Ich möchte Bianca Mothe, Carla Oseroff und Marie-France DelGuercio für ihre Einführung in immunologischen Assays und Maus Umgang danken. Die Arbeit im Labor von GA Splitter wird von NIH 1-RO1-AI-073558, GLRCE gewähren 1-U54-AI-057153, und BARD US-3829-06 finanziert.

1. Hervorrufen Target-spezifische Effektor CTLs durch Immunisierung (Tag 0) <ol><li> Zielsystem: Bevor Sie beginnen, auf eine spezifische Effektor entscheiden. In diesem Beispiel einer klassischen in vivo CTL-Assay verwenden wir ein gereinigtes Peptidimmunisierung spezifische CD8 + T-Zellen auslösen. Das gleiche Peptid wird verwendet, um den syngenen Zielzellen Puls, die Schaffung einer spezifischen Effektor: Target-System. Alternativ können Sie wählen, um ganze Erreger oder Protein benutzen, um Ihre spezifischen Effektoren und Targets zu erhalten. </li><li> Dekontaminieren die Arbeit im Inneren eines Bio Haube, um die Sterilität der Spritzen zu gewährleisten. Bereiten Sie die Peptid-Immunisierung durch das erste Inverkehrbringen 50 ug des gereinigten Peptids in 100 ul 10% DMSO in PBS / Maus in eine 2 ml-Röhrchen enthalten. Als Nächstes fügen Sie eine gleiche Menge an sehr kalten inkomplettem Freund-Adjuvans und bis zu den 0,2 mL-Marke mit 1 mm Perlen zum Mischen. Von diesem Punkt halten Immunogen auf Eis, bis Injektion. Sichere Deckel mit Parafilm. </li><li> Verwenden Sie einen Bead Beater auf Maximum RPMs, die Emulsion für 3 Minuten mischen, sofort platzieren die Emulsion wieder auf dem Eis nach dem Mischen. Die daraus entstehende Emulsion sollte ähnlich in Konsistenz von Mayonnaise. Lassen Emulsion bei 4 ° C über Nacht sitzen, wenn es die Zeit erlaubt (bis zu einer Woche), Mischen weiter, wenn eine Trennung ist zur Kenntnis genommen. </li><li> Verwenden Sie eine 1 ml-Spritze und 20 oder 20,5-Gauge-Nadel nach oben zu ziehen Emulsion vorsichtig, um nicht nach oben ziehen jedes Kügelchen (beads sollte größer sein als Gradmesser für Nadel, aber immer noch bewusst sein). </li><li> Sichere Maus für die subkutane Injektion an der Basis des Schwanzes. Wir verwenden eine modifizierte Kunststoff 50 ml konischen Rohr sicher und schonend fixieren Sie die Maus zur Injektion. Spritzen Sie die Maus mit 0,2 ml Emulsion. </li></ol> 2. Harvesting Zielzellen (Tag 8) <ol><li> Sie müssen naiven syngenen Mäusen für Ihre Zielzellen. Euthanize naive Mäuse nach AAALAC Standards. </li><li> Desinfizieren Sie mit der Maus durch Sprühen 70% Ethanol, um die Wahrscheinlichkeit einer Kontamination von Maus Haare oder andere äußere Verunreinigungen zu minimieren. </li><li> Mit Dissektion Schere machen kleine snip durch die Haut auf der linken Seite der Maus. Pull Hautlappen an Peritonäalsack und der inneren Organe aufzudecken. </li><li> Snip durch das Bauchfell, dabei darauf achten, keine Kompromisse bei der zugrunde liegenden Organen. Die Milz liegt in der Nähe der Oberfläche und ähnelt in Farbe, um die Leber (dunkelrot), aber kleiner und Kidneybohnen in Form. Infizierte Mäuse zeigen in der Regel deutlich größeren Milz als nicht infizierte Kontrollen. </li><li> Ziehen Sie vorsichtig die Milz, wobei darauf zu sanft sein, um nicht die Milz in mehrere Teile zu brechen, um maximale Zellzahlen erholen. Anschließend wird der Milz in eine 15 ml konische mit 10 ml vorbereitet Medien auf Eis. Fahren Sie mit dem restlichen Mäuse. </li><li> Gießen Sie die Inhalte der 15 mL konischen Rohr (Medien-und Milz) in ein Glas Gewebe Mahlwerk. Grind der Milz mit viel Oberarm Stärke. Wenn alle Gewebe hat seine Farbe verloren Sie können davon ausgehen, dass Sie alle Zellen aus dem Bindegewebe erholt. </li><li> Gießen Sie die Inhalte der Mühle durch einen 70 um Zelle Filter in einen 50 mL konischen Rohr. Spülen Schleifmaschine mit frischen Medien und gießen Sie durch eine Zelle Sieb. </li><li> Zentrifuge bei 4 ° C, 1300 rpm für 7 Minuten. Überstand abgießen. </li><li> Resuspendieren in 10 ml vorbereitet Lysereagenz und bei 37 ° für 7 Minuten. </li><li> Fügen Sie bis zu 50 ml mit frischem Medium und Zentrifuge, wie oben. </li><li> Resuspendieren in 50 ml frisches Medium und Zentrifuge, wie oben (Waschschritt). * Hinweis: In dieser Zeit können Sie eine magnetische Zellseparation für einen bestimmten Zelltyp (dh Makrophagen) durchführen, wenn eine reine Zielzellpopulation für den Transfer in Empfängermäuse gewünscht wird. Auch in dieser Zeit könnten Sie führen eine experimentelle Behandlung in die Kultur, um die Auswirkungen dieser Behandlung auf zellspezifische Töten zu studieren. </li><li> Zählen von Zellen und Platten 1 x 10 7 pro Well in 100 &amp;mgr; l in einer 96-well Rundboden Gewebekulturen behandelte Platte. * Dies ist eine hohe Konzentration von Zellen / Well, sondern weil der Test ist sehr einfach und schnell, wir haben keinen Verlust der Lebensfähigkeit der Zellen bei dieser Konzentration gesehen. </li></ol> 3. Behandlung und Kennzeichnung von Zielzellen <ol><li> Pulse entsprechenden Zellen und repliziert mit Erreger spezifische Peptid oder irrelevanten Peptid-Steuerung: Add 1 pg / mL Peptid. Inkubation bei 4 ° C für 1,5 Stunden. Bewegen bis 37 ° C Inkubator für die letzten 30 Minuten. Während dieser Zeit bereiten CFSE Reagenz. </li><li> Bereiten CFSE nach Angaben des Herstellers gelöst und auf eine funktionierende Konzentration zwischen 0,5 und 25 um. Machen Sie &quot;low&quot;-Konzentration 10x niedriger als &quot;hoch&quot; Konzentration CFSE. Zum Beispiel für [low] mit 0,5 ul CFSE zu 10 ml PBS und für [high], mit 5 ul CFSE zu 10 ml PBS. Warm vorbereitet CFSE bis 37 ° C vor der Etikettierung. </li><li> Entfernen Platte von Zellen aus Inkubator und pEllet in einem Hochgeschwindigkeits-Zentrifuge bei Raumtemperatur, 1300 RPM für 7 Minuten. Flick zu entfernen Überstand. </li><li> Die Zellen in entweder [low] oder [hoch] CFSE durch Zugabe von 200 ul der entsprechenden Reagenz in die entsprechenden Wells und Inkubation für 15 Minuten bei 37 ° C. </li><li> Entfernen Platte von Zellen aus Inkubator wieder Pellet bei Raumtemperatur, 1300 RPM für 7 Minuten. Flick zu entfernen Überstand. </li><li> Die Zellen in 200 ul / Well erwärmt frisches Medium und Inkubation für 30 Minuten bei 37 ° C. </li><li> Entfernen Platte von Zellen aus Inkubator und Pellet bei Raumtemperatur, 1300 RPM für 7 Minuten. Flick zu entfernen Überstand. </li><li> Die Zellen in 100 ul / well PBS. </li><li> Für adoptiven Transfer, fügen Sie eine gut [low] markierten Zellen zu einem gut [high] markierten Zellen pro Maus. Resultierender Injektion sollte 50% spezifische Ziele und 50% irrelevant Zellen in 0,2 MLS werden. </li><li> Vergessen Sie nicht beiseite zu legen einige markierte Zellen als Ihr nicht übertragenen Kontrollen während der Datenanalyse und für den Betrieb am Durchflusszytometer zu handeln (siehe Abbildung 1). </li></ol> 4. Adoptiven Transfer <ol><li> Anesthetize zuvor Peptid immunisierten Mäuse (ab part # 1 dieses Protokolls) nach AAALAC Standards. </li><li> Inject 0,2 ml markierten Zielzellen über retroorbitalen Weg in Empfängermäuse. </li><li> Warten Sie 6 Stunden oder anderen vorgegebenen optimalen Zeitpunkt vor euthanizing der Empfänger Mäusen. </li></ol> 5. Harvesting markierten Zellen * Hinweis: Die Schritte 5.1 bis 5,11 sind die gleichen wie die Schritte durch 2,11 2,1 <ol start="12"><li> Zählen von Zellen und die Platte bei 1 x 10 6 pro Well in 100 ul in 96-Well Rundboden Gewebekulturen behandelte Platte. </li><li> Fix erholt und nicht übertragenen Zellen durch Zugabe von 100 ul 2% Paraformaldehyd in einer Endkonzentration von 1% Paraformaldehyd in alle Vertiefungen. </li></ol> 6. Durchflusszytometrie und Datenanalyse <ol><li> Run Proben am Durchflusszytometer und die Daten analysieren. </li></ol> 7. Repräsentative Ergebnisse Eine abnehmende Epitop spezifischen Gipfel auf der resultierende Histogramm ist ein Indikator für die zellspezifische Tötens (Abbildung 1). Verwenden Sie die folgenden Gleichungen (Anwendung in Tabelle 1), um den tatsächlichen Wert der Zelle spezifische Lyse zu bestimmen: Ratio = Irrelevant Anteil: Epitop-spezifischen Anteil ([low] Höhepunkt: [hoch] peak), Prozent spezifische Lyse = [1 - (Non-Übertragung Regelverhältnis / Experimental-Verhältnis)] x 100 <img src="/files/ftp_upload/2250/2250fig1.jpg" alt="Abbildung 1" /> Abbildung 1. CFSE Profil erholt BALB / c Maus-Splenozyten. Die Zahlen geben den Prozentsatz an Zellen des hohen oder niedrigen CFSE Phänotyp. A) Kontrolle Non-übertragen. Alle [hoch] CFSE Peaks stellen Epitop-spezifischen Zielzellen, während [low] CFSE Gipfel Ziele mit irrelevanten Peptid gepulst darstellen. B) CFSE markierten Zellen aus PBS immunisiert Empfängermaus (Control) erholt. C) CFSE markierten Zellen aus zwei verschiedenen Peptid-spezifische Empfängermäuse erholt, beachten Sie die verminderte Epitop spezifischen Peaks. <img src="/files/ftp_upload/2250/2250fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Grafische Darstellung der analysierten Daten. Drei Datenpunkte für jede Gruppe von Mäusen, PBS, Peptide A und Peptid B gezeigt <table border="1"><tbody><tr><td> Probe </td><td> CFSE HOHE% Epitop Pulsed </td><td> CFSE LOW% Irrelevant </td><td> Verhältnis Low: High </td><td> Prozent Epitop-spezifischen Töten </td></tr><tr><td> PBS </td><td> 42,8 </td><td> 57,2 </td><td> 1,34 </td><td> 16,97 </td></tr><tr><td> PBS </td><td> 43,1 </td><td> 56,9 </td><td> 1,32 </td><td> 15,94 </td></tr><tr><td> PBS </td><td> 44,3 </td><td> 55,7 </td><td> 1,26 </td><td> 11,74 </td></tr><tr><td> Peptid A </td><td> 39,7 </td><td> 60,3 </td><td> 1,52 </td><td> 32,56 </td></tr><tr><td> Peptid A </td><td> 38,6 </td><td> 61,4 </td><td> 1,59 </td><td> 35,61 </td></tr><tr><td> Peptid A </td><td> 40,6 </td><td> 59,4 </td><td> 1,46 </td><td> 29,99 </td></tr><tr><td> Peptide B </td><td> 38,7 </td><td> 61,3 </td><td> 1,58 </td><td> 24,68 </td></tr><tr><td> Peptide B </td><td> 38,5 </td><td> 61,5 </td><td> 1,60 </td><td> 25,32 </td></tr><tr><td> Peptide B </td><td> 39,3 </td><td> 60,7 </td><td> 1,54 </td><td> 22,76 </td></tr><tr><td></td><td></td><td></td><td></td><td></td></tr><tr><td> PBS NT- </td><td> 47,4 </td><td> 52,6 </td&gt; <td> 1,11 </td><td></td></tr><tr><td> Pep A NT- </td><td> 49,4 </td><td> 50,6 </td><td> 1,02 </td><td></td></tr><tr><td> Pep B NT- </td><td> 45,6 </td><td> 54,4 </td><td> 1,19 </td><td></td></tr></tbody></table> Tabelle 1. Analyse von repräsentativen Daten. &quot;NT&quot; ist nicht übertragen.

<ol>
	<li>Blattman, J. N., Wherry, E. J., Ha, S. J., van der Most, R. G., Ahmed, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+epitope+escape+on+PD-1+expression+and+CD8+T-cell+exhaustion+during+chronic+infection.">Impact of epitope escape on PD-1 expression and CD8 T-cell exhaustion during chronic infection.</a> J Virol. 83 (9), 4386-4394 (2009).</li><li>Jenkins, M. R., Tsun, A., Stinchcombe, J. C., Griffiths, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+strength+of+T+cell+receptor+signal+controls+the+polarization+of+cytotoxic+machinery+to+the+immunological+synapse.">The strength of T cell receptor signal controls the polarization of cytotoxic machinery to the immunological synapse.</a> Immunity. 31 (4), 621-631 (2009).</li><li>Ingulli, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracing+tolerance+and+immunity+in+vivo+by+CFSE-labeling+of+administered+cells.">Tracing tolerance and immunity in vivo by CFSE-labeling of administered cells.</a> Methods Mol Biol. 380, 365-376 (2007).</li><li>Durward, M. A., Harms, J., Magnani, D. M., Eskra, L., Splitter, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discordant+Brucella+melitensis+antigens+yield+cognate+CD8++T+cells+in+vivo.">Discordant Brucella melitensis antigens yield cognate CD8+ T cells in vivo.</a> Infect Immun. 78 (1), 168-176 (2010).</li><li>Lyons, A. B., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+lymphocyte+division+by+flow+cytometry.">Determination of lymphocyte division by flow cytometry.</a> J Immunol Methods. 171 (1), 131-137 (1994).</li><li>Weston, S. A., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+fluorescent+dyes+for+lymphocyte+migration+studies.+Analysis+by+flow+cytometry+and+fluorescence+microscopy.">New fluorescent dyes for lymphocyte migration studies. Analysis by flow cytometry and fluorescence microscopy.</a> J Immunol Methods. 133 (1), 87-97 (1990).</li><li>Akondy, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+yellow+fever+virus+vaccine+induces+a+broad+and+polyfunctional+human+memory+CD8++T+cell+response.">The yellow fever virus vaccine induces a broad and polyfunctional human memory CD8+ T cell response.</a> J Immunol. 183 (12), 7919-7930 (2009).</li><li>Wallace, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+antigen-driven+responses+by+flow+cytometry:+monitoring+proliferation+by+dye+dilution.">Tracking antigen-driven responses by flow cytometry: monitoring proliferation by dye dilution.</a> Cytometry A. 73 (11), 1019-1034 (2008).</li><li>Labadi, A., Balogh, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+preferences+in+serosal+homing+and+distribution+of+peritoneal+B-cell+subsets+revealed+by+in+situ+CFSE+labeling.">Differential preferences in serosal homing and distribution of peritoneal B-cell subsets revealed by in situ CFSE labeling.</a> Int Immunol. 21 (9), 1047-1056 (2009).</li><li>Suffner, 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+cells+support+homeostatic+expansion+of+Foxp3++regulatory+T+cells+in+Foxp3.LuciDTR+mice.">Dendritic cells support homeostatic expansion of Foxp3+ regulatory T cells in Foxp3.LuciDTR mice.</a> J Immunol. 184 (4), 1810-1820 .</li></ol>

Keine Interessenskonflikte erklärt. Tierversuche wurden in Übereinstimmung mit den Richtlinien und Vorschriften her durch die Institutional Animal Care und Verwenden Committee (IACUC) an der University of Wisconsin-Madison gesetzt durchgeführt.

Dieser Test kann geändert werden, um die proliferative Kapazität von Zellen, einschließlich klonale Expansion untersucht werden, weil CFSE teilt relativ gleichmäßig auf Nachkommenschaft 10,11. Es ist auch möglich, CFSE Kennzeichnung zu verwenden, um die Zellwanderung 6,12 untersuchen, obwohl es andere Zelle Tracing-Verfahren, die besser geeignet sein kann je nach Hypothese, z. B. Tetramere und Biolumineszenz 13, die ebenfalls mit CFSE Kennzeichnung kombiniert werden kann. <p cl...

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		<th>Catalogue Number</th>
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<tr>
<td>1 mm glass beads</td>
<td>&nbsp;</td>
<td>Biospec Products</td>
<td>11079110</td>
<td>&nbsp;</td>
<tr>
<td>70 &mu;m cell strainer</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>352350</td>
<td>&nbsp;</td>
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<td>96-well plate</td>
<td>&nbsp;</td>
<td>Costar</td>
<td>3799</td>
<td>&nbsp;</td>
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<td>Adjulite Incomplete Freund's Adjuvant</td>
<td>&nbsp;</td>
<td>Pacific Immunology</td>
<td>n/a</td>
<td>&nbsp;</td>
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<td>DMSO</td>
<td>&nbsp;</td>
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<td>D-5879</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
<td>GIBCO</td>
<td>16000-077</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>n/a</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
<td>Kontes</td>
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<td>&nbsp;</td>
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<td>Mini Beadbeater</td>
<td>&nbsp;</td>
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<td>Parafilm "M"</td>
<td>&nbsp;</td>
<td>Pechiney Plastic Packaging</td>
<td>PM992</td>
<td>&nbsp;</td>
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<td>Paraformaldehyde</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>157-8</td>
<td>&nbsp;</td>
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<td>PBS</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>10010-023</td>
<td>&nbsp;</td>
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<td>PharmLyse lysing reagent</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>555899</td>
<td>&nbsp;</td>
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<td>RPMI 1640</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>11875-093</td>
<td>&nbsp;</td>
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<td>Syringe</td>
<td>&nbsp;</td>
<td>B-D</td>
<td>309602</td>
<td>&nbsp;</td>
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<td>Vybrant CFSE Kit</td>
<td>&nbsp;</td>
<td>Invitrogen (Molecular Probes)</td>
<td>V12883</td>
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antigen specific in vivo killing assay using cfse labeled target cells

Carboxyfluoresceindiacetat Succinimidylester (CFSE) können verwendet werden, um einfach und schnell Etikett einer Zellpopulation von Interesse für die in vivo Untersuchung. Diese Kennzeichnung ist klassisch benutzt worden, um die Proliferation und Migration zu studieren. In dem hier vorgestellten Verfahren haben wir die Zeitachse nach adoptiven Transfer auf das Überleben und die Tötung von Epitop-spezifischen CFSE markierten Zielzellen 4-6 aussehen verkürzt. Die Höhe der spezifischen Tötung eines CD8 + T-Zell-Klon kann zeigen die Qualität der Antwort, da ihre Menge kann irreführend sein. Spezifischen CD8 + T-Zellen können sich funktionell erschöpft im Laufe der Zeit mit einem Rückgang der Produktion von Zytokinen und Töten 7,8. Auch können bestimmte CD8 + T-Zell-Klonen nicht so gut wie andere zu töten mit unterschiedlichen TCR Besonderheiten 9. Für eine effektive zellvermittelte Immunität (CMI), muss Antigene identifiziert, die produzieren nicht nur eine ausreichende Anzahl von T-Zellen reagieren, sondern auch funktionell robust reagieren T-Zellen. Hier bewerten wir die Prozent-Zell-spezifische Tötung von zwei Peptid-spezifischen T-Zell-Klone in BALB / c Mäusen.

Watch this Scientific Journal Video about Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells at JoVE.com

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Viele Infektionen auslösen eine starke CTL-Antwort, aber gelegentlich, die Menge der reagierenden Zellen nicht korrelieren mit der Bekämpfung von Krankheitserregern,<sup&gt; 1</sup&gt;. Ein Maß für CTL Qualität ist ihre Fähigkeit, spezifisch abtöten<sup&gt; 2</sup&gt;. CFSE Markierung von Zielzellen kann verwendet werden, um diese CTL-Antwort Qualität zu untersuchen<em&gt; In vivo</em<sup&gt; 3,4</sup&gt;.

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.  ...

antigen-specific-in-vivo-killing-assay-using-cfse-labeled-target-cells

Research

JoVE Journal

Immunology and Infection

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

19.5K Aufrufe.  University of Wisconsin-Madison. Viele Infektionen auslösen eine starke CTL-Antwort, aber gelegentlich, die Menge der reagierenden Zellen nicht korrelieren mit der Bekämpfung von Krankheitserregern, 1. Ein Maß für CTL Qualität ist ihre Fähigkeit, spezifisch abtöten 2. CFSE Markierung von Zielzellen kann verwendet werden, um diese CTL-Antwort Qualität zu untersuchen In vivo

Serie: Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Trans-vivo Delayed Type Hypersensitivity Assay for Antigen Specific Regulation

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 immune system has experienced in the recent past. DTH reactions are often divided into a sensitization phase, referring to the initial antigen experience, and a challenge phase, which usually follows several days after sensitization. The lack of a delayed-type hypersensitivity response to a recall Ag demonstrated by skin testing is often regarded as an evidence of anergy. The traditional DTH assay has been effectively used in diagnosing many microbial infections.
Despite sharing similar immune features such as lymphocyte infiltration, edema, and tissue necrosis, the direct DTH is not a feasible diagnostic technique in transplant patients because of the possibility of direct injection resulting in sensitization to donor antigens and graft loss. To avoid this problem, the human-to-mouse "trans-vivo" DTH assay was developed 1,2. This test is essentially a transfer DTH assay, in which human peripheral blood mononuclear cells (PBMCs) and specific antigens were injected subcutaneously into the pinnae or footpad of a na&iuml;ve mouse and DTH-like swelling is measured after 18-24 hr 3. The antigen presentation by human antigen presenting cells such as macrophages or DCs to T cells in highly vascular mouse tissue triggers the inflammatory cascade and attracts mouse immune cells resulting in swelling responses. The response is antigen-specific and requires prior antigen sensitization. A positive donor-reactive DTH response in the Tv-DTH assay reflects that the transplant patient has developed a pro-inflammatory immune disposition toward graft alloantigens.
The most important feature of this assay is that it can also be used to detect regulatory T cells, which cause bystander suppression. Bystander suppression of a DTH recall response in the presence of donor antigen is characteristic of transplant recipients with accepted allografts 2,4-14. The monitoring of transplant recipients for alloreactivity and regulation by Tv-DTH may identify a subset of patients who could benefit from reduction of immunosuppression without elevated risk of rejection or deteriorating renal function.
A promising area is the application of the Tv-DTH assay in monitoring of autoimmunity15,16 and also in tumor immunology 17.

We describe a valuable diagnostic assay that could potentially be used to decide the withdrawal of immunosuppression after transplant without elevated risk of graft rejection. The assay uses the principles of Delayed Type Hypersensitivity and provides accurate assessment of both donor specific effector and regulatory immune responses mounted by recipients.

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 ...

Forschung

Immunologie und Infektion

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 ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

Entwicklung von Stammzellen abgeleiteten Antigen-spezifische regulatorische T-Zellen gegen Autoimmunität

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

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 ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

Generation von diskriminierendem menschlichen monoklonalen Antikörpern aus seltenen Antigen-spezifische B-Zellen im Blut zirkulierenden

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

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 ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

Opsonophagocytic Tötung Assay Immunreaktionen gegen bakterielle Krankheitserreger zu bewerten

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...

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 ...

High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the spontaneous non-obese diabetes (NOD) mouse model, insulin is the primary target, and genetic manipulation of these animals to remove a single key insulin epitope prevents disease. Thus, selective elimination of professional antigen presenting cells (APCs) bearing this pathogenic epitope is an approach to inhibit the unwanted insulin-specific autoimmune responses, and likely has greater translational potential.
Chimeric antigen receptors (CARs) can redirect T cells to selectively target disease-causing antigens. This technique is fundamental to recent attempts to use cellular engineering for adoptive cell therapy to treat multiple cancers. In this protocol, we describe an optimized T-cell retrovirus (RV) transduction and in vitro expansion protocol that generates high numbers of functional antigen-specific CD8 CAR-T cells starting from a low number of naive cells. Previously multiple CAR-T cell protocols have been described, but typically with relatively low transduction efficiency and cell viability following transduction. In contrast, our protocol provides up to 90% transduction efficiency, and the cells generated can survive more than two weeks in vivo and significantly delay disease onset following a single infusion. We provide a detailed description of the cell maintenance and transduction protocol, so that the critical steps can be easily followed. The whole procedure from primary cell isolation to CAR expression can be performed within 14 days. The general method may be applied to any mouse disease model in which the target is known. Similarly, the specific application (targeting a pathogenic peptide/MHC class II complex) is applicable to any other autoimmune disease model for which a key complex has been identified.

This article describes a protocol for the generation of antigen-specific CD8 T cells, and their expansion in vitro, with the aim of yielding high numbers of functional T cells for use in vitro and in vivo.

Hocheffiziente Generierung von Antigen-spezifischen primären Maus-Zytotoxischen T-Zellen für Funktionstests in einem Autoimmun-Diabetes-Modell

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the ...