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

Este trabajo fue apoyado por el NIH Grant R01 CA104757 (MH Manjili). Agradecemos el apoyo de VCU Massey Cancer Center y la Fundación del Commonwealth para la Investigación del Cáncer.

1. Aislamiento de linfocitos 5 <ol><li> Aislar el tumor ganglios linfáticos de drenaje o el bazo de los portadores de tumores FVBN202 ratones transgénicos y preparar la suspensión de células individuales en el helado RPMI1640 suplementado con FBS al 10%. B / I de activación en 50 ml de polipropileno tubos cónicos de resultados en una mayor producción de células T en comparación con los tubos de poliestireno. La ketamina y xilazina se inyectan ip para la anestesia. Dislocación cervical se utiliza c...

<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. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-existent+immunity+to+the+HER-2/neu+oncogenic+protein+in+patients+with+HER-2/neu+overexpressing+breast+and+ovarian+cancer.">Pre-existent immunity to the HER-2/neu oncogenic protein in patients with HER-2/neu overexpressing breast and ovarian cancer.</a> Breast Cancer Res Treat. 62, 245-252 (2000).</li><li>Liu, S., Riley, J., Rosenberg, S., Parkhurst, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+common+gamma-chain+cytokines,+interleukin-2,+interleukin-7,+and+interleukin-15+for+the+in+vitro+generation+of+human+tumor-reactive+T+lymphocytes+for+adoptive+cell+transfer+therapy.">Comparison of common gamma-chain cytokines, interleukin-2, interleukin-7, and interleukin-15 for the in vitro generation of human tumor-reactive T lymphocytes for adoptive cell transfer therapy.</a> J. 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>

Expansión selectiva de las células T tumor-reactiva con efectores anti-tumor función se puede lograr mediante el protocolo propuesto con B / I activación y expansión ex vivo de las citoquinas gamma de la cadena de IL-2, IL-7 e IL-15. Mientras que la IL-2 es un factor de crecimiento de las células T que pueden apoyar la diferenciación y la expansión de las células T antígeno-específicas, IL-7 puede inhibir la apoptosis de las células T y apoyar su viabilidad durante la expansión. IL-15 puede soporta...

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		<div>
		<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>
		</div>

ex vivo expansion of tumor-reactive t cells by means of bryostatin 1/ionomycin and the common gamma chain cytokines formulation

Se informó que los pacientes de cáncer de mama se han pre-existentes respuesta inmune contra los tumores 1,2. Sin embargo, como la respuesta inmune no pueden proporcionar una protección completa contra el desarrollo o recurrencia del cáncer de mama. Para superar este problema mediante el aumento de la frecuencia de células T tumor-reactiva, la inmunoterapia adoptiva ha sido empleado. Una gran variedad de protocolos han sido utilizados para la expansión de células T específicas del tumor. Estos protocolos, sin embargo, están restringidos a la utilización de ex vivo tumor antígenos para la activación de los antígenos específicos de células T. Muy recientemente, las citoquinas cadena gamma común, como la IL-2, IL-7, IL-15 e IL-21 se han utilizado solo o en combinación para la mejora de la anti-tumor respuesta inmune 3. Sin embargo, no está claro cuál es la formulación que mejor funciona para la expansión de las células T tumor-reactiva. Aquí se presenta un protocolo para la activación selectiva y la expansión del tumor, las células T reactivas a partir del modelo de ratón transgénico FVBN202 de carcinoma de mama positivo de Her-2/neu para su uso en la terapia adoptiva de células T del cáncer de mama. El protocolo incluye la activación de células T con bryostatin-1/ionomycin (B / I) e IL-2 en ausencia de antígenos tumorales durante 16 horas. B / I imita la activación de señales intracelulares que dan lugar a la activación de células T, al aumentar la actividad de la proteína quinasa C y el calcio intracelular, respectivamente 4. Este protocolo se activa específicamente el tumor de células T específicas, mientras que matar a las células T irrelevantes. Las células B / I-T activadas son cultivadas con IL-7 e IL-15 durante 24 horas y luego pulsadas con IL-2. Después de 24 horas, las células T se lavan, se divide, y cultivadas con IL-7 + IL-15 por más de 4 días. Tumor de la especificidad y la eficacia antitumoral de la expansión ex vivo de células T se determina.

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

Un protocolo eficiente para la<em&gt; Ex vivo</em&gt; Expansión de las células T tumor-reactiva de los ganglios linfáticos que drenan tumores u otros tejidos linfoides secundarios, de los portadores de tumores anfitriones se describe. Este protocolo se expande de forma selectiva del tumor de células T específicas para el uso de la inmunoterapia adoptiva del cáncer de mama.

Expansión ex vivo de células T tumor-reactiva por medio de la 1/Ionomycin briostatina y la formulación de la cadena gamma común de citoquinas

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

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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

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