Die Autoren bedanken sich bei den Beiträgen von AM VanBuskirk zur Entwicklung unseres Verständnisses des regulierten DTH-Reaktion in Transplantationspatienten anzuerkennen. Diese Arbeit wurde unterstützt NIH Zuschüsse PO1AI084853 und R01AI066219-06, und von der EU-geförderten einer Studie.

1. Herstellung von Lymphozyten <ol><li> Sammle Blut in ACD (Säure Citrate Dextrose) Röhren. </li><li> Isolieren PBMC aus frischem menschlichem peripheren Blut-Lymphozyten mit Separation Medium nach Standardmethoden. </li><li> Waschen Sie die PBMC dreimal mit PBS zu verunreinigen Thrombozyten zu entfernen. Thrombozyten wurden gefunden, um mit trans-vivo DTH Assay stören. Maximal zulässige Thrombozyten Kontamination von PBMC Vorbereitung ist ≤ 1x10 7 / Injektion. </li><li> Wenn es eine auffallende rote Blutkörperchen Kontamination führen Lyse von roten Blutkörperchen mit ACK-Lysepuffer beim ersten Waschen. Entfernen ACK Puffer und dann Waschen 2-mal mit PBS. </li></ol> 2. Herstellung von Alloantigen <ol><li> Isolieren PBMC vom Spender peripheren Blut unter Verwendung des Verfahrens oben gezeigt. </li><li> Resuspendieren Spender PBMC in PBS in einer Konzentration von 120x10 6 Zellen / ml (4x10 6 Zellen / 30 ul). </li><li> 1 uM PMSF zu der Mischung, um Protein zu verhindernAbbau. </li><li> Beschallen der Zellsuspension mit sieben 1-Sekunden-Impulse mit einer 2 mm-Ultraschallsonde. (Hinweis: Bewahren Sie das Material kalt und vermeiden Sie Blasen Wenn Schäumen auftritt, lassen Sie die Zellsuspension auf Eis sitzen für 2-3 min.). </li><li> Überprüfen Sie die Störung von&gt; 90% der Zellen mit einer Zählkammer. </li><li> Zentrifuge die Mischung bei 14.000 rpm bei 4 ° C für 20 min in gekühlten Mikrofuge. </li><li> Überstand in ein neues 2,0 ml Safe-Lock-Rohr und Protein-Konzentration bestimmen. </li></ol> 3. Handy Vorbereitung für Injektionen <ol><li> Für jede Injektion Aliquot 7x10 6 PBMC in 2,0 ml Safe-Lock Tubes. </li><li> Zentrifuge bei 6.000 rpm für eine Minute in Mikrofuge und entfernen Sie den Überstand. </li><li> Resuspendieren der Zellen in PBS mit oder ohne Antigene. Passen Sie das Injektionsvolumen bis 35 ul mit PBS. Das folgende Schema ist für Injektionen verwendet: Negative Kontrolle: PBMC + PBS Positive Kontrolle: PBMC + TT / DT (25 &amp; mu; G / Injektion) Experimentelle Antigen-spezifische Antwort: PBMC + Test Ag (4-8 g / Injektion) Experimentelle Antigenspezifische Verordnung: PBMC + Test Ag + TT / DT </li></ol> 4. Pre-Messung, Injektion, und Post-Messung <ol><li> Anesthetize CB17 SCID-Maus mit Isofluran. Messen hinteren Fußballen Dicke mit einem gefederten Sattel. Setzen Bremssattel in der Mitte der Fußsohle, mit einer Kante berühren die letzte Walking Pad des Fußes, um eine Benchmark zu halten, der Messstelle konsistent bereitzustellen. Footpad Dicke wird aufgezeichnet, wenn Manometer-Anzeige stabilisiert hat. </li><li> Langsam injizieren Zellsuspensionen subkutan in die Fußsohlen der Mäuse mit ½ cc Insulinspritzen mit 28-Gauge-Nadel. Führen Injektion mit der Nadel zeigt in Richtung der Zehen, und die Schräge nach oben. (Hinweis: Stellen Sie sicher, dass keine Leckage). </li><li> 18-24 Stunden nach der Injektion betäuben Maus mit Isofluran und wiederholen Messung der Fußballen Schwellung. </li><li> Subtrhandeln die Dicke jeder Fußballen vor der Injektion aus der Nacheinspritzung Wert auf die Fußballen Schwellung Wert zu erhalten, drückt die Daten in Einheiten von 10 -4 Zoll. Berechnen Sie die Antigen-spezifische net Schwellung durch Subtraktion Kontrolle Fußballen Schwellung (PBMC + PBS) aus den Fußballen Schwellungen Werte aus den Behandlungen (PBMC + Spender Alloantigen, TT / DT oder Spender Alloantigen + TT / DT) erhalten. Eine positive Kontrolle Reaktion auf Antigen TT / DT von ≥ 25x10 -4 Zentimeter über Hintergrund Reaktion auf PBS erinnern wird, damit der Test als gültig betrachtet werden benötigt. </li><li> Bestimmen der Hemmung der Rückruf Reaktionen in Gegenwart von Spender-Antigenen durch Vergleichen der Netto Schwellung jeder Injektion unter Verwendung der folgenden Formel: <img alt="Gleichung 1" fo:src="/files/ftp_upload/4454/4454eq1highres.jpg" src="/files/ftp_upload/4454/4454eq1.jpg" /></li></ol>

<ol>
	<li>Carrodeguas, L., 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=Trans+vivo+analysis+of+human+delayed-type+hypersensitivity+reactivity.">Trans vivo analysis of human delayed-type hypersensitivity reactivity.</a> Hum. Immunol. 60, 640-651 (1999).</li><li>VanBuskirk, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+allograft+acceptance+is+associated+with+immune+regulation.">Human allograft acceptance is associated with immune regulation.</a> J. Clin. Invest. 106, 145-155 (2000).</li><li>Burlingham, W. J., Jankowska-Gan, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+strain+and+injection+site+are+crucial+for+detecting+linked+suppression+in+transplant+recipients+by+trans-vivo+DTH+assay.">Mouse strain and injection site are crucial for detecting linked suppression in transplant recipients by trans-vivo DTH assay.</a> Am. J. Transplant. 7, 466-470 (2007).</li><li>Burlingham, W. 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=Loss+of+tolerance+to+a+maternal+kidney+transplant+is+selective+for+HLA+class+II:+Evidence+from+trans-vivo+DTH+and+alloantibody+analysis.">Loss of tolerance to a maternal kidney transplant is selective for HLA class II: Evidence from trans-vivo DTH and alloantibody analysis.</a> Human Immunology. 61, 1395-1402 (2000).</li><li>Geissler, F., 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=Human+liver+allograft+acceptance+and+the+'tolerance+assay':+In+vitro+anti-donor+T+cell+assays+show+hyporeactivity+to+donor+cells+but,+unlike+DTH,+fail+to+detect+linked+suppression.">Human liver allograft acceptance and the 'tolerance assay': In vitro anti-donor T cell assays show hyporeactivity to donor cells but, unlike DTH, fail to detect linked suppression.</a> Transplantation. 72, 571-580 (2001).</li><li>Jankowska-Gan, 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=Human+liver+allograft+acceptance+and+the+'tolerance+assay'.+II.+donor+HLA-A,+-B+but+not+DR+antigens+are+able+to+trigger+regulation+of+DTH.">Human liver allograft acceptance and the 'tolerance assay'. II. donor HLA-A, -B but not DR antigens are able to trigger regulation of DTH.</a> Hum. Immunol. 63, 862 (2002).</li><li>Cai, 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=Minor+H+Antigen+HA-1-specific+Regulator+and+Effector+CD8++T+Cells,+and+HA-1+Microchimerism,+in+Allograft+Tolerance.">Minor H Antigen HA-1-specific Regulator and Effector CD8+ T Cells, and HA-1 Microchimerism, in Allograft Tolerance.</a> J. Exp. Med. 199, 1017-1023 (2004).</li><li>Rodriguez, D. S., 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=Immune+regulation+and+graft+survival+in+kidney+transplant+recipients+are+both+enhanced+by+human+leukocyte+antigen+matching.">Immune regulation and graft survival in kidney transplant recipients are both enhanced by human leukocyte antigen matching.</a> Am. J. Transplant. 4, 537-543 (2004).</li><li>Xu, 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=Human+CD4+CD25low+adaptive+T+regulatory+cells+suppress+delayed-type+hypersensitivity+during+transplant+tolerance.">Human CD4+CD25low adaptive T regulatory cells suppress delayed-type hypersensitivity during transplant tolerance.</a> J. Immunol. 178, 3983-3995 (2007).</li><li>Derks, R. A., Jankowska-Gan, E., Xu, Q., Burlingham, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cell+type+determines+the+mechanism+of+bystander+suppression+by+adaptive+T+regulatory+cells+specific+for+the+minor+antigen+HA-1.">Dendritic cell type determines the mechanism of bystander suppression by adaptive T regulatory cells specific for the minor antigen HA-1.</a> J. Immunol. 179, 3443-3451 (2007).</li><li>Jankowska-Gan, 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=Successful+reduction+of+immunosuppression+in+older+renal+transplant+recipients+who+exhibit+donor-specific+regulation.">Successful reduction of immunosuppression in older renal transplant recipients who exhibit donor-specific regulation.</a> Transplantation. 88, 533-541 (2009).</li><li>Jankowska-Gan, 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=Pretransplant+immune+regulation+predicts+allograft+outcome:+bidirectional+regulation+correlates+with+excellent+renal+transplant+function+in+living-related+donor-recipient+pairs.">Pretransplant immune regulation predicts allograft outcome: bidirectional regulation correlates with excellent renal transplant function in living-related donor-recipient pairs.</a> Transplantation. 93, 283-290 (2012).</li><li>Knechtle, S. 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=Early+and+limited+use+of+tacrolimus+to+avoid+rejection+in+an+alemtuzumab+and+sirolimus+regimen+for+kidney+transplantation:+clinical+results+and+immune+monitoring.">Early and limited use of tacrolimus to avoid rejection in an alemtuzumab and sirolimus regimen for kidney transplantation: clinical results and immune monitoring.</a> Am. J. Transplant. 9, 1087-1098 (2009).</li><li>Haynes, L. 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=Donor-specific+indirect+pathway+analysis+reveals+a+B-cell-independent+signature+which+reflects+outcomes+in+kidney+transplant+recipients.">Donor-specific indirect pathway analysis reveals a B-cell-independent signature which reflects outcomes in kidney transplant recipients.</a> Am. J. Transplant. 12, 640-648 (2012).</li><li>Burlingham, W. 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=IL-17-dependent+cellular+immunity+to+collagen+type+V+predisposes+to+obliterative+bronchiolitis+in+human+lung+transplants.">IL-17-dependent cellular immunity to collagen type V predisposes to obliterative bronchiolitis in human lung transplants.</a> J. Clin. Invest. 117, 3498-3506 (2007).</li><li>Bobadilla, J. L., 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=TH-17,+Monokines,+Collagen+Type+V,+and+Primary+Graft+Dysfunction+in+Lung+Transplantation.">TH-17, Monokines, Collagen Type V, and Primary Graft Dysfunction in Lung Transplantation.</a> Am. J. Respir. Crit. Care Med. 177 (6), (2008).</li><li>Olson, B. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+prostate+tumor+antigen-specific+CD8++regulatory+T+cells+are+inhibited+by+CTLA-4+or+IL-35+blockade.">Human prostate tumor antigen-specific CD8+ regulatory T cells are inhibited by CTLA-4 or IL-35 blockade.</a> J. Immunol. 189, 5590-5601 (2012).</li></ol>

Die Autoren dieses Manuskripts haben keine Interessenkonflikte offen zu legen.

Die trans-vivo DTH Test ein neuer diagnostischer Test mit einer möglichen klinischen Anwendung bei der Beurteilung zellvermittelte Antworten in der Transplantationsmedizin, Krebs und Autoimmunerkrankungen Patienten. Es ist wertvoll, weil es nicht nur nützlich bei der Überwachung Rückruf T Effektor Antworten ist, aber es kann auch regulatorische T-Antworten erkennen. Eine zuverlässige Möglichkeit, menschliche DTH Regulierung erkennen könnte Sicherheit von Immunsuppression Rückzug in Patienten, die Kandidaten für...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>ACD tube for blood collection</td>
			<td>BD</td>
			<td>02-684-26</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lymphocyte Separation Medium</td>
			<td>Cellgro</td>
			<td>25-072-CV</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dulbecco's Phosphate-Buffered Saline</td>
			<td>Cellgro</td>
			<td>21-031-CM</td>
			<td>Without calcium &amp; magnesium</td>
		</tr>
		<tr>
			<td>ACK Lysis Buffer</td>
			<td>BioWhittaker</td>
			<td>10-548E</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TT/DT or EBV</td>
			<td>Sanofi Pasteur Inc./ Meridian Life Science, Inc.</td>
			<td>&nbsp;</td>
			<td>TT/DT 25 &mu;g/injection EBV 8 &mu;g/injection</td>
		</tr>
		<tr>
			<td>Protease inhibitor PMSF</td>
			<td>Sigma-Aldrich</td>
			<td>78830</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Eosine for cell count</td>
			<td>Sigma-Aldrich</td>
			<td>E-6003</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alloantigen</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Purified HLA antigens, synthetic allopeptides can be used instead of donor cell-free lysates</td>
		</tr>
		<tr>
			<td>50 ml sterile centrifuge tubes</td>
			<td>Fisher Scientific</td>
			<td>06-443-18</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>10 ml pipettes and pipettor</td>
			<td>BD Falcon</td>
			<td>13-675-20</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>2 ml safe-lock tubes</td>
			<td>Costar</td>
			<td>3213</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1000 &mu;l, 100 &mu;l , 10 &mu;l pipettes with sterile tips</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Fisher</td>
			<td>02-671-10</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Full size centrifuge and microfuge</td>
			<td>Beckman Coulter/Eppendorf</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1/2cc or 1cc insulin syringes</td>
			<td>Becton Dickinson</td>
			<td>14-826-79</td>
			<td>28 gauge</td>
		</tr>
		<tr>
			<td>Vibracell sonicator</td>
			<td>Divtech Equipment Co. Sonocs&amp; Materials Inc</td>
			<td>&nbsp;</td>
			<td>2 mm probe</td>
		</tr>
		<tr>
			<td>Dial thickness gauge</td>
			<td>Mitutoyo, Japan</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SCID mice</td>
			<td>Harlan</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Piramal Healthcare</td>
			<td>&nbsp;</td>
			<td>Inhalant anesthesia</td>
		</tr>
	</tbody>
</table>

trans-vivo delayed type hypersensitivity assay for antigen specific regulation

Spättypallergie Antwort (DTH) ist ein schneller in vivo Manifestation der T-Zell-abhängige Immunantwort auf ein fremdes Antigen (Ag), dass das Immunsystem des Wirts hat in der jüngsten Vergangenheit erlebt. DTH-Reaktionen werden oft in einer Sensibilisierung Phase unterteilt, die sich auf der ersten Antigen Erfahrung und Herausforderung Phase, die in der Regel folgt einige Tage nach der Sensibilisierung. Das Fehlen einer Überempfindlichkeit vom verzögerten Typ Reaktion auf einen Rückruf Ag durch Hauttests nachgewiesen wird oft als Beweis für Anergie angesehen. Die traditionelle DTH-Assay wurde effektiv bei der Diagnose vieler mikrobieller Infektionen eingesetzt. Trotz, die ähnliche Funktionen wie immun Lymphozyteninfiltration, Ödeme und Nekrosen, ist die direkte DTH keine praktikable diagnostische Technik bei transplantierten Patienten, da die Möglichkeit der direkten Einspritzung, was zur Sensibilisierung auf Spender-Antigene und Verlust des Transplantats. Um dieses Problem zu vermeiden, das Brummenein-zu-Maus &quot;trans-vivo&quot; DTH-Assay wurde 1,2 entwickelt. Dieser Test ist im Wesentlichen eine Übertragung DTH Test, wobei in menschlichen peripheren mononukleären Blutzellen (PBMCs) und Antigene wurden subkutan in die Ohrmuschel injiziert oder Fußballen naiven Maus und DTH-wie Schwellung nach 18-24 h 3 gemessen. Die Antigen-Präsentation von humanen Antigen-präsentierenden Zellen wie Makrophagen oder T-Zellen, DCs in gefäßreiches Mausgewebe löst die entzündliche Kaskade und zieht Maus Immunzellen, was zu Schwellung Antworten. Die Antwort ist Antigen-spezifischen Antigen und erfordert eine vorherige Sensibilisierung. Ein positiver Spender-reaktiven DTH-Reaktion in der Tv-DTH-Assay spiegelt wider, dass die transplantierten Patienten hat eine pro-inflammatorische Immunantwort Disposition zur Graft Alloantigene entwickelt. Das wichtigste Merkmal dieses Assays ist, dass es auch verwendet werden, um regulatorische T-Zellen, die Zuschauer Unterdrückung verursachen detektieren. Bystander Unterdrückung einesDTH Rückruf Reaktion in Gegenwart von Donor-Antigen ist charakteristisch für Transplantationspatienten mit anerkannten Allotransplantate 2,4-14. Die Überwachung von Transplantationspatienten für Alloreaktivität und Regulierung durch Tv-DTH kann identifizieren eine Untergruppe von Patienten, die aus der Reduktion der Immunsuppression ohne erhöhtes Risiko einer Abstoßung oder sich verschlechternder Nierenfunktion profitieren könnten. Ein viel versprechender Bereich ist die Anwendung des Tv-DTH-Assay bei der Überwachung von Autoimmunität 15,16 und auch in Tumorimmunologie 17.

1. Auswertung der Nierentransplantation für Spender Antigen-spezifische Antwort mit Tv-DTH-Assay Um die Spender-reaktiven zelluläre Immunität in Nierentransplantierten testen wir injiziert PBMC von Patienten mit Spender-Antigenen alleine oder mit einem Recall-Antigen Tetanus Toxoid (TT). Als positive Kontrolle wurden die Zellen mit TT allein injiziert. Wir beobachten drei Muster der Überempfindlichkeit vom verzögerten Typ in Transplantationspatienten (Abbildung 1). Alle Pa...

Watch this Scientific Journal Video about Trans-vivo Delayed Type Hypersensitivity Assay for Antigen Specific Regulation at JoVE.com

Trans-vivo Delayed Type Hypersensitivity Assay for Antigen Specific Regulation

Wir beschreiben eine wertvolle diagnostische Assays, die möglicherweise verwendet werden könnten, um den Abzug der Immunsuppression nach der Transplantation ohne erhöhten Risiko einer Abstoßungsreaktion zu entscheiden. Der Test basiert auf den Grundsätzen der Überempfindlichkeit vom verzögerten Typ und liefert genaue Beurteilung der beiden Spender spezifischen Effektor-und regulatorischen Immunantworten durch Empfänger montiert.

Trans-vivo Überempfindlichkeit vom verzögerten Typ Assay für Antigen-spezifische Verordnung

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host ...

trans-vivo-delayed-type-hypersensitivity-assay-for-antigen-specific

Research

JoVE Journal

Immunology and Infection

16.0K Aufrufe.  University of Wisconsin-Madison, School of Medicine and Public Health. Wir beschreiben eine wertvolle diagnostische Assays, die möglicherweise verwendet werden könnten, um den Abzug der Immunsuppression nach der Transplantation ohne erhöhten Risiko einer Abstoßungsreaktion zu entscheiden. Der Test basiert auf den Grundsätzen der Überempfindlichkeit vom verzögerten Typ und liefert genaue Beurteilung der beiden Spender spezifischen Effektor-und regulatorischen Immunantworten durch Empfänger montiert.

Serie: Trans-vivo Delayed Type Hypersensitivity Assay for Antigen Specific Regulation

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation. This labeling has classically been used to study proliferation and migration. In the method presented here, we have shortened the timeline after adoptive transfer to look at survival and killing of epitope specific CFSE labeled target cells4-6. The level of specific killing of a CD8 + T cell clone can indicate the quality of the response, as their quantity may be misleading. Specific CD8+ T cells can become functionally exhausted over time with a decline in cytokine production and killing7,8. Also, certain CD8 + T cell clones may not kill as well as others with differing TCR specificities9. For effective Cell Mediated Immunity (CMI), antigens must be identified that produce not only adequate numbers of responding T cells, but also functionally robust responding T cells. Here we assess the percent cell specific killing of two peptide specific T cell clones in BALB/c mice.

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Many infections elicit a strong CTL response, but occasionally, the quantity of responding cells does not correlate to control of the pathogen1. One measure of CTL quality is their ability to kill specifically2. CFSE labeling of target cells can be used to investigate this CTL response quality in vivo3,4.

Antigen-spezifische In Vivo Killing-Assay mit CFSE markierten Zielzellen

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation.  ...

Forschung

Immunologie und Infektion

Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering (si)RNAs to inhibit the expression of complementary RNA transcripts is being exploited as a new class of therapeutics for a variety of diseases including HIV. Many previous reports have shown that novel RNAi-based anti-HIV/AIDS therapeutic strategies have considerable promise; however, a key obstacle to the successful therapeutic application and clinical translation of siRNAs is efficient delivery. Particularly, considering the safety and efficacy of RNAi-based therapeutics, it is highly desirable to develop a targeted intracellular siRNA delivery approach to specific cell populations or tissues. The HIV-1 gp120 protein, a glycoprotein envelope on the surface of HIV-1, plays an important role in viral entry into CD4 cells. The interaction of gp120 and CD4 that triggers HIV-1 entry and initiates cell fusion has been validated as a clinically relevant anti-viral strategy for drug discovery. 
Herein, we firstly discuss the selection and identification of 2'-F modified anti-HIV gp120 RNA aptamers. Using a conventional nitrocellulose filter SELEX method, several new aptamers with nanomolar affinity were isolated from a 50 random nt RNA library. In order to successfully obtain bound species with higher affinity, the selection stringency is carefully controlled by adjusting the conditions. The selected aptamers can specifically bind and be rapidly internalized into cells expressing the HIV-1 envelope protein. Additionally, the aptamers alone can neutralize HIV-1 infectivity. Based upon the best aptamer A-1, we also create a novel dual inhibitory function anti-gp120 aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. Further, we utilize the gp120 aptamer-siRNA chimeras for cell-type specific delivery of the siRNA into HIV-1 infected cells. This dual function chimera shows considerable potential for combining various nucleic acid therapeutic agents (aptamer and siRNA) in suppressing HIV-1 infection, making the aptamer-siRNA chimeras attractive therapeutic candidates for patients failing highly active antiretroviral therapy (HAART).

Several 2&#x2019;-Fluoro RNA aptamers against HIV-1Ba-L gp120 with nanomole affinity are isolated from a RNA library by in vitro SELEX procedure. A new dual inhibitory function anti-gp120 aptamer-siRNA chimera is created and shows considerable promise for systemic anti-HIV therapy.

Entwicklung von Zell-spezifischen Anti-HIV-gp120-Aptamere für siRNA

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering ...

Basophil Activation Test for Investigation of IgE-Mediated Mechanisms in Drug Hypersensitivity

Hypersensitivity reactions against non-steroidal anti-inflammatory drugs (NSAIDs) like propyphenazone (PP) and diclofenac (DF) can manifest as Type I-like allergic reactions 1. In clinical practice, diagnosis of drug hypersensitivity is mainly performed by patient history, as skin testing is not reliable and oral provocation testing bears life-threatening risks for the patient 2. Hence, evidence for an underlying IgE-mediated pathomechanism is hard to obtain. 
Here, we present an in vitro method based on the use of human basophils derived from drug-hypersensitive patients that mimics the allergic effector reaction in vivo. As basophils of drug-allergic patients carry IgE molecules specific for the culprit drug, they become activated upon IgE receptor crosslinking and release allergic effector molecules. The activation of basophils can be monitored by the determination of the upregulation of CD63 surface expression using flow cytometry 3. 
In the case of low molecular weight drugs, conjugates are designed to enable IgE receptor crosslinking on basophils. As depicted in Figure 1, two representatives of NSAIDs, PP and DF, are covalently bound to human serum albumin (HSA) via a carboxyl group reacting with the primary amino group of lysine residues. DF carries an intrinsic carboxyl group and, thus, can be used directly 4, whereas a carboxyl group-containing derivative of PP had to be organochemically synthesized prior to the study 1.
The coupling degree of the low molecular weight compounds on the protein carrier molecule and their spatial distribution is important to guarantee crosslinking of two IgE receptor molecules. The here described protocol applies high performance-size exclusion chromatography (HPSEC) equipped with a sequential refractive index (RI) and ultra violet (UV) detection system for determination of the coupling degree.
As the described methodology may be applied for other drugs, the basophil activation test (BAT) bears the potential to be used for the determination of IgE-mediated mechanisms in drug hypersensitivity. Here, we determine PP hypersensitivity as IgE-mediated and DF hypersensitivity as non-IgE-mediated by BAT.

Basophil activation test is a potent tool for the detection of IgE-dependent allergies in vitro. Here, an optimized protocol for basophil activation test is used to investigate drug hypersensitivity. A method for the efficient production of covalent drug-protein conjugates and their physicochemical characterization is described.

Basophilenaktivierungstest für die Untersuchung von IgE-vermittelten Mechanismen in Drug Überempfindlichkeit

Hypersensitivity reactions against non-steroidal anti-inflammatory drugs (NSAIDs) like propyphenazone (PP) and diclofenac (DF) can manifest as Type I-like ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

Durchflusszytometrische Isolation of Primary Murine Type II Alveolarepithelzellen für Funktionelle und Molekulare Studien

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

Killer Künstliche Antigen-präsentierenden Zellen (KaAPC) für effiziente<em&gt; In-vitro-</em&gt; Depletion von humanen Antigen-spezifischen T-Zellen

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

Detektion und Anreicherung von Rare Antigen-spezifische B-Zellen für die Analyse von Phänotypen und Funktion

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

In Vivo Test zum Nachweis von Antigen-spezifischen T-Zell Zytolyse-Funktion mit einer Impfung-Modell

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in human sera and in separate preparations of polyclonal intravenous immunoglobulin (IVIg). The protocol describes the methodological steps involved in sample preparation, printing of arrays, assay procedure, and data analysis. In addition, this protocol could be further developed to incorporate diverse clinical samples including plasma and cell culture supernatants. We show how protein microarray can be used to determine a combination of isotype (IgG, IgA, IgM), subclass (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2), and strain-specific antibodies to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B-only expressing strain (CCUG 20309), a precursor form of a B fragment of binary toxin, pCDTb, ribotype-specific whole surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). During the experiment, microarrays are probed with sera from individuals with C. difficile infection (CDI), individuals with cystic fibrosis (CF) without diarrhea, healthy controls (HC), and from individuals pre- and post-IVIg therapy for the treatment of CDI, combined immunodeficiency disorder, and chronic inflammatory demyelinating polyradiculopathy. We encounter significant differences in toxin neutralization efficacies and multi-isotype specific antibody levels between patient groups, commercial preparations of IVIg, and sera before and following IVIg administration. Also, there is a significant correlation between microarray and enzyme-linked immunosorbent assay (ELISA) for antitoxin IgG levels in serum samples. These results suggest that microarray could become a promising tool for profiling antibody responses to C.difficile antigens in vaccinated or infected humans. With further refinement of antigen panels and a reduction in production costs, we anticipate that microarray technology may help optimize and select the most clinically useful immunotherapies for C. difficile infection in a patient-specific manner.

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

This article describes a simple protein microarray method for profiling humoral immune responses to a 7-plex panel of highly purified Clostridium difficile antigens in human sera. The protocol can be extended for the determination of specific antibody responses in preparations of polyclonal intravenous immunoglobulin.

Ein Protein-Microarray-Assay für die serologische Bestimmung der Antigen-spezifische Antikörper Antworten folgende Clostridium Difficile Infektion

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in ...

A DNA/Ki67-Based Flow Cytometry Assay for Cell Cycle Analysis of Antigen-Specific CD8 T Cells in Vaccinated Mice

The cell cycle of antigen-specific T cells in vivo has been examined by using a few methods, all of which possess some limitations. Bromodeoxyuridine (BrdU) marks cells that are in or recently completed S-phase, and carboxyfluorescein succinimidyl ester (CFSE) detects daughter cells after division. However, these dyes do not allow identification of the cell cycle phase at the time of analysis. An alternative approach is to exploit Ki67, a marker that is highly expressed by cells in all phases of the cell cycle except the quiescent phase G0. Unfortunately, Ki67 does not allow further differentiation as it does not separate cells in S-phase that are committed to mitosis from those in G1 that can remain in this phase, proceed into cycling, or move into G0.
Here, we describe a flow cytometric method for capturing a &#34;snapshot&#34; of T cells in different cell cycle phases in mouse secondary lymphoid organs. The method combines Ki67 and DNA staining with major histocompatibility complex (MHC)-peptide-multimer staining and an innovative gating strategy, allowing us to successfully differentiate between antigen-specific CD8 T cells in G0, in G1 and in S-G2/M phases of the cell cycle in the spleen&#160;and draining lymph nodes of mice after vaccination with viral vectors carrying the model antigen gag of human immunodeficiency virus (HIV)-1.
Critical steps of the method were the choice of the DNA dye and the gating strategy to increase the assay sensitivity and to include highly activated/proliferating antigen-specific T cells that would have been missed by current criteria of analysis. The DNA dye, Hoechst 33342, enabled us to obtain a high-quality discrimination&#160;of the G0/G1 and G2/M DNA peaks, while preserving membrane and intracellular staining. The method has great potential to increase knowledge about T cell response in vivo and to improve immuno-monitoring analysis.

Clonal expansion is a key feature of antigen-specific T cell response. However, the cell cycle of antigen-responding T cells has been poorly investigated, partly because of technical limitations. We describe a flow cytometric method to analyze clonally expanding antigen-specific CD8 T cells in spleen and lymph nodes of vaccinated mice.

Ein DNA/Ki67-basierter Durchflusszytometrie-Assay zur Zellzyklusanalyse von Antigen-spezifischen CD8-T-Zellen bei geimpften Mäusen

The cell cycle of antigen-specific T cells in vivo has been examined by using a few methods, all of which possess some limitations. Bromodeoxyuridine ...