Wir danken Drs.. Robert Bonneau und Neil Christensen für hilfreiche Kommentare. Diese Arbeit wurde von der Pennsylvania State University College of Medicine Fonds unterstützt.

1. Isolierung von T-Zellen aus Milz und Lymphknoten von Mäusen BDC2.5 <ol><li> Verwenden Sie 6 Wochen alten Pre-diabetischen weiblichen Mäuse BDC2.5 als Spender von diabetogene CD4 + T-Zellen. Mäuse sollten Diabetes-frei durch Uringlucose Messung (siehe unten) bestimmt. </li><li> Euthanize jede Maus mit CO 2-Erstickung und entfernen Sie die Milz, Achsel-und brachial Lymphknoten unter sterilen Bedingungen. Um die Milz zu entfernen, tränken Sie das Fell mit 70% Ethanol, dann geschnitten und einfahren Haut. Die Milz wird sichtbar als dunkelrotes Organ auf der linken Seite der Maus. Machen Sie eine 1-Zoll-Schnitt im Bauchfell mit den kleinen Schere und sanft greifen die Milz in der Mitte mit einem Paar kleine Pinzette. Sorgfältig schneiden das Bindegewebe und die angeschlossenen Fett so weit wie möglich zu entfernen und die Milz. </li><li> Sammeln Sie die Milz und die Lymphknoten in 10 ml Dulbecco&#39;s-modifiziertem Eagle-Medium (DMEM) in einem 15 ml konischen Röhrchen auf Eis. </li><li> Bereiten Sie eine einzelne Zelle Suspension mit the Ende einer sterilen 10 ml Spritze Kolben vorsichtig auf die lymphatischen Organen durch 70 um Zelle Siebe (eine pro Milz Sieb und 4-6 Lymphknoten pro Sieb) in den gleichen 50 ml konischen Röhrchen. </li></ol> Während des Prozesses, spülen jedes Sieb mit 1 ml DMEM mehrmals, um die Gewinnung von Zellen aus dem Sieb zu maximieren. <ol start="5"><li> Übertragen der gesammelten Zellen von dem 50 ml konischen Röhrchen in ein 15 ml konischen Röhrchen und zentrifugieren bei 300-400 xg für 7 min bei RT. </li><li> Um rote Blutzellen lysieren, den Überstand verwerfen, wieder die Zellen in 5 ml Ammonium-Chlorid-Kalium (ACK)-Puffer (pH 7,2) und inkubieren Sie die ACK-Zell-Suspension bei RT für 5 min. </li><li> 10 ml DMEM dem ACK-Zell-Suspension und zentrifugieren wie oben. Waschen Sie das Zellpellet einmal in 10 ml DMEM. </li></ol> 2. Fluorescence-Activated Cell Sorting von diabetogene CD4 + T-Zellen von Mäusen BDC2.5 <ol><li> Re-suspend Zellen in 5 ml FACS sTaining Puffer und zählen die Anzahl der lebensfähigen Zellen (mit einem Phasenkontrast-Mikroskop und einer Zählkammer) durch Trypanblau Ausgrenzung. </li><li> Mit FACS-Puffer, passen die Zellsuspension Volumen bis 5 x 10 7 Zellen / ml. Entfernen ~ 1 x 10 6 Zellen pro Färbung Kontrolle (1 keine Flecken, 3 Einzelzimmer Flecken). Färben der Probe auf nicht-aktivierten transgenen CD4 + T-Zellen mit anti-CD4 (APC), anti-TCR Vβ4 (FITC) und Anti-CD62L (PE) monoklonaler Antikörper (mAb) in einem 15 ml-Röhrchen. Führen Zellfärbung mit den mAb Konzentrationen vom Hersteller für 20-30 min schlug bei 4 ° C im Dunkeln. </li><li> Waschen Sie die Probe und einzigen Fleck Kontrollen mit FACS-Puffer bei&gt; 3X der Färbereaktion Volumen. Zentrifugieren bei 300-400 xg, entfernen Sie den Überstand und resuspendieren die Zellen in FACS-Puffer (1-2 x 10 7 Zellen / ml für die Sortierung von Probe und 300 ul für einzelne Fleck Kontrollen) und auf Eis lagern bis zum nächsten Schritt. </li><li> Übergeben Sie die Zelle Probe durch eine 35 um ZelleSieb Haubenrohr zu entfernen Zellklumpen. Ordnen Sie die Proben für CD4 + TCR Vβ4 + CD62L + Zellen auf einem Cell Sorter mit einem geschulten Bediener. Sammeln Sie die sortierten Zellen in 3 ml DMEM in einer 15 ml Tube. Erlauben 2,5-3 Std., einschließlich Setup, bis 1,5 x 10 8 Zellen (die ungefähre Anzahl von Zellen aus 3 Donormäusen erhalten) an einer Art von 2 x 10 4 insgesamt events / sec sortieren. Erwarten ~ 2,5 x 10 6 nicht aktivierten transgenen CD4 + T-Zellen pro Maus nach cell sorting. </li><li> Notieren Sie sich die absolute Zahl der sortierten Zellen am Ende der Sortierung und zentrifugieren für 7 min bei 300-400 g x. Entsorgen Sie den Überstand und resuspendieren die Zellen (2 x 10 6 Zellen / ml) in steriler Phosphat-gepufferter Kochsalzlösung (PBS) (Mg 2 + / Ca 2 +-frei) für die T-Zell-Transfer Adoptiveltern. </li></ol> 3. Adoptiven Transfer von CD4 + T diabetogene Zellen aus BDC2.5 Mäuse <ol><li> Mit einer 1-ml-Spritze und ein 18-1 ½-Gauge-Nadel, sanft resuspendieren die FACS-gereinigten CD4 +T-Zellen und laden Sie die 1 ml Spritze. In Vorbereitung zur Injektion, ersetzen Sie den 18-1 ½-Gauge-Nadel mit einer 27-Gauge-Nadel ½. </li><li> Expose 6-8 Wochen alte weibliche NOD.SCID Empfängermäuse einer Wärmelampe, bis sie Pflege Verhalten zeigen. </li><li> Zurückhalten Empfängermäuse in einem Verzögerer und wischen dem Schwanz mit 70% (v / v) Ethanol, um die Injektionsstelle zu desinfizieren. </li><li> Spritzen 1-2 x 10 6 FACS sortierten Zellen / Maus (in bis zu 500 ul PBS) entweder in der seitlichen Schwanzvenen. </li></ol> Nicht mit Gewalt den Kolben. Wenn die Nadel richtig in der Vene liegt, wird die Einspritzung fast ohne Widerstand zu nehmen. 4. Überwachung Empfänger Mäuse für Hyperglykämie und T1D <ol><li> Beginnend fünf Tage nach der T-Zell-Übertragung werden NOD.SCID Empfängermäuse täglich erhöhten Urin Glucose unter Verwendung von Teststreifen (Bayer Diastix) gemäß den Anweisungen des Herstellers überwacht. Mäuse, die mit zwei aufeinanderfolgenden urine Blutzuckerwerte&gt; 250 mg / dl gelten als Diabetiker. </li></ol>

<ol>
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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Syngeneic+transfer+of+autoimmune+diabetes+from+diabetic+NOD+mice+to+healthy+neonates.+Requirement+for+both+L3T4++and+Lyt-2++T+cells.">Syngeneic transfer of autoimmune diabetes from diabetic NOD mice to healthy neonates. Requirement for both L3T4+ and Lyt-2+ T cells.</a> J. Exp. Med. 166, 823-833 (1987).</li><li>Haskins, K., McDuffie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acceleration+of+diabetes+in+young+NOD+mice+with+a+CD4++islet-specific+T+cell+clone.">Acceleration of diabetes in young NOD mice with a CD4+ islet-specific T cell clone.</a> Science. 249, 1433-1436 (1990).</li><li>Christianson, S. W., Shultz, L. D., Leiter, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adoptive+transfer+of+diabetes+into+immunodeficient+NOD-scid/scid+mice.+Relative+contributions+of+CD4++and+CD8++T-cells+from+diabetic+versus+prediabetic+NOD.NON-Thy-1a+donors.">Adoptive transfer of diabetes into immunodeficient NOD-scid/scid mice. Relative contributions of CD4+ and CD8+ T-cells from diabetic versus prediabetic NOD.NON-Thy-1a donors.</a> Diabetes. 42, 44-55 (1993).</li><li>Wicker, L. S., Todd, J. A., Peterson, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+control+of+autoimmune+diabetes+in+the+NOD+mouse.">Genetic control of autoimmune diabetes in the NOD mouse.</a> Annual Reviews in Immunology. 13, 179-200 (1995).</li><li>Serreze, D. V., 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=MHC+class+I-mediated+antigen+presentation+and+induction+of+CD8++cytotoxic+T-cell+responses+in+autoimmune+diabetes-prone+NOD+mice.">MHC class I-mediated antigen presentation and induction of CD8+ cytotoxic T-cell responses in autoimmune diabetes-prone NOD mice.</a> Diabetes. 45, 902-908 (1996).</li><li>Milton, M. J., Poulin, M., Mathews, C., Piganelli, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation,+maintenance,+and+adoptive+transfer+of+diabetogenic+T-cell+lines/clones+from+the+nonobese+diabetic+mouse.">Generation, maintenance, and adoptive transfer of diabetogenic T-cell lines/clones from the nonobese diabetic mouse.</a> Methods Mol. Med. 102, 213-225 (2004).</li><li>Kurrer, M. O., Pakala, S. V., Hanson, H. L., Katz, J. 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D., Wang, B., Haskins, K., Benoist, C., Mathis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Following+a+diabetogenic+T+cell+from+genesis+through+pathogenesis.">Following a diabetogenic T cell from genesis through pathogenesis.</a> Cell. 74, 1089-1100 (1993).</li><li>Stadinski, B. D., 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=Chromogranin+A+is+an+autoantigen+in+type+1+diabetes.">Chromogranin A is an autoantigen in type 1 diabetes.</a> Nat. Immunol. 11, 225-231 (2010).</li><li>Luhder, F., Chambers, C., Allison, J. P., Benoist, C., Mathis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pinpointing+when+T+cell+costimulatory+receptor+CTLA-4+must+be+engaged+to+dampen+diabetogenic+T+cells.">Pinpointing when T cell costimulatory receptor CTLA-4 must be engaged to dampen diabetogenic T cells.</a> Proc. Natl. Acad. Sci. U.S.A. 97, 12204-12209 (2000).</li><li>Tang, Q., 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=In+vitro-expanded+antigen-specific+regulatory+T+cells+suppress+autoimmune+diabetes.">In vitro-expanded antigen-specific regulatory T cells suppress autoimmune diabetes.</a> J. Exp. Med. 199, 1455-1465 (2004).</li><li>Calderon, B., Suri, A., Pan, X. O., Mills, J. C., Unanue, E. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+autoimmune+diabetes+locus+Idd9+regulates+development+of+type+1+diabetes+by+affecting+the+homing+of+islet-specific+T+cells.">The autoimmune diabetes locus Idd9 regulates development of type 1 diabetes by affecting the homing of islet-specific T cells.</a> J. Immunol. 176, 5455-5462 (2006).</li><li>Verdaguer, 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=Spontaneous+autoimmune+diabetes+in+monoclonal+T+cell+nonobese+diabetic+mice.">Spontaneous autoimmune diabetes in monoclonal T cell nonobese diabetic mice.</a> J. Exp. Med. 186, 1663-1676 (1997).</li><li>Thomas, D. C., Mellanby, R. J., Phillips, J. 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Alle Mäuse wurden an der Penn State College of Medicine specific pathogen-freie (SPF)-Anlage in Übereinstimmung mit den Richtlinien der Penn State Institutional Animal Care und Verwenden Ausschuss untergebracht. Die Autoren erklären, dass sie keine finanziellen Interessen haben.

T1D in Empfängermäuse bei unterschiedlichen Wirkungsgrade durch adoptiven Transfer von ganzen Milzzellen oder T-Zell-Untergruppen von diabetischen NOD-Mäusen oder Mäusen transgenen TCR von diabetogene T-Zell-Klone abgeleitet induziert werden. Wir berichten hier über ein reproduzierbares Verfahren zur Induktion in Empfängermäuse innerhalb von zwei Wochen T1D bei 100% Inzidenz durch Übertragung FACS-gereinigten CD62L + BDC2.5 transgenen CD4 + T-Zellen in Mäusen NOD.SCID. Besondere Vor...

Die NOD-Maus entwickelt Autoimmundiabetes spontan und wurde vielfach als Tiermodell für die menschliche T1D 1,2 verwendet. Pathogenese der T1D in NOD-Mäusen durch Infiltration, beginnend bei 3-4 Wochen alt, der pankreatischen Langerhans-Inseln von dendritischen Zellen und Makrophagen durch T und B-Zellen folgte. Diese Phase der zerstörungsfreien peri-Insulitis führt zu einem langsamen, fortschreitenden Zerstörung der Insulin-produzierenden β-Zellen der Bauchspeicheldrüse, was zu manifester Diabetes von 4-6 Monaten 3 Jahren. Übertragen von Splenozyten 4,5, CD4 + oder CD8 + 6,7 8,9 T-Zellen von diabetischen NOD-Mäusen haben gezeigt, dass Diabetes bei immungeschwächten Mäusen NOD vermitteln, was anzeigt, dass Insel-reaktiven T-Zellen eine zentrale Rolle in T1D Pathogenese spielen. Abhängig von den experimentellen Bedingungen, entwickelt Diabetes in Empfängermäuse langsam, über mehrere Wochen in diesen Studien. Ebenso verschiedenen T-Zell-Klone, von Zeit-und kostenintensive abgeleitetKultivieren von diabetogenen T-Zellen wurde berichtet, Diabetes mehrere Wochen nach der Überführung in Empfängermäuse 7,10 vermitteln. Mit der Verfügbarkeit von transgenen Mäusen, die TCRs von CD4-oder CD8-restringierte T-Zell-Klone diabetogen, mehreren Laboratorien abgeleitet wurden später gezeigt, dass Milz-T-Zellen aus diesen Mäusen in der Lage, Diabetes Empfänger 11-13 übertragen wurden. Insbesondere sind BDC2.5 NOD-Mäuse transgen für das BDC2.5 TCR, die spezifisch für Chromogranin A, ein Protein in pankreatischen beta-Zellen 14-16 ist. Übertragen von in vitro-aktivierten oder un-aktivierte ganz oder fraktionierte Milzzellen offen Diabetiker oder prediabetic BDC2.5 Mäuse übertragen Diabetes bei Neugeborenen oder immungeschwächten NOD-Mäuse in unterschiedlichen Wirkungsgrade 11,17-19. Wir beschreiben eine einfache Methode, die gereinigt transgenen CD4 + T-Zellen nutzt von Pre-diabetischen Mäusen BDC2.5 in Empfängermäuse bei hohem Wirkungsgrad und consiste T1D induzierenncy. Eine große Anzahl von naiven Insel Antigen-spezifischen CD4 + T-Zellen aus diesen Mäusen durch Fluoreszenz-aktivierte Zellsortierung (FACS) für CD4 + CD62L + T Zellen, die das transgene TCR Vβ4 Kette isoliert. Gereinigtes transgenen T-Zellen werden dann ohne Aktivierung in NOD.SCID Mäusen, die Funktions-T-und B-Zellen fehlt und Insulitis-und Diabetes-freie 20 übertragen. Die Empfänger-Mäuse werden für erhöhte Konzentrationen von Urin Glucose T1D anzeigt, die sich schnell entwickelt sich innerhalb von zwei Wochen nach der T-Zell-Transfer überwacht. Im Gegensatz zu anderen Methoden, die übertragen diabetogene T-Zellen mit heterogenen Besonderheiten, unser Protokoll FACS-sortierten CD4 + T-Zellen, die fast ausschließlich Ausdruck der diabetogene BDC2.5 TCR verwendet. Aufgrund ihrer Homogenität, sind nur eine kleine Anzahl von T-Zellen übertragen (~ 1x10 6 Zellen / Maus) für die schnelle Entwicklung T1D innerhalb von 2 Wochen bei 100% Inzidenz erforderlich. Ein weiterer Vorteil ist, dass unser Protokoll irradiation des Empfängers Mäusen ist nicht notwendig, da es für einige andere Methoden. Ein möglicher Nachteil dieser Methode ist, dass es nicht zulassen, die Untersuchung der Beteiligung von CD4 und CD8 T-Zell-Untergruppen oder insbesondere CD8-T-Zellen bei Diabetes. Die beschriebene Protokoll wird nützlich für die Untersuchung schneller T1D Entwicklung von naiven monospezifischen CD4 + T-Zellen, sowie therapeutische Strategien vermittelt, um in Homing von Inselzellen-Antigen-spezifische Th-Zellen in das Zielorgan eingreifen.

<table border="1" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>BDC 2.5 TCR transgenic NOD mice (NOD.Cg-Tg(Tcr&alpha;BDC 2.5, Tcr&beta;BDC 2.5)</td>
			<td>JAX</td>
			<td>004460</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NOD.SCID mice (NOD.CB17-Prkdcscid/J)</td>
			<td>JAX</td>
			<td>001303</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dulbecco's Modified Eagle's Medium (DMEM)</td>
			<td>Themo Scientific</td>
			<td>SH30022.01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bayer Diastix</td>
			<td>Fisher Scientific</td>
			<td>AM2803</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>15 ml conical tubes</td>
			<td>Falcon</td>
			<td>352095</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>50 ml conical tubes</td>
			<td>Falcon</td>
			<td>352070</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sterile surgical tweezers</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sterile small pair scissors</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sterile large pair scissors</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>70 &mu;m cell strainers</td>
			<td>Fisher Scientific</td>
			<td>22363548</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>35 &mu;m cell strainer cap tubes</td>
			<td>BD Biosciences</td>
			<td>352235</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ammonium-Chloride-Potassium (ACK) buffer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>0.15 M NH4Cl, 1 mM KHCO3, 0.1 mM Na2EDTA, pH 7.2 in dH2O</td>
		</tr>
		<tr>
			<td>BD FACSFlowTM sheath fluid</td>
			<td>BD Biosciences</td>
			<td>342003</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FACS staining buffer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>PBS, 0.2 mM EDTA, 0.5% BSA/FCS, filter sterilized</td>
		</tr>
		<tr>
			<td>Phase contrast microscope</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-CD4 (APC) mAb</td>
			<td>Biolegend</td>
			<td>1005616</td>
			<td>clone RM4-5</td>
		</tr>
		<tr>
			<td>Anti-TCR V&beta;4 (FITC) mAb</td>
			<td>BD Biosciences</td>
			<td>553365</td>
			<td>clone KT4</td>
		</tr>
		<tr>
			<td>Anti-CD62L (PE) mAb</td>
			<td>BD Biosciences</td>
			<td>553151</td>
			<td>clone MEL-14</td>
		</tr>
		<tr>
			<td>Cell sorter</td>
			<td>BD Biosciences</td>
			<td>&nbsp;</td>
			<td>e.g. BD FACSAria III</td>
		</tr>
		<tr>
			<td>Heat lamp</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mouse restrainer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1 ml syringes</td>
			<td>Becton Dickinson</td>
			<td>309602</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>18-1&frac12; gauge needles (sterile)</td>
			<td>Becton Dickinson</td>
			<td>305196</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>27&frac12; gauge needles (sterile)</td>
			<td>Becton Dickinson</td>
			<td>305109</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

accelerated type 1 diabetes induction in mice by adoptive transfer of diabetogenic cd4+ t cells

Die nonobese diabetischen (NOD) Maus spontan entwickelt Autoimmun-Diabetes nach 12 Wochen alt und ist der am intensivsten untersuchten Tiermodell der menschlichen Typ-1-Diabetes (T1D). Zelltransfer Studien in bestrahlte Mäuse Empfänger haben festgestellt, dass T-Zellen sind entscheidende in T1D Pathogenese in diesem Modell. Wir beschreiben hier ein einfaches Verfahren, um schnell durch adoptiven Transfer von gereinigtem T1D induzieren primäre CD4 + T-Zellen von Pre-diabetischen NOD-Mäusen transgen für die Insel-spezifischen T-Zell-Rezeptor (TCR) BDC2.5 in NOD.SCID Empfängermäuse. Die wesentlichen Vorteile dieser Technik sind, dass Isolation und adoptiven Transfer von T-Zellen diabetogene abgeschlossen werden kann innerhalb des gleichen Tages, Bestrahlung der Empfänger nicht erforderlich ist, und eine hohe Inzidenz von T1D ist innerhalb von 2 Wochen nach entlockte T-Zell-Transfer werden. So können Untersuchungen der Pathogenese und therapeutische Interventionen in T1D mit einer schnelleren Rate als bei Methoden, die auf heterogenen T-Zell-Populationen oder Klonen verlassen gehenabgeleitet von diabetischen NOD-Mäusen.

Unsere Ergebnisse zeigen, die Isolierung von transgenen BDC2.5 exprimieren CD62L, die kritisch für die T-Zellen, nach Hause zu sekundären lymphatischen Organe wie Lymphknoten an ist. Unsere Ergebnisse zeigen weiterhin die starke Fähigkeit dieses monospezifische T-Zell-Population, um schnell und effizient zu übertragen NOD.SCID Empfängermäuse T1D. Isolierung von diabetogenen CD4 + T-Zellen von Mäusen BDC2.5 ist in Abbildung 2 dargestellt. Ungefähr 5 x 10 7 ...

Watch this Scientific Journal Video about Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells at JoVE.com

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

Wir bieten eine reproduzierbare Methode, um Typ-1-Diabetes (T1D) bei Mäusen innerhalb von zwei Wochen zu induzieren durch den adoptiven Transfer von Insel-Antigen-spezifischen, primären CD4 + T-Zellen.

Accelerated Typ 1 Diabetes Induktion in Mäusen durch adoptiven Transfer von CD4 + T diabetogene Cells

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of ...

accelerated-type-1-diabetes-induction-mice-adoptive-transfer

Research

JoVE Journal

Immunology and Infection

16.6K Aufrufe.  Pennsylvania State University College of Medicine. Wir bieten eine reproduzierbare Methode, um Typ-1-Diabetes (T1D) bei Mäusen innerhalb von zwei Wochen zu induzieren durch den adoptiven Transfer von Insel-Antigen-spezifischen, primären CD4 + T-Zellen.

Serie: Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

Herstellung und Verwendung von HIV-1 infizierten primären CD4 + T-Zellen als Zielzellen in der natürlichen Killerzellen Zytotoxische Assays

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

Forschung

Immunologie und Infektion

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

Beurteilung der Innate Sensing von HIV-1-infizierten CD4<sup&gt; +</sup&gt; T-Zellen durch Plasmazytoide Dendritische Zellen unter Verwendung eines<em&gt; Ex-vivo-</em&gt; Co-Kultursystem.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

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

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

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

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

Die Induktion von Murine Darmentzündungen durch adoptiven Transfer von Effektor CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; Hoch</sup&gt; T-Zellen in immundefizienten Mäusen

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

Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

The methods described herein for activation of na&#239;ve CD4+ T cells in suspension and their adherence in coverslips for confocal microscopy analysis allow the spatial localization and visualization of gangliosides involved in CD4+ T cell activation, that complement expression profiling experiments such as flow cytometry, western blotting or real-time PCR. The quantification of ganglioside expression through flow cytometry and their cellular localization through microscopy can be obtained by the use of anti-ganglioside antibodies with high affinity and specificity. Nonetheless, an adequate handling of cells in suspension involves the treatment of culture plates to promote the necessary adherence required for fluorescence or confocal microscopy acquisition. In this work, we describe a protocol for determining GD3 and GD2 ganglioside expression and colocalization with the TCR during na&#239;ve CD4+ T cell activation. Also, real-time PCR experiments using &#60;40,000 cells are described for the determination of the GD3 and GM2/GD2 synthase genes, demonstrating that gene analysis experiments can be performed with a low number of cells and without the need of additional low input RNA kits.

Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

We describe a standard antibody staining protocol for use in microscopy to determine the membrane expression and localization of gangliosides in resting and activated human na&#239;ve CD4+ T cells. Also described are real-time PCR experiments using &#60;40,000 cells that do not require additional low input RNA kits.

Herstellung von CD4<sup&gt; +</sup&gt; T-Zellen für die Analyse von GD3 und GD2 Gangliosid Membran-Expression durch Mikroskopie

The methods described herein for activation of na&#239;ve CD4+ T cells in suspension and their adherence in coverslips for confocal microscopy analysis ...

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

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing autoimmune diseases. Clinical approaches to adoptively transfer Tregs, or to deplete Tregs in cancer, are underway with promising first outcomes. 
Because the number of naturally occurring Tregs (nTregs) is very limited, studying certain Treg features using in vitro induced Tregs (iTregs) can be advantageous. To date, the best although not absolutely specific protein marker to delineate Tregs is the transcription factor FOXP3. Despite the importance of Tregs including non-redundant roles of peripherally induced Tregs, the protocols to generate iTregs are currently controversial, particularly for human cells. This protocol therefore describes the in vitro differentiation of human CD4+FOXP3+ iTregs from human na&#239;ve T cells using a range of Treg-inducing factors (TGF-&#946; plus IL-2 only, or their combination with retinoic acid, rapamycin or butyrate) in parallel. It also describes the phenotyping of these cells by flow cytometry and qRT-PCR. 
These protocols result in reproducible expression of FOXP3 and other Treg signature genes and enable the study of general FOXP3-regulatory mechanisms as well as protocol-specific effects to delineate the impact of certain factors. iTregs can be utilized to study various phenotypic aspects as well as molecular mechanisms of Treg induction. Detailed molecular studies are facilitated by relatively large cell numbers that can be obtained. 
A limitation for the application of iTregs is the relative instability of FOXP3 expression in these cells compared to nTregs. iTregs generated by these protocols can also be used for functional assays such as studying their suppressive function, in which iTregs induced by TGF-&#946; plus retinoic acid and rapamycin display superior suppressive activity. However, the suppressive capacity of iTregs can differ from nTregs and the use of appropriate controls is crucial.

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

This protocol describes the reproducible generation and phenotyping of human induced regulatory T cells (iTregs) from na&#239;ve CD4+ T cells in vitro. Different protocols for FOXP3 induction allow for the study of specific iTreg phenotypes obtained with respective protocols.

In vitro Differenzierung von humanen CD4 + FOXP3 + Induced regulatorischer T - Zellen (iTregs) von Naïve CD4 + T - Zellen unter Verwendung eines TGF-β-enthaltende Protokoll

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

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human central nervous system (CNS) autoimmune demyelinating disease, creation of an AQP4-targeted model with both clinical and histologic manifestations of CNS autoimmunity has proven challenging. Immunization of wild-type (WT) mice with AQP4 peptides elicited T cell proliferation, although those T cells could not transfer disease to na&#239;ve recipient mice. Recently, two novel AQP4 T cell epitopes, peptide (p) 135-153 and p201-220, were identified when studying immune responses to AQP4 in AQP4-deficient (AQP4-/-) mice, suggesting T cell reactivity to these epitopes is normally controlled by thymic negative selection. AQP4-/- Th17 polarized T cells primed to either p135-153 or p201-220 induced paralysis in recipient WT mice, that was associated with predominantly leptomeningeal inflammation of the spinal cord and optic nerves. Inflammation surrounding optic nerves and involvement of the inner retinal layers (IRL) were manifested by changes in serial optical coherence tomography (OCT). Here, we illustrate the approaches used to create this new in vivo model of AQP4-targeted CNS autoimmunity (ATCA), which can now be employed to study mechanisms that permit development of pathogenic AQP4-specific T cells and how they may cooperate with B cells in NMO pathogenesis.

Here, we present a protocol to induce paralysis and opticospinal inflammation by transfer of aquaporin-4 (AQP4)-specific T cells from AQP4-/- mice into WT mice. In addition, we demonstrate how to use serial optical coherence tomography to monitor visual system dysfunction.

Induktion von Lähmung und visuelles System Verletzungen bei Mäusen durch T-Zellen spezifisch für Neuromyelitis Optica Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...

Isolation and Adoptive Transfer of High Salt Treated Antigen-presenting Dendritic Cells

Excess dietary salt intake contributes to inflammation and plays a vital role in the development of hypertension. We previously found that antigen-presenting dendritic cells (DCs) can sense elevated extracellular sodium leading to the activation of the NADPH oxidase and formation of isolevuglandin (IsoLG)-protein adducts. These IsoLG-protein adducts react with self-proteins and promote an autoimmune-like state and hypertension. We have developed and optimized state-of-the-art methods to study DC function in hypertension. Here, we provide a detailed protocol for isolation, in vitro treatment with elevated sodium, and adoptive transfer of murine splenic CD11c+ cells into recipient mice to study their role in hypertension.

Here, we present a protocol to isolate dendritic cells from murine spleens by magnetic cell sorting and subsequent adoptive transfer into na&#239;ve mice. The model of high-salt activated dendritic cells was chosen to explain the step-by-step procedures of adoptive transfer and flow cytometry.

Isolation und Adoptiveltern Übertragung von hohen Salz behandelt Antigen-präsentierende dendritische Zellen

Excess dietary salt intake contributes to inflammation and plays a vital role in the development of hypertension. We previously found that ...

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear cells (PBMCs). The development of stable, non-toxic, fluorescent dyes such as carboxyfluorescein succinimidyl ester (CFSE) allows for rare, antigen-specific T cells to be distinguished from bystanders by diminution in fluorescent staining, as detected by flow cytometry. This method has the following advantages over alternative approaches: (i) it is very sensitive to low-frequency T cells, (ii) no knowledge of the antigen or epitope is required, (iii) the phenotype of the responding cells can be analyzed, and (iv) viable, responding cells can be sorted and used for further analysis, such as T cell cloning.

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Presented here is a protocol for measuring proliferating CD4+ T cells in response to antigenic proteins or peptides using dye dilution. This assay is particularly sensitive to rare antigen-specific T cells and can be modified to facilitate cloning of antigen-specific cells.

Quantifizierung von proliferierenden humanen Antigen-spezifischen CD4+ T-Zellen mit Carboxyfluorescein Succinimidylester

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear ...

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase (G6PI)-Induced RA Mice

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T (iNKT) cells. Patients with active RA present fewer iNKT cells, defective cell function, and excessive polarization of Th1. In this study, an RA animal model was established using a mixture of hGPI325-339 and hGPI469-483 peptides. The iNKT cells were obtained by in vivo induction and in vitro purification, followed by infusion into RA mice for adoptive immunotherapy. The in vivo imaging system (IVIS) tracking revealed that iNKT cells were mainly distributed in the spleen and liver. On day 12 after cell therapy, the disease progression slowed down significantly, the clinical symptoms were alleviated, the abundance of iNKT cells in the thymus increased, the proportion of iNKT1 in the thymus decreased, and the levels of TNF-&#945;, IFN-&#947;, and IL-6 in the serum decreased. Adoptive immunotherapy of iNKT cells restored the balance of immune cells and corrected the excessive inflammation of the body.

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase G6PI-Induced RA Mice

This protocol uses G6PI mixed peptides to construct rheumatoid arthritis models that are closer to that of human rheumatoid arthritis in CD4+ T cells and cytokines. High purity invariant natural killer T cells (mainly iNKT2) with specific phenotypes and functions were obtained by in vivo induction and in vitro purification for adoptive immunotherapy.

Adoptive Immuntherapie von iNKT-Zellen in Glucose-6-Phosphat-Isomerase (G6PI)-Induzierte RA-Mäuse

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T ...

A High-Throughput Multiplexed Screening for Type 1 Diabetes, Celiac Diseases, and COVID-19

An ongoing clinical trial, Autoimmunity Screening for Kids (ASK), is the first screening study in the general population for type 1 diabetes (T1D) and celiac disease in the United States. With the coronavirus disease 2019 (COVID-19) pandemic, the epidemiology of COVID-19 in the general population and knowledge about the association between COVID-19 infection and T1D development are urgently needed. The currently standard screening method of the radio-binding assay (RBA) has met two great challenges: low efficiency with a single assay format and low disease specificity with a large proportion of low-affinity antibodies generated in screening. With the platform of the multiplex electrochemiluminescence (ECL) assay we established previously, a novel 6-Plex ECL assay was developed that combines, in a single well, all four islet autoantibodies (IAbs) to insulin, glutamic acid decarboxylase (GAD65), insulinoma antigen 2 (IA-2), and Zinc transporter 8 (ZnT8) for T1D, transglutaminase autoantibodies (TGA) for celiac disease, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor-binding domain (RBD) antibodies for COVID-19. The assay was validated in blind using 880 samples from the ASK study, including 325 positive samples and 555 all antibody-negative samples, and compared with the standard RBAs and a single ECL assay. With the advantages of high efficiency, low cost, and low serum volume, this assay has been accepted as the primary screening tool for the ASK study.

In line with the urgent need for screening for type 1 diabetes, celiac disease, and coronavirus disease 2019, we developed a high throughput 6-Plex electrochemiluminescence assay to simultaneously detect all four islet autoantibodies, tissue transglutaminase autoantibodies, and antibodies to the receptor binding domain of severe acute respiratory syndrome coronavirus 2.

Ein Hochdurchsatz-Multiplex-Screening auf Typ-1-Diabetes, Zöliakie und COVID-19

An ongoing clinical trial, Autoimmunity Screening for Kids (ASK), is the first screening study in the general population for type 1 diabetes (T1D) and ...