CK est soutenu par Columbia University Graduate programme. UB est Fellow de la Société de leucémie et lymphome d&#39;Amérique, le destinataire de bourse de nouveau chercheur de la Fondation recherche sur la leucémie et est soutenu par l&#39;Université de Columbia New Faculté des fonds de démarrage.

1. Splénique B-Lymphocyte isolement et de stimulation <ol><li> Récolter les rates de souris déficientes AID-12 entre 2-3 mois d&#39;âge. L&#39;isolement de la rate est effectué dans des conditions stériles et les organes sont temporairement conservés dans une solution saline de tampon phosphate froid (PBS) contenant 15% de sérum fœtal bovin (FBS). </li><li> La rate est ensuite transférée à une hotte stérile et la culture de tissus homogénéisés dans 5 ml de PBS additionné de 2,5% de FBS. Transférer le broyat dans un tube de 15 ml faucon. </li><li> Centrifuger l&#39;échantillon de tissu homogénéisé la rate à 1000rpm pendant 10 minutes à 4 ° C pour culotter les cellules. </li><li> Décanter le surnageant après centrifugation. Resuspendre la cellule dans 10 ml de cellules rouges du sang de pellets (RBC) du tampon de lyse à température ambiante pendant 7 minutes pour éviter toute contamination par les globules rouges. </li><li> Centrifuger l&#39;échantillon à 1000 rpm pendant 10 minutes et décanter le surnageant et remettre en suspension les cellules dans 10 ml de PBS contenant 2,5% de FBS. </li><li> Retirer 100 aliquot uL et compter les cellules à une dilution de 1:1000 en utilisant un hématimètre standard en utilisant le bleu trypan pour exclure toutes les cellules mortes. Environ 20-30 millions de cellules peut être obtenue par rate. </li><li> Centrifuger l&#39;échantillon à 1000rpm pendant 10 minutes et décanter le surnageant. </li><li> Reprendre le culot dans 0,5% de BSA / PBS contenant 2 mM d&#39;EDTA à une concentration de 10 7 cellules par 90 pi. </li><li> Ajouter anti-CD43 billes magnétiques recouvertes d&#39;anticorps aux cellules de se lier spécifiquement les cellules non-B. Utiliser 10 perles ul par 90 pi (10 7 cellules). Bien mélanger et incuber à 4 ° C pendant 20 minutes. </li><li> Une fois les cellules ont été étiquetés, lavez-les en ajoutant 2,5% de FBS / PBS en haut du tube. Centrifuger l&#39;échantillon à 1000 rpm pendant 10 minutes. </li><li> Décanter le surnageant. Resuspendre les cellules dans 0,1% de BSA / PBS à une concentration de 10x10 7 cellules par ml. </li><li> Monter une colonne de tri magnétique sur un séparateur magnétique. Placer un tube conique directement sous la colonne pour recueillir les accréditives et charger la colonne avec 3 mL de 0,5% BSA / PBS au premier et au lavage. </li><li> Une fois le liquide de lavage a traversé, le remplacer par un tube de collecte de nouvelles. Si l&#39;échantillon est inférieur à 1 ml, ajuster le volume d&#39;échantillon à 1 ml avec 0,5% BSA / PBS / EDTA et de charger l&#39;échantillon dans la colonne. Chargez seulement 1 ml maximum de cellules par colonne et utiliser plusieurs colonnes si nécessaire. </li><li> Laisser les cellules passivement circulation dans la colonne et de recueillir les cellules non liée dans le tube conique sous la colonne. Laver la colonne 4 fois avec 3 ml de 0,5% BSA / PBS tout en continuant à collecter les actions accréditives. </li><li> Collecter toutes accréditives dans lequel CD43 négative des cellules B au repos sera enrichie. Jeter la colonne. Centrifugeuse accréditives à 1000 rpm pendant 10 minutes. </li><li> Égoutter le surnageant et ajouter 10 ml de milieu (15% FCS / + glutamine, Pen / Strep, non-acides aminés essentiels, 50 uM Β-ME) </li><li> Compter les cellules et la culture à 10 6 cellules par ml. </li><li> Afin de stimuler la croissance des cellules B et la prolifération, de compléter le milieu avec des anticorps recombinants IL-4 et anti-CD40 de telle sorte que les concentrations finales sont de 20 pg / ml et 1 pg / mL, respectivement, et le transfert des cellules d&#39;un flacon de culture. </li><li> Culture des cellules pendant la nuit. Compter les cellules et diluer à 10 6 cellules par ml, ajouter l&#39;IL-4 et CD40, le cas échéant. </li><li> Cellules en culture pendant 72 heures avant l&#39;infection virale. </li></ol> 2. La transfection des cellules productrices Utiliser des protocoles établis pour la transfection en utilisant soit des lipides cationiques ou de phosphate de calcium. 10cm Transfecter 3 plaque par construction d&#39;ADN, l&#39;ADN en utilisant 100 ug de plasmide par plaque. Nous utilisons régulièrement la méthode de transfection de phosphate de calcium 13, 14 et note élevée titre viral lorsqu&#39;ils sont complétés par la chloroquine. <ol><li> Utiliser 60% -70% de confluence des cellules Phoenix comme les cellules productrices d&#39;être transfectées. A ce nombre de cellules confluent devrait être de 5 millions de cellules par plaque de 10 cm. </li><li> Une heure avant la transfection, remplacez le support avec des produits frais média complète pour la croissance cellulaire Phoenix. </li><li> Dans un tube stérile eppendorpf, mélanger 100 ug d&#39;emballage construction d&#39;ADN, 250 ul de 0,5 M CaCl 2, et ajuster le volume final à 500 ul d&#39;eau stérile </li><li> En utilisant une pipette de 1 mL, mélanger énergiquement cette solution avec 500 PNH 2X uL dans un tube en polypropylène de 15 ml </li><li> Laisser ce mélange reposer à température ambiante pendant 8-10 minutes. Pendant ce temps, un précipité se forme. </li><li> Ajouter le mélange de transfection goutte à goutte pour les cellules tout en remuant le moyen de répartir uniformément le mélange à travers. </li><li> Incuber les cellules à 37 ° C pendant 12 heures. </li><li> 12 heures post-transfection, de remplacer le milieu par le milieu B 8ml la croissance cellulaire et le retour complet aux cellules de l&#39;incubateur à 37 ° C. </li></ol> 3. L&#39;infection de cellules stimulées B spléniques <ol><li> Retirer le surnageant des cellules Phoenix 48h l&#39;après-transfection et passer le surnageant à travers un filtre de 0,2 micron pour éliminer les débris cellulaires. </li><li> Mélangez 3 mL de surnageant contenant le virus avec 3 mL de cellules B pré-activé (comme dans l&#39;étape 1). Afin d&#39;améliorer adsorption virale dans les cellules, ajouter polybrène à une concentration finale de 16 pg par ml. </li><li> Infectent les cellules par centrifugation à 2500 rpm pendant 90 minutes à 30 ° C. </li><li> Immédiatement après le spin, incuber pendant une autre de 48 à 72 heures à 37 ° C pour permettre la croissance et la prolifération cellulaire. </li></ol> 4. Cytométrie en flux <ol><li> L&#39;utilisation d&#39;un cytomètre en flux, analyser les cellules pour l&#39;expression en surface d&#39;IgG1 B220 et de surface. La présence d&#39;IgG1 à la surface des cellules indique que la commutation isotypique est survenue à la suite de sauvetage réussi par la complémentation rétrovirale du gène de l&#39;AID. </li></ol> 5. Les résultats représentatifs Nous avons réussi à sauver la déficience de la déaminase protéine de facteur d&#39;activation induite cytidine (AID) de l&#39;AID-déficiente (AID-/ -) des lymphocytes B en utilisant transduction rétrovirale. En bref, nous avons généré des rétrovirus recombinants exprimant SECOURS souris (MAID) de protéines par transfection de cellules Phoenix avec PMX-MAID-GFP 15-18. Les virus récoltés ont été infectées en cellules B AID-déficient obtenu de l&#39;AID des souris déficientes en ex vivo la commutation isotypique (CSR) des conditions de stimulation 19. Les cellules B infectées ont ensuite été analysés pour la RSE au IgG1 après 72hrs en culture en utilisant une analyse par cytométrie de flux. La figure 2 montre la détection d&#39;IgG1 à la surface des cellules B avec l&#39;aide, mais pas la construction de vide, indiquant que la RSE est survenue à la suite du sauvetage d&#39;expression de l&#39;AID. <img src="/files/ftp_upload/2371/2371fig1.jpg" alt="Figure 1" /> Figure 1: Schéma d&#39;encapsidation rétrovirale dans des cellules producteur: gène d&#39;intérêt, représenté par Ψ, a été sous-cloné dans PMX-IRES-GFP, comme décrit précédemment 15. Le plasmide a été transfecté dans une ligne d&#39;emballage écotrope virale cellulaire capable de produire gag-pol et protéine d&#39;enveloppe (Phoenix, Orbigen), en utilisant la méthode du phosphate de calcium 13, 14. Quarante-huit heures après la transfection, le surnageant contenant les particules virales ont été récoltés pour l&#39;infection dans les cellules cibles B primaires provenant de souris. <img src="/files/ftp_upload/2371/2371fig2.jpg" alt="Figure 2" /> Figure 2: les cellules B SECOURS déficiente, stimulées par l&#39;anti-CD40 et IL-4 ont été infectés par un rétrovirus contenant PMX-MAID-GFP ou un rétrovirus exprimant GFP seule. Le niveau de la RSE pour IgG1 a été évaluée par analyse FACS.

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Introduction to Genetic Analysis. , (2000).</li><li>Gondo, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next-generation+gene+targeting+in+the+mouse+for+functional+genomics.">Next-generation gene targeting in the mouse for functional genomics.</a> BMB Rep. 42, 315-3123 (2009).</li><li>Plachy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proviruses+selected+for+high+and+stable+expression+of+transduced+genes+accumulate+in+broadly+transcribed+genome+areas.">Proviruses selected for high and stable expression of transduced genes accumulate in broadly transcribed genome areas.</a> J Virol. 84, 4204-4211 (2010).</li><li>Felice, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+factor+binding+sites+are+genetic+determinants+of+retroviral+integration+in+the+human+genome.">Transcription factor binding sites are genetic determinants of retroviral integration in the human genome.</a> PLoS One. 4, e4571-e4571 (2009).</li><li>Karavanas, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+targeting+by+murine+retroviral+vectors.">Cell targeting by murine retroviral vectors.</a> Crit Rev Oncol Hematol. 28, 7-30 (1998).</li><li>Noelle, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+39-kDa+protein+on+activated+helper+T+cells+binds+CD40+and+transduces+the+signal+for+cognate+activation+of+B+cells.">A 39-kDa protein on activated helper T cells binds CD40 and transduces the signal for cognate activation of B cells.</a> Proc Natl Acad Sci U S A. 89, 6550-6554 (1992).</li><li>Muramatsu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Class+switch+recombination+and+hypermutation+require+activation-induced+cytidine+deaminase+(AID),+a+potential+RNA+editing+enzyme.">Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme.</a> Cell. 102, 553-563 (2000).</li><li>Yelle, J., Dion, M., Hamelin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+transfection+of+mammalian+cells+with+viral+DNA+in+optimal+culture+conditions.">Efficient transfection of mammalian cells with viral DNA in optimal culture conditions.</a> J Virol Methods. 7, 321-326 (1983).</li><li>Chen, C., Okayama, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+transformation+of+mammalian+cells+by+plasmid+DNA.">High-efficiency transformation of mammalian cells by plasmid DNA.</a> Mol Cell Biol. 7, 2745-2752 (1987).</li><li>Fagarasan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+class+switching+and+differentiation+to+IgA-producing+cells+in+the+gut+lamina+propria.">In situ class switching and differentiation to IgA-producing cells in the gut lamina propria.</a> Nature. 413, 639-6343 (2001).</li><li>Basu, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+AID+antibody+diversification+enzyme+is+regulated+by+protein+kinase+A+phosphorylation.">The AID antibody diversification enzyme is regulated by protein kinase A phosphorylation.</a> Nature. 438, 508-5011 (2005).</li><li>McBride, K. 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Transduction rétrovirale de cellules B spléniques comme décrit ici et comme le montre la figure 1 est une approche génétique qui est utile dans l&#39;étude des lymphocytes B en raison de nombreux événements de développement dans lymphopoesis sont contrôlés par la régulation transcriptionnelle 1, 2. Dans les derniers stades de la maturation des cellules B, déclenchant via CD40L est essentiel pour l&#39;induction de la croissance des cellules B, l&#39;entrée dans le cycle cellulai...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>FCS</td>
<td>&nbsp;</td>
<td>Atlanta Biologicals</td>
<td>S11550</td>
<td>&nbsp;</td>
<tr>
<td>RPMI</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>22400</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>20012</td>
<td>&nbsp;</td>
<tr>
<td>Red Blood Cell (RBC) lysis buffer</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>R7757</td>
<td>&nbsp;</td>
<tr>
<td>CD43 beads</td>
<td>&nbsp;</td>
<td>Miltenyi</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>B Cell Complete Media</td>
<td>&nbsp;</td>
<td>Various components</td>
<td>Various Components</td>
<td>RPMI, 15% FCS, 1% Non-Essential Amino Acids, 1% Sodium Pyruvate, 1% HEPES, 1% Pen-Strep, 50&mu;M &beta;-Mercaptoethanol</td>
</tr>
<tr>
<td>IL-4</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Anti-CD40</td>
<td>&nbsp;</td>
<td>BD Pharmigen</td>
<td>553787</td>
<td>&nbsp;</td>
<tr>
<td>polybrene</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>107689 &#x2028;</td>
<td>&nbsp;</td>
<tr>
<td>Chloroquine diphosphate salt</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>C6628</td>
<td>Used at 100mM</td>
</tr>
<tr>
<td>Phoenix Eco cells (Murine)</td>
<td>&nbsp;</td>
<td>Orbigen</td>
<td>RVC-10002</td>
<td>&nbsp;</td>
<tr>
<td>PE-Cy5-&alpha;-mouse-CD45R (B220)</td>
<td>&nbsp;</td>
<td>eBioscience</td>
<td>15-0452-81</td>
<td>&nbsp;</td>
<tr>
<td>PE-&alpha;-mouse-IgG1</td>
<td>&nbsp;</td>
<td>BD Pharmigen</td>
<td>A85-1</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

recombinant retroviral production and infection of b cells

L&#39;expression des gènes transgéniques dans les cellules eucaryotes est un puissant démarche de génétique inverse dans lequel un gène d&#39;intérêt est exprimé sous le contrôle d&#39;un système d&#39;expression hétérologue pour faciliter l&#39;analyse du phénotype résultant. Cette approche peut être utilisée pour exprimer un gène qui n&#39;est pas normalement trouvé dans l&#39;organisme, d&#39;exprimer une forme mutante d&#39;un produit du gène, ou de sur-expriment une forme dominante-négative du produit du gène. Il est particulièrement utile dans l&#39;étude du système hématopoïétique, où la régulation transcriptionnelle est un mécanisme de contrôle majeur dans le développement et la différenciation des cellules B 1, a examiné en 2-4. Génétique de la souris est un outil puissant pour l&#39;étude des gènes humains et des maladies. Une analyse comparative de la souris et le génome humain révèle conservation de la synténie dans plus de 90% du génome 5. En outre, la plupart des technologies utilisées dans les modèles de souris est applicable à l&#39;étude des gènes humains, par exemple, les perturbations des gènes et de remplacement allélique 6 . Toutefois, la création d&#39;une souris transgénique nécessite beaucoup de ressources de nature à la fois financière et technique. Plusieurs projets ont commencé à compiler les bibliothèques d&#39;assommer souches de souris (KOMP, EUCOMM, NorCOMM) ou de la mutagenèse induite souches (RIKEN), qui nécessitent de vastes efforts et de collaboration 7. Par conséquent, il est souhaitable d&#39;étudier d&#39;abord le phénotype d&#39;un gène désiré dans un modèle de culture cellulaire de cellules primaires avant de passer à un modèle de souris. L&#39;ADN rétroviral s&#39;intègre dans l&#39;ADN de l&#39;hôte, de préférence au sein ou à proximité des unités de transcription ou îlots CpG, résultant en stable et héritable l&#39;expression du gène d&#39;intérêt emballé, tout en évitant le silencing transcriptionnel 8 9. Les gènes sont ensuite transcrits sous le contrôle d&#39;un promoteur à haute efficacité antirétrovirale, résultant en un rendement élevé de la transcription et la production de protéines. Par conséquent, l&#39;expression rétroviral peut être utilisé avec les cellules qui sont difficiles à transfecter, fourni les cellules sont dans un état actif lors de la mitose. Parce que les gènes de structure du virus sont contenus dans la lignée cellulaire d&#39;emballage, les vecteurs d&#39;expression utilisée pour cloner le gène d&#39;intérêt ne contiennent pas de gènes de structure du virus, ce qui élimine la possibilité à la fois de révertants virale et augmente la sécurité de travailler avec des surnageants viraux car aucune virions infectieux sont produits 10. Nous présentons ici un protocole pour la production recombinante rétroviraux et l&#39;infection ultérieure des cellules B spléniques. Après isolement, les cellules cultivées rate sont stimulées par Th. lymphokines dérivées et anti-CD40, ce qui induit un éclatement de la prolifération cellulaire et la différenciation B 11. Ce protocole est idéal pour l&#39;étude des événements survenus en fin de développement des cellules B et la différenciation, les cellules B sont isolées de la rate initiales suivantes évènements hématopoïétiques, mais avant la stimulation antigénique à induire la différenciation plasmocytaire.

Watch this Scientific Journal Video about Recombinant Retroviral Production and Infection of B Cells at JoVE.com

Recombinant Retroviral Production and Infection of B Cells

Un système efficace de structure et de l&#39;analyse fonctionnelle d&#39;un gène dans une<em&gt; Ex vivo</em&gt; Culture des lymphocytes B spléniques est décrite. Cette méthode tire parti de la production recombinante dans un aide-rétroviraux gratuits, ligne de conditionnement de cellules écotrophique. Stable, héréditaires expression d&#39;un gène d&#39;intérêt au sein des lymphocytes primaires est réalisée conduisant à la génération d&#39;anticorps de surface sur les cellules B subissant la commutation isotypique.

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 ...

recombinant-retroviral-production-and-infection-of-b-cells

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Immunology and Infection

14.0K vues.  Columbia University College of Physicians and Surgeons. Un système efficace de structure et de l'analyse fonctionnelle d'un gène dans une Ex vivo Culture des lymphocytes B spléniques est décrite. Cette méthode tire parti de la production recombinante dans un aide-rétroviraux gratuits, ligne de conditionnement de cellules écotrophique. Stable, héréditaires expression d'un gène d'intérêt au sein des lymphocytes primaires est réalisée conduisant à la génération d'anticorps de su...

Vidéo: Recombinant Retroviral Production and Infection of B Cells

Murine Model of CD40-activation of B cells

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

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

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 ...

Recherche

Immunologie et infection

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

Transduction rétrovirale des récepteurs de cellules T dans les lymphocytes T de souris

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Production and Titering of Recombinant Adeno-associated Viral Vectors

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently being tested in human clinical trials. Wild-type AAV is a non-pathogenic member of the parvoviridae family and inherently replication-deficient. The broad transduction profile, low immune response as well as the strong and persistent transgene expression achieved with these vectors has made them a popular and versatile tool for in vitro and in vivo gene delivery. rAAVs can be easily and cheaply produced in the laboratory and, based on their favourable safety profile, are generally given a low safety classification. Here, we describe a method for the production and titering of chimeric rAAVs containing the capsid proteins of both AAV1 and AAV2. The use of these so-called chimeric vectors combines the benefits of both parental serotypes such as high titres stocks (AAV1) and purification by affinity chromatography (AAV2). These AAV serotypes are the best studied of all AAV serotypes, and individually have a broad infectivity pattern. The chimeric vectors described here should have the infectious properties of AAV1 and AAV2 and can thus be expected to infect a large range of tissues, including neurons, skeletal muscle, pancreas, kidney among others. The method described here uses heparin column purification, a method believed to give a higher viral titer and cleaner viral preparation than other purification methods, such as centrifugation through a caesium chloride gradient. Additionally, we describe how these vectors can be quickly and easily titered to give accurate reading of the number of infectious particles produced.

Recombinant adeno-associated virus (rAAVs) vectors are becoming increasingly valuable for in vivo studies in animals. We describe how rAAVs can be produced in the laboratory and how these vectors can be titered to give an accurate reading of the number of infectious particles produced.

Production et titrage des adéno-associé recombinant vecteurs viraux

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently ...

Efficient Recombinant Parvovirus Production with the Help of Adenovirus-derived Systems

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 which regulate the expression of non-structural (NS1 and NS2) and capsid proteins (VP1 and VP2) respectively1. They attract high interest as anticancer agents for their oncolytic and oncosuppressive abilities while being non-pathogenic for humans2. NS1 is the major effector of viral cytotoxicity3. In order to further enhance their natural antineoplastic activities, derivatives from these vectors have been generated by replacing the gene encoding for the capsid proteins with a therapeutic transgene (e.g. a cytotoxic polypeptide, cytokine, chemokine, tumour suppressor gene etc.)4. The recombinant parvoviruses (recPVs) vector retains the NS1/2 coding sequences and the PV genome telomeres which are necessary for viral DNA amplification and packaging. Production of recPVs occurs only in the producer cells (generally HEK293T), by co-transfecting the cells with a second vector (pCMV-VP) expressing the gene encoding for the VP proteins (Fig. 1)4. The recPV vectors generated in this way are replication defective. Although recPVs proved to possess enhanced oncotoxic activities with respect to the parental viruses from which they have been generated, their production remains a major challenge and strongly hampers the use of these agents in anti-cancer clinical applications.
We found that introduction of an Ad-5 derived vector containing the E2a, E4(orf6) and the VA RNA genes (e.g. pXX6 plasmid) into HEK293T improved the production of recPVs by more than 10 fold in comparison to other protocols in use. Based on this finding, we have constructed a novel Ad-VP-helper that contains the genomic adenoviral elements necessary to enhance recPVs production as well as the parvovirus VP gene unit5. The use of Ad-VP-helper, allows production of rec-PVs using a protocol that relies entirely on viral infection steps (as opposed to plasmid transfection), making possible the use of cell lines that are difficult to transfect (e.g. NB324K) (Fig. 2). We present a method that greatly improves the amount of recombinant virus produced, reducing both the production time and costs, without affecting the quality of the final product5. In addition, large scale production of recPV (in suspension cells and bioreactors) is now conceivable.

Here we describe a protocol based only on cell infection, which improves the efficiency of recombinant parvovirus production by more than 100 fold in comparison to other protocols in use. This protocol relies on the use of a novel adenovirus 5-based helper containing the parvovirus VP transcription unit (Ad-VP).

Efficacité de la production Parvovirus recombinant avec l&#39;aide d&#39;adénovirus dérivés Systems

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 ...

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, ...

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCR&#945; and TCR&#946; chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR.

We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice.

Rétrovirale transduction des cellules progénitrices de la moelle osseuse pour générer des cellules T Receptor Retrogenic Souris

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident STIM1 regulates the activity of PM Orai1 channels in a process known as store operated calcium (Ca2+) entry which is the principal Ca2+ signaling process that drives the immune response. STIM1 undergoes post-translational N-glycosylation at two luminal Asn sites within the Ca2+ sensing domain of the molecule. However, the biochemical, biophysical, and structure biological effects of N-glycosylated STIM1 were poorly understood until recently due to an inability to readily obtain high levels of homogeneous N-glycosylated protein. Here, we describe the implementation of an in vitro chemical approach which attaches glucose moieties to specific protein sites applicable to understanding the underlying effects of N-glycosylation on protein structure and mechanism. Using solution nuclear magnetic resonance spectroscopy we assess both efficiency of the modification as well as the structural consequences of the glucose attachment with a single sample. This approach can readily be adapted to study the myriad glycosylated proteins found in nature.

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Biochemical and structural analyses of glycosylated proteins require relatively large amounts of homogeneous samples. Here, we present an efficient chemical method for site-specific glycosylation of recombinant proteins purified from bacteria by targeting reactive Cys thiols.

Ciblage des Thiols de cystéine pour in Vitro in situ Glycosylation des protéines recombinantes

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident ...

"Liver-on-a-Chip" Cultures of Primary Hepatocytes and Kupffer Cells for Hepatitis B Virus Infection

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally infected chimpanzees, most in vitro models require several hundreds to thousands of viral genomes per cell in order to initiate a transient infection. Additionally, static 2D cultures of primary human hepatocytes (PHH) allow only short-term studies due to their rapid dedifferentiation. Here, we describe 3D liver-on-a-chip cultures of PHH, either in monocultures or in cocultures with other nonparenchymal liver-resident cells. These offer a significant improvement to studying long-term HBV infections with physiological host cell responses. In addition to facilitating drug efficacy studies, toxicological analysis, and investigations into pathogenesis, these microfluidic culture systems enable the evaluation of curative therapies for HBV infection aimed at eliminating covalently closed, circular (ccc)DNA. This presented method describes the set-up of PHH monocultures and PHH/Kupffer cell co-cultures, their infection with purified HBV, and the analysis of host responses. This method is particularly applicable to the evaluation of long-term effects of HBV infection, treatment combinations, and pathogenesis.

&quot;Liver-on-a-Chip&quot; Cultures of Primary Hepatocytes and Kupffer Cells for Hepatitis B Virus Infection

The goal of this protocol is to provide a step-by-step guide to perform 3-D &#34;liver-on-a-chip&#34; infection experiments with the hepatitis B virus.

« Foie-on-a-Chip » Cultures d’hépatocytes primaires et des cellules de Kupffer pour Infection de Virus de l’hépatite B

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally ...

Retroviral Overexpression of CXCR4 on Murine B-1a Cells and Adoptive Transfer for Targeted B-1a Cell Migration to the Bone Marrow and IgM Production

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide further insight on cell function. B-1a cells are a unique B cell subset in mice that produce protective natural IgM antibodies against oxidation-specific epitopes that arise during health and disease. B-1a cell IgM production differs depending on B-1a cell location, and therefore it becomes useful from a therapeutic standpoint to target B-1a localization to niches supportive of high antibody production. Here we describe a method to target B-1a cell migration to the bone marrow by retroviral-mediated overexpression of the C-X-C motif chemokine receptor 4 (CXCR4). Gene induction in primary murine B cells can be challenging and typically yields low transfection efficiencies of 10-20% depending on technique. Here we demonstrate that retroviral transduction of primary murine B-1a cells results in 30-40% transduction efficiency. This method utilizes adoptive cell transfer of transduced B-1a cells into B cell-deficient recipient mice so that donor B-1a cell migration and localization can be visualized. This protocol can be modified for other retroviral constructs and can be used in diverse functional assays post-adoptive transfer, including analysis of donor cell or host cell phenotype and function, or analysis of soluble factors secreted post B-1a cell transfer. The use of distinct donor and recipient mice differentiated by CD45.1 and CD45.2 allotype and the presence of a GFP reporter within the retroviral plasmid could also enable detection of donor cells in other, immune-sufficient mouse models containing endogenous B cell populations.

Here we describe a method for retroviral overexpression and adoptive transfer of murine B-1a cells to examine in vivo B-1a cell migration and localization. This protocol can be extended for diverse downstream functional assays including quantification of donor B-1a cell localization or analysis of donor cell-derived secreted factors post-adoptive transfer.

Surexpression rétrovirale de CXCR4 sur les cellules murine B-1a et transfert adoptif pour la migration ciblée de cellules B-1a à la moelle osseuse et à la production d’IgM

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide ...