Ringraziamo il Dott. Clemens Wendtner per fornire tmuCD40-Ligand-HeLa linea cellulare.

Il protocollo per la generazione di murino CD40 delle cellule B attivate (mCD40B) da splenociti è diviso in due parti: la parte A dimostra la preparazione di murino CD40 ligando (CD40L) che esprime cellule HeLa (tmuCD40L HeLa), che sarà usato come piastra legato cellule nutrici. Parte B descrive l&#39;attuale murino B CD40-cultura. A. Preparazione di cellule di alimentazione (tmuCD40L HeLa) Il HeLa tmuCD40L è un aderente linea di cellule epiteliali umane, che non dovrebbe mai diventare completamente confluenti. Le cellule sono quindi suddivisi due volte alla settimana. Coltura per più di 6 settimane non è raccomandato. <ol><li> Rimuovere medio vecchio dalla coltura primaria con una pipetta sterile e lavare le cellule con 10 ml di PBS 1x. Aspirare il PBS dopo il lavaggio. </li><li> Aggiungere 4 ml di 1x tripsina / EDTA in 75 2 centimetri a riposo per 10-15 minuti a 37 ° C. Usa picchiettando leggermente per staccare le cellule. </li><li> Aggiungere 10 ml di medium di tipo selvatico e girevole delicatamente per fermare la digestione con tripsina. </li><li> Trasferire la sospensione cellulare in un tubo da 50 ml con una pipetta sterile e girare le celle in basso a 265 xg per 7 minuti. </li><li> Rimuovere il supernatante e risospendere il pellet cellulare in 10 ml di medium di tipo selvatico. Contare il numero di cellulare di una aliquota della sospensione cellulare e preparare tre da 50 mL con il numero appropriato di cellule: <ol><li> 2,5 x 10 6 cellule per la subcultura </li><li> 0,4 x 10 6 cellule / pozzetto di irradiazione utilizzate nel murino CD40-B colture cellulari </li><li> resto di congelare (se necessario). </li></ol></li><li> Spin le cellule verso il basso a 265 xg per 7 minuti. </li><li> Rimuovere il surnatante. <ol><li> Per subcolture: risospendere 2,5 x 10 6 cellule in 10 ml di medie selezione in un centimetro 75 2 pallone di coltura cellulare (cellule densità di 2,5 x 10 5 cellule / ml) e incubare le cellule a 37 ° C con 5% di CO 2. Dividere le celle due volte alla settimana. </li><li> Per la murino CD40-B coltura cellulare: Avete bisogno di 2,4 x 10 6 cellule per un 6-pozzetti. Risospendere le cellule tmuCD40L HeLa wild-type in media ad una densità di 0,2 x 10 6 cellule / ml e irradiare li a 78 Gy. Tavola 2 ml di sospensione cellulare in ciascun pozzetto e incubare loro a 37 ° C con 5% di CO 2. Utilizzare questa piastre preparate per la stimolazione delle cellule B quando tmuCD40L cellule HeLa sono aderenti (almeno 4 ore: controllare l&#39;aderenza con il microscopio, non aspettare più di 24 ore per iniziare la stimolazione delle cellule B). (Continua con B.) </li></ol></li></ol> B. murini CD40-B colture cellulari I. Preparazione di splenociti per CD40-stimolazione (giorno 0): Attenzione: prima di continuare a constatare che le cellule feeder siano aderenti. Aggiungere sempre soluzioni fresche di murini immediatamente interleuchina-4, β-mercaptoetanolo e ciclosporina A per il mezzo di crescita prima dell&#39;uso. <ol><li> Sezionare milza di C57BL / 6 topi (aplotipo H-2b) e lavarlo due volte in 10-20 ml DMEM integrato con Gentamicina [15μg/mL] trasferendo con una punta da 10 ml pipetta da un tubo ad un altro. Evitare il trasferimento di contaminanti (ad esempio capelli) del mouse. Tritare milza passando attraverso un colino cella (100μm) e risciacquare con un altro filtro ml 10 di media. Spin down splenociti a 265 xg per 7 minuti a temperatura ambiente, li risospendere in 7 ml muCD40 media delle cellule-B e linfociti separati per centrifugazione densità Ficoll. Per fare questo strato di cellule, il 5 ml Mouse-Pancoll (preriscaldato a temperatura ambiente) in una provetta da 15 ml e centrifugare a 450 xg per 30 min, senza freno a temperatura ambiente. </li><li> Erogare la maggior parte dello strato superiore, lasciando il livello dei linfociti indisturbato a livello di interfaccia. Trasferimento strato linfociti ad un nuovo tubo da 50 ml, lavare una volta con DMEM 30mL con Gentamicina [15μg/mL] riducendo la velocità a 265 xg per 7 minuti per eliminare altre cellule. Eliminare il supernatante e risospendere le cellule in 10 ml di mCD40-B medio di lavaggio. Determinare il numero di cellule in una parte aliquota della sospensione cellulare. Determinare il numero delle cellule contando una aliquota della sospensione cellulare. </li><li> Spin down la quantità necessaria di cellule a 265 xg per 7 minuti. Per un 6-pozzetti 5 x 10 6 cellule / pozzetto sono necessari, quindi 30 x 10 6 cellule per piastra. </li><li> Rimuovere il surnatante e risospendere i linfociti a 1,25 x 10 6 cellule / ml in mCD40-B terreno di coltura fresco integrato con 1 U / mL di interleuchina-4 come fattore di crescita, 100 mM β-mercaptoetanolo e 0,63 mg / ml ciclosporina A per prevenire conseguenza di cellule T (concentrazioni indicati si riferiscono a un mezzo ml cultura!). </li><li> Eliminare il surnatante dal 6 pozzetti pre-incubate con tmuCD40L cellule HeLa. </li><li> Delicatamente aggiungere 4 ml di sospensione linfocitaria (1,25 x 10 6 cellule / ml) in ciascun pozzetto della piastra ben 6. </li><li> Incubare la piastra a 37 ° C con 5% di CO 2. </li&gt; <li> Il giorno 3 reculture le cellule (Continua con II.1.). </li></ol> II. Sottocultura di cellule mCD40B (giorno 3 e poi due volte a settimana): <ol><li> Grappolo di cellule raccolta mCD40B da vigorosamente pipettando su e giù con una pipetta da 10 mL e la piscina in un tubo da 50 ml. </li><li> Spin down a 265 xg per 7 minuti e sostituire completamente il sopranatante con mCD40-B medio di lavaggio. Mentre conta la quantità delle cellule di una aliquota, girare la celle in basso a 265 xg per 7 minuti. Risospendere le cellule in mCD40B mCD40-B terreno di coltura ad una concentrazione di 0,75 x 10 6 cellule / ml. </li><li> Aggiungi soluzioni fresche di murini interleuchina-4 in una concentrazione di 1 U / mL, 100 mM β-mercaptoetanolo e 0,63 mg / ml ciclosporina A per la media. </li><li> Rimuovere il surnatante da un 6-pozzetti pre-incubate con irradiati tmuCD40L cellule HeLa. </li><li> Delicatamente aggiungere 4 ml di sospensione cellulare mCD40B (0,75 x 10 6 cellule / ml) in ciascun pozzetto della piastra ben 6. </li><li> Incubare le piastre a 37 ° C con 5% di CO 2. </li><li> Anche in questo caso le cellule sottocultura ogni 3-4 giorni per finire con elevata purezza murino CD40 delle cellule B attivate dopo un totale di 14 giorni. </li></ol> C. Soluzione dei problemi: cosa succede se le cellule B CD40-non crescono? <ol><li> Hai controllato di espressione ligando CD40 delle cellule alimentatore? </li><li> Sono legato piastra cellule alimentatore utilizzato per la stimolazione più di 24 ore? </li><li> C&#39;è una contaminazione con micoplasma? </li><li> È stata la interleuchina-4 soluzione utilizzata per la supplementazione appena scongelati e ce l&#39;ha l&#39;attività biologica appropriata? </li><li> Era β-mercaptoetanolo e ciclosporina A aggiunti nella concentrazione giusta? </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>

Gli esperimenti sono stati eseguiti in conformità con le linee guida nazionali ed europee per il mantenimento degli animali da laboratorio. Riferendosi alla legge tedesca sul benessere degli animali gli animali erano controllati regolarmente dalle autorità competenti.

Le modifiche di questo protocollo sono possibili, soprattutto per quanto riguarda l&#39;origine di CD40-stimolo e il tipo di ceppo di topi utilizzati, anche cedendo nella generazione efficiente di murini CD40 delle cellule B attivate. Ahmadi et al. hanno mostrato risultati simili per i fibroblasti di topo murine transfettate con CD40-ligando. In questa presentazione contesto di xeno-antigene può essere esclusa con certezza, ma studi approfonditi in materia di sicurezza e tossicità in vivo non ha dato segni di...

A. Preparazione dei supporti: <table border="1"><tbody><tr><td> Alimentatore cellule wild type media </td><td> Alimentatore cellulare medio selezione </td><td></td></tr><tr><td> Reagente </td><td> Concentrazione </td><td> Reagente </td><td> Concentrazione </td></tr><tr><td> RPMI 1640 </td><td></td><td> RPMI 1640 </td><td></td></tr><tr><td> L-Glutammina </td><td> 300 mg / ml </td><td> L-Glutammina </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> Hygromycin B </td><td> 0,2 mg / ml </td></tr></table><table border="1"><tbody><tr><td> mCD40-B lavaggio medio </td><td> mCD40-B terreno di coltura </td></tr><tr><td> Reagente </td><td> Concentrazione </td><td> Reagente </td><td> Concentrazione </td></tr><tr></tr><tr><td> D-MEM </td><td> </td><td> D-MEM </td><td> </td></tr><tr><td> L-Glutammina </td><td> 580 mg / ml </td><td> L-Glutammina </td><td> 580 mg / ml </td></tr><tr><td> Glucosio </td><td> 4,5 mg / ml </td><td> Glucosio </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> 0.1mm (1x) </td></tr><tr><td> </td><td> </td><td> FBS </td><td> 10% </td></tr></tbody></table> B. Varie Reagenti: <table border="1"><tr><td> Reagente </td><td> Azienda </td><td> Il decreto n. </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> Nessun gatto H15-011 </td></tr><tr><td> Gencin </td><td> Delta Select </td><td> Art. n. 7395800 </td></tr><tr><td> FBS </td><td> LONZA </td><td> Nessun gatto DE14-802C </td></tr><tr><td> HEPES Buffer </td><td> PAA </td><td> Nessun gatto S11-001 </td></tr><tr><td> MEM NEAA </td><td> Invitrogen Gibco </td><td> REF 11140 </td></tr><tr><td> Hygromycin B </td><td> PAA </td><td> Nessun gatto P02-015 </td></tr><tr><td> Ricombinante murino interleuchina-4 </td><td> Immunotools </td><td> Nessun gatto 12340045 </td></tr><tr><td> Ciclosporina A </td><td> Novartis </td><td> Nessun gatto NDC 0078-0109-01 </td></tr><tr><td> Tripsina / EDTA (10x) </td><td> Invitrogen Gibco </td><td> REF 15400-054 </td></tr></table> C. Varie Forniture: <table border="1"><tbody><tr><td> Reagente </td><td> Azienda </td><td> Il decreto n. </td></tr><tr><td> Puntali sterili </td><td> Sarstedt </td><td></td></tr><tr><td> 6 pozzetti </td><td> NUNC </td><td> Nessun gatto 140675 </td></tr><tr><td> 50mL tubo conico </td><td> BD Falcon ™ </td><td> Nessun gatto 352070 </td></tr><tr><td> Tessuto cultura fiasco </td><td> Sarstedt </td><td> Nessun gatto 83.1813.002 </td></tr></tbody></table>

murine model of cd40-activation of b cells

La ricerca sulle cellule B CD40 ha dimostrato che l&#39;attivazione di migliorare la loro capacità di presentazione dell&#39;antigene. Se stimolati con interleuchina-4 e CD40 ligando (CD40L), le cellule B umane può essere ampliato senza difficoltà da piccole quantità di sangue periferico entro 14 giorni di tempo per grandi quantità di altamente puri-CD40 delle cellule B (&gt; 10<sup&gt; 9</sup&gt; Cellule per paziente) da donatori sani e pazienti affetti da cancro<sup&gt; 1-4</sup&gt;. Cellule CD40-B esprimere importanti molecole nodo homing linfa e in grado di attrarre le cellule T<em&gt; In vitro</em<sup&gt; 5</sup&gt;. Inoltre, essi occupano in modo efficiente, elaborare e presentare antigeni alle cellule T<sup&gt; 6,7</sup&gt;. Cellule CD40-B hanno dimostrato di non solo ingenuo primo, ma anche di espandere le cellule T di memoria<sup&gt; 8,9</sup&gt;. Pertanto CD40-attivate le cellule B (linfociti B CD40-) sono stati studiati come fonte alternativa di immuno-stimolatorie cellule presentanti l&#39;antigene (APC) per i cellulari basati su immunotherapy1, 5,10. Per studiare ulteriormente se le cellule CD40-B inducono efficace le risposte delle cellule T in vivo e studiare il meccanismo di base abbiamo stabilito un sistema di coltura cellulare per la generazione di murino CD40 delle cellule B attivate. Utilizzando splenociti depurati o linfociti B di topi C57BL / 6 per CD40-attivazione, le condizioni ottimali sono stati identificati come segue: A partire da splenociti di C57BL / 6 topi (aplotipo H-2b) linfociti sono purificati mediante centrifugazione a gradiente di densità e di co-coltura con HeLa cellule che esprimono ricombinante murino ligando CD40 (tmuCD40L HeLa)<sup&gt; 11</sup&gt;. Le cellule sono recultured ogni 3-4 giorni e componenti chiave, quali CD40L, interleuchina-4,-mercaptoetanolo e ciclosporina A sono reintegrati. In questo protocollo dimostriamo come ottenere pienamente attivato murino CD40 delle cellule B (mCD40B) con simili APC-fenotipo di umani CD40 delle cellule B (Fig. 1a, b). CD40-stimolazione porta ad una conseguenza rapida espansione e di elevata purezza (&gt; 90%) delle cellule CD19 + B entro 14 giorni di coltura cellulare (Fig. 1c, d). Per evitare la contaminazione con le cellule non transfettate, espressione del ligando CD40 murino sulla trasfettate deve essere controllata regolarmente (Fig. 2). Murino CD40-attivate le cellule B possono essere utilizzati per studiare l&#39;attivazione delle cellule B e la differenziazione così come per indagare il loro potenziale di funzionare come APC in vitro e in vivo. Inoltre, essi rappresentano uno strumento promettente per stabilire la vaccinazione terapeutica o preventiva contro i tumori e contribuirà a rispondere a domande riguardanti la sicurezza e l&#39;immunogenicità di questo approccio<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

In questo video, dimostriamo la procedura di CD40-attivazione e l&#39;espansione delle cellule B da splenociti murini di C57BL / 6 topi, che può essere utilizzato come modello di una cellula presentanti l&#39;antigene (APC) per studiare l&#39;induzione di immunità.

Modello murino di CD40-attivazione delle cellule 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

12.6K 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.

Induzione e Valutazione della ricombinazione Classe Switch in depurata cellule B murini

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 ...

Ricerca

Immunologia e infezioni

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.

Produzione ricombinante retrovirali e infezione delle cellule 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.

Modello murino di asma indotta da allergene

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.

Saggio di placca per Norovirus Murine

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.

Rilevazione di True IgE-esprimono mouse B Cells Lineage

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.

Generazione di ricombinante umano IgG anticorpi monoclonali da immortalizzate cellule ordinati B

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 modello murino di Gruppo B<em&gt; Streptococcus</em&gt; Colonizzazione vaginale

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.

Rilevamento e l&#39;arricchimento di Rare antigene-specifiche cellule B per l&#39;analisi del fenotipo e funzione

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.

Analisi di taglio indotta da flusso di migrazione delle cellule di B di zona marginale murino 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.

In Vitro modello di differenziazione delle cellule di memoria umana normale B a plasmacellule longevo

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 ...