Name: generation of human cd40-activated b cells
Uploaded: 2009-10-16T23:30:00
Description: Watch this Scientific Journal Video about Generation of Human CD40-activated B cells Video at JoVE.com

<p class="jove_content"> Il protocollo per la generazione di umani CD40 delle cellule B attivate da PBMC è diviso in due parti: la parte A dimostra la preparazione di ligando CD40 esprimere NIH/3T3 celle, che sarà usato come piatto legato cellule nutrici. Parte B descrive l&#39;attuale B CD40-cultura. </p><p class="jove_title"> A. Preparazione di cellule di alimentazione (tCD40L NIH/3T3) </p><p class="jove_content"> Il NIH/3T3 tCD40L è un aderente murino linea cellulare di fibroblasti, che non dovrebbe mai diventare completamente confluenti....

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	<li>Schultze, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40-activated+human+B+cells:+an+alternative+source+of+highly+efficient+antigen+presenting+cells+to+generate+autologous+antigen-specific+T+cells+for+adoptive+immunotherapy.">CD40-activated human B cells: an alternative source of highly efficient antigen presenting cells to generate autologous antigen-specific T cells for adoptive immunotherapy.</a> <em style='font-style: italic'>J Clin Invest</em>. <b>100</b>, 2757-2765 (1997).</li><li>Schultze, J. L., Grabbe, S., vonBergwelt-Baildon, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DCs+and+CD40-activated+B+cells:+current+and+future+avenues+to+cellular+cancer+immunotherapy.">DCs and CD40-activated B cells: current and future avenues to cellular cancer immunotherapy.</a> <em style='font-style: italic'>Trends Immunol</em>. <b>25</b>, 659-664 (2004).</li><li>Bergwelt-Baildon, M. v. o. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40-activated+B+cells+express+full+lymph+node+homing+triad+and+induce+T-cell+chemotaxis:+potential+as+cellular+adjuvants.">CD40-activated B cells express full lymph node homing triad and induce T-cell chemotaxis: potential as cellular adjuvants.</a> <em style='font-style: italic'>Blood</em>. <b>107</b>, 2786-2789 (2006).</li><li>Wiesner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+immortalization+of+human+B+cells+by+CD40+ligation.">Conditional immortalization of human B cells by CD40 ligation.</a> <em style='font-style: italic'>PLoS ONE</em>. <b>3</b>, 1464-14 (2008).</li><li>Urashima, M., Chauhan, D., Uchiyama, H., Freeman, G. J., Anderson, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40+ligand+triggered+interleukin-6+secretion+in+multiple+myeloma.">CD40 ligand triggered interleukin-6 secretion in multiple myeloma.</a> <em style='font-style: italic'>Blood</em>. <b>85</b>, 1903-1912 (1995).</li><li>Lapointe, R., Bellemare-Pelletier, A., Housseau, F., Thibodeau, J., Hwu, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40-stimulated+B+lymphocytes+pulsed+with+tumor+antigens+are+effective+antigen-presenting+cells+that+can+generate+specific+T+cells.">CD40-stimulated B lymphocytes pulsed with tumor antigens are effective antigen-presenting cells that can generate specific T cells.</a> <em style='font-style: italic'>Cancer Res</em>. <b>63</b>, 2836-2843 (2003).</li><li>von Bergwelt-Baildon, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+primary+and+memory+cytotoxic+T+lymphocyte+responses+are+efficiently+induced+by+means+of+CD40-activated+B+cells+as+antigen-presenting+cells:+potential+for+clinical+application.">Human primary and memory cytotoxic T lymphocyte responses are efficiently induced by means of CD40-activated B cells as antigen-presenting cells: potential for clinical application.</a> <em style='font-style: italic'>Blood</em>. <b>99</b>, 3319-3325 (2002).</li><li>Kondo, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40-activated+B+cells+can+be+generated+in+high+number+and+purity+in+cancer+patients:+analysis+of+immunogenicity+and+homing+potential.">CD40-activated B cells can be generated in high number and purity in cancer patients: analysis of immunogenicity and homing potential.</a> <em style='font-style: italic'>Clin Exp Immunol</em>. <b>155</b>, 249-256 (2009).</li><li>Mason, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-loaded+CD40-activated+B+cells+stimulate+antigen-specific+T-cell+responses+in+dogs+with+spontaneous+lymphoma.">RNA-loaded CD40-activated B cells stimulate antigen-specific T-cell responses in dogs with spontaneous lymphoma.</a> <em style='font-style: italic'>Gene Ther</em>. <b>15</b>, 955-965 (2008).</li></ol>

<p class="jove_title"> A. Preparazione dei supporti: </p><table border="1"cellspacing="0"><tbody><tr><td colspan="2"> <strong>Alimentatore cellule wild type media</strong> </td><td colspan="2"> <strong>Alimentatore cellulare medio selezione</strong> </td></tr><tr><td> <strong>Reagente</strong> </td><td> <strong>Concentrazione</strong> </td><td> <strong>Reagente</strong> </td><td> <strong>Concentrazione</strong> </td></tr><tr><td> DMEM-Ham / F12 </td><td></td><td> DMEM-Ham / F12 </td><td></td></tr><tr><td> L-Glutammina </td><td> 365 mg / ml </td><td> L-Glutammina </td><td> 365 mg / ml </td></tr><tr><td> FBS </td><td> 10% </td><td> FBS </td><td> 10% </td></tr><tr><td> HEPES </td><td> 10 mM </td><td> HEPES </td><td> 10 mM </td></tr><tr><td> Gentamicina </td><td> 15 mg / ml </td><td> Gentamicina </td><td> 15 mg / ml </td></tr><tr><td></td><td></td><td> G-418 </td><td> 0,7 mg / ml </td></tr></table><table cellspacing="0" border="1"><tbody><tr><td colspan="2"> <strong>CD40-B lavaggio medio</strong> </td><td colspan="2"> <strong>CD40-B terreno di coltura</strong> </td></tr><tr><td> <strong>Reagente</strong> </td><td> <strong>Concentrazione</strong> </td><td> <strong>Reagente</strong> </td><td> <strong>Concentrazione</strong> </td></tr><tr></tr></tbody><tbody><tr><td> IMDM </td><td><br /></td><td> IMDM </td><td><br /></td></tr></tbody><tbody><tr><td> L-Glutammina </td><td> 584 mg / ml </td><td> L-Glutammina </td><td> 584 mg / ml </td></tr></tbody><tbody><tr><td> HEPES </td><td> 25 mM </td><td> HEPES </td><td> 25 mM </td></tr></tbody><tbody><tr><td> Gentamicina </td><td> 15 mg / ml </td><td> Gentamicina </td><td> 15 mg / ml </td></tr></tbody><tbody><tr><td><br /></td><td><br /></td><td> rh transferrina </td><td> 50 mg / mL </td></tr></tbody><tbody><tr><td><br /></td><td><br /></td><td> L&#39;insulina rh </td><td> 5 mcg / ml </td></tr></tbody><tbody><tr><td><br /></td><td><br /></td><td> AB-Humanserum </td><td> 10% </td></tr></tbody></table><p class="jove_title"> B. Varie Reagenti: </p><table cellspacing="0" border="1"><tbody><tr><td> <strong>Reagente</strong> </td><td> <strong>Azienda</strong> </td><td> <strong>Il decreto n.</strong> </td></tr><tr></tr><tr><td> DMEM / Ham F12 </td><td> PAA </td><td> Cat No: E15-813 </td></tr><tr><td> IMDM </td><td> Invitrogen Gibco ® </td><td> REF 21980-032 </td></tr><tr><td> Dulbecco PBS (10x) </td><td> PAA </td><td> Nessun gatto H15-011 </td></tr><tr><td> AB-siero umano </td><td> Invitrogen </td><td> Nessun gatto 34005100 </td></tr><tr><td> olo-transferrina umana </td><td> Sigma </td><td> Nessun gatto T0665 </td></tr><tr><td> Insulina umana </td><td> Sigma </td><td> Nessun gatto I2643 </td></tr><tr><td> Gencin ® </td><td> Delta Select </td><td> Art. n. 7395800 </td></tr><tr><td> Ricombinante umano interleuchina-4 </td><td> Immunotools </td><td> Nessun gatto 11130045 </td></tr><tr><td> Ciclosporina A </td><td> Novartis </td><td> Nessun gatto NDC 0078-0109-01 </td></tr><tr><td> FBS </td><td> LONZA </td><td> Nessun gatto DE14-802C </td></tr><tr><td> HEPES Buffer </td><td> PAA </td><td> Nessun gatto S11-001 </td></tr><tr><td> G-418 Solfato </td><td> PAA </td><td> Nessun gatto P02-012 </td></tr><tr><td> Tripsina / EDTA (10x) </td><td> Invitrogen Gibco ® </td><td> REF 15400-054 </td></tr></tbody></table><p class="jove_title"> C. Varie Forniture: </p><table cellspacing="0" border="1"><tbody><tr><td> <strong>Reagente</strong> </td><td> <strong>Azienda</strong> </td><td> <strong>Il decreto n.</strong> </td></tr><tr></tr><tr><td> Puntali sterili </td><td> Sarstedt </td><td></td></tr><tr><td> 6 pozzetti </td><td> NUNC </td><td> Nessun gatto 140675 </td></tr><tr><td> 50mL tubo conico </td><td> BD Falcon ™ </td><td> Nessun gatto 352070 </td></tr><tr><td> Tessuto cultura fiasco </td><td> Sarstedt </td><td> Nessun gatto 83.1813.002 </td></tr></tbody></table>

generation of human cd40-activated b cells

<p class="jove_content"> CD40-attivate le cellule B (linfociti B CD40-) sono stati identificati come fonte alternativa di immuno-stimolatorie cellule presentanti l&#39;antigene (APC) per l&#39;immunoterapia del cancro <sup>1-3.</sup> Rispetto alle cellule dendritiche (DC), il miglior APC caratterizzato, le cellule CD40-B hanno diverse proprietà distinte biologici e tecnici. Simile alla DC, le cellule B mostrano un aumento dell&#39;espressione di MHC e molecole co-stimolatorie (Fig.1b), mostrano una forte capacità migratoria e presentazione dell&#39;antigene presente in modo efficiente alle cellule T, dopo stimolazione con il ligando dell&#39;interleuchina-4 e CD40 (CD40L). Tuttavia, a differenza di DC immature o mature, le cellule B CD40-esprimono la piena triade nodo homing linfa composto da CD62L, CCR7/CXCR4, e la funzione dei leucociti antigene-1 (LFA1, CD11a/CD18), necessaria per homing di organi linfatici secondari (Fig.1a) <sup>3.</sup> Cellule CD40-B può essere generato senza difficoltà da piccolissime quantità di sangue periferico che può essere ulteriormente espanse <em>in vitro</em> di grandi quantità di altamente puri-CD40 delle cellule B (&gt; 10 <sup>9</sup> cellule per paziente) da donatori sani e come il cancro pazienti (Fig.1c, d) <sup>1,4.</sup> </p><p class="jove_content"> In questo protocollo dimostriamo come ottenere pienamente attivato le cellule B CD40-da PBMC umane. Molecole chiave per la coltura cellulare sono ligando CD40, l&#39;interleuchina-4 (IL-4) e ciclosporina A (CsA), che viene rifornito in un ciclo di 3-4 cultura giorno. Per scopi di laboratorio CD40-stimolazione è fornito da NIH/3T3 cellule che esprimono CD40 ligando ricombinante umano (tCD40L NIH/3T3) <sup>5.</sup> Per evitare la contaminazione con le cellule non transfettate, espressione del ligando CD40 umano sulla trasfettate deve essere controllata regolarmente (Fig.2). </p><p class="jove_content"> Dopo 14 giorni CD40-B colture cellulari costituiti da oltre il 95% delle cellule B puro e una espansione del CD40 delle cellule B con più di 65 giorni è spesso possibile senza alcuna perdita di funzione <sup>1, 4.</sup> Le cellule B CD40-in modo efficiente raccogliere, trasformare e antigeni presenti alle cellule T <sup>6.</sup> Non solo naϊve primo, ma anche di espandere le cellule T di memoria <sup>7,8.</sup> CD40-attivate le cellule B possono essere utilizzati per studiare l&#39;attivazione delle cellule B, la differenziazione e la funzione. Inoltre, essi rappresentano uno strumento promettente per la vaccinazione terapeutica o preventiva contro i tumori <sup>9.</sup> </p>

Watch this Scientific Journal Video about Generation of Human CD40-activated B cells Video at JoVE.com

Generation of Human CD40-activated B cells Video (Video) | JoVE

<p class="jove_content">In questo video vi presentiamo il<em&gt; Ex vivo</em&gt; Generazione ed espansione dei diritti umani CD40 delle cellule B attivate (CD40-B) dalle cellule mononucleari del sangue periferico (PBMC) attraverso la stimolazione con il ligando CD40 e l&#39;interleuchina-4.</p>

Generazione di Human CD40 delle cellule B attivate

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

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