Nous remercions le Dr Clemens gentiment Wendtner pour fournir lignée cellulaire tmuCD40-Ligand-HeLa.

Le protocole pour la génération de cellules murines B CD40-activé (mCD40B) à partir de splénocytes est divisé en deux parties: la partie A montre la préparation d&#39;murin CD40 ligand (CD40L) exprimant des cellules HeLa (HeLa tmuCD40L), qui sera utilisé comme plate- liés cellules nourricières. Partie B décrit la réelle murin CD40-B de la culture. A. Préparation de cellules nourricières (tmuCD40L HeLa) Les cellules HeLa tmuCD40L est une adhérente lignée humaine de cellules épithéliales, qui ne devrait jamais devenir complètement confluentes. Les cellules sont donc découpé deux fois par semaine. La culture sur plus de six semaines n&#39;est pas recommandée. <ol><li> Retirer ancien milieu de la culture primaire avec une pipette stérile et laver les cellules avec 10 ml de PBS 1x. Aspirer le PBS après le lavage. </li><li> Ajouter 4 mL de 1x trypsine / EDTA dans un flacon de 75 cm2 pendant 10-15 minutes à 37 ° C. Utilisez tapotant doucement pour détacher les cellules. </li><li> Ajouter 10 ml de milieu de type sauvage et pivotant doucement pour arrêter la digestion à la trypsine. </li><li> Transférer la suspension cellulaire dans un tube de 50 ml avec une pipette stérile et le spin des cellules vers le bas à 265 xg pendant 7 minutes. </li><li> Retirer le surnageant et remettre le culot cellulaire dans 10 mL de milieu de type sauvage. Comptez le nombre de cellules d&#39;une aliquote de la suspension cellulaire et préparer trois tubes de 50 ml avec le nombre approprié de cellules: <ol><li> 2,5 x 10 6 cellules pour une sous-culture </li><li> 0,4 x 10 6 cellules / puits pour l&#39;irradiation utilisée pour la culture de cellules CD40-B murines </li><li> restant à geler (si nécessaire). </li></ol></li><li> Faites tourner les cellules vers le bas à 265 xg pendant 7 minutes. </li><li> Enlever le surnageant. <ol><li> Pour repiquage: resuspendre 2,5 x 10 6 cellules dans 10 ml milieu de sélection dans un flacon de 75 cm2 de culture de cellules (2,5 x densité cellulaire 10 5 cellules / ml) et incuber les cellules à 37 ° C avec 5% de CO 2. Fractionner les cellules deux fois par semaine. </li><li> Pour la culture de cellules CD40-B murines: Vous avez besoin de 2,4 x 10 6 cellules pour une plaque de 6 puits. Resuspendre les cellules HeLa tmuCD40L en milieu sauvage à une densité de 0,2 x 10 6 cellules / ml et les irradier à 78 Gy. Planche 2 mL de la suspension cellulaire dans chaque puits et les incuber à 37 ° C avec 5% de CO 2. Utilisez cette assiettes préparées pour la stimulation des lymphocytes B lors tmuCD40L cellules HeLa sont adhérentes (au moins 4 heures: vérifier l&#39;adhérence à l&#39;aide du microscope, ne pas attendre plus de 24 h pour commencer la stimulation des lymphocytes B). (Continuer avec B.) </li></ol></li></ol> B. murin CD40-culture de cellules B I. Préparation des splénocytes pour CD40-stimulation (jour 0): S&#39;il vous plaît noter: avant de continuer de s&#39;assurer que les cellules nourricières sont adhérentes. Toujours ajouter de nouvelles solutions d&#39;murin immédiatement interleukine-4, β-mercaptoéthanol et cyclosporine A au milieu de croissance avant l&#39;utilisation. <ol><li> Disséquer la rate de souris C57BL / 6 (haplotype H-2b) et il laver deux fois dans 10-20 ml de DMEM complété avec Gentamycine [15μg/mL] en le transférant avec une pointe de pipette 10 ml d&#39;un tube à l&#39;autre. Évitez les contaminants transfert (par exemple les cheveux) de la souris. Hacher la rate en passant à travers une passoire cellulaire (100 microns) et rincez avec un autre filtre de 10 mL de milieu. Isoler splénocytes à 265 xg pendant 7 min à température ambiante, les remettre en suspension dans 7mL muCD40-B milieu cellulaire, et les lymphocytes séparés par centrifugation de densité de Ficoll. Pour ce faire des cellules couche sur 5 ml souris Pancoll (préchauffé à la température ambiante) dans un tube de 15 ml et centrifuger à 450 xg pendant 30 min sans frein à la température ambiante. </li><li> Prélever la plupart de la couche supérieure, laissant intacte la couche de lymphocytes à l&#39;interface. Transfert couche de lymphocytes dans un nouveau tube de 50 ml, laver une fois avec 30 ml de DMEM avec Gentamycine [15μg/mL] en faisant tourner le bas à 265 xg pendant 7 min pour éliminer d&#39;autres cellules. Jeter le surnageant et remettre en suspension les cellules dans 10 ml de milieu B mCD40-linge. Déterminer le nombre de cellules dans une aliquote de la suspension cellulaire. Déterminer le nombre de cellules en comptant une aliquote de la suspension cellulaire. </li><li> Isoler le montant requis de cellules à 265 xg pendant 7 minutes. Pour une plaque à 6 puits 5 x 10 6 cellules / puits sont nécessaires, donc 30 x 10 6 cellules par plaque. </li><li> Retirer le surnageant et remettre les lymphocytes à 1,25 x 10 6 cellules / ml dans mCD40-B milieu de culture fraîchement complété avec 1 U / ml d&#39;interleukine-4 comme facteur de croissance, 100 M β-mercaptoéthanol et 0,63 g / mL cyclosporine A pour prévenir la excroissance de cellules T (concentrations données se référer à un milieu de culture ml!). </li><li> Retirer le surnageant de la plaque de 6 puits de pré-incubées avec des cellules HeLa tmuCD40L. </li><li> Ajouter délicatement 4 mL de la suspension lymphocytaire (1,25 x 10 6 cellules / ml) dans chaque puits de la plaque de 6 puits. </li><li> Incuber la plaque à 37 ° C avec 5% de CO 2. </li&gt; <li> Au jour 3 reculture les cellules (Continuer avec II.1.). </li></ol> II. Repiquage de cellules mCD40B (3 jours et ensuite deux fois par semaine): <ol><li> Grappe des piles à Moisson mCD40B par vigoureusement aspiration et refoulement avec une pipette de 10 ml et de les mettre dans un tube de 50 ml. </li><li> Isoler à 265 xg pendant 7 minutes et remplacer complètement le surnageant avec mCD40-B milieu de lavage. Alors que le comptage des cellules du montant d&#39;une aliquote, tourner les cellules vers le bas à 265 xg pendant 7 minutes. Resuspendre les cellules dans mCD40B mCD40-B milieu de culture à une concentration de 0,75 x 10 6 cellules / ml. </li><li> Ajouter de nouvelles solutions d&#39;murin de l&#39;interleukine-4 à une concentration de 1 U / ml, 100 M β-mercaptoéthanol et 0,63 g / mL cyclosporine A dans le milieu. </li><li> Retirer le surnageant dans une plaque de 6 puits de pré-incubées avec des cellules HeLa irradiées tmuCD40L. </li><li> Ajouter délicatement 4 mL de la suspension cellulaire mCD40B (0,75 x 10 6 cellules / ml) dans chaque puits de la plaque de 6 puits. </li><li> Incuber les plaques à 37 ° C avec 5% de CO 2. </li><li> Encore une fois les cellules sous-culture tous les 3-4 jours pour finir avec très pure murin cellules B CD40-activé après un total de 14 jours. </li></ol> C. Dépannage: Que faire si le CD40-B cellules ne poussent pas? <ol><li> Avez-vous vérifié l&#39;expression du ligand CD40 des cellules nourricières? </li><li> Les cellules de la plaque liée alimentation utilisée pour la stimulation de plus de 24h? </li><li> Yat-il une contamination par des mycoplasmes? </li><li> Était la solution interleukine-4 utilisé pour la supplémentation fraîchement dégelé et at-elle avoir l&#39;activité biologique approprié? </li><li> Etait-β-mercaptoéthanol et la cyclosporine A a ajouté de la concentration correcte? </li></ol><img src="/files/ftp_upload/1734/1734fig1a.jpg" alt="Figure 1 une" /> 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>

Les expériences ont été réalisées en conformité avec les directives nationales et européennes pour garder des animaux de laboratoire. Se référant à la loi allemande du bien-être des animaux les animaux ont été contrôlés régulièrement par les autorités compétentes.

Modifications de ce protocole sont possibles, notamment sur l&#39;origine du CD40-stimulus et le type de souche de souris utilisée, également dans la production à rendement efficace de cellules murines B CD40-activé. Ahmadi et al. ont montré des résultats similaires pour les fibroblastes de souris transfectées avec CD40-ligand murin. Dans ce contexte de présentation xéno-antigènes peut être exclu avec certitude, mais des études intensives concernant la sécurité et la toxicité in vivo a donné auc...

A. Préparation des médias: <table border="1"><tbody><tr><td> Cellules nourricières type de support sauvages </td><td> Milieu de sélection de cellules Feeder </td><td></td></tr><tr><td> Réactifs </td><td> Concentration </td><td> Réactifs </td><td> Concentration </td></tr><tr><td> RPMI 1640 </td><td></td><td> RPMI 1640 </td><td></td></tr><tr><td> L-Glutamine </td><td> 300 pg / mL </td><td> L-Glutamine </td><td> 300 pg / 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> Gentamycine </td><td> 15 ug / ml </td><td> Gentamycine </td><td> 15 ug / ml </td></tr><tr><td></td><td></td><td> Hygromycine B </td><td> 0,2 mg / ml </td></tr></table><table border="1"><tbody><tr><td> mCD40-B milieu de lavage </td><td> mCD40-B milieu de culture </td></tr><tr><td> Réactifs </td><td> Concentration </td><td> Réactifs </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 pg / ml </td><td> L-Glutamine </td><td> 580 pg / ml </td></tr><tr><td> Le glucose </td><td> 4,5 mg / ml </td><td> Le 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> Gentamycine </td><td> 15 ug / ml </td><td> Gentamycine </td><td> 15 ug / 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. Réactifs Divers: <table border="1"><tr><td> Réactifs </td><td> Société </td><td> Ordonnance 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> AAP </td><td> Cat No H15-011 </td></tr><tr><td> Gencin </td><td> Sélectionnez Delta </td><td> Art Pas 7395800 </td></tr><tr><td> FBS </td><td> LONZA </td><td> Aucun chat DE14-802C </td></tr><tr><td> Tampon HEPES </td><td> AAP </td><td> Cat No S11-001 </td></tr><tr><td> MEM NEAA </td><td> Invitrogen Gibco </td><td> REF 11140 </td></tr><tr><td> Hygromycine B </td><td> AAP </td><td> Aucun chat P02-015 </td></tr><tr><td> Recombinant murin interleukine-4 </td><td> Immunotools </td><td> Cat No 12340045 </td></tr><tr><td> Cyclosporine A </td><td> Novartis </td><td> Cat No 0078-0109-01 NDC </td></tr><tr><td> Trypsine / EDTA (10x) </td><td> Invitrogen Gibco </td><td> REF 15400-054 </td></tr></table> C. Fournitures diverses: <table border="1"><tbody><tr><td> Réactifs </td><td> Société </td><td> Ordonnance n </td></tr><tr><td> Stérile pipette </td><td> Sarstedt </td><td></td></tr><tr><td> 6-même la plaque </td><td> NUNC </td><td> Cat No 140675 </td></tr><tr><td> 50mL tube conique </td><td> BD Falcon ™ </td><td> Cat No 352070 </td></tr><tr><td> Flacon de culture tissulaire </td><td> Sarstedt </td><td> Aucun chat 83.1813.002 </td></tr></tbody></table>

murine model of cd40-activation of b cells

La recherche sur les cellules B CD40 a montré que l&#39;activation améliore leur capacité de présentation antigénique. Lorsqu&#39;ils sont stimulés par l&#39;interleukine-4 et CD40 ligand (CD40L), les lymphocytes B humains peut être étendu sans difficulté à partir de petites quantités de sang périphérique dans les 14 jours pour très grandes quantités de très-pur-CD40 des cellules B (&gt; 10<sup&gt; 9</sup&gt; Cellules par patient) provenant de donneurs sains ainsi que des patients atteints de cancer<sup&gt; 1-4</sup&gt;. Cellules CD40-B expriment des molécules importantes ganglion homing et peuvent attirer les cellules T<em&gt; In vitro</em<sup&gt; 5</sup&gt;. En outre, ils prennent efficacement, le processus et présentent les antigènes aux cellules T<sup&gt; 6,7</sup&gt;. Cellules CD40-B ont été montrés pour non seulement naïve écoute, mais également d&#39;élargir la mémoire des lymphocytes T<sup&gt; 8,9</sup&gt;. Par conséquent CD40-activé des cellules B (CD40-cellules B) ont été étudiés comme une source alternative de l&#39;immuno-stimulation des cellules présentatrices d&#39;antigènes (APC) à base de cellules immunotherapy1, 5,10. Afin d&#39;étudier plus avant si les cellules CD40-B induisent des réponses efficaces cellules T in vivo et d&#39;étudier le mécanisme sous-jacent, nous avons établi un système de culture cellulaire pour la génération de cellules murines B CD40-activé. Utiliser splénocytes ou purifiée cellules B de souris C57BL / 6 pour les CD40-activation, les conditions optimales ont été identifiées comme suit: A partir de splénocytes de souris C57BL / 6 (haplotype H-2b) de lymphocytes sont purifiées par centrifugation sur gradient de densité et de co-cultivées avec des cellules HeLa cellules exprimant recombinant murin CD40-ligand (tmuCD40L HeLa)<sup&gt; 11</sup&gt;. Les cellules sont repiquées tous les 3-4 jours et les composants clés tels que CD40L, l&#39;interleukine-4,-mercaptoéthanol et la cyclosporine A sont reconstituées. Dans ce protocole, nous montrons comment obtenir pleinement activé murin CD40-cellules B (mCD40B) avec APC-phénotype similaire aux humains CD40 des cellules B (figure 1a, b). CD40-stimulation conduit à une excroissance rapide et l&#39;expansion de la très pure (&gt; 90%) CD19 + cellules B dans les 14 jours de culture cellulaire (figure 1c, d). Pour éviter la contamination par des cellules non transfectées, l&#39;expression du ligand CD40 murin sur les transfectants doit être contrôlée régulièrement (figure 2). Murin CD40-activé des cellules B peut être utilisé pour étudier activation des cellules B et la différenciation ainsi que d&#39;étudier leur potentiel de fonctionner comme APC in vitro et in vivo. Par ailleurs, ils représentent un outil prometteur pour l&#39;établissement de la vaccination thérapeutique ou préventive contre les tumeurs et aideront à répondre aux questions concernant la sécurité et l&#39;immunogénicité de cette approche<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

Dans cette vidéo, nous démontrons la procédure d&#39;activation de CD40-et à l&#39;expansion des cellules B murines à partir de splénocytes de souris C57BL / 6, qui peut être utilisé comme un modèle de cellule présentatrice d&#39;antigène (APC) pour étudier l&#39;induction de l&#39;immunité.

Modèle murin de CD40-activation des cellules 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 vues.  University Hospital of Cologne. Dans cette vidéo, nous démontrons la procédure d'activation de CD40-et à l'expansion des cellules B murines à partir de splénocytes de souris C57BL / 6, qui peut être utilisé comme un modèle de cellule présentatrice d'antigène (APC) pour étudier l'induction de l'immunité.

Vidéo: 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.

Induction et d&#39;évaluation de la recombinaison commutateur de classe dans purifiée cellules B murines

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 ...

Recherche

Immunologie et infection

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 production et infection rétrovirale des cellules 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.

Modèle murin de l&#39;asthme induit par les allergènes

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.

Détection de True IgE exprimant Souris B cellules de la lignée

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.

Génération de Recombinant Human IgG anticorps monoclonaux à partir de cellules immortalisées Classé 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 modèle murin du Groupe B<em&gt; Streptococcus</em&gt; Vaginal Colonisation

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.

Détection et enrichissement des rares cellules B spécifiques de l&#39;antigène pour l&#39;analyse des Phénotype et fonction

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.

Analyse de cisaillement induite par le flux de Migration des cellules B Zone marginale murin 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.

Dans modèle de différenciation in Vitro des cellules de la mémoire humaine normale B à longue durée de vie des cellules de Plasma

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.

Isolement des cellules mononucléaires Lamina Propria du colon murine à l'aide de la collagène 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 ...