investigate the role of miRNA in TEC development and function, we generatedmice (Foxn1-Cre::Dicerflox/flox) with TEC that are deficient for Dicer andhence miRNA expression. These mice reveal severe morphological changesin the composition and architecture of the thymic microenvironment initially per-taining to the thymic medulla but later also affecting the cortex. These changesinfluence intrathymic Tcell differentiation including a reduction in positive thymo-cyte selection and a selection of a T cell repertoire able to elicit autoimmunity. Inaged Foxn1-Cre::Dicerflox/flox mice, thymopoiesis is almost completelyreplaced by in-situ B cell maturation and the thymic microenvironment ofthese mutant mice is marked by morphological features typical of secondarylymphoid tissue. In summary, our data demonstrate the importance of miRNAexpression in TECs for the regular postnatal maintenance of a microenvironmentcompetent to instruct normal thymopoiesis.

WS/PP-062-04 The role of MicroRNAs in anti-listerial immuneresponses 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 Listeriamonocytogenes, a Gram-positive facultative intracellular bacterium.L. monocytogenes induces host cell transcriptional responses via membrane-bound/Toll-like receptors (TLR) and cytosolic/nuclear oligomerisation domain-like receptors (NLR), which lead to the production of proinflammatory cytokinesand antimicrobial mediators. However, our knowledge on the posttranscriptionalregulation of gene expression in anti-bacterial immune responses of macro-phages is far from comprehensive.

MicroRNAs are small noncoding RNAs that bind to mRNAs resulting in mRNAdegradation or translational inhibition. To investigate the potential role ofmiRNAs to posttranscriptionally regulate the response to bacterial infection,we first determined the expression of 585 murine microRNAs in bone marrowderived macrophages (BMDMs) infected in vitro with L. monocytogenes, byTaqMan low density arrays. We identified 14 miRNAs that are significantly upre-gulated at 6 hours postinfection, including miR-155 and miR-146a, which arepredicted to target components of receptor-induced signallingcascades.Interestingly, expression of miR-155 and other selected miRNAswas highly induced upon infection with L. monocytogenes deletion mutantDhly, which in macrophages is unable to escape from the phagosome andinduce cytosolic immune response in contrast to infection with wildtype bac-teria. Our findings support a role of miRNAs in the early vacuolar-dependentanti-listerial response of macrophages. Ongoing work focuses on the interactionof specific TLR signalling components and NFkappaB transcription factor withlisteria-induced microRNAs using macrophages from relevant transgenicmice. The results of our studies contribute to our understanding on the molecu-lar mechanisms regulating host-pathogen interactions.

WS/PP-062-05 Kinetics of microRNA action in innate immunecells 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, KeioUniversity School of Medicine, Tokyo, Japan, 3Babraham Institute,Cambridge, United Kingdom

MicroRNAs (miRNAs) are short non-coding RNAs involved in post-transcriptional gene regulation through base pairing with target mRNAs. It iswell established that microRNAs play important roles in both the innate andadaptive immune responses. Here, we examined how miRNAs can modulategene expression patterns in critical innate immune cells during early inflamma-tory responses. MicroRNA-mediated transcriptomic regulation is expected to bequite complex, thus, examining the change of the repertoire of target genes iscrucial for understanding the kinetics of miRNA action during dynamicimmune responses. By utilizing reverse genetics on cells from specific miRNAknockout mice, it might be possible to look for aberrant target genes of themiRNA of interest. An example of our proposed study models is miR-155, aso-called inducible immune miRNA. Adopting miR-155 KO mouse line, weexamined the impact of miR-155 induction on the gene expression profiles indendritic cells and/or macrophages during inflammation. Monitoring the geneexpression kinetics in miR-155-deficient DCs and/or Macrophages revealedthat the biological action of miR-155 might be quite broad as the repertoiresof miR-155 targets dramatically change at the different time points of inflamma-tory reaction. Our results hopefully bring us one step closer to revealing the roleof microRNAs in the immune system.

WS/PP-062-06 MicroRNA gene regulation is associated withCD8+ 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,4University of Pittsburgh, Pittsburgh, PA, United States

Mammalian microRNAs (miRNAs) are important regulators of gene expressionand are pivotal in both adaptive and innate immunity, including controlling thedifferentiation of immune cell subsets as well as their immunological functions.In the present study, we aimed to assess gene expression profiles and their regu-latory mechanisms by miRNAs, on CD8+ Tcells from patients with renal cell carci-noma (RCC), a disease where Tcell immune dysfunctions have been reported. Wecompared autologous and allogenic CD8+T-cell responses against RCC line gen-erated from RCC patients and their HLA-matched healthy donors, after mixed lym-phocyte/tumor cell stimulation (MLTC). On tumor-specific CD8+ Tcells generated,we analyzed the gene expression profiles by microarray approach and the mol-ecular mechanisms of gene regulation by miRNAs analysis.

Comparison of gene expression data in allogenic vs autologous RCC-reactiveCD8+ T cells demonstrated differential expression of genes involved in apoptosisand regulation of cell proliferation. Among these genes, JAK3 and MCL-1 geneswere down-regulated in patient CD8+Tcells versus their healthy counterpart withevi-dence for defective suppressor activity of miR-29b and miR-198 in regulating theirgene expression. Transfection experiments on Jurkat cells using Anti-hsa-miR-29band Anti-hsa-miR-198 inhibitors revealed a significant up-regulation of both proteinsconfirming 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 regulatingimmune-mediated mechanisms by interfering in CD8+ T cells gene expressionof JAK3 and MCL-1 and may have important implications for therapeutic strat-egies in RCC.

WS/PP-062-07 MicroRNA -146a is a significant brake onautoimmunity, 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, UnitedStates

Excessive or inappropriate activation of the immune system can be deleteriousto the organism, warranting multiple molecular mechanisms to control and prop-erly terminate immune responses. MicroRNAs, �22 nucleotide long noncodingRNAs, have recently emerged as key post-transcriptional regulators controllingdiverse biological processes, including responses to non-self.

We have examined the biological role of miRNA-146a using genetically engin-eered mice and showed that mutation of this gene, whose expression is stronglyupregulated following immune cell maturation and/or activation, results in anumber of immune-related phenotypes. First, lack of miRNA-146a expressionresults in hyperresponsiveness of macrophages to LPS and leads to an exag-gerated inflammatory response in endotoxin-challenged mice.

Later on in life, miR-146a null mice develop a spontaneous autoimmune-likedisorder, characterized by splenomegaly, lymphoadenopathy, and multiorganinflammation; as a result many die prematurely. Mechanistically, autoimmunityin 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 ofimmune cell proliferation - miR-146a null mice displayed excessive productionof myeloid cells and developed frank tumors suggesting that miR-146a canfunction as a tumor suppressor in the context of the immune system.

Using a combination of gain- and loss-of-function approaches, we have con-firmed TRAF6 and IRAK1 proteins as bona fide miR-146a targets, whose dere-pression in miR-146a knockout mice might account for some of the observedphenotypes. Taken together, our findings suggest that miR-146a plays a keyrole as a molecular brake on inflammation, myeloid proliferation and oncogenictransformation.

PP-062 MicroRNAs in immune development/responses

PP-062-08 Cellular oncomiR orthologue in EBV oncogenesis

S. G. Babu, S. Saxena. Department of Biotechnology, BabasahebBhimrao Ambedkar University, Lucknow, India

microRNAs (miRNAs) are a class of small, non coding and endogenous RNAsthat regulate gene expression at multiple levels as per the degree of comple-mentarity to the 3′ UTR of targeted mRNAs. They are now known to be keyplayers in the regulation of genes involved in diverse processes such as devel-opment, differentiation, apoptosis and proliferation. The recent discovery ofvirally encoded miRNAs (vmiRNAs), mostly in the family of oncogenic herpesviruses, has attracted immense attention towards their suggested role in viralreplication and pathogenesis. Recently, the kaposi’s-sarcoma-associatedherpes virus (KSHV or HHV8) has been shown to encode a miRNA that functionsas an orthologue of cellular miR-155. Keeping the same view, here, we extendthe analysis of miRNA-homology search to the Epstein-Barr virus (EBV) withrespect to humans. Our In silico analyses shows EBV encoded miR-BART-5has significant ‘seed’ sequence homology to human cellular miR-18(miR-17-92polycistron). Further, the computational predictions of human genes involvedin 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 downregulated by hsa-miR-18. With the above computational results we can specu-late that vmirBART-5 may be orthologous to hsa-miR-18.

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PP-062-09 Toll-like receptor stimulation by different bacteriastrains and microbial components trigger different expressionprofiles of miRNAs in murine bone marrow derived dendritic cells

S. B. Metzdorff, G. M. Weiss, H. Frøkiær. Department of Basic Sciencesand Environment, Frederiksberg C, Denmark

Dendritic cells respond to microorganism through Toll-like receptor binding ofspecific microbial compounds. Several miRNAs are rapidly expressed afterToll-like receptor activation and rising evidence shows that miRNAs might playen important role in regulating the innate immunity. Recently we have demon-strated that Lactobacillus acidophilus NCFM is capable of inducing IFN-b inmurine bone marrow derived dendritic cells. The IFN-b induction was TLR2dependent and also due to the dependency of an endocytotic event. In con-trast, the TLR2 ligand Pam3Cys did not induce IFN-b. We are now investigatingthree miRNAs; miR-155, miR-146a and miR-147 and their potential role in regu-lation of the observed IFN-b induction triggered by L. acidophilus NCFM. Ourresults reveal that each of the three miRNAs have their own expression profileover time upon stimulation with different TLR ligands and bacteria strains,including L. acidophilus NCFM. Moreover the expression patterns of the threemiRNAs are different for L. acidophilus NCFM stimulation compared to theother stimuli indicating that miRNAs may be involved in modulation of the cyto-kine response activated by L. acidophilus NCFM.

PP-062-10 Dicer-dependent miRNA deficiency leads to loss ofLangerhans cells in vivo

F. Schnorfeil1, H. Kuipers1, H. Fehling2, H. Bartels3, T. Brocker1.1Institute for Immunology, Munich, Germany, 2Institute of Immunology,Ulm, Germany, 3Institute for Anatomy, Munich, Germany

Dendritic cells (DCs) are central for the induction of T cell immunity and toler-ance. Fundamental for DCs to control the immune system is their differentiationfrom precursors into various DC subsets with distinct functions and locations inlymphoid organs and tissues. In contrast to the differentiation of epidermalLangerhans cells (LCs) and their seeding into the epidermis, LC maintenanceand homeostasis is strictly dependent on the presence of Dicer, which gener-ates mature microRNAs (miRNAs). Absence of miRNAs caused a strongly dis-turbed steady-state homeostasis of LCs by increasing their turnover andapoptosis rate leading to progressive ablation of LCs with age. Dicer-deficientLCs showed largely increased cell sizes and reduced expression levels of theC-type lectin receptor Langerin, resulting in the lack of Birbeck granules. Inaddition, LCs failed to upregulate MHC class II, CD40 and CD86 surface mol-ecules upon stimulation, which are critical hallmarks of functional DC maturation.The failure to maintain LCs populating the epidermis was accompanied by apro-apoptotic gene expression signature. Taken together, Dicer-dependent gen-eration of miRNAs affects homeostasis of epidermal LCs.

PP-062-11 The role of microRNAs in dendritic cells

M. Frietsch1, H. Kayo1, Y. Fukuda1, M. Tanabe2, T. Fukao1.1Max-Planck-Institute for Immunobiology, Freiburg, Germany, 2KeioUniversity, School of Medicine, Tokyo, Japan

Arising from previous work it is clear that there are many distinct dendritic cell(DC) subtypes that differ in location, migratory pathways and detailed immuno-logical function. Hereby, the cellular identity of each DC subset should bedefined by an unique gene expression pattern linked with their respective rolein the immune system. To study the impact of microRNAs in defining cellularidentities we utilized a comprehensive system biological approach.

Transcriptome and miRNA expression profiling in plasmacytoid DCs, conven-tional DCs and their common DC progenitor showed subset specific variationsin the microRNA signature along differentiation. There was a significant corre-lation between the dynamic change of the microRNome and alterations inmRNA levels during subset specification.

Hence, we identified in DCs several microRNAs that are regulated in a differ-entiation and subset specific manner. Furthermore, microRNA expression corre-lated with mRNA levels of putative target genes. Exploiting reverse genetics DCsubsets obtained from knockout mice lacking some of those differentiallyexpressed microRNAs showed aberrant transcriptome patterns. This points toa crucial impact of the dynamic microRNome during subset specification onsubsequent DC effector functions.

PP-062-12 Promiscuous gene expression in medullary thymicepithelial cells is under network regulation involving Aire andmicroRNA-mRNA interactions

G. A. Passos1,2, C. Macedo1, A. F. Evangelista1, T. A. Fornari1,E. T. Sakamoto-Hojo1, E. A. Donadi1, L. L. Lacerda1,3, W. Savino3.1Molecular Immunogenetics Group, Ribeirao Preto, Brazil, 2University ofSao Paulo, Ribeirao Preto, Brazil, 3Oswaldo Cruz Institute, Rio deJaneiro, Brazil

The expression of self-antigens in the thymus by medullary thymic epithelialcells (mTECs) is essential for the negative selection of autoreactive thymocytes.Due to heterogeneity of self-antigen representation, this phenomenon has beentermed promiscuous gene expression (PGE), in which the autoimmune regulator

(Aire) gene plays a role as a transcription factor in great part of these genes.However, a set of self-antigens seem to be Aire independent and their controlremains elusive. Thus, Aire is not sufficient to explain the regulation of PGE.To further investigate PGE, we hypothesized that self-antigens might be undernetwork regulation: 1) Aire might indirectly modulate self-antigen genes in cas-cade; 2) microRNAs might regulate self-antigens through microRNA-mRNAinteraction. Reconstruction of networks based on microarray expression dataof cultured mTEC 3.10 line, featured Gucy2d gene (an olfactory neuron self-antigen) interacting with downstream genes. Silencing Aire in mTECs bysiRNA, Gucy2d was down regulated demonstrating that is Aire-dependent.AIRE binding sites upstream to ATG codon of Gucy2d supports this effect. Inthis condition Gucy2d established larger number of interactions with self-antigen genes. Nevertheless, mTECs silenced to Dicer, a key ribonucleaseimplicated in maturation of microRNAs, featured down regulation of several ofthem, as expected. In such condition, Gucy2d and several other mRNAs weredetected in higher levels compared to control mTECs, suggesting a role formature microRNAs regulating self-antigens. This allowed demonstration thatPGE may be regulated at post-transcriptional level. Indirect participation ofAire in cascade and the effect of microRNAs represent additional mechanismsregulating PGE. (FAPESP)

PP-062-13 Inducible expression of microRNA-155 andmicroRNA-146a tightly regulate host antiviral immune response

P. Wang, X. Cao. Institute of Immunology, Second Military MedicalUniversity, Shanghai, China

Effective recognition of viral infection and subsequent triggering of appropriateantiviral immune response are essential to avoid virus infected disease and overactivated immune response. To exploit the role of miRNAs in this process, weexamined the expression profile of miRNAs in macrophages under vesicularstomatitis virus (VSV) challenge via microarray technology and identified arange of miRNAs changed significantly, of which miR-155 and miR-146a weremost up-regulated. We demonstrated that inducible miR-146a feedback inhib-ited RIG-I-dependent type I IFN production by targeting TRAF6, IRAK1, andIRAK21, and that inducible expression of miR-155 strengthened antiviral func-tion of type I IFN through targeting SCOS1 in macrophages. So host antiviralresponse was tightly regulated at miRNA level, both positively and negatively.

PP-062-14 MicroRNAs: Modulators of CD4+ T cell phenotype inmultiple sclerosis

K. M. Smith, M. Guerau-de-Arellano, M. K. Racke, A. E. Lovett-Racke,C. C. Whitacre. Ohio State University, Columbus, OH, United States

Multiple sclerosis (MS) is an inflammatory disease of the central nervous systemmediated by autoreactive CD4+ T cells of a TH1 and TH17 phenotype.Identifying novel factors that control TH differentiation will lead to a better under-standing of disease pathogenesis and the development of new therapeuticstrategies. MicroRNAs (miRs) are a class of small non-coding RNAs that nega-tively regulate post-transcriptional gene expression. MiRs have an establishedrole in immune system homeostasis, and profiling studies have found differentialexpression of miRs in autoimmune diseases, including MS. Dicer-deficientCD4+ T cells that lack miRs produce more cytokine upon stimulation.Together these data indicate the miR(s) are responsible for controlling cytokineexpression and act as negative regulators of inflammation. However, thespecific miRs responsible for modulating inflammatory cytokine productionand TH programming have not been defined. Using target prediction algor-ithms, we have identified miRs that are predicted to target factors that mediateTH1 specification and function. We have validated these miR-target pairs usinggain- and loss-of-function experiments in human TH1-polarized cells. We havealso profiled the expression of these miRs in naıve CD4+ T cells from MSpatients relative to healthy controls. Our results identify miRs that are key playersin the developmental programming of TH cells and are able to modulate T celleffector function. These miRs may be useful immunomodulatory treatments orbiomarkers in TH1-mediated disease such as MS.

PP-062-15 miR-124a is a regulator of proliferation and MCP1secretion in fibroblast like synoviocytes from patients withrheumatoid arthritis

Y. Nakamachi1, S. Kawano1, Y. Noguchi1, S. Kasagi2, S. Kumagai2.1Department of clinical labolatory, Kobe University Hospital, kobe, Japan,2Department of Clinical Pathology and Immunology, Kobe UniversityGraduate School of Medicine, kobe, Japan

Purpose: Micro RNAs (miRNAs) are small (�22 nt) endogenous noncodingRNAs that regulate the stability or the translational efficiency of target messen-ger RNAs. Our aim was to investigate the extent to which specific miRNAs areinvolved in the pathogenesis of rheumatoid arthritis (RA).

Methods: Fibroblast-like synoviocytes (FLS) from RA patients were comparedto those from osteoarthritis (OA) patients for their expression of a panel of 156miRNAs with quantitative stem-loop RT-PCR. We transfected the precursor(pre-miR) of specific miRNA into FLS, and performed XTT assay for proliferation,PI staining assay for cell cycle, and ELISA for cytokine production. Then, itseffect on predicted target protein/mRNA was examined by western blot analysis

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and luciferase reporter assay. Furthermore, methylation status of CpG islands atmiR-124a gene loci in RA-FLS was examined by bisulfate sequencing.

Results: We found five miRNAs were more strongly expressed in RA-FLS thanOA-FLS, and miR-124a was the only miRNA which significantly decreased inRA-FLS as compared to those from OA. Transfection of pre-miR-124a intoRA-FLS suppressed their proliferation significantly and arrested the cell cycleat G1 phase, and suppressed the expression of CDK2, CDK6 and MCP1protein. Luciferase reporter assay demonstrated miR-124a specifically sup-pressed the reporter activity driven by the 3′UTRs of CDK2, CDK6 and MCP1mRNA. However, miR-124a CpG islands in RA-FLSs were not methylated.

Conclusion: These results suggest that decreasing miR-124a expression inRA-FLS is involved in the pathogenesis of RA. Further studies are required toreveal the mechanism of miR-124a down-regulation in RA-FLS.

WS-063 New technology in immunologyChairpersons: Hiroyuki Kishi, Christopher Parish

WS/PP-063-01 Proteome-wide screening of antigens targeted bycell mediated immune responses

F. C. Cardoso, A. Trieu, P. Groves, D. L. Doolan. Queensland Institute ofMedical Research, Brisbane, Australia

Vaccine development against malaria and other complex diseases remains achallenge for the scientific community. The recent elucidation of the genome,proteome and transcriptome of important parasites including P. falciparum pro-vide the basis for rational vaccine design by identifying on a proteome-widescale novel target antigens that are recognized by T cells and antibodies fromexposed individuals. Generation of Plasmodium proteins using conventionalprotein expression platforms has been problematic but cell-free in vitro tran-scription translation (IVTT) strategies have recently proved very successful.Accordingly, we are developing high throughput assays using proteins pro-duced by IVTT to identify the T cell targets of complex pathogens. We are con-ducting a series of in vitro and in vivo experiments using IVTT proteinsexpressed in a customized vector and used as unpurified, affinity purifiedthrough Nickel resin or magnetic beads, or mixed with polybeads. In vitrostudies in humans with CMV, EBV, and Influenza A proteins have shown antigen-specific cytokine production in intracellular cytokine staining, ELIspot and CBAassays. In vitro and in vivo studies in mice immunized with the Plasmodium yoeliicircumsporozoite DNA vaccine with or without IVTT protein boost have alsoshown antigen-specific cytokine production against unpurified or purified IVTTproduced antigen. We anticipate that this work will facilitate high-throughputscreening of antigens involved in cell mediated immunity of complex infectiousdiseases that remain a threat to public health.

WS/PP-063-02 Single T cell analysis system for rapid cloningand functional evaluation of antigen-specific T cell receptors

E. Kobayashi, S. Hatakeyama, M. Horii, K. Tajiri, T. Ozawa, H. Kishi,A. Muraguchi. University of Toyama, Toyama, Japan

Antigen (Ag)-specific T-cell therapy or T-cell receptor (TCR) gene therapy is apromising immunotherapy for infectious diseases as well as cancers. Highthroughput screening system of Ag-specific T-cells and TCR repertoire is requi-site for controlling infectious diseases and cancers. Either TCR beta chain oralpha chain repertoire is currently analyzed: however, the availability of a suit-able screening system for analyzing both Ag-specific TCR alpha/beta pairsfrom single T cell is limited. In our laboratory, we are developing high throughputscreening system for Ag-specific T-cells, which is based on immunospot arrayassay on a chip (ISAAC). In this study, we established a rapid cloning and func-tional evaluation system of TCR alpha/beta cDNA pairs derived from Ag-specificmouse single T cells. TCR alpha/beta cDNAs were amplified from an isolatedAg-specific T cell by single cell 5′ RACE method. cDNAs encoding the TCRalpha and beta chains were cloned into expression vectors by homologousrecombination and introduced into the TCR-negative T cell hybridoma, TG40,to express TCR alpha/beta chains on the cell surface. Ag-specificity of clonedTCR was assessed by incubating TCR-expressing TG40 cells with T cell-depleted splenocytes that was loaded with the specific antigen and analyzingCD69-expression. We could obtain Ag-specific TCR alpha/beta cDNA pairwithin 14 days after detecting Ag-responding T-cells. Further promotion of effi-ciency of this system is being developed. The system will provide the suitablesystem for analyzing human T-cell repertoires in diseases and provide candi-date TCR genes for future TCR gene therapy.

WS/PP-063-03 Mass Cytometry: compensation-free, 35-plusparameter, single cell analysis for proteomic dissection ofimmune function

S. C. Bendall1, E. F. Simonds1, O. Ornatsky2, D. Bandura2, V. Baranov2,S. Tanner2, R. Balderas3, G. P. Nolan1. 1Stanford University - BaxterLaboratory In Stem Cell Biology, Stanford, CA, United States, 2DVSSciences, Toronto, ON, Canada, 3BD Biosciences, San Diego, CA,United States

Classical four-color fluorescence flow cytometry helped define the major cellsubsets of the immune system that we understand today (i.e. T-cells, B-cells,macrophages). Machines with eight or more colors brought characterizationof rare immune subsets and stem cells. With intracellular staining, higher par-ameter measurements lead to examination of regulatory signaling networksand patient stratification with clinical outcomes. However, this progression hasnow been stymied by the limit of fluorescence parameters measurable, realisti-cally capped at 12-15 due to boundaries in instrumentation and spectral overlapconsiderations in fluorophore-based tagging methods.

Now, a novel combination of elemental mass spectrometry with single cellanalysis (mass cytometry) offers examination of 30-50 parameters (theoreticallyup to 100) without fluorescent agents or interference from spectral overlap.Instead, it utilizes non-biological, elemental isotopes as reporters. By exploitingthe resolution, sensitivity, and dynamic range of elemental mass spectrometry,on a time-scale that allows the measurement of 1000 individual cells persecond, this device offers a much-simplified alternative for ultra-high contentcytometric analysis.

At Stanford, using the world’s first commercial version of this instrument(CyTOF), we have applied simple modifications to protocols already establishedin our lab for quantization of cellular signaling events in immunological sub-types. Already measuring 20+ intracellular antigens (phosphorylations) in con-junction with 10+ cell surface markers we detail the approaches we are takingtowards an unprecedented profile of cytokine/immune responses of all majorcell types in human blood and bone marrow. We will present these studiesand demonstrate the detailed systems-level view of immune function theyreveal.

WS/PP-063-04 Dramatical improvement of human blood lineagecells in humanized mouse by expression of human cytokines

Q. Chen1, M. Khoury2,1, J. Chen1,2. 1Singapore-MIT Alliance in Researchand Technology (SMART), Singapore, Singapore, 2Koch Institute,Massachusetts Institute of Technology, Cambridge, MA, United States

Adoptive transfer of human hematopoietic stem cells (HSCs) into mice lacking T,B and natural killer (NK) cells leads to development of human blood lineagecells in the recipient mice (humanized mice). Although human B cell reconstitu-tion is robust and T cell reconstitution is reasonable in the recipient mice, recon-stitution of NK cells and myeloid cells is generally poor or undetectable. Here,we show that the poor reconstitution is mainly due to a deficiency of appropriatehuman cytokines that are necessary for the development and maintenance ofthese cell lineages. When plasmid DNA encoding human interleukin (IL) 15and Flt-3/Flk-2 ligand were delivered into humanized mice by hydrodynamictail vein injection, the expression of the human cytokine lasted for 2 to 3weeks and elevated levels of NK cells were induced for more than a month.The cytokine-induced NK cells expressed both activation and inhibitory recep-tors, killed target cells in vitro, and responded robustly to a virus infection in vivo.Similarly, expression of human granulocyte macrophage colony stimulatingfactor (GM-CSF) and IL-4, macrophage colony stimulating factor (M-CSF), orerythropoietin (EPO) and IL-3 resulted in significantly enhanced reconstitutionof dendritic cells, monocytes/macrophages or erythrocytes, respectively.Thus, human cytokine gene expression by hydrodynamic delivery is a simpleand efficient method to improve reconstitution of specific human blood celllineages in humanized mice, providing an important tool for studying humanimmune responses and disease progression in a small animal model.

WS/PP-063-05 IL-12/IL-23p40 small interfering RNA (siRNA)establish protective immunity against autoimmune disease

J. Bruck, I. Glocova, S. Pascolo, K. Fuchs, K. Ghoreschi, M. Rocken.Department of Dermatology, Tubingen, Germany

Experimental autoimmune encephalomyelitis (EAE) is a prototypic organ-specific autoimmune disease, mediated by pro-inflammatory T helper (Th)cells that are directed against myelin peptides. Destructive inflammationwithin the central nervous system is initiated by the infiltration ofIFN-g-producing Th1 cells and IL-17-producing Th17 cells. IL-12 and IL-23are heterodimers sharing the IL-12/IL-23p40 subunit. In contrast to anti-bodies, siRNA is short lived and interferes with immune responses for verylimited periods of time. We therefore asked, whether siRNA allows establish-ing protective immunity when applied at the time of DC maturation. We syn-thesized various siRNA directed against IL-12/IL-23p40 and tested theircapacity to inhibit IL-12/IL-23p40 production by DC in vitro after stimulationthrough 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, anddeviated the development of Th1/Th17 responses into a Th2 phenotype invivo. Simultaneously, immunization in the presence of IL-12/IL-23p40 siRNAsignificantly improved the clinical course of EAE. After active immunizationonly 20% developed EAE in the siRNA group, while in the control group100% developed the disease. Thus, in vivo immunization with CFA in thepresence of IL-12/23p40 siRNA prevented induction of pro-inflammatoryTh1/Th17 immune response and deviated the cells into an anti-inflammatoryTh2 phenotype. It IL-12/23p40 siRNA can be used to deviate the develop-ment of harmful Th1/h17 responses into Th2 responses and to establish pro-tective immunity against encephalomyelitis. This may provide the basis fornew vaccination strategies that establish protective immunity against inflam-matory autoimmune diseases.

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