Name: t cells capture bacteria by transinfection from dendritic cells
Uploaded: 2016-01-13T13:00:00
Description: Watch this Scientific Journal Video about T Cells Capture Bacteria by Transinfection from Dendritic Cells at JoVE.com

This work was supported by grants BFU2011-29450, BFU2008-04342/BMC from the Spanish Ministry of Science and Innovation and PIES201020I046 from Consejo Superior de Investigaciones Cient&#236;ficas (CSIC).

Bemærk: Eksperimentelle procedurer blev godkendt af Udvalget for Forskning Etik af Universidad Autónoma de Madrid og gennemføres under tilsyn af Universidad Autónoma de Madrid Leder af dyrevelfærd og sundhed i overensstemmelse med spanske og europæiske retningslinjer. Mus blev opdrættet i specifik patogen fri (SPF) hus, og de ​​blev aflivet af uddannet og kvalificeret personale ved hjælp af kuldioxid (CO 2) indånding metode. 1. Mus knoglemarv-afledte DC&#39;er Different...

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T-celler eller T-lymfocytter er en type af leukocytter, som spiller en central rolle i cellemedieret immunitet og tilhører immunrespons adaptive 26. T-celler er resistente over for at blive smittet in vitro, men nogle rapporter tyder på, at de kan blive inficeret in vivo 14,15. De intime kontakter APC og T-celler under immune synapse fungere som en platform for udveksling af biologisk materiale 13, herunder nogle vira som HIV 11. Det blev for nylig vist, at...

Når et patogen inficerer sin vært, er der normalt en aktivering af de medfødte og adaptive immunrespons, som er nødvendige for bakteriel clearance. Medfødte immunforsvar er den første linje i forsvaret, der forhindrer de fleste infektioner. Medfødte immunforsvar skelner i et præcist elementer, der er bevaret blandt brede grupper af mikroorganismer (patogen-associeret molekylære mønstre, PAMPS) 1. Mekanismerne i medfødte immunitet omfatter fysiske barrierer, såsom hud, kemikalier barrierer (antimikrobielle peptider, lysozym) og de medfødte leukocytter, som omfatter fagocytter (makrofager, neutrofiler og dendritiske celler), mastceller, eosinofiler, basofiler og naturlige dræberceller 2. Disse celler identificere og fjerne patogener, enten ved at angribe dem gennem kontakt eller via fagocytose, som omfatter patogen oversvømmer og drab. Dette system tillader ikke livslang forsvar, i modsætning til adaptiv immunitet, som giver immunologisk hukommelse mod pathogens. Det adaptive immunsystem er den anden linje i forsvaret og er i stand til at genkende og reagere på specifikke antigener af multipel mikrobiel og ikke-mikrobielle stoffer 3. De vigtigste komponenter i det adaptive immunsystem er de lymfocytter, som omfatter B- og T-celler. B-celler er involveret i det humorale respons, der secernerer antistoffer mod patogener eller exogene proteiner. Imidlertid repræsenterer T-celler celle-medieret immunitet, modulering af immunresponset med cytokiner sekretion eller dræbe patogene-inficerede celler 4. Antigenpræsenterende celler (APC&#39;er), herunder dendritiske celler eller makrofager, bestanddele af det medfødte immunsystem, kan genkende fagocytere patogener og proces bakterielle bestanddele i antigener, der præsenteres på celleoverfladen af Major Histocompatibility Complex (MHC) 5-7. Efter APC har fagocyteres patogener, de normalt migrere til dræning lymfeknuder, hvor de interagerer med Tceller. T-lymfocytter kan genkende specifikke peptid-MHC-komplekser med deres T-cellereceptorer. Immun synapse (IS) forekommer i grænsefladen mellem et antigen-loaded APC og en lymfocyt under antigenpræsentation 8,9. Nogle bakterier kan overleve fagocytose og formidle systematisk inden APC. I denne opfattelse, inficerede APC&#39;er tjener som bakterielle reservoirer eller &quot;trojanske heste&quot;, som letter bakteriel spredning 10. Den intime kontakt mellem APC og lymfocytter, der finder sted i løbet af IS-dannelse fungerer også som en platform for udveksling af en del af membraner, genetisk materiale og exosomer og kan kapret for nogle virus til inficerer T-celler; denne proces kaldes transinfection 11-13. Nogle sygdomsfremkaldende bakterier (Listeria monocytogenes, Salmonella enterica og Shigella flexneri) er i stand til at invadere T-lymfocytter in vivo og ændre deres adfærd 14-16. Vi harfor nylig beskrevet, at T-lymfocytter er også i stand til at fange bakterier ved transinfection fra tidligere inficerede dendritiske celler (DC&#39;er) i løbet af antigenpræsentation 16. T-celle bakteriel indfange transinfection overordentlig mere effektiv (1,000-4,000x) end direkte infektioner. T-celler capture patogen og ikke-patogen bakterier indikerer end transinfection er en proces, der drives af T-celler. Slående, transinfected T (TIT) celler hurtigt dræbte de erobrede bakterier og gjorde det mere effektivt end professionelle fagocytter 16. Disse resultater, der bryder et dogme for immunologi, viser, at cellerne i adaptiv immunitet kan udføre funktioner, der var angiveligt eksklusive den medfødte immunitet. Endvidere viste vi, at Tit celler udskiller store mængder af pro-inflammatoriske cytokiner og beskytte mod bakterielle infektioner in vivo. Her præsenterer vi de forskellige protokoller, der anvendes til at studere den bakterielle transinfection procesen musemodel. Denne model er baseret på brugen af CD4 + T-celler fra transgene OTII mus, der bærer en TCR specifik for peptid 323-339 af OVA (OVAp) i forbindelse med I-Ab 17, der interagerer specifikt med bakteriel-inficerede knogle marrow- afledte DC&#39;er (BMDCs) 18,19 lastet med OVAp, danner stabile immune synapser. T-celle transinfection kan visualiseres og spores ved hjælp af fluorescensmikroskopi. Derudover flowcytometri kan anvendes til påvisning af inficerede celler ved at drage fordel af fluorescens udsendt af bakterier, der udtrykker grønt fluorescerende protein (GFP) 16,20. Endvidere kan T-celle transinfection kvantificeres ved en mere følsom metode, gentamicin overlevelse assay, der tillader måling af en lang række arrangementer. Gentamicin er et antibiotikum, ikke kan trænge eukaryote celler. Derfor bruger dette antibiotikum muliggør differentiering af intracellulære bakterier, der overlevede den antibiotiske tilsætning frOM ekstracellulære dem, der blev dræbt 21.

<table border="0" cellpadding="0" cellspacing="0" width="743">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">RPMI</td>
			<td style="width:131px;">Fisher Scientific</td>
			<td style="width:104px;">SH3025501</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">r-GMCSF</td>
			<td style="width:131px;">Peprotech</td>
			<td style="width:104px;">315-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">LPS</td>
			<td style="width:131px;">SIGMA</td>
			<td style="width:104px;">L2630-10mg</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Na Pyruvate</td>
			<td style="width:131px;">Thermo Scientific</td>
			<td style="width:104px;">SH3023901</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">2-ME</td>
			<td style="width:131px;">Gibco</td>
			<td style="width:104px;">31350-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">OVAp OTII (323&#8211;339)</td>
			<td style="width:131px;">GenScript</td>
			<td style="width:104px;">---</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Cell Strainer 70uM</td>
			<td style="width:131px;">BD</td>
			<td align="right" style="width:104px;">352350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;30uM Syringe Filcons Sterile</td>
			<td>BD</td>
			<td align="right">340598</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">AutoMacs Classic</td>
			<td style="width:131px;">Miltenyi Biotec</td>
			<td style="width:104px;">130-088-887</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Gentamicin</td>
			<td style="width:131px;">Normon</td>
			<td style="width:104px;">624601.6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Transwell</td>
			<td style="width:131px;">Costar</td>
			<td align="right" style="width:104px;">3415</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">LB</td>
			<td style="width:131px;">Pronadisa</td>
			<td align="right">1231</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Agar</td>
			<td style="width:131px;">Pronadisa</td>
			<td align="right">1800</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:341px;">Paraformaldehyde 16%</td>
			<td style="width:131px;">Electron Microscopy Sciences</td>
			<td align="right" style="width:104px;">15710</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Triton X-100</td>
			<td style="width:131px;"></td>
			<td style="width:104px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">CD8 biot</td>
			<td style="width:131px;">BD Biosciences</td>
			<td align="right" style="width:104px;">553029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">IgM Biot</td>
			<td style="width:131px;">ImmunoStep</td>
			<td style="width:104px;">Clone RMM-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">B220 Biot</td>
			<td style="width:131px;">BD Biosciences</td>
			<td align="right" style="width:104px;">553086</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">CD19 biot</td>
			<td style="width:131px;">BD Biosciences</td>
			<td align="right">553784</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">MHC-II Biot (I-A/I-E)</td>
			<td style="width:131px;">BD Biosciences</td>
			<td align="right">553622</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">CD11b biot</td>
			<td style="width:131px;">Immunostep</td>
			<td style="width:104px;">11BB-01mg</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">CD11c biot</td>
			<td style="width:131px;">Immunostep</td>
			<td>11CB3-01mg</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">DX5 biot</td>
			<td style="width:131px;">BD Biosciences</td>
			<td align="right" style="width:104px;">553856</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Gr-1 biot</td>
			<td style="width:131px;">BD Biosciences</td>
			<td align="right" style="width:104px;">553125</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">CD16/CD32</td>
			<td style="width:131px;">ImmunoStep</td>
			<td>M16PU-05MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">anti Salmonella</td>
			<td style="width:131px;">ABD Serotec</td>
			<td style="width:104px;">8209-4006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">CD11cPE</td>
			<td style="width:131px;">BD Biosciences</td>
			<td align="right" style="width:104px;">553802</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">CD4-APC</td>
			<td style="width:131px;">Tonbo Biosciences</td>
			<td style="width:104px;">20-0041-U100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gr-1 APC</td>
			<td style="width:131px;">BD Biosciences</td>
			<td align="right">553129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MHC-II (I-A/I-E) FITC</td>
			<td style="width:131px;">BD Biosciences</td>
			<td align="right">553623</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa-Fluor 647 Goat Anti-Rabbit IgG (H+L) Antibody, highly cross-adsorbed</td>
			<td>Invitrogen</td>
			<td>A-21245</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">CMAC (7-amino-4-chloromethylcoumarin)</td>
			<td style="width:131px;">Life technologies</td>
			<td>C2110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">BSA</td>
			<td style="width:131px;">SIGMA</td>
			<td style="width:104px;">A7030-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Streptavidin MicroBeads</td>
			<td style="width:131px;">Miltenyi Biotec</td>
			<td style="width:104px;">130-048-101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">BD FACSCanto II</td>
			<td style="width:131px;">BD Biosciences</td>
			<td style="width:104px;">---</td>
			<td></td>
		</tr>
	</tbody>
</table>

t cells capture bacteria by transinfection from dendritic cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Heri beskrev vi, hvordan du udfører murine T-celle bakteriel transinfection fra inficerede knoglemarv afledt-DCs og hvordan man måler bakteriel transinfection via to forskellige tilgange: flowcytometri og gentamicin overlevelse assay Figur 1 opsummerer procedure for at opnå cellerne.. DC&#39;er genereres ved inkubation af knoglemarvsceller med GM-CSF i 9 dage. Derefter er DC&#39;er modnes med LPS for at øge MHCII på membranen for at indlæse dem på et senere tidspu...

Watch this Scientific Journal Video about T Cells Capture Bacteria by Transinfection from Dendritic Cells at JoVE.com

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Her en protokol præsenteres til at måle bakteriel fange af CD4 + T-celler, som opstår i løbet af antigen præsentation via transinfection fra præ-inficerede dendritiske celler (DC). Vi viser, hvordan du udfører de nødvendige skridt: isolering af primærelementer, infektion af DC / T-celle-konjugat dannelse, og måling af bakteriel T-celle transfektion.

T-celler Capture bakterier ved Transinfection fra dendritiske celler

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

Research

JoVE Journal

Immunology and Infection

Following Cell-fate in E. coli After Infection by Phage Lambda

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Isolation of Mouse Lung Dendritic Cells

Isolering af Mouse Lung dendritiske celler

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

Generation of Induced Regulatory T Cells from Primary Human NaÃƒÆ’Ã‚Â¯ve and Memory T Cells

Generering af inducerede regulatoriske T-celler fra primære humane naive og memory T-celler

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Isolering og karakterisering af dendritiske celler og makrofager fra mus Tarmen

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches

Isolering og immunfarvning Lymfocytter og dendritiske celler fra murine Peyerske plaks

Peyer's  patches (PPs) are integral components of the gut-associated lymphoid tissues  (GALT) and play a central role in intestinal immunosurveillance and ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

Isolering af myeloide dendritiske celler og epitelceller fra human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Vurdering af Innate Sensing af HIV-1 inficerede CD4<sup&gt; +</sup&gt; T-celler ved plasmacytoide dendritiske celler ved hjælp af en<em&gt; Ex vivo</em&gt; Co-dyrkningssystem.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Afledning af T-celler<em&gt; In Vitro</em&gt; Fra mus embryonale stamceller

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

Generation af human alloantigen-specifikke T-celler fra perifert blod

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

Automatiseret Cell Berigelse af cytomegalovirus-specifikke T-celler til kliniske applikationer ved hjælp af Cytokin-capture system

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection ...