Name: ex vivo expansion of tumor-reactive t cells by means of bryostatin 1/ionomycin and the common gamma chain cytokines formulation
Uploaded: 2011-01-14T17:00:00
Description: Watch this Scientific Journal Video about Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation at JoVE.com

Diese Arbeit wurde vom NIH R01 CA104757 Grant (MH Manjili) unterstützt. Wir danken für die Unterstützung der VCU Massey Cancer Center und dem Commonwealth Foundation for Cancer Research.

1. Isolierung von Lymphozyten 5 <ol><li> Isolieren Tumor-drainierenden Lymphknoten oder Milz von tumortragenden FVBN202 transgenen Mäusen und bereiten einzelne Zellsuspension in eiskaltem RPMI1640 mit 10% FBS. B / I-Aktivierung in 50-ml-Polypropylen-konische Gefäße führt zu einer größeren T-Zell-Ausbeute auf Polystyrol-Röhrchen verglichen. Ketamin und Xylazin injiziert ip für die Anästhesie. Genickbruch ist als eine Methode der Euthanasie verwendet. </li><li> Kultur der Zellen (10 6 Zell...

<ol>
	<li>Goodell, V., Waisman, J., Salazar, L. G., de la Rosa, C., Link, J., Coveler, A. L., Childs, J. S., Fintak, P. A., Higgins, D. M., Disis, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Level+of+HER-2/neu+protein+expression+in+breast+cancer+may+affect+the+development+of+endogenous+HER-2/neu-specific+immunity.">Level of HER-2/neu protein expression in breast cancer may affect the development of endogenous HER-2/neu-specific immunity.</a> Mol Cancer Ther. 7, 449-454 (2008).</li><li>Disis, M. L., Knutson, K. L., Schiffman, K., Rinn, K., McNeel, D. 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Immunother. 29, 284-293 (2006).</li><li>Bear, H. D., Roberts, J., Cornell, D., Tombes, M. B., Kyle, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adoptive+immunotherapy+of+cancer+with+pharmacologically+activated+lymph+node+lymphocytes:+a+pilot+clinical+trial.">Adoptive immunotherapy of cancer with pharmacologically activated lymph node lymphocytes: a pilot clinical trial.</a> Cancer Immunol Immunother. 5, 269-274 (2001).</li><li>Morales, J. K., Kmieciak, M., Graham, L., Feldmesser, M., Bear, H. D., Manjili, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adoptive+transfer+of+HER2/neu-specific+T+cells+expanded+with+alternating+gamma+chain+cytokines+mediate+tumor+regression+when+combined+with+the+depletion+of+myeloid-derived+suppressor+cells.">Adoptive transfer of HER2/neu-specific T cells expanded with alternating gamma chain cytokines mediate tumor regression when combined with the depletion of myeloid-derived suppressor cells.</a> Cancer Immunol Immunother. 58, 941-953 (2009).</li><li>Cha, E., Graham, L., Manjili, M. H., Bear, H. D., Guy, C. T., Webster, M. A., Schaller, M., Parsons, T. J., Cardiff, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-7+++IL-15+are+superior+to+IL-2+for+the+ex+vivo+expansion+of+4T1+mammary+carcinoma-specific+T+cells+with+greater+efficacy+against+tumors+in+vivo.">IL-7 + IL-15 are superior to IL-2 for the ex vivo expansion of 4T1 mammary carcinoma-specific T cells with greater efficacy against tumors in vivo.</a> Breast Cancer Res Treat. 89, 10578-10582 (2009).</li><li>Kmieciak, M., Morales, J. K., Morales, J., Bolesta, E., Grimes, M., Manjili, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Danger+signals+and+nonself+entity+of+tumor+antigen+are+both+required+for+eliciting+effective+immune+responses+against+HER-2/neu+positive+mammary+carcinoma:+implications+for+vaccine+design.">Danger signals and nonself entity of tumor antigen are both required for eliciting effective immune responses against HER-2/neu positive mammary carcinoma: implications for vaccine design.</a> Cancer Immunol Immunother. 57, 1391-1398 (2008).</li><li>Stern, J. B., Smith, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-2+induction+of+T-cell+G1+progression+and+c-myb+expression.">Interleukin-2 induction of T-cell G1 progression and c-myb expression.</a> Science. 233, 203-206 (1986).</li><li>Kittipatarin, C., Khaled, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ex+vivo+expansion+of+memory+CD8+T+cells+from+lymph+nodes+or+spleen+through+in+vitro+culture+with+interleukin-7.">ex vivo expansion of memory CD8 T cells from lymph nodes or spleen through in vitro culture with interleukin-7.</a> J Immunol Methods. 344, 45-57 (2009).</li><li>Kokaji, A. I., Hockley, D. L., Kane, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-15+transpresentation+augments+CD8++T+cell+activation+and+is+required+for+optimal+recall+responses+by+central+memory+CD8++T+cells.">IL-15 transpresentation augments CD8+ T cell activation and is required for optimal recall responses by central memory CD8+ T cells.</a> J Immunol. 180, 4391-4401 (2008).</li><li>Le, H. K., Graham, L., Miller, C. H., Kmieciak, M., Manjili, M. H., Bear, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incubation+of+antigen-sensitized+T+lymphocytes+activated+with+bryostatin+1+++ionomycin+in+IL-7+++IL-15+increases+yield+of+cells+capable+of+inducing+regression+of+melanoma+metastases+compared+to+culture+in+IL-2.">Incubation of antigen-sensitized T lymphocytes activated with bryostatin 1 + ionomycin in IL-7 + IL-15 increases yield of cells capable of inducing regression of melanoma metastases compared to culture in IL-2.</a> Cancer Immunol Immunother. 58, 1565-1576 (2009).</li></ol>

Selektiver Ausbau von Tumor-reaktiven T-Zellen mit Effektorfunktionen Anti-Tumor-Funktion kann durch das vorgeschlagene Protokoll mit B erreicht werden / I Aktivierung und ex vivo Expansion mit der gamma-Kette Zytokine IL-2, IL-7 und IL-15. Während IL-2 ist ein T-Zell-Wachstumsfaktor, der die Differenzierung und Expansion von Antigen-spezifischen T-Zellen unterstützt, können IL-7 inhibiert die Apoptose von T-Zellen und unterstützen ihre Lebensfähigkeit während der Expansion. IL-15 unterstützt Memory T-Ze...

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		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Bryostatin 1</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>B7431-10ug</td>
<td>&nbsp;</td>
<tr>
<td>Ionomycin</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>407950</td>
<td>&nbsp;</td>
<tr>
<td>Mouse IL-7</td>
<td>&nbsp;</td>
<td>PeproTech</td>
<td>217-17</td>
<td>&nbsp;</td>
<tr>
<td>Mouse IL-15</td>
<td>&nbsp;</td>
<td>PeproTech</td>
<td>210-15</td>
<td>&nbsp;</td>
<tr>
<td>Human IL-2</td>
<td>&nbsp;</td>
<td>PeproTech</td>
<td>200-02</td>
<td>&nbsp;</td>
<tr>
<td>RPMI1640</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11875</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Gemini</td>
<td>100-106</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>30-002-CI</td>
<td>&nbsp;</td>
<tr>
<td>L- glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25030081</td>
<td>&nbsp;</td>
<tr>
<td>&beta;- mercaptoethanol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M7522</td>
<td>&nbsp;</td>
<tr>
<td>anti-CD16/32 antibody</td>
<td>&nbsp;</td>
<td>Biolegend</td>
<td>101302</td>
<td>&nbsp;</td>
<tr>
<td>Annexin V-FITC Apoptosis Detection Kit</td>
<td>&nbsp;</td>
<td>BD Pharmingen</td>
<td>556547</td>
<td>&nbsp;</td>
<tr>
<td>FITC-CD4</td>
<td>&nbsp;</td>
<td>Biolegend</td>
<td>100406</td>
<td>&nbsp;</td>
<tr>
<td>PE-CD8</td>
<td>&nbsp;</td>
<td>Biolegend</td>
<td>100708</td>
<td>&nbsp;</td>
<tr>
<td>anti-c-Erb2/c&#x2013;Neu</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>OP16</td>
<td>&nbsp;</td>
<tr>
<td>PE- anti mouse IgG</td>
<td>&nbsp;</td>
<td>Biolegend</td>
<td>405307</td>
<td>&nbsp;</td>
<tr>
<td>formaldehyde</td>
<td>&nbsp;</td>
<td>Polysciences</td>
<td>04018</td>
<td>&nbsp;</td>
<tr>
<td>Hemocytometer</td>
<td>&nbsp;</td>
<td>Hycor</td>
<td>87144</td>
<td>&nbsp;</td>
<tr>
<td>Light microscope</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>V200073</td>
<td>&nbsp;</td>
<tr>
<td>Mouse IFN-&gamma; ELISA set</td>
<td>&nbsp;</td>
<td>BD Pharmingen</td>
<td>555138</td>
<td>&nbsp;</td>
<tr>
<td>Cell culture flasks</td>
<td>&nbsp;</td>
<td>Greiner</td>
<td>658175</td>
<td>&nbsp;</td>		</tbody>
		</table>
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ex vivo expansion of tumor-reactive t cells by means of bryostatin 1/ionomycin and the common gamma chain cytokines formulation

Es wurde berichtet, dass Brustkrebs-Patientinnen bereits bestehende Immunantworten gegen ihren Tumoren 1,2 haben. Allerdings scheitern solche Immunantwort auf einen vollständigen Schutz gegen die Entwicklung oder Wiederauftreten von Brustkrebs bieten. Zur Lösung dieses Problems durch die Erhöhung der Häufigkeit von Tumor-reaktiven T-Zellen hat adoptiven Immuntherapie eingesetzt. Eine Vielzahl von Protokollen für die Expansion von Tumor-spezifischen T-Zellen verwendet wurden. Diese Protokolle sind jedoch die Verwendung von Tumor-Antigenen ex vivo für die Aktivierung von Antigen-spezifischen T-Zellen beschränkt. Vor kurzem haben gemeinsame gamma-Kette Zytokine wie IL-2, IL-7, IL-15 und IL-21 wurden allein oder in Kombination zur Verbesserung der Anti-Tumor-Immunantwort 3 verwendet. Es ist jedoch nicht klar, was Formulierung am besten für die Expansion von Tumor-reaktiven T-Zellen. Hier präsentieren wir ein Protokoll für die selektive Aktivierung und Expansion von Tumor-reaktiven T-Zellen aus dem FVBN202 transgenen Mausmodell der HER-2/neu positiven Mammakarzinoms für den Einsatz in adoptiven T-Zell-Therapie von Brustkrebs. Das Protokoll umfasst die Aktivierung von T-Zellen mit bryostatin-1/ionomycin (B / I) und IL-2 in Abwesenheit von Tumor-Antigenen für 16 Stunden. B / I Aktivierung imitiert intrazelluläre Signale, die sich in T-Zell-Aktivierung durch die Erhöhung Proteinkinase C Aktivität und den intrazellulären Kalzium-, bzw. 4. Dieses Protokoll ausdrücklich aktiviert tumor-spezifische T-Zellen, während das Töten irrelevant T-Zellen. Die B / I-aktivierten T-Zellen werden mit IL-7 und IL-15 für 24 Stunden kultiviert und anschließend mit IL-2 gepulst. Nach 24 Stunden, T-Zellen werden gewaschen, halbiert und mit kultivierten IL-7 + IL-15 für weitere 4 Tage. Tumor-Spezifität und anti-Tumor-Wirksamkeit des ex vivo expandierten T-Zellen bestimmt.

Watch this Scientific Journal Video about Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation at JoVE.com

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

Ein effizientes Protokoll für die<em&gt; Ex vivo</em&gt; Ausbau der Tumor-reaktiven T-Zellen aus Tumor-drainierenden Lymphknoten oder anderen sekundären lymphatischen Gewebe des tumortragenden Gastgeber beschrieben. Dieses Protokoll selektiv erweitert tumor-spezifische T-Zellen für den Einsatz in adoptiven Immuntherapie von Brustkrebs.

Ex vivo Expansion von Tumor-reaktiven T-Zellen mittels Bryostatin 1/Ionomycin und der Gemeinsamen gamma-Kette Zytokine Formulation

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide ...

Research

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Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

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

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Ein<em&gt; Ex-vivo-</em&gt; Modell eines Oligodendrozyten-gerichtete T-Cell Attack in akuten Hirnschnitten

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