Name: in vitro differentiation of mouse granulocyte-macrophage-colony-stimulating factor (gm-csf)-producing t helper (thgm) cells
Uploaded: 2018-09-10T14:00:00
Description: Watch this Scientific Journal Video about In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor GM-CSF-producing T Helper THGM Cells at JoVE.com

Denne studien ble støttet av tilskudd fra den National University Health System of Singapore (T1-2014 oktober-12 og T1-2015 Sep-10).

Alle mus brukes i denne protokollen var på C57BL/6 genetisk bakgrunn og plassert under bestemte patogen-gratis forhold ved National University of Singapore. Alle eksperimentene ble utført ved hjelp av protokoller godkjent av institusjonelle Animal Care og bruk komiteen av National University of Singapore.
1. reagenser og materiale forberedelse
<ol><li>Forberede 500 mL fosfat-bufret saltvann (PBS) som inneholder 2% fosterets bovin serum (FBS) og 1 mM ethylenediaminetetraacetic syre (EDTA).<...

<ol>
	<li>Zhu, J., Paul, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4+T+cells:+fates,+functions,+and+faults.">CD4 T cells: fates, functions, and faults.</a> Blood. 112, 1557-1569 (2008).</li><li>Kara, E. E., 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=Tailored+immune+responses:+novel+effector+helper+T+cell+subsets+in+protective+immunity.">Tailored immune responses: novel effector helper T cell subsets in protective immunity.</a> PLoS Pathogens. 10, e1003905 (2014).</li><li>Bou Nasser Eddine, F., Ramia, E., Tosi, G., Forlani, G., Accolla, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+Immunology+meets...Immunology:+Modified+cancer+cells+as+professional+APC+for+priming+naive+tumor-specific+CD4++T+cells.">Tumor Immunology meets...Immunology: Modified cancer cells as professional APC for priming naive tumor-specific CD4+ T cells.</a> Oncoimmunology. 6, e1356149 (2017).</li><li>Dong, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TH17+cells+in+development:+an+updated+view+of+their+molecular+identity+and+genetic+programming.">TH17 cells in development: an updated view of their molecular identity and genetic programming.</a> Nature Reviews. Immunology. 8, 337-348 (2008).</li><li>Korn, T., Bettelli, E., Oukka, M., Kuchroo, V. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-17+and+Th17+Cells.">IL-17 and Th17 Cells.</a> Annual Review of Immunology. 27, 485-517 (2009).</li><li>Sheng, W., 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=STAT5+programs+a+distinct+subset+of+GM-CSF-producing+T+helper+cells+that+is+essential+for+autoimmune+neuroinflammation.">STAT5 programs a distinct subset of GM-CSF-producing T helper cells that is essential for autoimmune neuroinflammation.</a> Cell Research. 24, 1387-1402 (2014).</li><li>Codarri, L., 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=RORgammat+drives+production+of+the+cytokine+GM-CSF+in+helper+T+cells,+which+is+essential+for+the+effector+phase+of+autoimmune+neuroinflammation.">RORgammat drives production of the cytokine GM-CSF in helper T cells, which is essential for the effector phase of autoimmune neuroinflammation.</a> Nature Immunology. 12, 560-567 (2011).</li><li>El-Behi, M., 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=The+encephalitogenicity+of+T(H)17+cells+is+dependent+on+IL-1-+and+IL-23-induced+production+of+the+cytokine+GM-CSF.">The encephalitogenicity of T(H)17 cells is dependent on IL-1- and IL-23-induced production of the cytokine GM-CSF.</a> Nature Immunology. 12, 568-575 (2011).</li><li>Ivanov, I. I., 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=The+orphan+nuclear+receptor+RORgammat+directs+the+differentiation+program+of+proinflammatory+IL-17++T+helper+cells.">The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17+ T helper cells.</a> Cell. 126, 1121-1133 (2006).</li><li>Zhang, J., 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=A+novel+subset+of+helper+T+cells+promotes+immune+responses+by+secreting+GM-CSF.">A novel subset of helper T cells promotes immune responses by secreting GM-CSF.</a> Cell Death and Differentiation. 20, 1731-1741 (2013).</li><li>Croxford, A. L., Spath, S., Becher, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GM-CSF+in+Neuroinflammation:+Licensing+Myeloid+Cells+for+Tissue+Damage.">GM-CSF in Neuroinflammation: Licensing Myeloid Cells for Tissue Damage.</a> Trends in Immunology. 36, 651-662 (2015).</li></ol>

Her beskrev vi protokollen en i vitro THGM differensiering fra musen naiv CD4 + celler, etterfulgt av en analyse av differensierte celler å validere metoden. Av notatet, kan både milten og lymfeknutene brukes for naiv CD4 + T celle rensing og THGM differensiering. Cytokin uttrykket bestemmes av intracellulær cytokin flekker kombinert med flowcytometri viste at rundt 55% av cellene ble indusert bli GM-CSF-uttrykke celler under forutsetning av THGM (figur 1A...

CD4 + T hjelper (TH) celler er viktige komponenter i immunsystemet, har avgjørende roller i vert forsvar mot mikrobielle patogener, kreft overvåking og i autoimmunitet1,2,3. Ved T-celle reseptor (TCR) aktivering, naiv CD4 + T celler kan differensieres til TH1 TH2, TH 17 eller forskriftsmessige T (Treg) celler under påvirkning av ulike cytokin miljøer2,4, 5. nylig en ny gruppe TH celler, som hovedsakelig produserer GM-CSF, ble identifisert og navngitt THGM6. Differensiering av THGM celler er drevet av IL-7 gjennom aktivering av en signal svinger og aktivator transkripsjon 5 (STAT5). Disse celler uttrykke mye GM-CSF samtidig ha en lav uttrykk for andre TH-celle signatur cytokiner som IFNγ og IL-176. GM-CSF ble funnet for å spille en avgjørende rolle i utviklingen av CD4 + T celle-mediert neuroinflammation7,8. Sammenlignet med IFNγ - eller IL-17-uttrykke autoreactive T celler, celler GM-CSF-uttrykke T overført til vill-type (WT) mus forårsaket en tidligere sykdommen utbruddet og høyere sykdommens alvorlighetsgrad. I tillegg er Csf2- / - T celler mislyktes å indusere eksperimentelle autoimmune immunsviktvirus (EAE) etter blir adoptively overført til WT mottakere, mens T-celler mangler IFNγ eller IL-17A beholdt evnen til å megle EAE7. Videre ameliorated en blokade av GM-CSF bruker nøytraliserende antistoffer EAE sykdom alvorlighetsgrad8. Videre resulterte mangel på STAT5 i T-celler i mus i en redusert THGM generasjon, og dermed i en motstand av mus EAE utvikling6. Disse funnene understreker betydningen av GM-CSF-uttrykke TH celler i autoimmune neuroinflammatory sykdom. Dermed vil etablere en metode for å skille GM-CSF-uttrykke TH celler fra naiv CD4 + T celler være viktig i studiet av patogenesen av autoimmune neuroinflammation og T-celle-mediert immunreaksjoner. Men en protokoll som effektivt genererer THGM celler fra murine naiv CD4 + ikke er fastslått.
Her presenterer vi en metode som skiller murine THGM celler fra naiv CD4 + T celler. Denne protokollen beskriver hele prosedyren, inkludert utvinning av milten fra musa, utarbeidelse av en enkeltcelle suspensjon, CD4 positiv valg, fluorescens-aktivert cellen sortering (FACS) og TH cellen differensiering og analyse. Differensiert T hjelper celler analyseres av intracellulær cytokin flekker kombinert med flowcytometri å bestemme cytokin uttrykket på encellede nivå, av en kvantitativ sanntid PCR å bestemme cytokin uttrykket på mRNA nivå, og med ELISA å vurdere cytokin uttrykket på protein nivå. Denne metoden kan brukes til studier av THGM cellebiologi under ulike forhold, for eksempel EAE, der GM-CSF spiller en viktig rolle i patogenesen.

<table>
	<tbody>
		<tr>
			<td>RPMI 1640</td>
			<td>Biowest</td>
			<td>L0500-500</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS Heat Inactivated</td>
			<td>Capricorn</td>
			<td>FBS-HI-12A</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>10x PBS</td>
			<td>1st Base</td>
			<td>BUF-2040-10</td>
			<td>Diluted to 1x PBS in sterile water</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>1st Base</td>
			<td>BUF-1053-100ml-pH8.0</td>
			<td></td>
		</tr>
		<tr>
			<td>10X ACK buffer</td>
			<td>Biolegend</td>
			<td>420301</td>
			<td>diluted in sterile water</td>
		</tr>
		<tr>
			<td>cell strainer</td>
			<td>SPL Life Sciences</td>
			<td>93070</td>
			<td></td>
		</tr>
		<tr>
			<td>CD4 microbeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-049-201</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma</td>
			<td>M7522</td>
			<td></td>
		</tr>
		<tr>
			<td>LS column</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>magnetic stand</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>1ml syringe</td>
			<td>Terumo</td>
			<td>SS+01T</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>Eppendorf 5810R</td>
			<td></td>
		</tr>
		<tr>
			<td>Sony SY3200 cell sorter</td>
			<td>Sony</td>
			<td>Sony SY3200 cell sorter</td>
			<td></td>
		</tr>
		<tr>
			<td>50ml conical centrifuge tube</td>
			<td>Greiner Bio-One</td>
			<td>210261</td>
			<td></td>
		</tr>
		<tr>
			<td>15m conical centrifuge tube</td>
			<td>Greiner Bio-One</td>
			<td>188271</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS tube</td>
			<td>Corning</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr>
			<td>24 well cell culture plate</td>
			<td>Greiner Bio-One</td>
			<td>662160</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse CD4 PerCP-eFluor 710</td>
			<td>eBioscience</td>
			<td>46-0041-82</td>
			<td></td>
		</tr>
		<tr>
			<td>PE conjugated anti-mouse CD25 (IL-2Ra, p55)</td>
			<td>eBioscience</td>
			<td>12-0251-82</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-human/mouse CD44 APC</td>
			<td>eBioscience</td>
			<td>17-0441-82</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse CD62L FITC</td>
			<td>eBioscience</td>
			<td>11-0621-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer-improved counting chamber</td>
			<td>Marienfeld</td>
			<td>640010</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyclone Trypan Blue Solution</td>
			<td>GE healthcare Life Sciences</td>
			<td>SV30084.01</td>
			<td></td>
		</tr>
		<tr>
			<td>microscope</td>
			<td>Nikon</td>
			<td>Nikon Elipse TS100</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified&#160;anti-mouse CD3e&#160;antibody</td>
			<td>Biolegend</td>
			<td>100314</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified hamster anti-mouse CD28</td>
			<td>BD Biosciences</td>
			<td>553295</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified Rat&#160;anti-mouse IFN&#947; &#160;</td>
			<td>eBioscience</td>
			<td>16-7312-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified anti-mouse IL-4 Antibody</td>
			<td>Biolegend</td>
			<td>504102</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse IL-7 Protein</td>
			<td>R&#38;D system</td>
			<td>407-ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse IL-6</td>
			<td>R&#38;D system</td>
			<td>406&#8209;ML</td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant human TGF-beta</td>
			<td>R&#38;D system</td>
			<td>240-B-010</td>
			<td></td>
		</tr>
		<tr>
			<td>PMA</td>
			<td>Merck Millipore</td>
			<td>19-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Ionomycin</td>
			<td>Sigma</td>
			<td>I0634</td>
			<td></td>
		</tr>
		<tr>
			<td>GolgiPlug protein transport inhibitor</td>
			<td>BD Biosciences</td>
			<td>51-2301KZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Intracellular Fixation&#38;Permeabilization buffer set</td>
			<td>eBioscience</td>
			<td>88-8824-00</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse GM-CSF PE</td>
			<td>eBioscience</td>
			<td>12-7331-82</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC anti-mouse IL-17A</td>
			<td>Biolegend</td>
			<td>506908</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse IFN-gamma</td>
			<td>Biolegend</td>
			<td>505810</td>
			<td></td>
		</tr>
		<tr>
			<td>GO Taq qPCR master mix</td>
			<td>Promega</td>
			<td>A6002</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse GM-CSF ELISA Ready-SET-Go!</td>
			<td>invitrogen</td>
			<td>88-7334-88</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IL-17A Uncouted ELISA</td>
			<td>invitrogen</td>
			<td>88-7371-22</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro differentiation of mouse granulocyte-macrophage-colony-stimulating factor (gm-csf)-producing t helper (thgm) cells

De granulocytt-macrophage-koloni-stimulerende faktoren (GM-CSF)-produserer T helper (THGM) celle er et nylig identifisert T hjelper celle delsett som hovedsakelig skiller GM-CSF uten å produsere interferon (IFN) γ eller interleukin (IL) -17 er å spille en viktig rolle i den autoimmune neuroinflammation. En metode for isolering av naiv CD4 + T celler fra en enkeltcelle suspensjon av splenocytes og THGM generasjon fra naiv CD4 + T celler ville være en nyttig teknikk i studiet av T celle-mediert immunitet og autoimmune sykdommer. Her beskriver vi en metode som skiller musen naiv CD4 + T celler i THGM celler fremmes av IL-7. Utfallet av differensiering ble vurdert av analyse av cytokiner uttrykket i ulike teknikker, inkludert intracellulær cytokin flekker kombinert med flowcytometri, en kvantitativ sanntids polymerasekjedereaksjons (PCR), og enzym knyttet immunosorbent analyser (ELISA). Bruker THGM differensiering protokollen som beskrevet her, uttrykte om lag 55% av cellene GM-CSF med en minimal uttrykk for IFNα eller IL-17. Dominerende uttrykket av GM-CSF av THGM celler ble ytterligere bekreftet av analyse av uttrykk for GM-CSF, IFNα og IL-17 både mRNA og protein. Dermed denne metoden kan brukes til å skille naiv CD4 + T celler til THGM celler i vitro, som vil være nyttig i studiet av THGM cellebiologi.

Naive CD4 + T celler isolert fra to 8-uke-gamle mannlige C57BL/6 mus ble delt i tre deler. En del av cellene var differensiert i THGM celler følger protokollen beskrevet. En annen del ble kultivert under en THGM tilstand i nærvær av anti-IL-4 antistoff (10 µg/mL) for å teste påvirkning av en IL-4 blokaden i differensiering av THGM. Den siste delen ble kultivert under en TH17 differensiering tilstand (3 µg/mL anti-CD3e, 1 µg/mL anti-CD28,...

Watch this Scientific Journal Video about In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor GM-CSF-producing T Helper THGM Cells at JoVE.com

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor GM-CSF-producing T Helper THGM Cells

In Vitro Differensiering av mus granulocytt-macrophage-koloni-stimulerende faktor (GM-CSF)-produserende T Helper (THGM) celler

Her presenterer vi en protokoll for å skille murine granulocyte-macrophage-colony-stimulating-factor-producing T hjelper (THGM) celler fra naiv CD4 + T celler, inkludert isolering av naiv CD4 + T celler, differensiering av THGM, og analyse av differensierte THGM celler. Denne metoden kan brukes til studier av regulering og funksjon av THGM celler.

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (THGM) cell is a newly identified T helper cell subset that predominantly ...

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