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 at JoVE.com

<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 ...

Research

JoVE Journal

Biology

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture ...

Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing ...

Robust Generation of Hepatocyte-like Cells from Human Embryonic Stem Cell Populations

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical ...

Phenotypic and Functional Characterization of Endothelial Colony Forming Cells Derived from Human Umbilical Cord Blood

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of ...

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

Generation of Dispersed Presomitic Mesoderm Cell Cultures for Imaging of the Zebrafish Segmentation Clock in Single Cells

Segmentation is a  periodic and sequential morphogenetic process in vertebrates. This rhythmic  formation of blocks of tissue called somites along the ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...