<p class="jove_content">The protocol for the generation of human CD40-activated B cells from PBMC is divided into two parts: Part A demonstrates the preparation of CD40 ligand expressing NIH/3T3 cells, which will be used as plate-bound feeder cells. Part B describes the actual CD40-B culture.</p>
<p class="jove_title">A. Preparation of feeder cells (tCD40L NIH/3T3)</p>
<p class="jove_content">The tCD40L NIH/3T3 is an adherent murine fibroblast cell line, which should never become completely confluent. The cells are therefore splitted twice per ...

<ol>
	<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. Preparation of Media:</p>
<table border="1"cellspacing="0">
<tbody>
  <tr>
    <td colspan="2"><strong>Feeder cell wild type medium</strong></td>
    <td colspan="2"><strong>Feeder cell selection medium</strong></td>
  </tr>
  <tr>
    <td><strong>Reagent</strong></td>
    <td><strong>Concentration</strong></td>
    <td><strong>Reagent</strong></td>
    <td><strong>Concentration</strong></td>
  </tr>
  <tr>
    <td>DMEM-Ham's / F12</td>
    <td>&nbsp;</td>
    <td>DMEM-Ham's / F12</td>
    <td>&nbsp;</td>
  </tr>
  <tr>
    <td> L-Glutamine</td>
    <td>365 &mu;g/mL</td>
    <td> L-Glutamine</td>
    <td>365 &mu;g/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>Gentamycin</td>
    <td>15 &mu;g/mL</td>
    <td>Gentamycin</td>
    <td>15 &mu;g/mL</td>
  </tr>
  <tr>
    <td>&nbsp;</td>
    <td>&nbsp;</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 washing medium</strong></td>
      <td colspan="2"><strong>CD40-B culture medium</strong></td>
    </tr>
    <tr>
      <td><strong>Reagent</strong></td>
      <td><strong>Concentration</strong></td>
      <td><strong>Reagent</strong></td>
      <td><strong>Concentration</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-Glutamine</td>
      <td>584 	&mu;g/mL</td>
      <td>L-Glutamine</td>
      <td>584 	&mu;g/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>Gentamycin</td>
      <td>15 	&mu;g/mL</td>
      <td>Gentamycin</td>
      <td>15 	&mu;g/mL</td>
    </tr>
  </tbody>
  <tbody>
    <tr>
      <td><br />
      </td>
      <td><br />
      </td>
      <td>rh 	Transferrin</td>
      <td>50 	&mu;g/mL</td>
    </tr>
  </tbody>
  <tbody>
    <tr>
      <td><br />
      </td>
      <td><br />
      </td>
      <td>rh 	Insulin</td>
      <td>5 	&mu;g/mL</td>
    </tr>
  </tbody>
  <tbody>
    <tr>
      <td><br />
      </td>
      <td><br />
      </td>
      <td>AB-Humanserum </span> </td>
      <td>10 	%</td>
    </tr>
  </tbody>
</table>
<p class="jove_title">B. Miscellaneous Reagents:</p>
<table cellspacing="0" border="1">
<tbody>
  <tr>
    <td><strong>Reagent</strong></td>
    <td><strong>Company</strong></td>
    <td><strong>Order No</strong></td>
  </tr>
  <tr></tr>
  <tr>
    <td>DMEM / Ham's F12</td>
    <td>PAA</td>
    <td>Cat No: E15-813</td>
  </tr>
  <tr>
    <td>IMDM</td>
    <td>Invitrogen Gibco&reg;</td>
    <td>REF 21980-032</td>
  </tr>
  <tr>
    <td>Dulbecco's PBS (10x)</td>
    <td>PAA</td>
    <td>Cat No H15-011</td>
  </tr>
  <tr>
    <td>AB-Human Serum</td>
    <td>Invitrogen</td>
    <td>Cat No 34005100</td>
  </tr>
  <tr>
    <td>holo-Transferrin human</td>
    <td>Sigma</td>
    <td>Cat No T0665</td>
  </tr>
  <tr>
    <td>Insulin human</td>
    <td>Sigma</td>
    <td>Cat No I2643</td>
  </tr>
  <tr>
    <td>Gencin&reg;</td>
    <td>Delta Select</td>
    <td>Art No 7395800</td>
  </tr>
  <tr>
    <td>Recombinant Human Interleukin-4</td>
    <td>Immunotools</td>
    <td>Cat No 11130045</td>
  </tr>
  <tr>
    <td>Cyclosporin A</td>
    <td>Novartis</td>
    <td>Cat No NDC 0078-0109-01</td>
  </tr>
  <tr>
    <td>FBS</td>
    <td>LONZA</td>
    <td>Cat No DE14-802C</td>
  </tr>
  <tr>
    <td>HEPES Buffer</td>
    <td>PAA</td>
    <td>Cat No S11-001</td>
  </tr>
  <tr>
    <td>G-418 Sulphate</td>
    <td>PAA</td>
    <td>Cat No P02-012</td>
  </tr>
  <tr>
    <td>Trypsin/EDTA (10x)</td>
    <td>Invitrogen Gibco&reg;</td>
    <td>REF 15400-054</td>
  </tr>
  </tbody>
</table>
<p class="jove_title">C. Miscellaneous Supplies:</p>
<table cellspacing="0" border="1">
  <tbody>
  <tr>
    <td><strong>Reagent</strong></td>
    <td><strong>Company</strong></td>
    <td><strong>Order No</strong></td>
  </tr>
  <tr></tr>
  <tr>
    <td>Sterile pipette tips</td>
    <td>Sarstedt</td>
    <td>&nbsp;</td>
  </tr>
  <tr>
    <td>6-well plate</td>
    <td>NUNC</td>
    <td>Cat No 140675</td>
  </tr>
  <tr>
    <td>50mL conical tube</td>
    <td>BD Falcon&trade;</td>
    <td>Cat No 352070</td>
  </tr>
  <tr>
    <td>Tissue culture flask</td>
    <td>SARSTEDT</td>
    <td>Cat No 83.1813.002</td>
  </tr>
  </tbody>
</table>

generation of human cd40-activated b cells

<p class="jove_content">CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy <sup>1-3</sup>. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) <sup>3</sup>. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded <em>in vitro</em> to very large amounts of highly-pure CD40-B cells (&gt;10<sup>9</sup> cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) <sup>1,4</sup>.</p> 
<p class="jove_content">In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) <sup>5</sup>. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2).</p> 
<p class="jove_content">After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function <sup>1, 4</sup>. CD40-B cells efficiently take up, process and present antigens to T cells <sup>6</sup>. They do not only prime na&#970;ve, but also expand memory T cells <sup>7,8</sup>. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors <sup>9</sup>.</p>

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

Generation of Human CD40-activated B cells

<p class="jove_content">In this video we present the <em>ex vivo</em> generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.</p>

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

generation-of-human-cd40-activated-b-cells

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JoVE Journal

Biology

15.1K visualizações.  University Hospital of Cologne, Department I of Internal Medicine. Neste vídeo, apresentamos a Ex vivo Geração e expansão de células CD40-activated B (CD40-B) a partir de células mononucleares do sangue periférico (PBMC) de estimulação com CD40 ligante e interleucina-4.