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 denna studie gavs möss hölls i en specifik patogen-fri anläggning av Yonsei Laboratory Animal Research Center av Yonsei University. Alla djurförsök utfördes i enlighet med de riktlinjer som koreanska Food and Drug Administration med hjälp av protokoll som godkänts av International Animal Care och användning kommitté Yonsei Laboratory Animal Research Center vid Yonsei University. 1. Beredning av lösningar <ol><li> Förbereda 2% RPMI medier genom att späda fetalt bovint serum (...

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Även om det finns bara ett litet antal av T-reg-celler i möss och människor, är det viktigt att förstå deras funktion eftersom de spelar en avgörande roll i regleringen av immunförsvaret och bibehålla immuntolerans. Siffer och undertryckande funktioner T reg celler ökar under en kronisk virusinfektion 15-20 samt cancerutveckling 13,14. Detta är troligen på grund av fortsatt antigenstimulering. För att utvärdera T reg celler fungera under antigen uthål...

Regulatoriska T (T reg) celler uttrycker forkhead box P3 (Foxp3) som en transkriptionsfaktor för deras utveckling och funktion 1. Dessutom T reg-celler uttrycker olika andra molekyler såsom CD25 2, lymfocyt-aktivering gen 3 (LAG-3) 3, glukokortikoid-inducerad tumömekrosfaktor-receptor 4, och cytotoxisk T-lymfocyt-associerat protein 4 (CTLA-4) 5 på sin yta eller intracellulära regionen. Under kronisk infektion med olika typer av patogener, såsom virus 6,7, bakterier 8,9 och parasiter 10-12, eller i samband med cancerutveckling 13,14, blir T reg-celler differentieras till aktiverade celler, visar förbättrad undertryckande funktion inriktnings effektor CD4 + och CD8 + T-celler. Ett antal artiklar har föreslagit att expanderade och aktiverade T-reg-celler bidrar till osäkra CD8 + T-cells response under vän retrovirus (FV) infektion 15-17. FV-inducerade T reg-celler inhiberar IFN-γ eller granzym B-uttryck och cytotoxiska reaktivitet av CD8 + T-celler 15-17. Dessutom i ett herpes simplex-virus infektionsmodell, rapporterades det att utarmning av CD4 + CD25 + T-reg-celler resulterade i expansion av virusspecifika CD8 + T-celler och allvarlig vävnadsskada genom infiltrering av immunopathogenic CD4 + T-celler 18-20. Möss infekterade kroniskt med klon 13-stammen av lymfocytisk koriomeningitvirus (LCMV CL13) 21-24 har ofta använts för att karakterisera fenotypen och funktionen av effektor-T-celler (T eff) och T reg-celler under kronisk virusinfektion. Under ihållande LCMV infektion, virus-specifika T eff celler förlorar gradvis sin effektorfunktion och blir utmattad T (T exh) celler. Å andra sidan, Treg-celler förstärker deras förmåga att undertrycka virusspecifikt T-cellsvar 25. Minskningen i den fungerande kapacitet av T eff celler kan förklaras av flera faktorer, såsom uppreglering av hämmande receptorer på T eff celler, förändrad funktion av antigenpresenterande celler, produktion av immunregulatoriska cytokiner, och ökad frekvens eller ökad funktion av T-reg celler 26. Bland de faktorer som är involverade i T-cellundertryckning, programmerad celldöd protein-1 (PD-1) uttryckande T EXH celler och T-reg-celler har allmänt betraktas som kännetecken av antigen uthållighet och undertryckande miljö. Nyligen rapporterades det att blockaden av PD-1 vägen och ablation av T-reg-celler leder till förbättrad funktion T-cell och minskad virusbelastning under LCMV kronisk infektion 27. Vidare är T reg-celler aktiveras under kronisk infektion av möss med LCMV 23,25 </supp&gt; och deras undertryckande funktion stärks 25. PD-1 uttrycks kraftigt på T reg-celler såväl som T EXH celler, och nivån på PD-1 uttrycks av T reg-celler korrelerar med styrkan av sin suppressiva funktionen att inhibera T-cellsproliferation 25. Här beskriver vi en metod för att jämföra egenskaperna hos aktiverade T-reg-celler som isolerats från möss infekterade med LCMV CL13 och vilande T reg celler som isolerats från naiva möss. Dessutom förklarar vi en rad processer för att separera aktiverade T-reg-celler och undersöka deras ex vivo fenotyp, samt mäta deras undertryckande 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

Regulatoriska T (T reg) celler, vilka uttrycker Foxp3 som en transkriptionsfaktor, är undergrupper av CD4 + T-celler. T reg celler spelar avgörande roller i immuntolerans och homeostas underhåll genom att reglera immunsvaret. Den primära rollen av T-reg-celler är att undertrycka proliferationen av effektor-T (T eff) celler och produktionen av cytokiner såsom IFN-γ, TNF-α, och IL-2. Det har visats att T reg cellernas förmåga att inhibera funktionen av T-eff celler förstärks under ihållande patogen infektion och utveckling av cancer. För att förtydliga funktionen av T-reg celler under vila eller inflammerade villkor, ett flertal in vitro-undertrycknings analyser med användning av mus eller humant T reg-celler har uppfunnits. Huvudsyftet med denna studie är att utveckla en metod för att jämföra skillnaderna i fenotyp och undertryckande funktion mellan vilande och aktiverade T regceller. För att isolera aktiverade T-reg-celler, möss infekterade med lymfocytär koriomeningitvirus (LCMV) klon 13 (CL13), en kronisk stam av LCMV. T reg-celler isolerade från mjälten hos LCMV CL13-infekterade möss uppvisade både den aktiverade fenotypen och förbättrad suppressiv aktivitet jämfört med vilande T reg celler som isolerats från naiva möss. Här beskriver vi den grundläggande protokollet för ex vivo fenotyp analys att skilja aktiverade T-reg-celler från vilande T reg-celler. Dessutom beskriver vi ett protokoll för mätning av den undertryckande aktiviteten hos helt aktiverade T-reg-celler.

Vi genererade möss med motståndskraftigt virus infektion genom att injicera dem med 2 x 10 6 PFU av LCMV CL13 intravenöst. För att undersöka de fenotypiska förändringar i T reg-celler och T conv celler under kronisk virusinfektion, var mjältlymfocyter erhållna från naiva och infekterade möss färgades med olika antikroppar och analyserades med flödescytometri. Vid 16 d pi, var PD-1 uppregleras i både Foxp3 - CD4 + T <sub...

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 och funktionell analys av aktiverade regulatoriska T-celler isolerade från kronisk lymfatisk koriomeningitvirus-infekterade möss

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 ...

The overall goal of this procedure is to evaluate the in vitro suppressive function of regulatory T cells, isolated from chronic virus infected mice. This method can help answer questions about the phenotype and in vitro suppressive function of activated regulatory T cells during a chronic virus infection. This technique can evaluate the direct effect of regulatory T cells on the individual with a positive response to T cells in vitro.This is a great advantage of this technique. Begin by transferring five times ten to the fifth total splenic lymphocytes in 50 microliters of facs buffer into each well of a new U bottom 96 well plate, followed by 150 microliters of facs buffer. Centrifuge the plate and discard the supernatant.Then agitate the cell pellets and wash the cells two more times in 200 microliters of facs buffer per well. After the second wash, resuspend the pellets in 50 microliters of antibody cocktail per well, for 20 minutes in the dark at four degrees Celsius. Then wash the cells two times in facs buffer, and decant the supernatant.Next, fix the cells for 20 minutes in the dark at four degrees Celsius with 100 microliters of fixation buffer, followed by two washes with permeabilization wash buffer. After the second wash, resuspend the cells in 50 microliters of anti-FOXP3 antibody. To prepare the anti CD3 CD28 coated beads, transfer the appropriate volume of magnetic beads into a 15 milliliter tube, and add an equal volume of PPS.Gently mix the solution by pipetting, and centrifuge the magnetic beads. At the end of the wash, discard the supernatant, and dilute the beads in 50 microliters of complete medium per well. Next, seed 50 microliters of one times ten to the fifth CD4 positive CD25 positive regulatory T cells per well in a 96 well U bottom plate, followed by 50 microliters of one times ten to the fifth CD8 positive responder T cells per well.When all of the cells have been plated, add 50 microliters of the diluted anti CD3 CD28 coated beads to each well, and add up to 50 microliters of medium to bring the total volume of each well up to 200 microliters. Then cover the plate with foil and place it at 37 degrees Celsius in a carbon dioxide incubator for 72 hours. To analyze the CD8 positive T cell cytokine production, after three days of culture, transfer the supernatants into another 96 well plate and analyze the supernatants by ELISA, according to the manufacturer's instructions.To analyze the proliferation of the CD8 positive T cells, wash the plate of cells with facs buffer three times. Then resuspend the pellets in 50 microliters of an appropriate antibody cocktail against proliferated CD8 positive T cells for 20 minutes in the dark at four degrees Celsius. At the end of the incubation, wash the cells two times in facs buffer and fix them for 20 minutes in the dark at four degrees Celsius with 100 microliters of fixation buffer.Finally, wash the cells two more times in facs buffer and resuspend the pellets in 200 microliters of facs buffer. Then measure the percentage of phylate dilabelled CD8 positive T cell proliferation by flow cytometry. In splenic lymphocytes obtained from naive and infected mice, PD-1 is specifically up regulated in both conventional FOXP3 negative CD4 positive and FOXP3 negative CD8 positive cells of LCMV clone 13 infected mice.The frequency of FOXP3 positive CD4 positive regulatory T cells is two times higher in LCMV clone 13 infected mice than in naive mice, with most of the FOXP3 positive CD4 positive cells also displaying an activated PD-1 high phenotype. CD8 positive responder T cells can be separated successfully without significant contamination by other immune cells. After regulatory T cell isolation, a dominant population of CD25 positive CD4 positive T cells with more than 80 percent purity can be obtained.The stimulation of isolated regulatory and responder T cells by anti CD3 CD28 coated beads results in the inhibition of CD8 positive responder T cells in a dose dependent manner. Indeed, when CD8 positive responder T cells are co-cultured with chronic regulatory T cells at a one to one ratio, the CD8 positive responder T cell proliferation is significantly inhibited. Interferon gamma production by CD8 positive responder cells is also significantly inhibited when CD8 positive responder T cells are co-cultured with activated regulatory T cells from chronically infected mice, compared to co-culture with resting regulatory T cells from naive mice.We have found this technique can be completed in 80 hours if it is performed properly. While you are attempting this procedure, it is important to remember to use freshly isolated regulatory T cells immediately after their isolation. After this development, this technique paved the way for researchers in the field of medicine to explore the function of regulatory T cells in a chronic inflammatory disease.After watching this video, you should have a good understanding of how to set up an in vitro regulatory T cell suppression procedure, using a regulatory and responder T cell co-culture system.

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 Na&#239;ve and Memory T Cells

Generering av Inducerade regulatoriska T-celler från primära humana naiva och 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

Nya verktyg för att expandera reglerande T-celler från HIV-1-infekterade 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 ...

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

Lymfocytisolering från human hud för fenotypisk analys och<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 ...

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

Bestämning av biofilm Initiation på virusinfekterade celler av bakterier och svampar

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

Functional Characterization of Regulatory Macrophages That Inhibit Graft-reactive Immunity

Funktionell karaktärisering av regulatoriska makrofager som inhiberar graftreaktiv immunitet

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

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Utveckling av stamcellshärledda antigen-specifika regulatoriska T-celler mot autoimmunitet

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Na&#239;ve CD4+ T Cells Using a TGF-&#946;-containing Protocol

In vitro-differentiering av humana CD4 + Foxp3 + Induced regulatoriska T-celler (iTregs) från naiva CD4 + -T-celler med ett TGF-β-innehållande protokoll

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing ...

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Encelliga kvantitering av mRNA och ytan proteinuttryck i Simian Immunodeficiency Virus-infekterade CD4+ T celler isolerade från Rhesus makaker

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be ...

In Vivo Augmentation of Gut-Homing Regulatory T Cell Induction

In vivo augmentation av Gut-homing reglerande T-cell induktion

Inflammatory bowel disease (IBD) is an inflammatory chronic disease in the gastrointestinal tract (GUT). In the United States, there are approximately 1.4 ...