Name: phenotypic and functional analysis of activated regulatory t cells isolated from chronic lymphocytic choriomeningitis virus-infected mice
Uploaded: 2016-06-22T15:00:00
Description: Watch this Scientific Journal Video about Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice at JoVE.com

This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2015R1A6A3A01020610 to HJP) and a grant from the Korean Health Technology R&amp;D Project, Ministry for Health, Welfare and Family Affairs, Republic of Korea (HI15C0493 to SJH).

I denne studien ble musene holdt i en bestemt patogen fritt anlegg av Yonsei Laboratory Animal Research Center of Yonsei University. Alle dyreforsøk ble utført i samsvar med Korean Food and Drug Administration retningslinjer som bruker protokoller godkjent av International Animal Care og bruk komité Yonsei Laboratory Animal Research Center ved Yonsei University. 1. Utarbeidelse av Solutions <ol><li> Fremstille 2% RPMI media ved å fortynne føtalt bovint serum (FBS) til 2% og penicillin...

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Selv om bare et lite antall av T-reg-celler eksisterer i mus og mennesker, er det viktig å forstå deres funksjon som de spiller en viktig rolle i reguleringen av immunrespons og opprettholdelse av immuntoleranse. Antall og undertrykkende funksjoner av T reg cellene øker i løpet av en kronisk virusinfeksjon 15-20 samt kreft progresjon 13,14. Dette er trolig på grunn av fort antigen stimulering. For å evaluere T-celler reg arbeide under antigen persistens og syk...

Regulatoriske T (T reg) celler uttrykker gaffelhode boksen P3 (foxp3) som en transkripsjonsfaktor for deres utvikling og funksjon 1. I tillegg T-reg-celler uttrykker forskjellige andre molekyler slik som CD25 2, lymfocytt-aktivering gen 3 (LAG-3) 3, glukokortikoid-indusert tumor nekrose faktor reseptor 4, og cytotoksisk T-lymfocytt-assosiert protein 4 (CTLA-4) 5- på sin overflate eller intracellulær region. Under kronisk infeksjon med ulike typer patogener som virus 6,7, bakterier 8,9 og parasitter 10-12, eller i løpet av kreftutvikling 13,14, blir T reg celler differensiert i aktiverte celler, viser forbedret undertrykkende funksjon målretting effektor CD4 + og CD8 + T-celler. En rekke aviser har antydet at utvidet og aktiverte T-reg celler bidra til svekket CD8 + T-celle response under venn retrovirus (FV) infeksjon 15-17. FV-induserte T reg celler hemme IFN-γ eller granzyme B uttrykk og cytotoksiske reaktivitet av CD8 + T-celler 15-17. Videre, i en herpes simplex virus-infeksjon-modellen, ble det rapportert at utarming av CD4 + CD25 + -T-reg-celler resulterte i utvidelse av virus-spesifikke CD8 + T-celler og alvorlige skader vev ved infiltrasjon av immunopathogenic CD4 + T-celler 18-20. Mus smittet kronisk med klone 13 stamme av lymfatisk choriomeningittvirus (LCMV CL13) 21-24 har vært mye brukt for å karakterisere fenotype og funksjon av effektor T-celler (T EFF) og T-reg cellene under kronisk virusinfeksjon. Under vedvarende LCMV infeksjon, virus-spesifikke T-eff celler gradvis mister sin effektor funksjon og bli utmattet T (T exh) celler. På den annen side, Treg celler styrke sin evne til å undertrykke virus-spesifikke T-celle respons 25. Nedgangen i den fungerende kapasiteten av T eff-celler kan forklares av flere faktorer slik som oppregulering av inhibitoriske reseptorer på T eff-celler, endret funksjon av antigen-presenterende celler, produksjon av immunoregulatoriske cytokiner, og økt frekvens eller forsterket funksjon av T reg celler 26. Blant de som er involvert i T-celle undertrykkelse faktorer, programmert celledød protein-1 (PD-1) -expressing T exh celler og T-reg celler har blitt ansett som kjennetegnene ved antigen utholdenhet og undertrykkende miljø. Nylig ble det rapportert at blokade av PD-1 reaksjonsvei og ablasjon av T-reg-celler fører til økt T-cellefunksjonen og nedsatt virusbelastningen i løpet av LCMV kronisk infeksjon 27. Videre er T reg cellene aktiveres under kronisk infeksjon av mus med LCMV 23,25 </sopp&gt; og deres undertrykkende funksjon styrkes 25. PD-1 er sterkt uttrykt på T reg-celler, så vel som T-celler exh, og nivået av PD-1 uttrykkes av T-reg-celler korrelerer med styrken av sin undertrykkende funksjon for å inhibere T-celleproliferasjon 25. Her beskriver vi en metode for å sammenligne egenskapene til aktiverte T-reg celler isolert fra mus infisert med LCMV CL13 og hvile T reg celler isolert fra naive mus. Videre forklarer vi en rekke prosesser for å skille aktiverte T-celler og reg undersøke deres ex vivo fenotype, samt måle deres undertrykkende aktivitet in vitro.

<table border="0" cellpadding="0" cellspacing="0" width="976">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">FITC Rat Anti-Mouse CD4</td>
			<td style="width:67px;">RM4-5</td>
			<td style="width:173px;">BD Biosciences</td>
			<td style="width:104px;">553047</td>
			<td style="width:249px;">Please determine appropriate concentration. In this protocol, this reagent was diluted 100X in FACS buffer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytofix/Cytoperm</td>
			<td></td>
			<td>BD Biosciences</td>
			<td>554714</td>
			<td>Use this reagent for cell surface staining.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">U-Bottom Tissue Culture Plates</td>
			<td></td>
			<td>BD Biosciences</td>
			<td>353077</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fixation buffer</td>
			<td></td>
			<td>BD Biosciences</td>
			<td align="right">554655</td>
			<td>Use this reagent for cell surface staining.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FITC Rat Anti-Mouse CD25</td>
			<td>7D4</td>
			<td>BD Biosciences</td>
			<td>553072</td>
			<td>Please determine appropriate concentration. In this protocol, this reagent was diluted 100X in FACS buffer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell strainer, 70mm</td>
			<td></td>
			<td>BD Biosciences</td>
			<td>352350</td>
			<td>Use this strainer for grinding the whole spleen.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell strainer, 40mm</td>
			<td></td>
			<td>BD Biosciences</td>
			<td>352340</td>
			<td>Use this strainer for filtering the cells before column enrichment.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Brilliant Violet 421 Anti-mouse CD279 (PD-1)</td>
			<td>29F.1A12</td>
			<td>BioLegend</td>
			<td>135217</td>
			<td>Please determine appropriate concentration. In this protocol, this reagent was diluted 100X in FACS buffer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Brilliant Violet 605 Anti-Mouse CD4</td>
			<td>RM4-5</td>
			<td>Biolegend</td>
			<td align="right">100547</td>
			<td>Please determine appropriate concentration. In this protocol, this reagent was diluted 100X in FACS buffer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">APC Anti-Mouse/Rat Foxp3&#160;</td>
			<td>FJK-16s</td>
			<td>eBioscience</td>
			<td>17-5773</td>
			<td>Please determine appropriate concentration. In this protocol, this reagent was diluted 100X in FACS buffer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Foxp3 / Transcription Factor Staining Buffer Set</td>
			<td></td>
			<td>eBioscience</td>
			<td>00-5223</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PerCP-Cyanine5.5 Anti-Mouse CD8a</td>
			<td>53-6.7</td>
			<td>eBiosicence</td>
			<td>45-0081</td>
			<td>Please determine appropriate concentration. In this protocol, this reagent was diluted 100X in FACS buffer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse IFN-gamma Platinum ELISA</td>
			<td></td>
			<td>eBiosicence</td>
			<td>BMS606</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640</td>
			<td></td>
			<td>GE Life Sciences</td>
			<td>SH30027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS (1X)</td>
			<td></td>
			<td>GE Life Sciences</td>
			<td>SH30256</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ACK Lysing Buffer</td>
			<td></td>
			<td>Gibco</td>
			<td>A10492-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Glutamine, 200mM solution</td>
			<td></td>
			<td>Gibco&#160;</td>
			<td>25030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin, 10,000U/mL</td>
			<td></td>
			<td>Gibco&#160;</td>
			<td>10378-016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit</td>
			<td></td>
			<td>Life technologies</td>
			<td>L-34975</td>
			<td>Please determine appropriate concentration. In this protocol, this reagent was diluted 500X in FACS buffer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD8a+ T Cell Isolation Kit, mouse</td>
			<td></td>
			<td>Miltenyibiotec</td>
			<td>130-104-075</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD4+CD25+ Regulatory T Cell Isolation Kit, mouse</td>
			<td></td>
			<td>Miltenyibiotec</td>
			<td>130-091-041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MACS Separation Columns, LD columns</td>
			<td></td>
			<td>Miltenyibiotec</td>
			<td>130-042-901</td>
			<td>Use this column for Treg cell isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MACS Separation Columns, LS columns</td>
			<td></td>
			<td>Miltenyibiotec</td>
			<td>130-042-401</td>
			<td>Use this column for CD8+ T cell and Treg cell isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA, 0.5M (pH 8.0)</td>
			<td></td>
			<td>Promega</td>
			<td>V4231</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-Mercaptoethanol</td>
			<td></td>
			<td>Sigma Life Science</td>
			<td>M7522</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td></td>
			<td>Thermo Fisher Scientific</td>
			<td>SH30919.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CellTrace Violet Cell Proliferation Kit</td>
			<td></td>
			<td>Thermo Fisher Scientific</td>
			<td>C34557</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD Canto II flowcytometer</td>
			<td></td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Flow cytometer*</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flowjo</td>
			<td></td>
			<td>TreeStar</td>
			<td></td>
			<td>Flow cytometry software&#8224;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hematocytomer</td>
			<td></td>
			<td>Marienfeld superior</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

phenotypic and functional analysis of activated regulatory t cells isolated from chronic lymphocytic choriomeningitis virus-infected mice

Regulatoriske T (T reg) celler som uttrykker foxp3 som en transkripsjonsfaktor, er undergrupper av CD4 + T-celler. T reg-celler spiller avgjørende roller i immuntoleranse og homeostase vedlikehold ved å regulere immunresponsen. Den primære rolle av T-reg-celler, er å undertrykke proliferasjon av effektor-T-T (eff) celler, og produksjon av cytokiner så som IFN-γ, TNF-α, og IL-2. Det har blitt vist at T reg-cellenes evne til å hemme funksjonen av T-celler eff er forbedret i løpet av vedvarende patogen infeksjon og kreftutvikling. For å klargjøre funksjon av T-reg celler under hvile eller betente forhold, en rekke in vitro undertrykkelse analyser ved hjelp av mus eller humane T reg celler har blitt utviklet. Hovedformålet med denne studien er å utvikle en metode for å sammenligne forskjeller i fenotype og undertrykkende funksjon mellom hvile og aktiverte T regceller. For å isolere aktiverte T-reg celler, ble mus infisert med lymfatisk choriomeningittvirus (LCMV) klon 13 (CL13), en kronisk belastning av LCMV. T reg-celler isolert fra milten av LCMV CL13-infiserte mus oppviste både den aktiverte fenotype og forbedret undertrykkende aktivitet i forhold til hvilende T-reg-celler isolert fra naive mus. Her beskriver vi grunnleggende protokollen for ex vivo fenotype analyse for å skille aktiverte T-reg celler fra hvilende T reg celler. Videre beskriver vi en protokoll for måling av den undertrykkende aktivitet av fullt aktiverte T-reg-celler.

Vi generert mus med vedvarende virusinfeksjon ved å injisere dem med 2 x 10 6 pfu av LCMV CL13 intravenøst. For å undersøke de fenotypiske endringer i T-reg celler og T-conv celler under kronisk virusinfeksjon, ble miltlymfocytter hentet fra naive og infiserte mus farget med forskjellige antistoffer og analysert ved flowcytometri. På 16 d pi, ble PD-1 oppregulert i både foxp3 - CD4 + T conv (figur 1A,</strong...

Watch this Scientific Journal Video about Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice at JoVE.com

Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

Here, we describe a protocol to analyze the phenotype of regulatory T (Treg) cells isolated from na&#239;ve and chronic lymphocytic choriomeningitis virus-infected mice. In addition, we provide a process to evaluate the suppressive activity of the Treg cells.

Fenotypisk og funksjonell analyse av Aktivert regulatoriske T-celler isolert fra kronisk lymfatisk choriomeningittvirus-infiserte mus

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance ...

Research

JoVE Journal

Immunology and Infection

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 av Utførte regulatoriske T-celler fra primære humane naiv og Minne T-celler

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

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

Nye verktøy for å utvide Regulatoriske T-celler fra HIV-1-infiserte individer

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE

Systemisk Injeksjon av Neural Stem / stamceller i Mus med kronisk EAE

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Avledning av T-celler<em&gt; In Vitro</em&gt; Fra Mouse Embryonic Stem Cells

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 of Human alloantigen spesifikke 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 ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Lymfocyttisolasjonsmetoder fra menneskelig hud for fenotypeanalyser og<em&gt; Ex Vivo</em&gt; Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

T celler Capture Bakterier av Transinfection fra dendrittiske 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 ...

Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi

Bestemmelse av Biofilm Initiation på virusinfiserte celler av bakterier og sopp

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with ...