Name: functional characterization of regulatory macrophages that inhibit graft-reactive immunity
Uploaded: 2017-06-07T12:30:00
Description: Watch this Scientific Journal Video about Functional Characterization of Regulatory Macrophages That Inhibit Graft-reactive Immunity at JoVE.com

Vi anerkender de tekniske bidrag fra Flow Cytometry, Microsurgery og Bio-repository / Patologi Centers of Excellence for Forskning på Mount Sinai. Dette arbejde blev understøttet af COST Action BM1305: Handling til at fokusere og fremskynde celletolerogen terapi (A Fact), Mount Sinai Recanati / Miller Transplantation Institute udviklingsfondene, Ministerio de Ciencia e Innovacion SAF2013-48834-R og SAF2016-80031-R JO

<html> I denne undersøgelse er musene anbragt i overensstemmelse med De Forenede Nationers Department of Agriculture retningslinjer og anbefalingerne fra Public Health Service Guide til pleje og brug af laboratoriedyr. Alle eksperimentelle teknikker, der involverer dyreanvendelse, blev udført i overensstemmelse med Inocular Animal Care and Utilization Committee (IACUC) godkendte protokoller fra Mount Sinai School of Medicine. 1. Medieforberedelse <ol><li> Forbered komplet RPMI 1640 medium...

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	<li>Jose, M. D., Ikezumi, Y., van Rooijen, N., Atkins, R. C., Chadban, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+act+as+effectors+of+tissue+damage+in+acute+renal+allograft+rejection.">Macrophages act as effectors of tissue damage in acute renal allograft rejection.</a> Transplantation. 76 (7), 1015-1022 (2003).</li><li>Oberbarnscheidt, M. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-self+recognition+by+monocytes+initiates+allograft+rejection.">Non-self recognition by monocytes initiates allograft rejection.</a> J Clin Invest. 124 (8), 3579-3589 (2014).</li><li>Garcia, M. 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(70), e4287 (2012).</li><li>Poujol, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+evaluation+of+lymphocyte+transformation+test+based+on+5-ethynyl-2'deoxyuridine+incorporation+as+a+clinical+alternative+to+tritiated+thymidine+uptake+measurement.">Flow cytometric evaluation of lymphocyte transformation test based on 5-ethynyl-2'deoxyuridine incorporation as a clinical alternative to tritiated thymidine uptake measurement.</a> J Immunol Methods. 415, 71-79 (2014).</li><li>Last'ovicka, J., Budinsky, V., Spisek, R., Bartunkova, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+lymphocyte+proliferation:+CFSE+kills+dividing+cells+and+modulates+expression+of+activation+markers.">Assessment of lymphocyte proliferation: CFSE kills dividing cells and modulates expression of activation markers.</a> Cell Immunol. 256 (1-2), 79-85 (2009).</li><li>Corry, R. J., Winn, H. J., Russell, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primarily+vascularized+allografts+of+hearts+in+mice.+The+role+of+H-2D,+H-2K,+and+non-H-2+antigens+in+rejection.">Primarily vascularized allografts of hearts in mice. The role of H-2D, H-2K, and non-H-2 antigens in rejection.</a> Transplantation. 16 (4), 343-350 (1973).</li><li>Liu, F., Kang, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotopic+heart+transplantation+in+mice.">Heterotopic heart transplantation in mice.</a> J Vis Exp. (6), e238 (2007).</li><li>Ochando, J., Conde, P., Bronte, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocyte-Derived+Suppressor+Cells+in+Transplantation.">Monocyte-Derived Suppressor Cells in Transplantation.</a> Curr Transplant Rep. 2 (2), 176-183 (2015).</li><li>Gallina, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumors+induce+a+subset+of+inflammatory+monocytes+with+immunosuppressive+activity+on+CD8++T+cells.">Tumors induce a subset of inflammatory monocytes with immunosuppressive activity on CD8+ T cells.</a> J Clin Invest. 116 (10), 2777-2790 (2006).</li><li>Huang, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gr-1+CD115++immature+myeloid+suppressor+cells+mediate+the+development+of+tumor-induced+T+regulatory+cells+and+T-cell+anergy+in+tumor-bearing+host.">Gr-1+CD115+ immature myeloid suppressor cells mediate the development of tumor-induced T regulatory cells and T-cell anergy in tumor-bearing host.</a> Cancer Res. 66 (2), 1123-1131 (2006).</li><li>Luan, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocytic+myeloid-derived+suppressor+cells+accumulate+in+renal+transplant+patients+and+mediate+CD4(+)+Foxp3(+)+Treg+expansion.">Monocytic myeloid-derived suppressor cells accumulate in renal transplant patients and mediate CD4(+) Foxp3(+) Treg expansion.</a> Am J Transplant. 13 (12), 3123-3131 (2013).</li><li>Tario, J. D., Muirhead, K. A., Pan, D., Munson, M. E., Wallace, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+immune+cell+proliferation+and+cytotoxic+potential+using+flow+cytometry.">Tracking immune cell proliferation and cytotoxic potential using flow cytometry.</a> Methods Mol Biol. 699, 119-164 (2011).</li></ol>

Denne protokol beskriver de metoder, vi anvendte til immunokarakterisering af graft-infiltrerende myeloidcelleundergrupper i en eksperimentel murint model af hjerte-transplantation, som også er anvendelig for andre væv i forskellige murine eksperimentelle modeller. Lavtrykscelle sortering ved 20 psi var den foretrukne metode til at isolere et godt udbytte af rene celle undergrupper. Opretholdelse af renheden af ​​hver myeloid delmængde er kritisk til at fastslå afgørende resultater af den undertrykkende kapacit...

Denne protokol beskriver in vitro- teknikker til at studere funktionen af ​​væv-infiltrerende makrofager isoleret fra hjerteallotografer i overensstemmelse med deres evne til at modulere T-celle responser. Bredt beskrevet i litteraturen er fluorescerende cellesporingsfarvestoffer i kombination med flowcytometri kraftige værktøjer til at studere den undertrykkende funktion af specifikke celletyper in vitro og in vivo. Vores protokol følger karboxyfluoresceinsuccinimidylester-metoden (CFSE) til overvågning af lymfocytproliferation in vitro . Når en CFSE-mærket celle opdeles, køber sin afkom halve antallet af carboxyfluorescein-mærket molekyler 6 . Det tilsvarende fald i cellefluorescens ved hjælp af flowcytometri kan anvendes til at vurdere celledeling, overvågning af kapaciteten af ​​undertrykkende makrofager til modulering af T-celleimmunrespons. Da CFSE er et fluoresceinbaseret farvestof, er det kompatibelt med en brOad rækkevidde af andre fluorokromer, hvilket gør den anvendelig til flervalsflowcytometri. Det passende valg af fluorochromer til fænotyping er også vigtigt for at undgå overdreven spektral overlapning og manglende evne til at genkende antistof-positive celler, især med synlige proteinfarvestoffer, såsom CFSE 7 . Der er mange fordele ved at anvende fluorescerende farvestoffer over alternative teknikker, der måler celleproliferation, såsom tymidininkorporationsassayet, der anvender radioaktivt mærket thymidin (TdR) 8 . Dette assay udnytter tritieret thymidin (3H-TdR), som er inkorporeret i nye tråde af kromosomalt DNA under mitotisk celledeling. En sikkerhedsproblemer forbundet med dette assay er brugen af ​​radioisotoper, da en scintillation beta-tæller anvendes til at måle radioaktiviteten i DNA, der er udvundet fra cellerne for at bestemme omfanget af celledeling. Metodisk er det tritierede thymidin-assAy er ikke fleksibel nok til at passe vigtige kliniske laboratoriebegrænsninger såsom lavt antal celler og forsinket analyse efter farvning. Tværtimod har CFSE-farvning vist sig at forhindre celleproliferation og at interferere med kritiske aktiveringsmarkører, såsom CD69, HLA-DR og CD25 9 . Derfor er forståelse fordele og begrænsninger af hver metode, især i flerfarvede undersøgelser, hvor flere farvestoffer anvendes til at spore forskellige celletyper, afgørende for at opnå præcise og reproducerbare resultater.

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functional characterization of regulatory macrophages that inhibit graft-reactive immunity

Makrofagophopning i transplanterede organer er længe blevet anerkendt som et træk ved allograftafvisning 1 . Immunogene monocytter infiltrerer allograften tidligt efter transplantationen, monterer et transplantatreaktivt respons mod det transplanterede organ og initierer organafstødning 2 . Nylige data tyder på, at undertrykkende makrofager letter vellykket langtidstransplantation 3 og er nødvendige for induktion af transplantationstolerance 4 . Dette antyder et multidimensionelt koncept for makrofage ontogeni, aktivering og funktion, som kræver en ny køreplan for isolering og analyse af makrofagfunktion 5 . På grund af makrofagernes plasticitet er det nødvendigt at tilvejebringe en metode til at isolere og karakterisere makrofager afhængigt af vævsmiljøet og at definere deres funktioner i henhold til forskellige scenarier. Her beskriver viVære en protokol for immunkarakterisering af transfusionsinfiltrerende makrofager og de metoder, vi tidligere anvendte til funktionelt at evaluere deres evne til at inhibere CD8 + T proliferation og fremme CD4 + Foxp3 + Treg ekspansion in vitro .

De repræsentative resultater viser gatingstrategien beskrevet i ovennævnte protokol. Resultaterne viser også analysen af ​​T-celleproliferationsaktiviteten efter co-kulturen med transplantat-infiltrerende makrofager. Den in vitro- undertrykkende kapacitet af makrofagundergrupper blev analyseret i figur 4 . Resultaterne indikerer, at Ly6C lo Ly6G - makrofagerne opnået fra tolererede modtagere er undertrykkende. Resultaterne viser ogs...

Watch this Scientific Journal Video about Functional Characterization of Regulatory Macrophages That Inhibit Graft-reactive Immunity at JoVE.com

Functional Characterization of Regulatory Macrophages That Inhibit Graft-reactive Immunity

Makrofager er plastceller i det hæmatopoietiske system, der spiller en afgørende rolle i beskyttende immunitet og homeostase. I denne rapport beskriver vi optimerede in vitro teknikker til fænotypisk og funktionelt karakterisering af graft-infiltrerende regulatoriske makrofager, som akkumuleres i det transplanterede organ under tolerogene betingelser.

Funktionel karakterisering af regulatoriske makrofager, der hæmmer graftreaktiv immunitet

Macrophage accumulation in transplanted organs has long been recognized as a feature of allograft rejection1. Immunogenic monocytes infiltrate the ...

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Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-&#947;-Dependent Cell Autonomous Immunity

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Forward Genetik skærme med makrofager til at identificere<em&gt; Toxoplasma gondii</em&gt; Gener Vigtigt for Modstand mod IFN-γ-Dependent Cell Autonome Immunitet

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within ...

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Using X-ray Crystallography, Biophysics, and Functional Assays to Determine the Mechanisms Governing T-cell Receptor Recognition of Cancer Antigens

Brug røntgenkrystallografi, Biofysik, og funktionelle assays til Bestem Mekanismer Governing T-celle receptor Anerkendelse af kræft antigener

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Isolation, karakterisering og rensning af makrofager fra væv ramt af fedme-relaterede Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

Isolation and Characterization of Neutrophil-derived Microparticles for Functional Studies

Isolering og karakterisering af neutrofile-afledte mikropartikler for funktionelle studier

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in ...

Flow Cytometric Measurement Of ROS Production In Macrophages In Response To Fc&#947;R Cross-linking

Flow Cytometric Measurement Of ROS Production In Macrophages In Response To FcÃƒÅ½Ã‚Â³R Cross-linking

Cytometric flowmåling af ROS produktion i makrofager i svar til FcγR tværbinding

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in ...