Name: in situ detection of autoreactive cd4 t cells in brain and heart using major histocompatibility complex class ii dextramers
Uploaded: 2014-08-01T14:00:00
Description: Watch this Scientific Journal Video about In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers at JoVE.com

Ce travail a été soutenu par l&#39;American Heart Association et de la Fondation cardiomyopathie des enfants (SDG2462390204001) et les Instituts nationaux de santé (de HL114669). CM est un bénéficiaire d&#39;une subvention de bourse de recherche postdoctorale décerné par la Fondation myocardite, NJ.

Déclaration d&#39;éthique: Toutes les procédures d&#39;animaux ont été effectuées en conformité avec les lignes directrices pour le soin et l&#39;utilisation des animaux de laboratoire et approuvés par l&#39;Université de Nebraska-Lincoln, Lincoln, NE. Une. Induction de l&#39;EAE <ol><li> Pour induire l&#39;EAE, préparer l&#39;émulsion de peptide / de l&#39;adjuvant complet de Freund (CFA), comme décrit dans 11 avec les modifications suivan...

<ol>
	<li>Cecconi, V., Moro, M., Del Mare, ., Dellabona, S., P, G., Casorati, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+MHC+class+II+tetramers+to+investigate+CD4++T+cell+responses:+problems+and+solutions.">Use of MHC class II tetramers to investigate CD4+ T cell responses: problems and solutions.</a> Cytometry A. 73 (11), 1010-1018 (2008).</li><li>Ferlin, W., Glaichenhaus, N., Mougneau, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Present+difficulties+and+future+promise+of+MHC+multimers+in+autoimmune+exploration.">Present difficulties and future promise of MHC multimers in autoimmune exploration.</a> Curr Opin Immunol. 12, 670-675 (2000).</li><li>Kwok, W. W., Ptacek, N. A., Liu, A. W., Buckner, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+class+II+tetramers+for+identification+of+CD4++T+cells.">Use of class II tetramers for identification of CD4+ T cells.</a> J Immunol Methods. 268, 71-81 (2002).</li><li>Landais, 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=New+design+of+MHC+class+II+tetramers+to+accommodate+fundamental+principles+of+antigen+presentation.">New design of MHC class II tetramers to accommodate fundamental principles of antigen presentation.</a> J Immunol. 183, 7949-7957 (2009).</li><li>Nepom, G. T. <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+II+tetramers.">MHC class II tetramers.</a> J Immunol. 188, 2477-2482 (2012).</li><li>Vollers, S. S., Stern, L. 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(88), e51654 (2014).</li><li>Massilamany, C., Steffen, D., Reddy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+epitope+from+Acanthamoeba+castellanii+that+cross-react+with+proteolipid+protein+139-151-reactive+T+cells+induces+autoimmune+encephalomyelitis+in+SJL+mice.">An epitope from Acanthamoeba castellanii that cross-react with proteolipid protein 139-151-reactive T cells induces autoimmune encephalomyelitis in SJL mice.</a> J Neuroimmunol. 219 (1-2), 17-24 (2010).</li><li>Donermeyer, D. L., Beisel, K. W., Allen, P. M., Smith, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocarditis-inducing+epitope+of+myosin+binds+constitutively+and+stably+to+I-Ak+on+antigen-presenting+cells+in+the+heart.">Myocarditis-inducing epitope of myosin binds constitutively and stably to I-Ak on antigen-presenting cells in the heart.</a> J Exp Med. 182 (5), 1291-1300 (1995).</li><li>Liao, L., Sindhwani, R., Leinwand, L., Diamond, B., Factor, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+alpha-myosin+heavy+chains+differ+in+their+induction+of+myocarditis.+Identification+of+pathogenic+epitopes.">Cardiac alpha-myosin heavy chains differ in their induction of myocarditis. Identification of pathogenic epitopes.</a> J Clin Invest. 92 (6), 2877-2882 (1993).</li><li>Cameron, T. O., Cochran, J. R., Yassine-Diab, B., Sekaly, R. P., Stern, L. 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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=Activated+human+epitope-specific+T+cells+identified+by+class+II+tetramers+reside+within+a+CD4high,+proliferating+subset.">Activated human epitope-specific T cells identified by class II tetramers reside within a CD4high, proliferating subset.</a> Int Immunol. 13 (6), 799-806 (2001).</li><li>Reddy, 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=Detection+of+autoreactive+myelin+proteolipid+protein+139-151-specific+T+cells+by+using+MHC+II+(IAs)+tetramers.">Detection of autoreactive myelin proteolipid protein 139-151-specific T cells by using MHC II (IAs) tetramers.</a> J Immunol. 170 (2), 870-877 (2003).</li><li>Skinner, P. J., Daniels, M. A., Schmidt, C. S., Jameson, S. C., Haase, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+edge:+In+situ+tetramer+staining+of+antigen-specific+T+cells+in+tissues.">Cutting edge: In situ tetramer staining of antigen-specific T cells in tissues.</a> J Immunol. 165 (2), 613-617 (2000).</li><li>Skinner, P. J., Haase, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+tetramer+staining.">In situ tetramer staining.</a> J Immunol Methods. 268 (1), 29-34 (2002).</li><li>Skinner, P. J., Haase, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+staining+using+MHC+class+I+tetramers.">In situ staining using MHC class I tetramers.</a> Curr Protoc Immunol. Chapter 17, Unit 17.14.11 (2004).</li></ol>

Contrairement tétramères CMH de classe I, l&#39;utilisation de tétramères CMH de classe II pour les applications de routine continue d&#39;être difficile, surtout pour énumérer les fréquences de précurseurs de cellules T CD4 autoréactifs de faible affinité, en partie à cause de leur dépendance à l&#39;égard d&#39;activation 6,15-17. Récemment, ce problème a été contourné par la création de la prochaine génération de tétramères, appelé &quot;dextramers,&quot; ce qui nous permet de dé...

L&#39;utilisation de complexe majeur d&#39;histocompatibilité (CMH) de classe II tétramères pour la détection de cellules spécifiques de l&#39;antigène T CD4 a été un défi 1-7. Pour améliorer la sensibilité de détection de tétramères CMH de classe II, l&#39;équipe de recherche a créé des tétramères de la prochaine génération, désignés &quot;dextramers&quot; 8. Dextramers contiennent des squelettes de dextrane dans lequel de multiples fragments de streptavidine fluorophore sont conjugués, tels que plusieurs monomères du CMH biotinylés, des peptides attachés peuvent être attachés à une molécule de dextran 9. Ainsi, de grands agrégats de dextramers CMH qui contiennent plusieurs complexes CMH-peptide peut interagir avec plusieurs récepteurs de cellules T (TCR). Ce groupe a récemment généré dextramers pour différents auto-antigènes et a démontré que la sensibilité de détection de dextramers était environ cinq fois plus que les tétramères 8. Les systèmes expérimentaux comprennent encéphalomyélite auto-immune expérimentale (EAE) induite par la protéine protéolipidique (PLP) 139-151 chez la souris SJL; EAE induite par la glycoprotéine de la myéline des oligodendrocytes (MOG) 35-55 dans C57BL / 6 souris; et la myocardite auto-immune expérimentale (EAM) induite avec la myosine cardiaque chaîne lourde α-(MyHC) 334-352 chez des souris A / J. Cette étude démontre l&#39;utilisation de PLP 139-151 - 334-352 dextramers MyHC et pour détecter les cellules spécifiques de l&#39;antigène T CD4 in situ avec une spécificité en développant un protocole de coloration directe, éliminant ainsi la nécessité pour amplifier les signaux en utilisant des anticorps à partir de fluorophores, un approcher couramment employé à l&#39;utilisation de tétramères. En outre, un procédé global de l&#39;évaluation des tissus ont également été mis au point pour énumérer de manière fiable les fréquences des cellules T CD4 spécifiques de l&#39;antigène in situ 10. La détection de cellules T spécifiques de l&#39;antigène in situ permet la délimitation des événements provoqués par des stimuli antigéniques spécifiques de ceux causés par l&#39;activation de l&#39;observateur. De même, la technique in situ également PROVIDes possibilités pour suivre la cinétique d&#39;infiltrations de cellules T spécifiques de l&#39;antigène dans les organes cibles.

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			<td height="25" style="height:25px;width:619px;">CFA</td>
			<td style="width:364px;">Sigma Aldrich, St Louis, MO</td>
			<td style="width:219px;">5881</td>
			<td style="width:167px;">Store at 4&#959;C</td>
		</tr>
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			<td height="25" style="height:25px;">MTB&#160; H37Rv extract&#160;</td>
			<td>Difco Laboratories, Detroit, MI</td>
			<td>231141</td>
			<td>Store at 4&#959;C</td>
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			<td height="25" style="height:25px;">PT</td>
			<td>List Biologicals Laboratories, Campbell, CA</td>
			<td>181</td>
			<td>Store at 4&#959;C</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">1x PBS&#160;</td>
			<td>Corning, Manassas, VA</td>
			<td>21-040-CV</td>
			<td>Store at 4&#959;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Female A/J mice&#160;</td>
			<td>Jackson Laboratories, Bar Harbor, ME</td>
			<td>646</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Female SJL/J mice</td>
			<td>Jackson Laboratories, Bar Harbor, ME</td>
			<td>686</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leur-lok sterile 1 ml syrringe</td>
			<td>BD, Franklin Lakes, NJ</td>
			<td>309628</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leur-lok sterile 3 ml syrringe</td>
			<td>BD, Franklin Lakes, NJ</td>
			<td>309657</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile needle, 18 G</td>
			<td>BD, Franklin Lakes, NJ</td>
			<td>305195</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile needle, 27 1/2 G</td>
			<td>BD, Franklin Lakes, NJ</td>
			<td>305109</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3-way stopcock&#160;</td>
			<td>Smiths Medical ASD, Inc. Dublin, OH</td>
			<td>MX5311L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kerlix gauze bandage rolls&#160;</td>
			<td>Covidien, Mansfield, MA</td>
			<td>6720</td>
			<td></td>
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			<td height="21" style="height:21px;">Kimwipes</td>
			<td>Kimberly-Clark Professional, Roswell, GA</td>
			<td>34155</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose, Low Melting Point, Analytical Grade</td>
			<td>Promega corporation, Madison, WI</td>
			<td style="width:219px;">V2111</td>
			<td></td>
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			<td height="25" style="height:25px;">Immunopure normal goat serum</td>
			<td>Thermo Scientific, Waltham, MA</td>
			<td style="width:219px;">31873</td>
			<td>Store at -20 &#959;C</td>
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		<tr height="25">
			<td height="25" style="height:25px;">Anti-mouse CD4 conjugated with PE dye</td>
			<td style="width:364px;">eBioscience, San Diego, CA&#160;</td>
			<td style="width:219px;">12004185</td>
			<td>Store at 4&#959;C</td>
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			<td height="25" style="height:25px;">Paraformaldehyde</td>
			<td>Electron Microscopy Sciences, Hatfield, PA</td>
			<td style="width:219px;">19202</td>
			<td>Store at 4&#959;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Faramount Aqueous Mounting Medium</td>
			<td>Dako, Carpinteria, CA</td>
			<td style="width:219px;">10073021</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Conical tube (15ml)</td>
			<td>BD Biosciences, San Jose, CA</td>
			<td style="width:219px;">352099</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Conical tube (50 ml)</td>
			<td>BD Biosciences, San Jose, CA</td>
			<td style="width:219px;">352070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24-well flat bottomed tissue culture plates</td>
			<td>BD Biosciences, San Jose, CA</td>
			<td>356775</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Water color #2 brushes</td>
			<td>Charles Leonard, Inc. Hauppauge, NY</td>
			<td style="width:219px;">73502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plain Microscope slides, size: 3&#34; x 1&#34; x 1.2 mm</td>
			<td>Fisher Scientific, Pittsburgh, PA</td>
			<td style="width:219px;">1255010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Premium Cover glass, 22 x 22-1</td>
			<td>Fisher Scientific, Pittsburgh, PA</td>
			<td style="width:219px;">12548B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable deep base mold histology cassettes</td>
			<td>Laboratory prodcust, Rochester, NY</td>
			<td style="width:219px;">M-475-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Loctite Super glue</td>
			<td>Henkel Corporation, Westlake, OH</td>
			<td style="width:219px;">1471879</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Razor blades</td>
			<td>Gillette SuperSilver</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Myhc-a 334-352 (DSAFDVLSFTAEEKAGVYK)</td>
			<td style="width:364px;">Neopeptide, Cambridge, MA</td>
			<td style="width:219px;"></td>
			<td>Store at 4&#959;C</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">PLP 139-151 (HSLGKWLGHPDKF)</td>
			<td style="width:364px;">Neopeptide, Cambridge, MA</td>
			<td style="width:219px;"></td>
			<td>Store at 4&#959;C</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">MHC class II/IAs PLP 139-151 or TMEV 70-86 dextramers conjugated with APC dye</td>
			<td></td>
			<td></td>
			<td>Store at 4&#959;C</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">MHC class II/IAk Myhc 334-352 or&#160; or RNase 40-56 dextramers conjugated with APC dye</td>
			<td></td>
			<td></td>
			<td>Store at 4&#959;C</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Dextran conjugated SA-APC</td>
			<td>&#160;Immundex, Copenhagen, Demark</td>
			<td style="width:219px;"></td>
			<td>Store at 4&#959;C</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Sterile surgical scissors and forceps</td>
			<td style="width:364px;">INOX tool Corporation</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro oven</td>
			<td style="width:364px;">GE Healthcare, Pittsuburgh, PA</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica VT 1200 Vibratome</td>
			<td>Leica Microsystems, Inc. Buffalo Grove, IL</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Olympus BX 60&#160; Laser scanning confocal microscope</td>
			<td>Olympus America, Inc. Center Valley, PA</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nikon A1-Eclipse 90i confocal microscope</td>
			<td>Nikon Inc. Melville, NY</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

in situ detection of autoreactive cd4 t cells in brain and heart using major histocompatibility complex class ii dextramers

This report demonstrates the use of major histocompatibility complex (MHC) class II dextramers for detection of autoreactive CD4 T cells in situ in myelin proteolipid protein (PLP) 139-151-induced experimental autoimmune encephalomyelitis (EAE) in SJL mice and cardiac myosin heavy chain-&#945; (Myhc) 334-352-induced experimental autoimmune myocarditis (EAM) in A/J mice. Two sets of cocktails of dextramer reagents were used, where dextramers+ cells were analyzed by laser scanning confocal microscope (LSCM): EAE, IAs/PLP 139-151 dextramers (specific)/anti-CD4 and IAs/Theiler&#8217;s murine encephalomyelitis virus (TMEV) 70-86 dextramers (control)/anti-CD4; and EAM, IAk/Myhc 334-352 dextramers/anti-CD4 and IAk/bovine ribonuclease (RNase) 43-56 dextramers (control)/anti-CD4. LSCM analysis of brain sections obtained from EAE mice showed the presence of cells positive for CD4 and PLP 139-151 dextramers, but not TMEV 70-86 dextramers suggesting that the staining obtained with PLP 139-151 dextramers was specific. Likewise, heart sections prepared from EAM mice also revealed the presence of Myhc 334-352, but not RNase 43-56-dextramer+ cells as expected. Further, a comprehensive method has also been devised to quantitatively analyze the frequencies of antigen-specific CD4 T cells in the &#8216;Z&#8217; serial images.

Dans ce rapport, la détection in situ de spécifiques de l&#39;antigène, auto-réactifs cellules T CD4 par coloration directe avec dextramers CMH de classe II en est décrite. Sections cérébrales fraîchement coupées ont été obtenues à partir de souris EAE et colorées avec des cocktails contenant PLP 139-151 (spécifique) ou TMEV 70-86 (contrôle) dextramers et anti-CD4. Les panneaux supérieurs de la figure 1 montre l&#39;analyse LSCM de sections cérébrales costained avec P...

Watch this Scientific Journal Video about In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers at JoVE.com

In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers

Le protocole pour détecter les cellules autoréactives T CD4 dans le cerveau et le cœur par coloration directe avec les grands complexes dextramers d&#39;histocompatibilité de classe II a été décrite dans le présent rapport. Pour une analyse complète, une méthode fiable pour énumérer les fréquences de cellules T CD4 + spécifiques de l&#39;antigène in situ est également conçu.

Détection in situ de autoréactifs T CD4 des cellules dans le cerveau et le cœur à l&#39;aide majeur d&#39;histocompatibilité Dextramers classe II

This report demonstrates the use of major histocompatibility complex (MHC) class II dextramers for detection of autoreactive CD4 T cells in situ in myelin ...

Research

JoVE Journal

Immunology and Infection

In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers

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