Name: induction of intestinal inflammation by adoptive transfer of cbir1 tcr transgenic cd4+ t cells to immunodeficient mice
Uploaded: 2021-12-16T14:30:00
Description: Watch this Scientific Journal Video about Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice at JoVE.com

Dette arbejde blev delvist støttet af National Institutes of Health-tilskud DK125011, AI150210 og DK124132, University of Texas System STARs-prisen (Y.C.) og James W. McLaughlin Fellowship Fund fra University of Texas Medical Branch i Galveston (W.Y.). Figur 1 blev oprettet med BioRender.com.

Alle dyreforsøg blev udført i henhold til University of Texas Medical Branch's Committee on the Use and Care of the Animals. CBir1 TCR Tg mus blev leveret af Dr. Charles Elson fra University of Alabama i Birmingham. CBir1 TCR Tg mus kan være hun- eller hanmus, men bør være på 8-12 uger. Rag1-/- mus på C57BL/6-baggrunden blev opnået fra Jackson Laboratory10. Rag1-/- mus skal være køns- og aldersmatchede, og enten han eller hun kan bruges, men skal ...

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Selvom hvert trin er afgørende for reproducerbarheden af denne colitis-model, er der flere kritiske trin. Modtageren Rag-/- mus skal modtage tilstrækkelig levedygtige naϊve CD4 + T-celler til at fremkalde tarmbetændelse. Vi brugte milt til isolering af naive CD4 + T-celler i stedet for MLN'er. Fordi udbyttet af naive CD4 + T-celler i MLN'er er meget lavere end i milt. CD62L udtrykkes stærkt i naive T-celler, og CD44 og CD25 er aktiveringsmarkørerne for T-cel...

Inflammatoriske tarmsygdomme (IBD), hovedsageligt herunder Crohns sygdom (CD) og ulcerøs colitis (UC), er karakteriseret ved kronisk, recidiverende-remitterende betændelse i mave-tarmkanalen, der påvirker millioner over hele verden1. Flere faktorer har været impliceret i udviklingen og patogenesen af IBD, herunder genetisk modtagelighed, tarmmikrobiota, immunresponser, kost og livsstil2. Den nøjagtige mekanisme for IBD er dog stadig ikke helt forstået.
En af de særlige interesser er samspillet mellem tarmmikrobiota og værtsimmunresponser i reguleringen af tarmbetændelse3. Tarmmikrobiota tilvejebringer en række immunstimulerende molekyler og antigener, som kan aktivere immunresponser4. Mens balancen mellem effektor T-celler og regulatoriske T-celler (Tregs) er kritisk for at opretholde intestinal homeostase, bidrager det overdrevne tarmslimhinde-CD4 + T-cellerespons på tarmmikrobiotaantigener til tarmbetændelse5,6,7. Som et immundominerende tarmmikrobiotaantigen har CBir1 flagellin været relateret til patogenesen af human CD8,9. Desuden inducerer overførsel af CBir1 TCR transgene (Tg) T-celler tarmbetændelse hos immunmangelfulde mus6, der ligner den humane IBD, hvilket indikerer, at denne T-celleoverførselsmodel hjælper med at undersøge mekanismerne for human IBD.
Dette arbejde beskriver den detaljerede protokol for inducering af colitis hos Rag1-/- mus ved adoptivoverførsel af CBir1 TCR Tg naϊve CD4+ T-celler og vurdering af sygdommens sværhedsgrad. Desuden vises de forventede resultater, og de kritiske trin i proceduren og fejlfinding diskuteres, hvilket vil hjælpe forskere med at undersøge mekanismerne for patogenese af tarmbetændelse og teste de potentielle lægemidler til behandling af IBD.

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		<tr>
			<td>0.22 &#181;m vacuum-driven disposable bottle top filter</td>
			<td>MilliporeSigma</td>
			<td>SCGPS05RE</td>
			<td></td>
		</tr>
		<tr>
			<td>100x Penicillin-Streptomycin</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>100-&#181;m strainer</td>
			<td>BD Biosciences</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>3-mL Transfer Pipette</td>
			<td>Fisherbrand</td>
			<td>13-711-9CM</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD16/32</td>
			<td>Biolegend</td>
			<td>101302</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD25-Percp/Cy5.5</td>
			<td>Biolegend</td>
			<td>102030</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD3-Percp/Cy5.5</td>
			<td>Biolegend</td>
			<td>100327</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD4 APC</td>
			<td>Biolegend</td>
			<td>100516</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD4 Magnetic Particles</td>
			<td>BD Biosciences</td>
			<td>551539</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD4-BV421</td>
			<td>Biolegend</td>
			<td>100544</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD62L-PE</td>
			<td>Biolegend</td>
			<td>104408</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse Foxp3-PE</td>
			<td>ThermoFisher</td>
			<td>12-5773-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse IFN&#947;-FITC</td>
			<td>Biolegend</td>
			<td>505806</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse IL-17A-PE/Cy7</td>
			<td>Biolegend</td>
			<td>506922</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated Cell Counter</td>
			<td>Bio-rad</td>
			<td>TC20</td>
			<td></td>
		</tr>
		<tr>
			<td>Brefeldin A</td>
			<td>BD Biosciences</td>
			<td>555029</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Fisher Bioreagents</td>
			<td>BP1600-1</td>
			<td></td>
		</tr>
		<tr>
			<td>C tube</td>
			<td>Miltenyi</td>
			<td>130-093-237</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Separation Magnet</td>
			<td>BD Biosciences</td>
			<td>552311</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase IV</td>
			<td>Sigma-Aldrich</td>
			<td>C5138</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissociator Machine</td>
			<td>Miltenyi</td>
			<td>130-096-427</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Corning</td>
			<td>46-034-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA (0.5 M, PH 8.0)</td>
			<td>Corning</td>
			<td>46-034-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>R&#38;D Systems</td>
			<td>S11550</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>BD Biosciences</td>
			<td>LSD Fortessa</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat Lamp</td>
			<td>CoverShield</td>
			<td>BR40</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin and Eosin (H&#38;E) Stain Kit</td>
			<td>Abcam</td>
			<td>ab245880</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin Syringes</td>
			<td>BD Biosciences</td>
			<td>329412</td>
			<td></td>
		</tr>
		<tr>
			<td>Ionomycin</td>
			<td>ThermoFisher</td>
			<td>I24222</td>
			<td></td>
		</tr>
		<tr>
			<td>Live/dead Fixable Near-IR Dead Cell Stain kit</td>
			<td>ThermoFisher</td>
			<td>L10119</td>
			<td></td>
		</tr>
		<tr>
			<td>MaxQ 6000 Incubated/Refrigerated Stackable Shakers</td>
			<td>ThermoFisher</td>
			<td>SHKE6000</td>
			<td></td>
		</tr>
		<tr>
			<td>NH4Cl</td>
			<td>Thermo Scientific</td>
			<td>A687-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll</td>
			<td>GE Healthcare</td>
			<td>17-0891-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Phorbol-12-myristate 13-acetate</td>
			<td>Sigma-Aldrich</td>
			<td>P8139</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium</td>
			<td>Cytiva HyClone</td>
			<td>SH3002702</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorter</td>
			<td>BD Biosciences</td>
			<td>Arial Fusion</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Automatic Processor</td>
			<td>ThermoFisher</td>
			<td>STP120</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Embedding/Processing Cassette</td>
			<td>Fisher Healthcare</td>
			<td>22048142</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Thermo Scientific</td>
			<td>BP154-1</td>
			<td></td>
		</tr>
		<tr>
			<td>True-Nuclear Transcription Factor Buffer Set (including Perm Buffer)</td>
			<td>Biolegend</td>
			<td>424401</td>
			<td></td>
		</tr>
	</tbody>
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induction of intestinal inflammation by adoptive transfer of cbir1 tcr transgenic cd4+ t cells to immunodeficient mice

Med stigningen i forekomsten pålægger inflammatoriske tarmsygdomme (IBD), som er kroniske sygdomme, der påvirker mave-tarmkanalen, en betydelig sundhedsmæssig og økonomisk byrde for enkeltpersoner og samfund. Derfor er det afgørende at undersøge de mekanismer, der ligger til grund for patogenesen og udviklingen af IBD. Her beskrives en tarmmikrobiota-antigenspecifik T-celleoverførselskolitismodel. CBir1 flagellin er blevet anerkendt som det immundominerende tarmbakterieantigen i eksperimentel colitis og patienter med Crohns sygdom. CBir1 TCR transgene naϊve CD4 + T-celler, der er specifikke for CBir1 flagellin, kan fremkalde kronisk colitis efter adoptiv overførsel til immunmangelfulde Rag1-/- mus. Sygdommens sværhedsgrad vurderes af histopatologi. CD4 + T-cellefænotyperne i colonic lamina propria bestemmes også. Denne model ligner meget udviklingen af IBD, som giver en ideel murinmodel til undersøgelse af de mekanismer, der driver patogenesen af IBD og tester de potentielle lægemidler til behandling af IBD.

Ca. 5 x 106 CBir1 TCR Tg naϊve CD4+ T-celler pr. milt blev isoleret fra en voksen CBir1 TCR Tg-mus. Overførsel af CBir1 TCR Tg naϊve CD4+ T-celler induceret kronisk colitis hos modtager Rag1-/- mus. Efter celleoverførsel blev kliniske tegn overvåget for at evaluere progressionen af tarmbetændelse, herunder vægttab, afføringskonsistens og bøjet kropsholdning. Som forventet begyndte mus at tabe sig omkring tre uger efter celleoverførsel, og vægten nåede omkri...

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Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

I denne protokol beskrives en tarmmikrobiota-antigenspecifik T-celleadoptel overførsel colitis-model. CD4+ T-celler isoleres fra CBir1 TCR-transgene mus. Disse er specifikke for et immundominerende tarmmikrobiota-antigen CBir1 flagellin, som overføres til modtager Rag1-/- mus, hvilket fører til tarmbetændelse.

Induktion af tarmbetændelse ved adoptivoverførsel af CBir1 TCR transgene CD4+ T-celler til immundefekte mus

With the increase of incidence, inflammatory bowel diseases (IBD), which are chronic diseases affecting the gastrointestinal tract, impose a considerable ...

Inflammatorisk tarmsygdom, eller IBD, pålægger enkeltpersoner og samfundet en betydelig sundhedsmæssig og økonomisk byrde. Denne colitis model er nyttig til at studere mekanismerne i IBD og evaluere behandlingerne for IBD. Denne immundominerende Cbir flagellin T-celle-specifik adoptiv transfer colitis model giver indsigt i, hvordan tarmbakterier antigen inducerer T-celle reaktioner for at inducere colitis.Efter aflivning af musen skal du lave et ca. en centimeter venstre abdominal snit og trække huden væk fra mavemuskelvævet. Lav derefter et ca. tre centimeter snit i mavemuskelvævet. Fjern derefter milten med steril saks og tang.Læg milten i en kulturskål, der indeholder fem ml forkølet vaskebuffer. Slib milten med den ru overflade af to sterile glasrutschebaner. Overfør cellesuspensionen til et 50 ml centrifugerør ved at føre det gennem en 100 mikrometer cellesil.Skyl glasglider og kulturskål med fem milliliter forkølet vaskebuffer, og overfør vaskebufferen ind i røret. Centrifugering af cellesuspensionen, kassér supernatanten og resuspend cellerne med fem milliliter forvarmet Tris ammoniumchloridlysebuffer pr. Milt. Inkuber i 10 minutter ved stuetemperatur.Tilsæt 10 ml forkølet vaskebuffer til røret. Centrifuger derefter cellesuspensionen, kassér supernatanten og resuspend cellerne med 10 ml forkølet isolationsbuffer. For at tælle cellerne blandes 10 mikroliter hver af cellesuspension og Trypan blå grundigt og lægger 10 mikroliter på et dias.Indsæt derefter diaset i den automatiserede celletæller for at opnå det levedygtige cellenummer. Centrifugering af den resterende cellesuspension og kassér alle supernatant. Vortex anti-mus CD4 magnetiske partikler, direkte tilføje 50 mikroliter af partiklerne pr. 10 millioner celler, og bland med cellepiller grundigt.Inkuber blandingen i 30 minutter ved fire grader Celsius. Overfør cellepartikelsuspensionen til et sterilt opsamlingsrør. Tilsæt 3,5 ml forkølet isolationsbuffer i røret.Placer røret på celleseparationsmagneten i otte minutter ved stuetemperatur. Derefter aspireres forsigtigt supernatanten af med en overførselspipette på tre milliliter. Fjern røret fra celleseparationsmagneten.Derefter suspenderes cellerne igen med 3,5 ml forkølet isolationsbuffer og anbringes røret på magneten i fire minutter ved stuetemperatur. Aspirer forsigtigt supernatanten med en pipette på tre milliliter. Endelig suspenderes cellerne igen i en milliliter forkølet FACS-buffer.For at overføre cellerne til modtagermusene skal du suspendere Cbir1 TCR transgene-naive CD4-positive T-celler til 5 millioner celler pr. Milliliter i 1x PBS. Varm musene op under en varmelampe, og hold musene tilbage ved hjælp af en musebegrænsning. Intravenøst injicere 200 mikroliter af cellesuspensionen i musens halevene.Efter aflivning af musen skal du lave et ca. en centimeter ventralt midterlinjehudsnit. Træk huden væk fra mavemuskelvævet. Lav derefter et tre centimeter snit i mavemuskelvævet.Identificer derefter coecum og fjern hele tyktarmen med steril saks og tang. Våd tyktarmen med forkølet PBS i en kulturskål. Brænd tyktarmen i længderetningen, og skyl den med forkølet PBS.Skær 1/3 af tyktarmen i længderetningen. Læg tyktarmsstrimlen i et papirhåndklæde med lumenalsiden opad. Udfør schweizisk rulning ved hjælp af et tandstikker.Placer tyktarmen Swiss i en kassette, og sæt kassetten i 10% bufret formalin i 24 timer. Dehydrer og paraffin-integrer tyktarmen med en automatiseret processor. Skær derefter fem mikrometer vævssektioner på et mikrotome.Monter vævet på dias og udfør hæmatoxylin og eosin farvning. Rag knockout-musene begyndte at tabe sig omkring tre uger efter celleoverførsel, og deres vægt nåede omkring 80 til 85% af deres oprindelige vægt seks uger efter celleoverførsel. Modtagermus viste kort tyktarmslængde seks uger efter celleoverførsel.Modtagermusene viste mere celleinfiltration i tarmlamina propria fire uger efter celleoverførsel. De viste bægercelletab og intestinal epitelcellehyperplasi fem uger efter celleoverførsel samt slimhindeerosion og inflammatorisk celleinfiltration i submucosa i tyktarmen seks uger efter celleoverførsel. De histopatologiske scorer steg betydeligt fra fire til seks uger.Samtidig var der ingen betændelse i Rag knockout-mus, der fik PBS alene. Ved hjælp af denne procedure kunne coloncytokinniveauer bestemmes af ELISA og qPCR.

Research

JoVE Journal

Medicine

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

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Fluorescens-medieret tomografi til påvisning og kvantificering af makrofag-relaterede Murine tarmbetændelse

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Nephrotic syndrome is the most extreme manifestation of proteinuric kidney disease and characterized by heavy proteinuria, hypoalbuminemia, and edema due ...