Amablemente las gracias al Dr. Clemens Wendtner para proporcionar tmuCD40-ligando línea celular HeLa.

El protocolo para la generación de murino CD40-linfocitos B activados (mCD40B) a partir de esplenocitos se divide en dos partes: la parte A demuestra la preparación de murino CD40 ligando (CD40L) que expresan las células HeLa (tmuCD40L HeLa), que se utiliza como placa- obligados células alimentadoras. La parte B describe la actual murino CD40-B cultura. A. Preparación de las células de alimentación (tmuCD40L HeLa) El HeLa tmuCD40L es un adherente línea humana de células epiteliales, lo que nunca debe ser totalmente confluente. Las células son por lo tanto, dividido dos veces por semana. El cultivo de más de más de 6 semanas no es recomendable. <ol><li> Quitar medio de edad de la cultura de primaria con una pipeta estéril y lavar las células con 10 ml de PBS 1x. Aspirar el PBS después del lavado. </li><li> Agregar 4 ml de 1x tripsina / EDTA en un matraz de 75 cm 2 durante 10-15 minutos a 37 ° C. Use suaves golpecitos para separar las células. </li><li> Agregar 10 ml de medio de tipo salvaje y giro con suavidad para detener la digestión con tripsina. </li><li> La transferencia de la suspensión de células en un tubo de 50 ml con una pipeta estéril y el giro de las células hacia abajo a 265 xg durante 7 minutos. </li><li> Eliminar el sobrenadante y resuspender el botón celular en 10 ml de medio de tipo salvaje. Cuente el número de células de una parte alícuota de la suspensión celular y preparar tres tubos de 50 ml con el número adecuado de células: <ol><li> 2.5 x 10 6 células de la subcultura </li><li> 0,4 x 10 6 células / pocillo de irradiación utilizadas para el cultivo de células murinas CD40-B </li><li> resto de congelar (si es necesario). </li></ol></li><li> Giro de las células hacia abajo a 265 xg durante 7 minutos. </li><li> Eliminar el sobrenadante. <ol><li> Para subcultivo: resuspender 2,5 x 10 6 células en 10 ml de medio de selección de 75 cm 2 frasco de cultivo celular (densidad celular de 2,5 x 10 5 células / ml) e incubar las células a 37 º C con CO2 al 5%. Dividir las células dos veces por semana. </li><li> Para el cultivo de células murinas CD40-B: Es necesario 2,4 x 10 6 células por uno de 6 pocillos. Resuspender las células HeLa tmuCD40L en el medio silvestre, con una densidad de 0,2 x 10 6 células / ml e irradiar a los 78 Gy. Placa de 2 ml de la suspensión celular en cada pocillo y se incuba a 37 ° C con 5% de CO 2. Utilice esta platos preparados para la estimulación de células B en células HeLa tmuCD40L son adherentes (por lo menos 4 horas: comprobar el cumplimiento con el microscopio, no espere más de 24 horas para empezar la estimulación de células B). (Continuar con B.) </li></ol></li></ol> B. murino CD40-B de cultivo celular I. Preparación de esplenocitos de CD40-estimulación (día 0): Tenga en cuenta: antes de continuar asegurarse de que las células alimentadoras son adherentes. Siempre agregue nuevas soluciones de forma inmediata murino interleucina-4, β-mercaptoetanol y la ciclosporina A para el medio de cultivo antes de su uso. <ol><li> Diseccionar el bazo de los ratones C57BL / 6 (haplotipo H-2b) y lavar dos veces en 10-20 ml de DMEM suplementado con gentamicina [15μg/mL] mediante la transferencia con una punta de pipeta 10 ml de un tubo a otro. Evitar la transferencia de contaminantes (por ejemplo, pelo) del ratón. Picar el bazo pasándolo por un colador celular (100μm) y enjuague con otro tamiz de 10 ml de medio. Centrifugar a 265 xg esplenocitos durante 7 minutos a temperatura ambiente, a volver a suspender en 7 ml muCD40-B medio celular, los linfocitos y por separado por centrifugación de densidad Ficoll. Para ello las células de la capa de 5 ml de ratón Pancoll (precalentado a temperatura ambiente) en un tubo de 15 ml y centrifugar a 450 xg durante 30 minutos sin freno a temperatura ambiente. </li><li> Extraiga la mayor parte de la capa superior, dejando la capa de linfocitos sin problemas en la interfaz. La transferencia de la capa de linfocitos a un tubo fresco mL 50, lavar una vez con 30 ml de DMEM con Gentamicina [15μg/mL] girando hacia abajo a 265 g durante 7 min para eliminar otras células. Eliminar el sobrenadante y resuspender las células en 10 ml de mCD40-B medio de lavado. Determinar el número de células en una parte alícuota de la suspensión celular. Determinar el número de células por contar una alícuota de la suspensión celular. </li><li> Decantar la cantidad necesaria de células a 265 xg durante 7 minutos. Para una placa de 6 pocillos 5 x 10 6 células / así se necesitan, por lo tanto 30 x 10 6 células por placa. </li><li> Eliminar el sobrenadante y resuspender los linfocitos a 1,25 x 10 6 células / ml en mCD40 B-medio de cultivo fresco suplementado con 1 U / mL de IL-4 como factor de crecimiento, 100 mM β-mercaptoetanol y 0,63 mg / ml de ciclosporina A para prevenir la consecuencia de las células T (las concentraciones indicadas se refieren a un medio de cultivo ml!). </li><li> Eliminar el sobrenadante de la placa de 6 pocillos pre-incubadas con células HeLa tmuCD40L. </li><li> Suavemente agregar 4 ml de la suspensión de linfocitos (1,25 x 10 6 células / ml) a cada pocillo de la placa de 6 pocillos. </li><li> Incubar la placa a 37 ° C con 5% de CO 2. </li&gt; <li> El día 3 reculture las células (Continúe con II.1.). </li></ol> II. Subcultura de células mCD40B (días 3 y luego dos veces por semana): <ol><li> Cosecha mCD40B grupo de células vigorosamente pipeteando arriba y abajo con una pipeta de 10 ml y la piscina que en un tubo de 50 ml. </li><li> Centrifugar a 265 xg durante 7 minutos y sustituir por completo el sobrenadante con mCD40-B medio de lavado. Al contar la cantidad de células de una parte alícuota, girar las células hacia abajo a 265 xg durante 7 minutos. Resuspender las células en mCD40B mCD40 B-medio de cultivo a una concentración de 0,75 x 10 6 células / ml. </li><li> Añadir nuevas soluciones de murino interleucina-4 en una concentración de 1 U / mL, 100 mM β-mercaptoetanol y 0,63 mg / ml de ciclosporina A en el medio. </li><li> Eliminar el sobrenadante de una placa de 6 pocillos pre-incubadas con células HeLa irradiados tmuCD40L. </li><li> Suavemente agregar 4 ml de la suspensión celular mCD40B (0,75 x 10 6 células / ml) a cada pocillo de la placa de 6 pocillos. </li><li> Incubar las placas a 37 ° C con 5% de CO 2. </li><li> Una vez más la subcultura de las células cada 3-4 días para terminar con alta pureza células murinas B CD40-activa después de un total de 14 días. </li></ol> C. Solución de problemas: ¿Qué pasa si CD40-células B no crecen? <ol><li> ¿Ha revisado para la expresión del ligando CD40 de células alimentadoras? </li><li> Son las células de alimentador de placas con destino utilizados para la estimulación de más de 24 horas? </li><li> ¿Existe una contaminación por micoplasma? </li><li> Era la solución de interleuquina-4 utilizado para la suplementación recién descongelado y lo tienen la actividad biológica apropiada? </li><li> Fue β-mercaptoetanol y ciclosporina A agregado en la concentración correcta? </li></ol><img src="/files/ftp_upload/1734/1734fig1a.jpg" alt="Figura 1 a" /> Figura 1a. <img src="/files/ftp_upload/1734/1734fig1b.jpg" alt="Figura 1 b" /> Figura 1b. <img src="/files/ftp_upload/1734/1734fig1d.jpg" alt="Figura 1 d" /> Figura 1d. <img src="/files/ftp_upload/1734/1734fig2.jpg" alt="Figura 2" /> Figura 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>

Los experimentos fueron realizados de acuerdo con las directrices nacionales y europeas para la cría de animales de laboratorio. Refiriéndose a la ley de bienestar animal alemán los animales fueron controlados periódicamente por las autoridades competentes.

Las modificaciones de este protocolo son posibles, especialmente en relación con el origen de CD40-estímulo y el tipo de cepa de ratón utilizada, también está dando en la generación eficiente de las células B CD40 murino activado. Ahmadi et al. han mostrado resultados similares para los fibroblastos de ratón transfectadas con CD40 murino-ligando. En este contexto de la presentación de xeno-antígeno puede ser excluido con certeza, pero los estudios intensivos sobre la seguridad y la toxicidad in vivo n...

A. Preparación de los medios de comunicación: <table border="1"><tbody><tr><td> Alimentador de medio celular de tipo salvaje </td><td> Alimentador de selección de células medianas </td><td></td></tr><tr><td> Reactivo </td><td> Concentración </td><td> Reactivo </td><td> Concentración </td></tr><tr><td> RPMI 1640 </td><td></td><td> RPMI 1640 </td><td></td></tr><tr><td> L-Glutamina </td><td> 300 mg / mL </td><td> L-Glutamina </td><td> 300 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> Higromicina B </td><td> 0,2 mg / ml </td></tr></table><table border="1"><tbody><tr><td> mCD40-B medio de lavado </td><td> mCD40 B-medio de cultivo </td></tr><tr><td> Reactivo </td><td> Concentración </td><td> Reactivo </td><td> Concentración </td></tr><tr></tr><tr><td> D-MEM </td><td> </td><td> D-MEM </td><td> </td></tr><tr><td> L-Glutamina </td><td> 580 mg / ml </td><td> L-Glutamina </td><td> 580 mg / ml </td></tr><tr><td> Glucosa </td><td> 4,5 mg / ml </td><td> Glucosa </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> Gentamicina </td><td> 15 mg / ml </td><td> Gentamicina </td><td> 15 mg / ml </td></tr><tr><td> </td><td> </td><td> MEM </td><td> 0,1 mm (1x) </td></tr><tr><td> </td><td> </td><td> FBS </td><td> 10% </td></tr></tbody></table> B. Otros reactivos: <table border="1"><tr><td> Reactivo </td><td> Empresa </td><td> El número de orden </td></tr><tr><td> RPMI 1640 </td><td> Invitrogen Gibco </td><td> REF 21875 </td></tr><tr><td> D-MEM </td><td> Invitrogen Gibco </td><td> REF 41965 </td></tr><tr><td> Dulbeccos PBS (10x) </td><td> PAA </td><td> No gato H15-011 </td></tr><tr><td> Gencin </td><td> Seleccione Delta </td><td> Arte No 7395800 </td></tr><tr><td> FBS </td><td> LONZA </td><td> Gato No DE14-802C </td></tr><tr><td> Tampón HEPES </td><td> PAA </td><td> No gato S11-001 </td></tr><tr><td> MEM AANE </td><td> Invitrogen Gibco </td><td> REF 11140 </td></tr><tr><td> Higromicina B </td><td> PAA </td><td> No gato P02-015 </td></tr><tr><td> Recombinante murino interleucina-4 </td><td> Immunotools </td><td> Gato No 12340045 </td></tr><tr><td> La ciclosporina A </td><td> Novartis </td><td> No NDC 0078-0109-01 gato </td></tr><tr><td> Tripsina / EDTA (10 veces) </td><td> Invitrogen Gibco </td><td> REF 15400-054 </td></tr></table> C. Otros materiales: <table border="1"><tbody><tr><td> Reactivo </td><td> Empresa </td><td> El número de orden </td></tr><tr><td> Puntas de pipeta estériles </td><td> Sarstedt </td><td></td></tr><tr><td> 6 y placa </td><td> NUNC </td><td> Gato No 140675 </td></tr><tr><td> Tubo cónico de 50 ml </td><td> BD Falcon ™ </td><td> Gato No 352070 </td></tr><tr><td> Frasco de cultivo de tejidos </td><td> Sarstedt </td><td> Gato No 83.1813.002 </td></tr></tbody></table>

murine model of cd40-activation of b cells

La investigación sobre células B CD40 ha demostrado que mejora la capacidad de activación de la presentación de antígenos. Cuando se estimula con la interleucina-4 y CD40 ligando (CD40L), las células B se puede ampliar sin problemas a partir de pequeñas cantidades de sangre periférica dentro de 14 días a cantidades muy grandes de muy pura de células B CD40-(&gt; 10<sup&gt; 9</sup&gt; Células por paciente) de donantes sanos, así como los pacientes con cáncer<sup&gt; 4.1</sup&gt;. Las células CD40-B expresan importantes moléculas mensajeras de los ganglios linfáticos y puede atraer a las células T<em&gt; In vitro</em<sup&gt; 5</sup&gt;. Además se ocupan de manera eficiente, procesar y presentar antígenos a los linfocitos T<sup&gt; 6,7</sup&gt;. Las células CD40-B mostraron no sólo ingenuo principal, sino también ampliar las células T de memoria<sup&gt; 8,9</sup&gt;. Por lo tanto-CD40 activan las células B (CD40-células B) se han estudiado como una fuente alternativa de inmuno-estimulantes de células presentadoras de antígeno (APC) para celulares basado en immunotherapy1, 5,10. Con el fin de estudiar más a fondo si las células CD40-B induce respuestas eficaces de las células T in vivo y para estudiar el mecanismo subyacente que estableció un sistema de cultivo celular para la generación de células B CD40 murino activado. Utilizando esplenocitos o purificada células B de ratones C57BL / 6 para la activación de CD40-, las condiciones óptimas fueron identificados de la siguiente manera: A partir de esplenocitos de ratones C57BL / 6 (haplotipo H-2b) los linfocitos se purificaron por centrifugación en gradiente de densidad y co-cultivadas con células HeLa células que expresan el ligando de CD40 murino recombinante (tmuCD40L HeLa)<sup&gt; 11</sup&gt;. Las células son recultured cada 3-4 días y los componentes clave, tales como CD40L, la interleucina-4,-mercaptoetanol y ciclosporina A se repone. En este protocolo se demuestra cómo obtener completamente activado murino CD40-células B (mCD40B) con similares APC-fenotipo en humanos CD40-células B (Fig. 1a, b). CD40-estimulación conduce a un resultado rápido y la expansión de alta pureza (&gt; 90%) las células B CD19 + en los 14 días de cultivo de células (Fig. 1c, d). Para evitar la contaminación con células no transfectadas, la expresión del ligando de CD40 murino en los transfectantes tiene que ser controlado regularmente (Fig. 2). Murino CD40-linfocitos B activados se pueden utilizar para estudiar activación de células B y la diferenciación, así como para investigar su potencial para funcionar como APC in vitro e in vivo. Además, representan una herramienta prometedora para el establecimiento de la vacunación terapéutica o preventiva contra los tumores y le ayudará a responder a las preguntas relativas a la seguridad y la inmunogenicidad de este enfoque<sup&gt; 12</sup&gt;.

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

En este video, que demuestre el procedimiento de activación CD40-y la expansión de las células B murinas a partir de esplenocitos de ratones C57BL / 6, que puede ser utilizado como un modelo de célula presentadora de antígeno (APC) para estudiar la inducción de la inmunidad.

Modelo murino de CD40-activación de las células B

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 vistas.  University Hospital of Cologne. En este video, que demuestre el procedimiento de activación CD40-y la expansión de las células B murinas a partir de esplenocitos de ratones C57BL / 6, que puede ser utilizado como un modelo de célula presentadora de antígeno (APC) para estudiar la inducción de la inmunidad.

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.

Inducción y evaluación de la recombinación de cambio de clase en las células murinas purificada B

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 ...

Investigación

Inmunología e Infección

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.

Recombinante de producción retrovirales y la infección de células B

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.

Modelo murino de asma inducida por alergenos

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, ...

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.

Detección de True IgE que expresan las células del linaje B de ratón

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.

Generación de recombinante humano IgG anticuerpos monoclonales a partir de células inmortalizadas B Ordenada

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.

Un modelo murino de Grupo B<em&gt; Streptococcus</em&gt; Colonización vaginal

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.

Detección y enriquecimiento de Rare células B específicas de antígeno para el análisis del fenotipo y la función

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.

Análisis de cortante inducida por el flujo de migración de las células B de zona Marginal de Murine 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, ...

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.

En Vitro modelo de diferenciación de células de memoria humana Normal B a células plasmáticas duradero

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 ...

Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous antigens and modulate responses to potent microbially derived immune stimuli. For this reason, the intestine is a major target site of immune dysregulation and inflammation in many diseases including but, not limited to inflammatory bowel diseases such as Crohn&#8217;s disease and ulcerative colitis, graft-versus-host disease (GVHD) after bone marrow transplantation (BMT), and many allergic and infectious conditions. Murine models of gastrointestinal inflammation and colitis are heavily used to study GI complications and to pre-clinically optimize strategies for prevention and treatment. Data gleaned from these models via isolation and phenotypic analysis of immune cells from the intestine is critical to further immune understanding that can be applied to ameliorate gastrointestinal and systemic inflammatory disorders. This report describes a highly effective protocol for the isolation of mononuclear cells (MNC) from the colon using a mixed silica-based density gradient interface. This method reproducibly isolates a significant number of viable leukocytes while minimizing contaminating debris, allowing subsequent immune phenotyping by flow cytometry or other methods.

The goal of this protocol is to isolate mononuclear cells that reside in the lamina propria of the colon by enzymatic digestion of the tissue using collagenase. This protocol allows for the efficient isolation of mononuclear cells resulting in a single cell suspension which in turn can be used for robust immunophenotyping.

Aislamiento de células mononucleares lamina Propria de Colon Murino usando colagenasa E

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous ...