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

Bu çalışma NIH R01 CA104757 Grant (MH Manjili) tarafından desteklenmiştir. Biz minnetle VCU Massey Kanser Merkezi ve Commonwealth Kanser Araştırma Vakfı desteğiyle kabul etmiş sayılırsınız.

1. Lenfositler 5 izolasyonu <ol><li> Isolate tümör drenaj lenf düğümleri veya FVBN202 tümör taşıyan transgenik farelerin dalak ve buz RPMI1640 tek hücre süspansiyonu hazırlamak% 10 FBS ile desteklenmiştir. Polistiren tüplere göre daha büyük bir T hücre verimi 50 ml polipropilen konik tüpler sonuçları B / I aktivasyon. Ketamin ve Xylazine anestezi için ip enjekte edilir. Servikal dislokasyon ötenazi bir yöntem olarak kullanılmaktadır. </li><li> Kültür bryostatin-1 (5 nM) ve ionomy...

<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>

Seçici genişleme efektör anti-tümör işlevi tümör-reaktif T hücrelerinin B kullanarak önerilen protokol ile elde edilebilir I / aktivasyonu ve gama zinciri sitokinler IL-2, IL-7 ve IL-15 ile ex vivo genişlemesi. IL-2, antijen-spesifik T hücrelerinin farklılaşması ve genişleme destek T hücre büyüme faktörü iken, IL-7, T hücreleri, apoptozis inhibe eder ve genişleme sırasında canlılığı destekleyecek. IL-15, uzun vadeli bir anti-tümör cevapların evlatlık T hücre tedavisi...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<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

Meme kanseri hastalarının, tümörler 1,2 karşı önceden var olan bağışıklık yanıtlarını olduğu bildirilmiştir . Ancak, bu tür bağışıklık yanıtlarını meme kanseri geliştirme veya nüks karşı tam koruma sağlamak için başarısız olur. Tümör-reaktif T hücrelerinin sıklığı artırarak bu sorunu aşmak için, evlatlık immünoterapi istihdam edilmiştir. Tümör spesifik T hücrelerinin genişlemesi için çeşitli protokoller kullanılmaktadır. Bu protokoller, ancak tümör antijenleri ex vivo antijen-spesifik T hücrelerinin aktivasyonu için kullanımı sınırlıdır. Çok yakın bir zamanda, ortak gama zinciri sitokinler, IL-2, IL-7, IL-15, ve IL-21 gibi, anti-tümör immün yanıtların 3 geliştirme için tek başına veya kombine olarak kullanılır olmuştur. Ancak, bu formülasyon, tümör-reaktif T hücrelerinin genişlemesi için en iyi çalışmalarınızı ne açık değildir. Burada HER-2/neu evlatlık T hücre tedavisi meme kanseri kullanmak için pozitif meme kanseri FVBN202 transgenik fare modelinde tümör-reaktif T hücrelerinin selektif aktivasyonu ve genişleme için bir protokol mevcut. Bu protokol, 16 saat boyunca tümör antijenleri yokluğunda bryostatin-1/ionomycin (B / I) ve IL-2 ile T hücrelerinin aktivasyonunu içerir. B / I aktivasyonu hücre içi sinyalleri taklit eden, sırasıyla 4, protein kinaz C aktivitesi ve hücre içi kalsiyum artırarak T hücre aktivasyonu ile sonuçlanabilir . Bu protokol ilgisiz T hücreleri öldürüyor iken, özellikle tümör spesifik T hücrelerini aktive eder. B / I-aktive T hücreleri, IL-7 ve IL-15 ile 24 saat boyunca kültür ve sonra IL-2 ile darbeli. 24 saat sonra, T hücreleri, split yıkanmış ve kültürlü, IL-7 + ek 4 gün IL-15. Ex vivo genişletilmiş T hücrelerinin tümör özgüllük ve anti-tümör etkinliği belirlenir.

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

Için verimli bir protokol<em&gt; Ex vivo</em&gt; Tümör-reaktif T hücrelerinin tümör drenaj lenf düğümlerine veya diğer ikincil lenfoid dokularda tümör taşıyan ana genişleme açıklanmıştır. Bu protokol, meme kanseri evlatlık immünoterapi kullanmak için seçici, tümör spesifik T hücrelerinin genişletir.

Bryostatin 1/Ionomycin Yoluyla Tümör-reaktif T hücrelerinin ex vivo Genişleme ve Ortak Gama Zinciri Sitokinler Formülasyon

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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HIV-1 Enfekte CD4 Doğuştan Algılama Değerlendirilmesi<sup&gt; +</sup&gt; Bir kullanılması plazmositoid dendritik hücreler tarafından T Hücreleri<em&gt; Ex vivo</em&gt; Co-kültür sistemi.

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