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 confluent. The cells are therefore splitted twice per week. Culturing over more than 6 weeks is not recommended.
<ol>
 <li>Remove old medium from the primary culture with a sterile pipette and wash cells with 10 mL of 1x PBS. Aspirate the PBS after washing.</li>
 <li>Add 4 mL of 1x Trypsin/EDTA in a 75 cm2 flask for 10-15 minutes at 37&deg;C. Use gentle tapping to detach the cells.</li>
 <li>Add 10 mL of wild type medium and swivel gently to stop digestion with trypsin.</li>
 <li>Transfer the cell suspension into a 50 mL tube with a sterile pipette and spin the cells down at 265 x g for 7 minutes.</li>
 <li>Remove the supernatant and resuspend the cell pellet in 10 mL of wild type medium. Count the cell number of an aliquot of the cell suspension and prepare three 50 mL tubes with the appropriate number of cells:
 <ol>
 <li>2.5 x 106 cells for subculture</li>
 <li>0.4 x 106 cells/well for irradiation used for the murine CD40-B cell culture</li>
 <li>remainder to freeze (if needed).</li>
 </ol>
 </li>
 <li>Spin the cells down at 265 x g for 7 minutes.</li>
 <li>Remove the supernatant.
 <ol>
 <li>For subculturing: resuspend 2.5 x 106 cells in 10 mL selection medium in a 75 cm2 cell culture flask (cell density 2.5 x 105 cells/mL) and incubate the cells at 37&deg;C with 5 % CO2. Split the cells twice per week.</li>
 <li>For the murine CD40-B cell culture: You need 2.4 x 106 cells for one 6-well plate. Resuspend the tmuCD40L HeLa cells in wild type medium at a density of 0.2 x 106 cells/mL and irradiate them at 78 Gy. Plate 2 mL of the cell suspension into each well and incubate them at 37&deg;C with 5 % CO2. Use this prepared plates for B-cell stimulation when tmuCD40L HeLa cells are adherent (at least 4 hours: check adherence using the microscope; do not wait more than 24 h to start B-cell stimulation). (Continue with B.)</li>
 </ol>
 </li>
</ol>
B. Murine CD40-B cell culture
I. Preparation of splenocytes for CD40-stimulation (day 0):
Please note: before you continue ascertain that feeder cells are adherent. Always add fresh solutions of murine interleukin-4, &beta;-Mercaptoethanol and cyclosporin A to the growth medium immediately before use.
<ol>
 <li>Dissect spleen of C57BL/6 mice (haplotype H-2b) and wash it twice in 10-20 mL DMEM supplemented with Gentamycin [15&mu;g/mL] by transferring it with a 10mL pipette tip from one tube to another. Avoid transferring contaminants (e.g. hair) of the mouse. Mince spleen by passing it through a cell strainer (100&mu;m) and rinse strainer with another 10 mL of medium. Spin down splenocytes at 265 x g for 7 min at room temperature, resuspend them in 7mL muCD40-B cell medium, and separate lymphocytes by Ficoll density centrifugation. To do so layer cells on 5 mL Mouse-Pancoll (preheated at room temperature) in a 15 mL tube and centrifuge at 450 x g for 30 min without brake at room temperature. </li>
 <li>Draw off most of the upper layer, leaving the lymphocyte layer undisturbed at the interface. Transfer lymphocyte layer to a fresh 50 mL tube, wash once with 30mL DMEM with Gentamycin [15&mu;g/mL] by spinning down at 265 x g for 7 min to remove other cells. Discard the supernatant and resuspend the cells in 10 mL of mCD40-B washing medium. Determine the cell number in an aliquot of the cell suspension. Determine cell number by counting an aliquot of the cell suspension.</li>
 <li>Spin down the required amount of cells at 265 x g for 7 minutes. For a 6-well plate 5 x 106 cells/well are needed, thus 30 x 106 cells per plate. </li>
 <li>Remove the supernatant and resuspend the lymphocytes at 1.25 x 106 cells/mL in mCD40-B culture medium freshly supplemented with 1 U/mL of interleukin-4 as growth factor, 100 &mu;M &beta;-Mercaptoethanol and 0.63 &mu;g/mL cyclosporin A to prevent outgrowth of T-cells (Given concentrations refer to one mL culture medium!). </li>
 <li>Remove the supernatant from the 6-well plate pre-incubated with tmuCD40L HeLa cells. </li>
 <li>Gently add 4 mL of the lymphocyte suspension (1.25 x 106 cells/mL) to each well of the 6-well plate. </li>
 <li>Incubate the plate at 37&deg;C with 5 % CO2. </li>
 <li>On day 3 reculture the cells (Continue with II.1.). </li>
</ol>
II. Subculture of mCD40B cells (day 3 and then twice a week):
<ol>
 <li>Harvest mCD40B cell cluster by vigorously pipetting up and down with a 10 mL pipette and pool them into a 50 mL tube.</li>
 <li>Spin down at 265 x g for 7 minutes and completely replace the supernatant with mCD40-B washing medium. While counting the cell amount of an aliquot, spin the cells down at 265 x g for 7 minutes. Resuspend mCD40B cells in mCD40-B culture medium at a concentration of 0.75 x 106 cells/mL.</li>
 <li>Add fresh solutions of murine interleukin-4 in a concentration of 1 U/mL, 100 &mu;M &beta;-Mercaptoethanol and 0.63 &mu;g/mL cyclosporin A to the medium.</li>
 <li>Remove the supernatant from a 6-well plate pre-incubated with irradiated tmuCD40L HeLa cells.</li>
 <li>Gently add 4 mL of the mCD40B cell suspension (0.75 x 106cells/mL) to each well of the 6-well plate.</li>
 <li>Incubate plates at 37&deg;C with 5 % CO2.</li>
 <li>Again subculture the cells every 3-4 days to end up with highly pure murine CD40-activated B cells after a total of 14 days. </li>
</ol>
C. Trouble-shooting: What if CD40-B cells do not grow?
<ol>
 <li>Have you checked for CD40 ligand expression of feeder cells?</li>
 <li>Are the plate-bound feeder cells used for the stimulation older than 24h?</li>
 <li>Is there a contamination with mycoplasma?</li>
 <li>Was the interleukin-4 solution used for supplementation freshly thawed and did it have the appropriate biological activity?</li>
 <li>Was &beta;-Mercaptoethanol and cyclosporin A added in the correct concentration?</li>
</ol>

<img src="/files/ftp_upload/1734/1734fig1a.jpg" alt="Figure 1 a" /> Figure 1a.
<img src="/files/ftp_upload/1734/1734fig1b.jpg" alt="Figure 1 b" /> Figure 1b.
<img src="/files/ftp_upload/1734/1734fig1d.jpg" alt="Figure 1 d" /> Figure 1d.
<img src="/files/ftp_upload/1734/1734fig2.jpg" alt="Figure 2" /> Figure 2.

<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

12.6K Views.  University Hospital of Cologne. In questo video, dimostriamo la procedura di CD40-attivazione e l'espansione delle cellule B da splenociti murini di C57BL / 6 topi, che può essere utilizzato come modello di una cellula presentanti l'antigene (APC) per studiare l'induzione di immunità.

Video: Murine Model of CD40-activation of B cells

Induction and Assessment of Class Switch Recombination in Purified Murine B Cells

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the immunoglobulin locus undergoes a series of genetic modifications to alter the binding capacity and effector function of secreted antibodies. This process is highlighted by a genomic recombination event known as class switch recombination (CSR) in which the default IgM antibody isotype is substituted for one of IgG, IgA, or IgE. Each isotype possesses distinct effector functions thereby making CSR crucial to the maintenance of immunity. Diversification of the immunoglobulin locus is mediated by the enzyme activation-induced cytidine deaminase (AID). A schematic video describing this process in detail is available online (http://video.med.utoronto.ca/videoprojects/immunology/aam.html). AID's activity and the CSR pathway are commonly studied in the assessment of B cell function and humoral immunity in mice. The protocol outlined in this report presents a method of B cell isolation from murine spleens and subsequent stimulation with bacterial lipopolysaccharide (LPS) to induce class switching to IgG3 (for other antibody isotypes see Table 1). In addition, the fluorescent cell staining dye Carboxyfluorescein succinimidyl ester (CFSE) is used to monitor cell division of stimulated cells, a process crucial to isotype switching 1, 2. The regulation of AID and the mechanism by which CSR occurs are still unclear and thus in vitro class switch assays provide a reliable method for testing these processes in various mouse models. These assays have been previously used in the context of gene deficiency using knockout mice 3. Furthermore, in vitro switching of B cells can be preceded by viral transduction to modulate gene expression by RNA knockdown or transgene expression 4-6. The data from these types of experiments have impacted our understanding of AID activity, resolution of the CSR reaction, and antibody-mediated immunity in the mouse.

Following antigen exposure, subpopulations of activated B cells undergo a process known as class switch recombination (CSR) to produce antibody isotypes with distinct effector functions. The protocol outlined in this report explains how CSR can be induced and analyzed in vitro for the purposes of studying B cell function.

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the ...

Recombinant Retroviral Production and Infection of B Cells

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control of a heterologous expression system to facilitate the analysis of the resulting phenotype. This approach can be used to express a gene that is not normally found in the organism, to express a mutant form of a gene product, or to over-express a dominant-negative form of the gene product. It is particularly useful in the study of the hematopoetic system, where transcriptional regulation is a major control mechanism in the development and differentiation of B cells 1, reviewed in 2-4.
Mouse genetics is a powerful tool for the study of human genes and diseases. A comparative analysis of the mouse and human genome reveals conservation of synteny in over 90% of the genome 5. Also, much of the technology used in mouse models is applicable to the study of human genes, for example, gene disruptions and allelic replacement 6. However, the creation of a transgenic mouse requires a great deal of resources of both a financial and technical nature. Several projects have begun to compile libraries of knock out mouse strains (KOMP, EUCOMM, NorCOMM) or mutagenesis induced strains (RIKEN), which require large-scale efforts and collaboration 7. Therefore, it is desirable to first study the phenotype of a desired gene in a cell culture model of primary cells before progressing to a mouse model. 
Retroviral DNA integrates into the host DNA, preferably within or near transcription units or CpG islands, resulting in stable and heritable expression of the packaged gene of interest while avoiding transcriptional silencing 8 9. The genes are then transcribed under the control of a high efficiency retroviral promoter, resulting in a high efficiency of transcription and protein production. Therefore, retroviral expression can be used with cells that are difficult to transfect, provided the cells are in an active state during mitosis. Because the structural genes of the virus are contained within the packaging cell line, the expression vectors used to clone the gene of interest contain no structural genes of the virus, which both eliminates the possibility of viral revertants and increases the safety of working with viral supernatants as no infectious virions are produced 10. 
Here we present a protocol for recombinant retroviral production and subsequent infection of splenic B cells. After isolation, the cultured splenic cells are stimulated with Th derived lymphokines and anti-CD40, which induces a burst of B cell proliferation and differentiation 11. This protocol is ideal for the study of events occurring late in B cell development and differentiation, as B cells are isolated from the spleen following initial hematopoetic events but prior to antigenic stimulation to induce plasmacytic differentiation.

An efficient system of structure and function analysis of a gene in an ex vivo culture of splenic B-lymphocytes is described. This method takes advantage of recombinant retroviral production in a helper free, ecotrophic packaging cell line. Stable, heritable expression of a gene of interest within primary lymphocytes is achieved leading to generation of surface antibodies on B cells undergoing class switch recombination.

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control ...

Murine Model of Allergen Induced Asthma

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, with the prevalence increasing.2 As with other diseases, animal models of allergic airway disease greatly facilitate understanding of the underlying pathophysiology, help identify potential therapeutic targets, and allow preclinical testing of possible new therapies. Models of allergic airway disease have been developed in several animal species, but murine models are particularly attractive due to the low cost, ready availability, and well-characterized immune systems of these animals.3 Availability of a variety of transgenic strains further increases the attractiveness of these models.4 Here we describe two murine models of allergic airway disease, both employing ovalbumin as the antigen. Following initial sensitization by intraperitoneal injection, one model delivers the antigen challenge by nebulization, the other by intratracheal delivery. These two models offer complementary advantages, with each mimicking the major features of human asthma.5
The major features of acute asthma include an exaggerated airway response to stimuli such as methacholine (airway hyperresponsiveness; AHR) and eosinophil-rich airway inflammation. These are also prominent effects of allergen challenge in our murine models,5,6 and we describe techniques for measuring them and thus evaluating the effects of experimental manipulation. Specifically, we describe both invasive7 and non-invasive8 techniques for measuring airway hyperresponsiveness as well as methods for assessing infiltration of inflammatory cells into the airways and the lung. Airway inflammatory cells are collected by bronchoalveolar lavage while lung histopathology is used to assess markers of inflammation throughout the organ. These techniques provide powerful tools for studying asthma in ways that would not be possible in humans.

Experimental mouse models of allergic asthma offer new possibilities for studying disease pathogenesis and developing new therapeutics. These models are well suited to measuring factors governing the allergic immune response, airway inflammation, and pulmonary pathophysiology.

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, ...

Plaque Assay for Murine Norovirus

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are observed during MNV-1 infection of murine dendritic cells or macrophages 1. This property of MNV-1 can be used to quantify the number of infectious particles in a given sample by performing a plaque assay 1. The plaque assay relies on the ability of MNV-1 to lyse cells and to form holes in a confluent cell monolayer, which are called plaques 3.
Multiple techniques can be used to detect viral infections in tissue culture, harvested tissue, clinical, and environmental samples, but not all measure the number of infectious particles (e.g. qRT-PCR). One way to quantify infectious viral particles is to perform a plaque assay 3, which will be described in detail below. A variation on the MNV plaque assay is the fluorescent focus assay, where MNV antigen is immunostained in cell monolayers 4. This assay can be faster, since viral antigen expression precedes plaque formation. It is also useful for titrating viruses unable to form plaques. However, the fluorescent focus assay requires additional resources beyond those of the plaque assay, such as antibodies and a microscope to count focus-forming units. Infectious MNV can also be quantified by determining the 50% Tissue Culture Infective Dose (TCID50) 3. This assay measures the amount of virus required to produce CPE in 50% of inoculated tissue culture cells by endpoint titration 5. However, its limit of detection is higher compared to a plaque assay 4.
In this article, we describe a plaque assay protocol that can be used to effectively determine the number of infectious MNV particles present in biological or environmental samples 1, 4, 6. This method is based on the preparation of 10-fold serial dilutions of MNV-containing samples, which are used to inoculate a monolayer of permissive cells (RAW 264.7 murine macrophage cells). Virus is allowed to attach to the cell monolayer for a given period of time and then aspirated before covering cells with a mixture of agarose and cell culture media. The agar enables the spread of viral progeny to neighboring cells while limiting spread to distantly located cells. Consequently, infected cells are lysed and form holes in the monolayer known as plaques. Upon sufficient spread of virus, plaques become visible following staining of cells with dyes, like neutral red, methylene blue, or crystal violet. At low dilutions, each plaque originates from one infectious viral particle and its progeny, which spread to neighboring cells. Thus, counting the number of plaques allows one to calculate plaque-forming units (PFU) present in the undiluted sample 3.

Here we describe a method to quantify infectious particles of murine norovirus (MNV), which is the only norovirus that efficiently replicates in cell culture. The plaque assay takes advantage of MNV&#x2019;s tropism for murine macrophages and can be adapted for use with biological or environmental samples containing MNV.

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are ...

Detection of True IgE-expressing Mouse B Lineage Cells

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH isotypes, such as IgA, IgE and IgG. Measurement of IgH CSR in vitro is a key method for the study of a number of biologic processes ranging from DNA recombination and repair to aspects of molecular and cellular immunology. In vitro CSR assay involves the flow cytometric measurement surface Ig expression on activated B cells. While measurement of IgA and IgG subclasses is straightforward, measurement of IgE by this method is problematic due to soluble IgE binding to Fc&#949;RII/CD23 expressed on the surface of activated B cells. Here we describe a unique procedure for accurate measurement of IgE-producing mouse B cells that have undergone CSR in culture. The method is based on trypsin-mediated cleavage of IgE-CD23 complexes on cell surfaces, allowing for detection of IgE-producing B lineage cells by cytoplasmic staining. This procedure offers a convenient solution for flow cytometric analysis of CSR to IgE.

In vitro analysis of class switch recombination in mice is challenging due to cytophilic IgE molecules bound to Fc receptors on the surface of B cells. We describe a method for IgE detection using trypsin-mediated cleavage of surface-bound IgE prior to fixation and permeabilization for cytoplasmic fluorescence staining.

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, MZBs must migrate up the shear force of blood flow. We present here a method for analyzing this flow-induced MZB migration in vitro. First, MZBs are isolated from the mouse spleen. Second, MZBs are settled on integrin ligands in flow chamber slides, exposed to shear flow, and imaged under a microscope while migrating. Third, images of the migrating MZBs are processed using the MTrack2 automatic cell tracking plugin for ImageJ, and the resulting cell tracks are quantified using the Ibidi chemotaxis tool. The migration data reveal how fast the cells move, how often they change direction, whether the shear flow vector affects their migration direction, and which integrin ligands are involved. Although we use MZBs, the method can easily be adapted for analyzing migration of any leukocyte that responds to the force of shear flow.

Marginal zone B cells (MZBs) respond to the force of shear flow by re-orienting their migration path up the flow. This protocol shows how to record and analyze the migration using a fluidics unit, pump, microscope imaging system, and free software.

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, ...

In Vitro Differentiation Model of Human Normal Memory B Cells to Long-lived Plasma Cells

Plasma cells (PCs) secrete large amounts of antibodies and develop from B cells that have been activated. PCs are rare cells located in the bone marrow or mucosa and ensure humoral immunity. Due to their low frequency and location, the study of PCs is difficult in human. We reported a B to PC in vitro differentiation model using selected combinations of cytokines and activation molecules that allow to reproduce the sequential cell differentiation occurring in vivo. In this in vitro model, memory B cells (MBCs) will differentiate into pre-plasmablasts (prePBs), plasmablasts (PBs), early PCs and finally, into long-lived PCs, with a phenotype close to their counterparts in healthy individuals.&#160;We also built an open access bioinformatics tools to analyze the most prominent information from GEP data related to PC differentiation. These resources can be used to study human B to PC differentiation and in the current study, we investigated the gene expression regulation of epigenetic factors during human B to PC differentiation.

Using multi-step culture systems, we report an in vitro B cell to plasma cell differentiation model.

Plasma cells (PCs) secrete large amounts of antibodies and develop from B cells that have been activated. PCs are rare cells located in the bone marrow or ...