Name: in vivo augmentation of gut-homing regulatory t cell induction
Uploaded: 2020-01-22T13:00:00
Description: Watch this Scientific Journal Video about In Vivo Augmentation of Gut-Homing Regulatory T Cell Induction at JoVE.com

Dette arbejde blev støttet af Kontoret for den assisterende forsvarsminister for Sundhedsanliggender gennem peer reviewed medicinsk forskning program under Award No. W81XWH-15-1-0240 (XT). Meninger, fortolkninger, konklusioner, og anbefalinger er dem af forfatteren og er ikke nødvendigvis godkendt af Department of Defense. Dette arbejde blev også delvist understøttet af forsknings innovations stipendier fra Institut for medicin på Loma Linda University (681207-2967 [XT og GG], 681205-2967 [XT] og 325491 [DJB]).

Alle in vivo dyreforsøg protokoller blev gennemgået og godkendt af Loma Linda University institutionel dyrepleje og brug udvalg (IACUC) samt Animal Care og use Review Office (ACURO) af den amerikanske hær medicinsk forskning og materiel Command (USAMRMC) af Forsvarsministeriet.
1. klargøring af lentivirus, der udtrykker både 1α-hydroxylase og RALDH2 (Lenti-CYP-ALDH-virus)
<ol><li>Dag 0: i den tidlige morgen, forberede 5 x 105 celler/ml af 293T celler i cm-10-D cellekulturmed...

<ol>
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C., Gaitan, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunomodulatory+effects+of+Vitamin+D+in+multiple+sclerosis.">Immunomodulatory effects of Vitamin D in multiple sclerosis.</a> Brain. 132, 1146-1160 (2009).</li><li>Iwata, 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=Retinoic+acid+imprints+gut-homing+specificity+on+T+cells.">Retinoic acid imprints gut-homing specificity on T cells.</a> Immunity. 21 (4), 527-538 (2004).</li><li>Steinman, R. M., Banchereau, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taking+dendritic+cells+into+medicine.">Taking dendritic cells into medicine.</a> Nature. 449 (7161), 419-426 (2007).</li><li>Vicente-Suarez, I., Brayer, J., Villagra, A., Cheng, F., Sotomayor, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TLR5+ligation+by+flagellin+converts+tolerogenic+dendritic+cells+into+activating+antigen-presenting+cells+that+preferentially+induce+T-helper+1+responses.">TLR5 ligation by flagellin converts tolerogenic dendritic cells into activating antigen-presenting cells that preferentially induce T-helper 1 responses.</a> Immunology Letters. 125 (2), 114-118 (2009).</li><li>Danova, K., 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=NF-kappaB,+p38+MAPK,+ERK1/2,+mTOR,+STAT3+and+increased+glycolysis+regulate+stability+of+paricalcitol/dexamethasone-generated+tolerogenic+dendritic+cells+in+the+inflammatory+environment.">NF-kappaB, p38 MAPK, ERK1/2, mTOR, STAT3 and increased glycolysis regulate stability of paricalcitol/dexamethasone-generated tolerogenic dendritic cells in the inflammatory environment.</a> Oncotarget. 6 (16), 14123-14138 (2015).</li><li>Liu, P. T., 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=Toll-like+receptor+triggering+of+a+vitamin+D-mediated+human+antimicrobial+response.">Toll-like receptor triggering of a vitamin D-mediated human antimicrobial response.</a> Science. 311 (5768), 1770-1773 (2006).</li><li>Cao, 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=Application+of+vitamin+D+and+vitamin+D+analogs+in+acute+myelogenous+leukemia.">Application of vitamin D and vitamin D analogs in acute myelogenous leukemia.</a> Experimental Hematology. 50, 1-12 (2017).</li><li>Anderson, A., 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=Lasting+Impact+of+Clostridium+difficile+Infection+in+Inflammatory+Bowel+Disease:+A+Propensity+Score+Matched+Analysis.">Lasting Impact of Clostridium difficile Infection in Inflammatory Bowel Disease: A Propensity Score Matched Analysis.</a> Inflammatory Bowel Disease. 23 (12), 2180-2188 (2017).</li><li>Tsai, J. 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=Association+of+Aneuploidy+and+Flat+Dysplasia+With+Development+of+High-Grade+Dysplasia+or+Colorectal+Cancer+in+Patients+With+Inflammatory+Bowel+Disease.">Association of Aneuploidy and Flat Dysplasia With Development of High-Grade Dysplasia or Colorectal Cancer in Patients With Inflammatory Bowel Disease.</a> Gastroenterology. 153 (6), 1492-1495 (2017).</li><li>Lee, H. 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I denne artikel beskriver vi brugen af DC-CYP-ALDH celler, for at forstærke induktion af Gut-homing Treg celler i perifert lymfevæv. Vores data har vist, at DC-CYP-ALDH-cellerne kan syntetisere lokalt høje koncentrationer af de Novo af både 1,25 (OH)2D og RA in vitro i nærværelse af tilsvarende substrater (dvs. 25 [Oh] d og retinol, hhv.). Da tilstrækkelige blodkoncentrationer på 25 (Oh) D og retinol let kan opnås gennem d-vitamin og et tillægsspørgsmål til henholdsvis patienter med mangler<sup cla...

Inflammatorisk tarmsygdom (IBD) er en inflammatorisk kronisk sygdom i mave-tarmkanalen (GUT). I USA er der ca. 1.400.000 IBD-patienter. Det er almindeligt anerkendt, at en dysreguleret immunrespons på tarm bakterier initierer sygdommen og forstyrrer slimhinde epitelial barrieren1,2. Af denne grund, i øjeblikket tilgængelige U.S. Food and Drug Administration (FDA)-godkendte lægemidler hæmmer funktionerne af inflammatoriske mediatorer eller blokere homing af immunceller i tarmen. Men, de inflammatoriske mediatorer og immunceller, der er målrettet er også nødvendige for immunforsvaret. Som et resultat, de inflammatoriske mediator hæmmere kompromittere systemiske immunforsvar og immuncelle homing blokkere svække tarm immunforsvar, som begge kan føre til alvorlige konsekvenser3,4. Desuden kan immuncelle homing blokkere også blokere for homing af regulerende T (Treg) celler i tarmen og dermed kan forværre den allerede kompromitterede tarm immuntolerance i IBD patienter. Desuden kan blokering af Treg-celle homing i tarmen også føre til systemisk immunsuppression på grund af ophobning af Treg celler i blodet5. Endelig virker inhibitorer og blokkere forbigående og kræver dermed hyppige administrationer. Hyppig administration af disse hæmmere og blokkere kan yderligere forværre de uheldige bivirkninger.
For nylig foreslog vi en ny strategi, der potentielt kan afbøde eller endda eliminere bivirkningerne forbundet med nuværende lægemidler til IBD behandling6. Denne strategi forstærker induktion af tarm-homing Treg celler i perifert lymfevæv6. Rationalet bag denne strategi er, at Gut-homing Treg celler specifikt hjem til tarmen og dermed ikke vil kompromittere systemiske immunforsvar. Desuden, da treg celler kan potentielt danne hukommelse7,8, Gut-homing treg celler kan potentielt give en stabil kontrol af kronisk tarmbetændelse i IBD patienter og dermed, behandling bør ikke behøver at blive administreret så hyppigt. Da denne strategi desuden forstærker induktion af tarmationstreg-celler in vivo, er der ingen bekymring for in vivo-ustabilitet i et stærkt proinflammatorisk miljø, der er forbundet med adoptiv overførsel af in vitro-genererede treg-celler9,10. I denne henseende, in vitro genereret treg celler er en af de foreslåede strategier for behandling af autoimmune sygdomme11,12,13 og transplantation afvisning14,15. Endelig, i denne strategi, dendritiske celler (DCs) er konstrueret til at producere lokalt høje koncentrationer af to molekyler de Novo: aktiv vitamin D (1,25-dihydroxyvitamin D eller 1,25 [OH]2D) og aktive vitamin a (retinsyre eller RA). Vi valgte 1,25 (Oh)2d og RA fordi 1,25 (Oh)2d kan inducere ekspression af regulerende molekyler (f. eks, forkhead box P3 [foxp3] og interleukin-10 [Il-10])16,17 og at RA kan stimulere ekspression af Gut-homing receptorer i T-celler18. Fordi både 1,25 (Oh)2D og RA også kan tolerize DCS28,29, vi grunden til, at de konstruerede DCS vil være stabilt vedligeholdt i en tolerogenic status in vivo og dermed omgå in vivo ustabilitet bekymringer, der er forbundet med in vitro genereret tolerogenic DCS (toldcs)19,20,21. I denne henseende er toldc'er også en af de foreslåede strategier for in vivo augmentation af treg celle funktioner19,20,21. For at støtte vores ræsonnement, har vi vist, at de konstruerede DCs, ved in vivo levering, kan forøge induktion af Gut-homing Treg celler i perifert lymfoid væv6.
En yderligere fordel ved vores foreslåede strategi er, at 1,25 (OH)2D har også andre funktioner, der potentielt kan gavne IBD patienter. Disse andre funktioner omfatter evnen af 1,25 (OH)2D at stimulere udskillelsen af antimikrobielle stoffer22 og undertrykker carcinogenese23. Infektioner og kræft er ofte forbundet med IBD24,25.
For at generere de DCs, der kan producere lokalt høje koncentrationer af både 1,25 (OH)2D og RA de Novo, bruger vi en lentiviral vektor til at ingeniør DCS til at over udtrykke to gener. Et gen er cytochrom P450 familie 27 familien B medlem 1 (CYP27B1), der koder 25-hydroxyvitamin D 1α-hydroxylase (1α-hydroxylase), som fysiologisk katalyserer syntesen af 1,25 (Oh)2D. Det andet gen er aldehyd dehydrogenase 1 familiemedlem a2 (ALDH1a2), der koder retinaldehyd dehydrogenase 2 (RALDH2), som fysiologisk katalyserer syntesen af RA6.
Fordi in vivo augmentation af Gut-homing Treg celle induktion er potentielt vigtigt i behandlingen af IBD, i den følgende protokol vil vi detaljeret de procedurer for generering af de 1α-hydroxylase-RALDH2-overudtrykker DCs (DC-CYP-ALDH celler), der kan anvendes til den fremtidige undersøgelse af Gut-homing Treg celler in vivo.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 mL syringes</td>
			<td>ThermoFisher Scientific</td>
			<td>Cat# 03-377-23</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm x 20 mm culture dishes</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# CLS430167</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well culture plates</td>
			<td>ThermoFisher Scientific</td>
			<td>Cat# 07-200-82</td>
			<td></td>
		</tr>
		<tr>
			<td>150 mm x 25 mm culture dishes</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# CLS430559</td>
			<td></td>
		</tr>
		<tr>
			<td>25-hydroxycholecalciferol (25[OH]D)</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# H4014</td>
			<td></td>
		</tr>
		<tr>
			<td>293T cells</td>
			<td>ATCC</td>
			<td>CRL-3216</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>ThermoFisher Scientific</td>
			<td>Cat#: 21985023</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well culture plates</td>
			<td>ThermoFisher Scientific</td>
			<td>Cat# 07-200-83</td>
			<td></td>
		</tr>
		<tr>
			<td>ALDEFLUOR kit</td>
			<td>Stemcell Technologies</td>
			<td>Cat# 01700</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CYP27B1</td>
			<td>Abcam</td>
			<td>Cat# ab95047</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSAria II</td>
			<td>BD Biosciences</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# C1016</td>
			<td></td>
		</tr>
		<tr>
			<td>CM-10-D cell culture medium</td>
			<td></td>
			<td></td>
			<td>DMEM medium containing 10% fetal bovine serum (FBS), 100 U/ml penicillin/streptomycin, 0.055 mM 2-mercaptoethanol (2-ME), 1 mM sodium pyruvate, 0.1 mM nonessential amino acid, and 2 mM L-glutamine.</td>
		</tr>
		<tr>
			<td>CM-10-R cell culture medium</td>
			<td></td>
			<td></td>
			<td>RPMI 1640 medium (no glutamine) containing 10% fetal bovine serum (FBS), 100 U/ml penicillin/streptomycin, 0.055 mM 2-mercaptoethanol (2-ME), 1 mM sodium pyruvate, 0.1 mM nonessential amino acid, and 2 mM L-glutamine.</td>
		</tr>
		<tr>
			<td>CM-4-D cell culture medium</td>
			<td></td>
			<td></td>
			<td>DMEM medium containing 4% fetal bovine serum (FBS), 100 U/ml penicillin/streptomycin, 0.055 mM 2-mercaptoethanol (2-ME), 1 mM sodium pyruvate, 0.1 mM nonessential amino acid, and 2 mM L-glutamine.</td>
		</tr>
		<tr>
			<td>Corning bottle-top vacuum filters, 0.22 mM, 500 mL</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# CLS430513</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning bottle-top vacuum filters, 0.45 mM, 500 mL</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# CLS430514</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting scissor</td>
			<td>ThermoFisher Scientific</td>
			<td>Cat# 08-940</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM medium</td>
			<td>ThermoFisher Scientific</td>
			<td>Cat# 11960044</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>ThermoFisher Scientific</td>
			<td>Cat# 16000044</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>ThermoFisher Scientific</td>
			<td>Cat# 22-327379</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco ACK lysing buffer</td>
			<td>ThermoFisher Scientific</td>
			<td>Cat# A1049201</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG</td>
			<td>Abcam</td>
			<td>Cat# ab205718</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Millipore</td>
			<td>Cat# 391340</td>
			<td></td>
		</tr>
		<tr>
			<td>Lenti-CYP-ALDH</td>
			<td>Custom-made</td>
			<td>1.6-kb mouse CYP27B1 and ALDH1a2 cDNAs were amplified by PCR using a plasmid containing the CYP27B1 cDNA and a plasmid containing the ALDH1a2 cDNA respectively (GeneCopoeia). The amplified CYP27B1 cDNA fragment with a 5&#39; KOZAK ribosome entry sequence was cloned into the pRRL-SIN.cPPt.PGKGFP.WPRE lentiviral vector (Addgene). The resulting construct was designated as lenti-CYP-GFP. The amplified ALDH1a2 cDNA fragment was cloned into the lenti-CYP-GFP to replace the GFP and was designated as lenti-CYP-ALDH. This bicistronic plasmid expresses CYP27B1 controlled by SFFV promoter and ALDH1a2 controlled by PGK promoter.</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>ThermoFisher Scientific</td>
			<td>Cat#25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipopolysaccharide</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# L3755</td>
			<td></td>
		</tr>
		<tr>
			<td>Murine GM-CSF</td>
			<td>Peprotech</td>
			<td>Cat# 315-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Murine IL-4</td>
			<td>Peprotech</td>
			<td>Cat# 214-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# NIST2186II</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>Needles</td>
			<td>ThermoFisher Scientific</td>
			<td>Cat# 14-841-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonessential Amino Acids</td>
			<td>ThermoFisher Scientific</td>
			<td>Cat#: 11140076</td>
			<td></td>
		</tr>
		<tr>
			<td>pCMVR8.74</td>
			<td>Addgene</td>
			<td>Plasmid# 22036</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>ThermoFisher Scientific</td>
			<td>Cat#15140148</td>
			<td></td>
		</tr>
		<tr>
			<td>Phoshate Balanced Solution (PBS)</td>
			<td>ThermoFisher Scientific</td>
			<td>Cat#: 20012027</td>
			<td></td>
		</tr>
		<tr>
			<td>PMD2G</td>
			<td>Addgene</td>
			<td>Plasmid# 12259</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene tube, 15 mL</td>
			<td>ThermoFisher Scientific</td>
			<td>Cat# AM12500</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene tube, 50 mL</td>
			<td>ThermoFisher Scientific</td>
			<td>Cat# AM12502</td>
			<td></td>
		</tr>
		<tr>
			<td>Protamine sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# P3369</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit polycloncal IgG isotype control</td>
			<td>Abcam</td>
			<td>Cat# ab171870</td>
			<td></td>
		</tr>
		<tr>
			<td>Radioimmunoassay for 1,25(OH)2D measurement</td>
			<td>Heartland Assays</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 medium, no glutamine</td>
			<td>ThermoFisher Scientific</td>
			<td>Cat# 21870076</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvat</td>
			<td>ThermoFisher Scientific</td>
			<td>Cat#: 11360070</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall Legend XTR Centrifuge</td>
			<td>ThermoFisher Scientific</td>
			<td>Cat# 75004521</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Cell strainers, 40 mm</td>
			<td>ThermoFisher Scientific</td>
			<td>Cat# 07-201-430</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile storage bottles, 500 mL</td>
			<td>ThermoFisher Scientific</td>
			<td>Cat# CLS431432</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo augmentation of gut-homing regulatory t cell induction

Inflammatorisk tarmsygdom (IBD) er en inflammatorisk kronisk sygdom i mave-tarmkanalen (GUT). I USA er der ca. 1.400.000 IBD-patienter. Det er almindeligt anerkendt, at en dysreguleret immunrespons på tarm bakterier initierer sygdommen og forstyrrer slimhinde epitelial barrieren. Vi har for nylig vist, at Gut-homing regulerende T (Treg) celler er en lovende behandling for IBD. Derfor indeholder denne artikel en protokol for in vivo augmentation af tarm-homing Treg celle induktion. I denne protokol er dendritiske celler konstrueret til at producere lokalt høje koncentrationer af to molekyler de Novo, aktiv vitamin D (1,25-dihydroxyvitamin D eller 1,25 [OH]2D) og aktiv vitamin a (retinsyre eller RA). Vi valgte 1,25 (OH)2d og RA baseret på tidligere resultater, der viser, at 1,25 (Oh)2d kan inducere ekspression af regulerende molekyler (f. eks. Forkhead box P3 og interleukin-10), og at RA kan stimulere ekspression af tarm-homing receptorer i T-celler. For at generere sådanne konstruerede dendritiske celler bruger vi en lentiviral vektor til at transduce dendritiske celler til at over udtrykke to gener. Et gen er cytochrom P450 familie 27 familien B medlem 1, der koder 25-hydroxyvitamin D 1α-hydroxylase, som fysiologisk katalyserer syntesen af 1,25 (Oh)2D. Det andet gen er aldehyd dehydrogenase 1 familiemedlem a2, der koder retinaldehyd dehydrogenase 2, som fysiologisk katalyserer syntesen af RA. Denne protokol kan anvendes til fremtidig undersøgelse af Gut-homing Treg celler in vivo.

DC-CYP-ALDH-celler udtrykte signifikant øget mængde af 1α-hydroxylase. For at afgøre, om DC-CYP-ALDH-celler genereret fra Bmdc'er udtrykte en signifikant forøget mængde på 1α-hydroxylase, blev Bmdc'er transduceret med Lenti-CYP-ALDH-virus til at producere knoglemarv afledte DC-CYP-ALDH-celler (BMDC-CYP-ALDH-celler). Efterfølgende blev BMDC-CYP-ALDH-cellerne undersøgt for ekspression af 1α-hydroxylase af FACS. Vores data viste, at BMDC-CYP-ALDH-cellerne, sammenlignet med forældre-Bmdc'erne, vi...

Watch this Scientific Journal Video about In Vivo Augmentation of Gut-Homing Regulatory T Cell Induction at JoVE.com

In Vivo Augmentation of Gut-Homing Regulatory T Cell Induction

Her præsenterer vi en protokol for in vivo augmentation af Gut-homing regulatoriske T-celle induktion. I denne protokol er dendritiske celler konstrueret til lokalt at producere høje koncentrationer af det aktive vitamin D (1,25-dihydroxyvitamin D eller 1,25 [OH]2D) og den aktive vitamin a (RETINSYRE eller RA) de Novo.

In vivo augmentation af Gut-homing regulerende T-celle induktion

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

Denne protokol beskriver en potentielt ny terapeutisk strategi, der let kan ændres til menneskelige anvendelser. Denne teknik direkte øger induktion af gut-homing regulerende T-celler i perifere lymfoide væv og kan stabilt gennemføres selv i meget pro-inflammatoriske miljøer. Disse teknikker kan også bruges til at undersøge den rolle, som aktive D-vitamin og vitamin A i det perifere immunsystem.Denne visuelle demonstration vil give trin for trin instruktioner til generering af høj kvalitet manipuleret dendritiske celler, der er afgørende for succes af denne teknik. Begynd med at så en gang 10 til de syv 293T-celler i 20 milliliter CM-10-D cellekulturmedium i 150 gange med 25 millimeter kulturplader til en 24 timers inkubation ved 37 grader Celsius og 5 % kuldioxid. Den næste dag, tilsættes 1,62 milliliter frisklavet dobbelt koncentreret HBS opløsning, 9,5 mikrogram kuvert plasmid, 17,5 mikrogram emballage plasmid, og 27 mikrogram af LENTI-CYP-ALDH plasmid til en 50 milliliter konisk rør.Rørindholdets endelige volumen bringes op til 3,0375 milliliter med vand efterfulgt af tilsætning af 202,5 mikroliter af to molarcalciumchlorid dråbevis. Lad DNA-udfældningsblandingen være ved stuetemperatur i 20 minutter med lejlighedsvis blanding, før der tilsættes 3,24 milliliter af opløsningen dråbevis pr. 293T-kultur, mens skålen forsigtigt hvirvles rundt. Pladerne hældes i inkubatoren i 24 timer, før hver plade vaskes forsigtigt, og cellerne fodres med CM-4-D dyringsmedie.Efter yderligere 24 timers kultur, høst supernatanterne i sterile flasker til opbevaring på fire til otte grader Celsius og fodre cellerne med frisk CM-4-D medium i yderligere 24 timer. På dag fire, høste supernatanter igen og stamme supernatanter fra begge dage tre og fire til en 0,45 mikrometer filter. Derefter koncentrere VSV-G pseudotyped virus ved centrifugering i 24 timer.Den næste dag dekantere supernatanter. Lad rørene løbe af på en køkkenrulle i et sterilt biosikkerhedsskab i flere minutter, hvortil pelletsene skal opslæmes i 33,3 mikroliter steril PBS, der indeholder 5% glycerol pr. kulturskål. For knoglemarv afledt dendritisk celle generation, placere skinneben og lårben, høstet fra BALB / c mus, i en 100 af 20 millimeter kultur parabol indeholder frisk kultur medium.Brug dissekere saks og saftæt til at fjerne væv fra knoglerne. Når alle knoglerne er blevet renset, skal du bruge saksen til at skære begge ender af hver knogle til at eksponere knoglemarvshulerne. Brug en 10 milliliter sprøjte udstyret med en 30 gauge nål til at skylle hver knogle med 10 milliliter frisk kultur medium, indsamle knoglemarven i en 15 milliliter rør.Når alle knoglemarven er indsamlet, sediment knoglemarven ved centrifugering og resuspenderet pellet i to milliliter røde blodlegemer lysis buffer. Efter fire til fem minutter ved stuetemperatur ved lejlighedsvis omrystning stoppes reaktionen med 10 milliliter cellekulturmedium, og cellerne opsamres ved centrifugering. Opslæm pellet i 10 milliliter CM-10-R kultur medium til optælling og justere cellerne for en gange 10 til de seks celler pr milliliter CM-10-R kultur medium koncentration.Dernæst tilsættes 100 enheder pr. milliliter rekombinant murine GM-CSF og 10 enheder pr. milliliter IL-4 til cellerne. Plade fire milliliter celler i individuelle brønde af en seks godt kultur plade for deres inkubation ved 37 grader Celsius og 5% kuldioxid i 48 timer. Ved slutningen af inkubationen, forsigtigt aspirere de ikke-klæbende celler.Tilsæt frisk medium suppleret med GM-CSF og IL-4 til kulturerne, før cellerne returneres til cellekulturinkubatoren i yderligere 48 timer. Ved afslutningen af den anden inkubation høstes de ikke-klæbende knoglemarv afledte dendritiske celler i 50 milliliter konisk rør for deres centrifugering. For at generere BMDC-CYP-ALDH celler, resuspend kulturerne på en gange ti til den sjette dendritiske celler pr 500 mikroliter CM-10-R medium pr brønd koncentration, og plade 500 mikroliter af celler i hver brønd af en ny seks godt kultur plade.Tilsæt 50 mikroliter LENTI-CYP-ALDH-virus og 8 mikrogram pr. milliliter protamin til hver brønd, og anbring derefter pladen i inkubatoren i 24 timer. Den næste dag, erstatte supernatanter med frisk kultur medium og returnere pladerne til cellekultur inkubator i yderligere 24 timer. Ved afslutningen af den anden inkubation kan cellerne aktiveres med 100 nanogram pr. milliliter lipopolysaccharid pr. brønd i 24 timer før høst til funktionelle analyser.Sammenlignet med forældrenes knoglemarv afledte dendritiske celler viser BMDC-CYP-ALDH-celler et forbedret udtryk for 1-alfa-hydroxylase. Koncentrationerne af den aktive form for D-vitamin i dyrknonatanterne af cellerne BMDC-CYP og BMDC-CYP-ALDH er ca. 20 gange højere end dem, der er observeret i forældrebensmarven afledt dendritisk celle og BMDC-ALDH-cellekulturerne. De gennemsnitlige fluorescerende intensiteter af BMDC-CYP-ALDH-celler, der behandles med substratet, er ca. seks gange højere end for de forældrebenmarvbaserede dendritiske celler, der er behandlet med substratet.Dette tyder på, at BMDC-CYP-ALDH celler udtrykke betydeligt forbedret retinale dehydehydrogenase-2 enzymatisk aktivitet. DC2.4 celler behandlet med 25-hydroxy D-vitamin og retinale ikke fremkalde en væsentlig ændring i den overflod af foxp3 positive CCR9 positive CD4 positive T-celler. I modsætning hertil fremkalder DC2.4 CYP-ALDH-celler behandlet med 25-hydroxy D-vitamin og retinal betydeligt højere antal gut-homing regulerende T-celler på en koncentrationsafhængig måde.Sammenlignet med intraperitonealt injicerede kontrolceller fremkalder DC CYP-ALDH-celler desuden en signifikant stigning i antallet af foxp3 positive CCR9 positive CD4 positive T-celler efter intraperitoneal injektion i BALB/c recipientdyr. Procedurens succes afhænger af forberedelsen af sunde T-celler og en korrekt forberedt DNA-blanding.

Research

JoVE Journal

Immunology and Infection

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Induktion af graft-versus-host sygdom og<em&gt; In Vivo</em&gt; T Cell Monitoring Brug en MHC-matchet murin model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

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 ...

piggyBac Transposon System Modification of Primary Human T Cells

piggyBac Transposon System Modification of Primary Human T Cells

piggyBac Transposon System Ændring af primære humane T-celler

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

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

Nye værktøjer til at udvide regulatoriske T celler fra HIV-1-inficerede personer

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

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

Anvendelse af fluorescerende Skud Arrays for Vurdering af T-celle respons<em&gt; In vivo</em

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of ...

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

En<em&gt; Ex vivo</em&gt; Model af en oligodendrocyt rettet T-celle Angrib akut Brain Skiver

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Induktion af murine tarmbetændelse ved adoptiv overførsel af effektor CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; Høj</sup&gt; T celler i immunsvækkede mus

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Mouse NaÃƒÆ’Ã‚Â¯ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Mus Naïve CD4<sup&gt; +</sup&gt; T Cell Isolation og<em&gt; In vitro</em&gt; Differentiering til T-celledelmængder

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...