We kindly thank Dr. Clemens Wendtner for providing tmuCD40-Ligand-HeLa cell line.

The protocol for the generation of murine CD40-activated B cells (mCD40B) from splenocytes is divided into two parts: Part A demonstrates the preparation of murine CD40 ligand (CD40L) expressing HeLa cells (tmuCD40L HeLa), which will be used as plate-bound feeder cells. Part B describes the actual murine CD40-B culture.
A. Preparation of feeder cells (tmuCD40L HeLa)
The tmuCD40L HeLa is an adherent human epithelial cell line, which should never become completely con...

<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> J Clin Invest. 100 (11), 2757-2765 (1997).</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> PLoS ONE. 3, e1464-e1464 (2008).</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=Efficient+generation+of+antigen-specific+cytotoxic+T+cells+using+retrovirally+transduced+CD40-activated+B+cells.">Efficient generation of antigen-specific cytotoxic T cells using retrovirally transduced CD40-activated B cells.</a> J Immunol. 169 (4), 2164-2171 (2002).</li><li>Liebig, T. M., Fiedler, A., Zoghi, S., Shimabukuro-Vornhagen, A., 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=Generation+of+human+CD40-activated+B+cells.">Generation of human CD40-activated B cells.</a> J Vis Exp. 32, (2009).</li><li>von Bergwelt-Baildon, M. <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> Blood. 107 (7), 2786-2789 (2006).</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> Cancer Res. 63 (11), 2836-2843 (2003).</li><li>Ahmadi, T., Flies, A., Efebera, Y., Sherr, D. H. <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-activated,+antigen-specific+B+cells+are+comparable+to+mature+dendritic+cells+in+presenting+protein+antigens+and+major+histocompatibility+complex+class+I-+and+class+II-binding+peptides.">CD40 Ligand-activated, antigen-specific B cells are comparable to mature dendritic cells in presenting protein antigens and major histocompatibility complex class I- and class II-binding peptides.</a> Immunology. 124 (1), 129-140 (2008).</li><li>Bergwelt-Baildon, M. S. 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=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> Blood. 99 (9), 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> Clin Exp Immunol. 155 (2), 249-256 (2009).</li><li>Schultze, J. L., Grabbe, S., 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=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> Trends Immunol. 25 (12), 659-664 (2004).</li><li>Mayr, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+translocations+are+associated+with+poor+prognosis+in+chronic+lymphocytic+leukemia.">Chromosomal translocations are associated with poor prognosis in chronic lymphocytic leukemia.</a> Blood. 107 (2), 742-751 (2006).</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> Gene Ther. 15 (13), 955-965 (2008).</li></ol>

The experiments were performed in accordance with the national and European guidelines for laboratory animal keeping. Referring the German animal welfare law the animals were controlled regularly by the proper authorities.

Modifications of this protocol are possible, especially regarding the origin of CD40-stimulus and the type of mouse strain used, also yielding in efficient generation of murine CD40-activated B cells. Ahmadi et al. have shown similar results for mouse fibroblasts transfected with murine CD40-Ligand. In this context presentation of xeno-antigen can be excluded with certainty, but intensive studies regarding security and toxicity in vivo gave no signs for inflammation or auto-immunity reaction in this setting usin...

A. Preparation of Media:
<table border="1">
<tbody>
 <tr>
 <td>Feeder cell wild type medium</td>
 <td>Feeder cell selection medium</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Reagent</td>
 <td>Concentration</td>
 <td>Reagent</td>
 <td>Concentration</td>
 </tr>
 <tr>
 <td>RPMI 1640</td>
<td>&nbsp;</td> 
 <td>RPMI 1640</td>
<td>&nbsp;</td> 
</tr>
 <tr>
 <td> L-Glutamine</td>
 <td>300 &mu;g/mL</td>
 <td> L-Glutamine</td>
 <td>300 &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>Hygromycin B</td>
 <td>0.2 mg/mL</td>
 </tr>
</table>
<table border="1">
 <tbody>
 <tr>
 <td>mCD40-B washing medium</td>
 <td>mCD40-B culture medium</td>
 </tr>
 <tr>
 <td>Reagent</td>
 <td>Concentration</td>
 <td>Reagent</td>
 <td>Concentration</td>
 </tr>
 <tr></tr>
 <tr>
 <td>D-MEM</td>
 <td> 
 </td>
 <td>D-MEM</td>
 <td> 
 </td>
 </tr>
 <tr>
 <td>L-Glutamine</td>
 <td>580 &mu;g/mL</td>
 <td>L-Glutamine</td>
 <td>580 &mu;g/mL</td>
 </tr>
 <tr>
 <td>Glucose</td>
 <td>4.5 mg/mL</td>
 <td>Glucose</td>
 <td>4.5 mg/mL</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> 
 </td>
 <td> 
 </td>
 <td>MEM</td>
 <td>0.1mM (1x)</td>
 </tr>
 <tr>
 <td> 
 </td>
 <td> 
 </td>
 <td>FBS</td>
 <td>10%</td>
 </tr>
 </tbody>
</table>
B. Miscellaneous Reagents:
<table border="1">
 <tr>
 <td>Reagent</td>
 <td>Company</td>
 <td>Order No</td>
 </tr>
 <tr>
 <td>RPMI 1640</td>
 <td>Invitrogen Gibco&#174;</td>
 <td>REF 21875</td>
 </tr>
 <tr>
 <td>D-MEM</td>
 <td>Invitrogen Gibco&#174;</td>
 <td>REF 41965</td>
 </tr>
 <tr>
 <td>Dulbecco&#180;s PBS (10x)</td>
 <td>PAA</td>
 <td>Cat No H15-011</td>
 </tr>
 <tr>
 <td>Gencin&#174;</td>
 <td>Delta Select</td>
 <td>Art No 7395800</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>MEM NEAA</td>
 <td>Invitrogen Gibco&#174;</td>
 <td>REF 11140</td>
 </tr>
 <tr>
 <td>Hygromycin B</td>
 <td>PAA</td>
 <td>Cat No P02-015</td>
 </tr>
 <tr>
 <td>Recombinant Murine Interleukin-4</td>
 <td>Immunotools</td>
 <td>Cat No 12340045</td>
 </tr>
 <tr>
 <td>Cyclosporin A</td>
 <td>Novartis</td>
 <td>Cat No NDC 0078-0109-01</td>
 </tr>
 <tr>
 <td>Trypsin/EDTA (10x)</td>
 <td>Invitrogen Gibco&#174;</td>
 <td>REF 15400-054</td>
 </tr>
</table>
C. Miscellaneous Supplies:
<table border="1">
<tbody>
 <tr>
 <td>Reagent</td>
 <td>Company</td>
 <td>Order No</td>
 </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>

murine model of cd40-activation of b cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

Watch this Scientific Journal Video about Murine Model of CD40-activation of B cells at JoVE.com

Murine Model of CD40-activation of B cells

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

murine-model-of-cd40-activation-of-b-cells

Research

JoVE Journal

Immunology and Infection

12.5K visualizações.  University Hospital of Cologne. Neste vídeo, demonstramos o procedimento de ativação CD40-e expansão de células B murino de esplenócitos de camundongos C57BL / 6, que pode ser usado como um modelo de células apresentadoras de antígenos (APC) para estudar a indução de imunidade.