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

Detta arbete stöddes av American Heart Association och Barnens Kardiomyopati Foundation (SDG2462390204001) och National Institutes of Health (HL114669). CM är en mottagare av en postdoktoral forskning stipendium bidrag som beviljas av Myokardit Foundation, NJ.

Etik uttalande: Alla djurförsök har utförts i enlighet med de riktlinjer för skötsel och användning av försöksdjur och godkänts av University of Nebraska-Lincoln, Lincoln, NE. 1. Induktion av EAE <ol><li> För att inducera EAE, framställa peptid / komplett Freunds adjuvans (CFA)-emulsion, såsom beskrivs i 11 med följande modifikationer: Steg 1.1:. Förbereda PLP 139-151 vid en koncentration av 1 mg / ml i 1 x fosfatbuffrad saltlös...

<ol>
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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. J. <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:+detection+of+antigen-specific+CD4++T+cells+by+HLA-DR1+oligomers+is+dependent+on+the+T+cell+activation+state.">Cutting edge: detection of antigen-specific CD4+ T cells by HLA-DR1 oligomers is dependent on the T cell activation state.</a> J Immunol. 166 (2), 741-745 (2001).</li><li>Novak, E. 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. 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Till skillnad från MHC klass I tetramers, fortsätter användningen av MHC klass II tetramers för rutinmässiga tillämpningar att vara en utmaning, särskilt för att räkna upp utgångsfrekvenserna för låg affinitet autoreaktiva CD4 T-celler, delvis på grund av deras aktivering beroende 6,15-17. Nyligen har detta problem kringgås genom att skapa nästa generationen av tetramerer, kallade &quot;dextramers&quot;, tillåter oss att detektera antigen-specifika CD4 T-celler för ett antal autoantigener, t....

Användningen av större histokompatibilitetskomplex (MHC) klass Il-tetramerer för detektion av antigen-specifika CD4 T-celler som har varit en utmaning 1-7. För att öka känsligheten för MHC klass II tetramers upptäckt, har forskargruppen skapat nästa generations tetramers, betecknade &quot;dextramers&quot; 8. Dextramers innehålla dextran huvudkedjor i vilka multipla streptavidin-fluorofor-grupper är konjugerade, exempelvis att flera peptid tjudrad, biotinylerade MHC-monomerer kan bindas till en dextranmolekyl 9. Sålunda kan stora aggregat av MHC dextramers som innehåller flera MHC-peptidkomplex interagera med flera T-cellreceptorer (TCR). Denna grupp nyligen genererade dextramers för olika självantigener och visat att känsligheten hos dextramers detektion var ca fem gånger mer än tetramerer 8. De experimentella system innefattar experimentell autoimmun encefalomyelit (EAE) inducerades med proteolipidprotein (PLP) 139-151 i SJL-möss; EAE inducerad med myelin oligodendrocyte glykoprotein (MOG) 35-55 i C57BL / 6 möss; och experimentell autoimmun myokardit (EAM) framkallad med hjärt myosin tung kedja-α (Myhc) 334-352 i A / J-möss. Denna studie demonstrerar användningen av PLP 139-151 - och Myhc 334-352 dextramers att detektera antigen-specifika CD4 T-celler in situ med specificitet genom att utveckla en direktfärgningsprotokollet, varigenom man eliminerar behovet av att förstärka signalerna från användning av fluorofor-antikroppar, en närma vanligtvis används med användning av tetramerer. Dessutom har en omfattande metod för att utvärdera vävnader också tagits fram för att på ett tillförlitligt sätt räkna upp de frekvenser av antigen-specifika CD4 T-celler på plats 10. Detektion av antigen-specifika T-celler in situ medger avgränsning av händelser som orsakas av specifika antigena stimuli från de som orsakas av bystander-aktivering. Likaså in situ teknik också provides möjligheter att spåra kinetiken av antigen-specifika T-cell infiltration i målorganen.

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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>
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			<td>Difco Laboratories, Detroit, MI</td>
			<td>231141</td>
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			<td>List Biologicals Laboratories, Campbell, CA</td>
			<td>181</td>
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			<td height="25" style="height:25px;">1x PBS&#160;</td>
			<td>Corning, Manassas, VA</td>
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			<td height="21" style="height:21px;">Female A/J mice&#160;</td>
			<td>Jackson Laboratories, Bar Harbor, ME</td>
			<td>646</td>
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			<td>Jackson Laboratories, Bar Harbor, ME</td>
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			<td height="21" style="height:21px;">Leur-lok sterile 1 ml syrringe</td>
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			<td>309657</td>
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			<td height="21" style="height:21px;">Sterile needle, 18 G</td>
			<td>BD, Franklin Lakes, NJ</td>
			<td>305195</td>
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			<td height="21" style="height:21px;">Sterile needle, 27 1/2 G</td>
			<td>BD, Franklin Lakes, NJ</td>
			<td>305109</td>
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			<td height="21" style="height:21px;">3-way stopcock&#160;</td>
			<td>Smiths Medical ASD, Inc. Dublin, OH</td>
			<td>MX5311L</td>
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			<td height="21" style="height:21px;">Faramount Aqueous Mounting Medium</td>
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			<td>BD Biosciences, San Jose, CA</td>
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			<td>BD Biosciences, San Jose, CA</td>
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			<td>BD Biosciences, San Jose, CA</td>
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			<td height="21" style="height:21px;">Water color #2 brushes</td>
			<td>Charles Leonard, Inc. Hauppauge, NY</td>
			<td style="width:219px;">73502</td>
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			<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>
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			<td height="21" style="height:21px;">Premium Cover glass, 22 x 22-1</td>
			<td>Fisher Scientific, Pittsburgh, PA</td>
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			<td height="21" style="height:21px;">Disposable deep base mold histology cassettes</td>
			<td>Laboratory prodcust, Rochester, NY</td>
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			<td height="21" style="height:21px;">Loctite Super glue</td>
			<td>Henkel Corporation, Westlake, OH</td>
			<td style="width:219px;">1471879</td>
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			<td>Gillette SuperSilver</td>
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			<td height="25" style="height:25px;">Myhc-a 334-352 (DSAFDVLSFTAEEKAGVYK)</td>
			<td style="width:364px;">Neopeptide, Cambridge, MA</td>
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			<td>Store at 4&#959;C</td>
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			<td height="25" style="height:25px;">PLP 139-151 (HSLGKWLGHPDKF)</td>
			<td style="width:364px;">Neopeptide, Cambridge, MA</td>
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			<td>Store at 4&#959;C</td>
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			<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>
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			<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>
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			<td height="25" style="height:25px;">Dextran conjugated SA-APC</td>
			<td>&#160;Immundex, Copenhagen, Demark</td>
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			<td>Store at 4&#959;C</td>
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			<td height="21" style="height:21px;">Sterile surgical scissors and forceps</td>
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			<td style="width:364px;">GE Healthcare, Pittsuburgh, PA</td>
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			<td></td>
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			<td height="21" style="height:21px;">Leica VT 1200 Vibratome</td>
			<td>Leica Microsystems, Inc. Buffalo Grove, IL</td>
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			<td></td>
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			<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>
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			<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>
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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.

I denna rapport, in situ bestämning av antigenspecifika, autoreaktiva CD4 T-celler genom direkt färgning med MHC klass II dextramers beskrivs. Nyklippt cerebrala avsnitt härrör från EAE-möss och färgas med cocktails som innehåller PLP 139-151 (specifik) eller TMEV 70-86 (kontroll) dextramers och anti-CD4. Den översta panelerna i Figur 1 visar LSCM analys av cerebrala sektioner costained med PLP 139-151 dextramers (röd) och CD4-antikropp (grön), där de dubbel positiva celler verkade ...

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

Protokollet för att upptäcka självreaktiva CD4 T-celler i hjärna och hjärta genom direkt färgning med MHC klass II dextramers har beskrivits i denna rapport. För omfattande analys, är en tillförlitlig metod för att räkna upp de frekvenser av antigen-specifika CD4 + T-celler på plats också fram.

In Situ Upptäckt av autoreaktiva CD4 T-celler i hjärna och hjärta Använda MHC klass 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 ...

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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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