autoreactive thymocytes. AIRE binding sites upstream to ATG codon of Gucy2d supports this effect. Fornari1. United States Workshops Workshops Dendritic cells (DCs) are central for the induction of T cell immunity and tolerance. Furthermore. We transfected the precursor (pre-miR) of specific miRNA into FLS. Identifying novel factors that control TH differentiation will lead to a better understanding of disease pathogenesis and the development of new therapeutic strategies. suggesting a role for mature microRNAs regulating self-antigens. Second Military Medical University. IRAK1. including L. Japan. Our results reveal that each of the three miRNAs have their own expression profile over time upon stimulation with different TLR ligands and bacteria strains. So host antiviral response was tightly regulated at miRNA level. mTECs silenced to Dicer. Savino3. 2University of Sao Paulo. kobe. Transcriptome and miRNA expression profiling in plasmacytoid DCs. and ELISA for cytokine production. H. Noguchi1. 2) microRNAs might regulate self-antigens through microRNA-mRNA interaction. the specific miRs responsible for modulating inflammatory cytokine production and TH programming have not been defined. kobe. Columbus. Munich. LCs failed to upregulate MHC class II. L. F. Germany. However. We have validated these miR-target pairs using gain. Reconstruction of networks based on microarray expression data of cultured mTEC 3. acidophilus NCFM. To exploit the role of miRNAs in this process. including MS. and that inducible expression of miR-155 strengthened antiviral function of type I IFN through targeting SCOS1 in macrophages. Brazil. Aire is not sufficient to explain the regulation of PGE. The IFN-b induction was TLR2 dependent and also due to the dependency of an endocytotic event. featured down regulation of several of them. Brocker1. Nakamachi1. Dicer-deficient LCs showed largely increased cell sizes and reduced expression levels of the C-type lectin receptor Langerin. miR-155. of which miR-155 and miR-146a were most up-regulated. M. Frietsch1. Several miRNAs are rapidly expressed after Toll-like receptor activation and rising evidence shows that miRNAs might play en important role in regulating the innate immunity. We demonstrated that inducible miR-146a feedback inhibited RIG-I-dependent type I IFN production by targeting TRAF6. Our aim was to investigate the extent to which specific miRNAs are involved in the pathogenesis of rheumatoid arthritis (RA). Donadi1. S. microRNA expression correlated with mRNA levels of putative target genes. Lovett-Racke. These miRs may be useful immunomodulatory treatments or biomarkers in TH1-mediated disease such as MS. Exploiting reverse genetics DC subsets obtained from knockout mice lacking some of those differentially expressed microRNAs showed aberrant transcriptome patterns. There was a significant correlation between the dynamic change of the microRNome and alterations in mRNA levels during subset specification. The failure to maintain LCs populating the epidermis was accompanied by a pro-apoptotic gene expression signature. LC maintenance and homeostasis is strictly dependent on the presence of Dicer. A. the TLR2 ligand Pam3Cys did not induce IFN-b. acidophilus NCFM stimulation compared to the other stimuli indicating that miRNAs may be involved in modulation of the cytokine response activated by L. G. Shanghai.

Machines with eight or more colors brought characterization of rare immune subsets and stem cells. CA. The system will provide the suitable system for analyzing human T-cell repertoires in diseases and provide candidate TCR genes for future TCR gene therapy. mediated by pro-inflammatory T helper (Th) cells that are directed against myelin peptides. which is based on immunospot array assay on a chip (ISAAC). respectively. In contrast to antibodies. Here. Already measuring 20+ intracellular antigens (phosphorylations) in conjunction with 10+ cell surface markers we detail the approaches we are taking towards an unprecedented profile of cytokine/immune responses of all major cell types in human blood and bone marrow. proteome and transcriptome of important parasites including P. providing an important tool for studying human immune responses and disease progression in a small animal model. Germany Experimental autoimmune encephalomyelitis (EAE) is a prototypic organspecific autoimmune disease. We synthesized various siRNA directed against IL-12/IL-23p40 and tested their capacity to inhibit IL-12/IL-23p40 production by DC in vitro after stimulation through TLR. Immunizing mice with PLP and CFA in the presence of IL-12/ IL-23p40 siRNA impaired IL-12 production by DC in vivo and in vitro. TG40. a novel combination of elemental mass spectrometry with single cell analysis (mass cytometry) offers examination of 30-50 parameters (theoretically up to 100) without fluorescent agents or interference from spectral overlap. P. S. In our laboratory. Although human B cell reconstitution is robust and T cell reconstitution is reasonable in the recipient mice. the availability of a suitable screening system for analyzing both Ag-specific TCR alpha/beta pairs from single T cell is limited. Stanford. K. 14th International Congress of Immunology . In this study. V. Results: We found five miRNAs were more strongly expressed in RA-FLS than OA-FLS. TCR alpha/beta cDNAs were amplified from an isolated Ag-specific T cell by single cell 5′ RACE method. WS/PP-063-05 IL-12/IL-23p40 small interfering RNA (siRNA) establish protective immunity against autoimmune disease J. Further studies are required to reveal the mechanism of miR-124a down-regulation in RA-FLS. H. 1Singapore-MIT Alliance in Research and Technology (SMART). reconstitution of NK cells and myeloid cells is generally poor or undetectable. Similarly. Muraguchi. Pascolo. Kishi. methylation status of CpG islands at miR-124a gene loci in RA-FLS was examined by bisulfate sequencing. Baranov2.Baxter Laboratory In Stem Cell Biology. Transfection of pre-miR-124a into RA-FLS suppressed their proliferation significantly and arrested the cell cycle at G1 phase. K. MA. and Influenza A proteins have shown antigenspecific cytokine production in intracellular cytokine staining. In vitro and in vivo studies in mice immunized with the Plasmodium yoelii circumsporozoite DNA vaccine with or without IVTT protein boost have also shown antigen-specific cytokine production against unpurified or purified IVTT produced antigen. A. Department of Dermatology. WS/PP-063-03 Mass Cytometry: compensation-free. M. Australia Vaccine development against malaria and other complex diseases remains a challenge for the scientific community. Ozawa. When plasmid DNA encoding human interleukin (IL) 15 and Flt-3/Flk-2 ligand were delivered into humanized mice by hydrodynamic tail vein injection. Furthermore. It IL-12/23p40 siRNA can be used to deviate the development of harmful Th1/h17 responses into Th2 responses and to establish protective immunity against encephalomyelitis. Groves. Bru¨ck. We are conducting a series of in vitro and in vivo experiments using IVTT proteins expressed in a customized vector and used as unpurified. Generation of Plasmodium proteins using conventional protein expression platforms has been problematic but cell-free in vitro transcription translation (IVTT) strategies have recently proved very successful. C. By exploiting the resolution. Ghoreschi. Nolan1. elemental isotopes as reporters. L. A. United States Adoptive transfer of human hematopoietic stem cells (HSCs) into mice lacking T.e. Brisbane. Chen1. we established a rapid cloning and functional evaluation system of TCR alpha/beta cDNA pairs derived from Ag-specific mouse single T cells. to express TCR alpha/beta chains on the cell surface. CDK6 and MCP1 mRNA. siRNA is short lived and interferes with immune responses for very limited periods of time. higher parameter measurements lead to examination of regulatory signaling networks and patient stratification with clinical outcomes. Cardoso. cDNAs encoding the TCR alpha and beta chains were cloned into expression vectors by homologous recombination and introduced into the TCR-negative T cell hybridoma. At Stanford. Singapore. The recent elucidation of the genome.2. and miR-124a was the only miRNA which significantly decreased in RA-FLS as compared to those from OA. We anticipate that this work will facilitate high-throughput screening of antigens involved in cell mediated immunity of complex infectious diseases that remain a threat to public health. B and natural killer (NK) cells leads to development of human blood lineage cells in the recipient mice (humanized mice). Doolan. S. P. we are developing high throughput screening system for Ag-specific T-cells. 2Koch Institute. human cytokine gene expression by hydrodynamic delivery is a simple and efficient method to improve reconstitution of specific human blood cell lineages in humanized mice. T. Christopher Parish WS/PP-063-01 Proteome-wide screening of antigens targeted by cell mediated immune responses F. G. affinity purified through Nickel resin or magnetic beads. Accordingly. immunization in the presence of IL-12/IL-23p40 siRNA significantly improved the clinical course of EAE. Singapore. macrophages). University of Toyama. F. Glocova. We could obtain Ag-specific TCR alpha/beta cDNA pair within 14 days after detecting Ag-responding T-cells. Tanner2. 35-plus parameter. We therefore asked. or erythropoietin (EPO) and IL-3 resulted in significantly enhanced reconstitution of dendritic cells. macrophage colony stimulating factor (M-CSF). Ag-specificity of cloned TCR was assessed by incubating TCR-expressing TG40 cells with T celldepleted splenocytes that was loaded with the specific antigen and analyzing CD69-expression. Queensland Institute of Medical Research. CA. ELIspot and CBA assays. 1Stanford University . and deviated the development of Th1/Th17 responses into a Th2 phenotype in vivo. or mixed with polybeads.1. Either TCR beta chain or alpha chain repertoire is currently analyzed: however. it utilizes non-biological. ON. Tajiri. sensitivity. this device offers a much-simplified alternative for ultra-high content cytometric analysis. J. Thus. D. Destructive inflammation within the central nervous system is initiated by the infiltration of IFN-g-producing Th1 cells and IL-17-producing Th17 cells. The cytokine-induced NK cells expressed both activation and inhibitory receptors. Instead. Khoury2. Simonds1. falciparum provide the basis for rational vaccine design by identifying on a proteome-wide scale novel target antigens that are recognized by T cells and antibodies from exposed individuals. United States. Now. expression of human granulocyte macrophage colony stimulating factor (GM-CSF) and IL-4. in vivo immunization with CFA in the presence of IL-12/23p40 siRNA prevented induction of pro-inflammatory Th1/Th17 immune response and deviated the cells into an anti-inflammatory Th2 phenotype. Bendall1. United States iii124 Wednesday Classical four-color fluorescence flow cytometry helped define the major cell subsets of the immune system that we understand today (i. Fuchs. Hatakeyama. 2DVS Sciences. Further promotion of efficiency of this system is being developed. Horii. Conclusion: These results suggest that decreasing miR-124a expression in RA-FLS is involved in the pathogenesis of RA. 3BD Biosciences. However. O. M. Trieu. we show that the poor reconstitution is mainly due to a deficiency of appropriate human cytokines that are necessary for the development and maintenance of these cell lineages. monocytes/macrophages or erythrocytes. B-cells. High throughput screening system of Ag-specific T-cells and TCR repertoire is requisite for controlling infectious diseases and cancers. Toyama. realistically capped at 12-15 due to boundaries in instrumentation and spectral overlap considerations in fluorophore-based tagging methods. Cambridge. I. IL-12 and IL-23 are heterodimers sharing the IL-12/IL-23p40 subunit. the expression of the human cytokine lasted for 2 to 3 weeks and elevated levels of NK cells were induced for more than a month. this progression has now been stymied by the limit of fluorescence parameters measurable. Toronto. We will present these studies and demonstrate the detailed systems-level view of immune function they reveal. on a time-scale that allows the measurement of 1000 individual cells per second. CDK6 and MCP1 protein. Canada. E. we are developing high throughput assays using proteins produced by IVTT to identify the T cell targets of complex pathogens. we have applied simple modifications to protocols already established in our lab for quantization of cellular signaling events in immunological subtypes. and suppressed the expression of CDK2. This may provide the basis for new vaccination strategies that establish protective immunity against inflammatory autoimmune diseases. killed target cells in vitro. T-cells. WS-063 New technology in immunology Chairpersons: Hiroyuki Kishi.Clinical Seminars Workshops Workshops Lunchtime Lectures Symposia Master Lectures 14th ICI Abstract book and luciferase reporter assay. D. and dynamic range of elemental mass spectrometry. Thus. Simultaneously. Kobayashi. Ro¨cken. With intracellular staining. WS/PP-063-04 Dramatical improvement of human blood lineage cells in humanized mouse by expression of human cytokines Q. In vitro studies in humans with CMV. single cell analysis for proteomic dissection of immune function S. San Diego. K. and responded robustly to a virus infection in vivo. Chen1. Tu¨bingen. miR-124a CpG islands in RA-FLSs were not methylated. Balderas3. C. Influenza WS/PP-063-02 Single T cell analysis system for rapid cloning and functional evaluation of antigen-specific T cell receptors E. Japan Antigen (Ag)-specific T-cell therapy or T-cell receptor (TCR) gene therapy is a promising immunotherapy for infectious diseases as well as cancers. S. R. After active immunization only 20% developed EAE in the siRNA group. M. using the world’s first commercial version of this instrument (CyTOF). Ornatsky2. while in the control group 100% developed the disease. EBV. Massachusetts Institute of Technology. However. whether siRNA allows establishing protective immunity when applied at the time of DC maturation. Luciferase reporter assay demonstrated miR-124a specifically suppressed the reporter activity driven by the 3′ UTRs of CDK2. Bandura2.