C.K. is supported by Columbia University Graduate program. U.B. is Fellow of the Leukemia and Lymphoma Society of America, the recipient of New Investigator Award from the Leukemia Research Foundation and is supported by Columbia University New Faculty start-up funds.

1. Splenic B-Lymphocyte Isolation and Stimulation 
<ol>
	<li>Harvest the spleens from AID-deficient mice 12 between 2-3 months of age. The isolation of spleen is performed in sterile conditions and the organs are temporarily kept in cold phosphate buffer saline (PBS) containing 15% fetal bovine serum (FBS).</li>
	<li>The spleen is then transferred to a sterile tissue culture hood and homogenized in 5 mL of PBS supplemented with 2.5% FBS. Transfer the homogenate to a 15 mL falcon tube.</li>
	<li>Centrifuge the homogenized splenic tissue sample at 1000rpm for 10 minutes at 4&deg;C to pellet the cells. </li>
	<li>Decant the supernatant after centrifugation. Resuspend the cell pellet in 10 mL red blood cell (RBC) lysis buffer at room temperature for 7 minutes to avoid contamination by red blood cells.</li>
	<li>Centrifuge sample at 1000 rpm for 10 minutes and decant the supernatant and resuspend the cells in 10 mL PBS containing 2.5% FBS.</li>
	<li>Remove 100 &mu;L aliquot and count the cells at a 1:1000 dilution using a standard hemacytometer using trypan blue to exclude any dead cells. Approximately 20-30 million cells can be obtained per spleen.</li>
	<li>Centrifuge sample at 1000rpm for 10 minutes and decant the supernatant.</li>
	<li>Resuspend the pellet in 0.5%BSA/PBS containing 2mM EDTA at a concentration of 107cells per 90 &mu;L.</li>
	<li>Add anti-CD43 antibody coated magnetic beads to the cells to specifically bind non-B cells. Use 10 &mu;L beads per 90 &mu;L (107cells). Mix well and incubate at 4&deg;C for 20 minutes.</li>
	<li>Once the cells have been labeled, wash them by adding 2.5% FBS/PBS to top of the tube. Centrifuge sample at 1000 rpm for 10 minutes.</li>
	<li>Decant the supernatant. Resuspend the cells in 0.1% BSA/PBS at a concentration of 10x107cells per ml.</li>
	<li>Mount a magnetic sorting column on a magnetic separator. Position a conical tube directly underneath the column to collect the flow-through and load the column with 3mL 0.5% BSA/PBS to prime and wash.</li>
	<li>Once the wash fluid has flowed through, replace with a new collection tube. If the sample is less than 1 mL, adjust sample volume to 1 mL with 0.5 %BSA/PBS/EDTA and load the sample in the column. Load only 1 mL maximum of cells per column and use multiple columns if necessary. </li>
	<li>Allow the cells to passively flow through the column and collect the unbound cells in the conical tube under the column. Wash the column 4 times with 3 mL 0.5% BSA/PBS while continuing to collect the flow-through.</li>
	<li>Collect all flow-through in which CD43 negative resting B cells will be enriched. Discard the column. Centrifuge flow-through at 1000 rpm for 10 minutes. </li>
	<li>Drain the supernatant and add 10 mL medium (15% FCS/ +glutamine, Pen/Strep, Non-Essential amino acids, 50 &mu;M &#914;-ME)</li>
	<li>Count the cells and culture at 106 cells per ml.</li>
	<li>In order to stimulate B cell growth and proliferation, supplement the medium with recombinant IL-4 and anti-CD40 antibody such that the final concentrations are 20 &mu;g/mL and 1 &mu;g/mL, respectively, and transfer the cells to a culture flask. </li>
	<li>Culture the cells overnight. Count cells and dilute to 106 cells per ml, adding IL-4 and CD40 as appropriate.</li>
	<li>Culture cells for 72 hours before viral infection.</li>
</ol>
2. Transfection of Producer Cells
Use established protocols for transfection using either cationic lipids or calcium phosphate. Transfect 10cm3 plate per DNA construct, using 100 &mu;g plasmid DNA per plate. We routinely use calcium phosphate transfection method 13, 14 and note high viral titer when supplemented with chloroquine.
<ol>
	<li>Use 60%-70% confluent Phoenix cells as producer cells to be transfected. At this confluence cell count should be 5 million cells per 10 cm plate. </li>
	<li>One hour prior to transfection, replace the media with fresh complete media for Phoenix cell growth. </li>
	<li>In a sterile eppendorpf tube, combine 100 &mu;g of packaging construct DNA, 250 &mu;L of 0.5M CaCl2, and adjust the final volume to 500 &mu;L with sterile water </li>
	<li>Using a 1 mL pipette, vigorously mix this solution with 500 &mu;L 2X HNP in a 15mL polypropylene tube </li>
	<li>Allow this mixture to rest at room temperature for 8-10 minutes. During this time a precipitate will form. </li>
	<li>Add the transfection mixture drop-wise to the cells while swirling the medium to evenly distribute the mixture throughout. </li>
	<li>Incubate the cells at 37&deg;C for 12 hours. </li>
	<li>12 hours post-transfection, replace the medium with 8ml complete B cell growth medium and return the cells to the 37&deg;C incubator.</li>
</ol>
3. Infection of Stimulated Splenic B Cells
<ol>
	<li>Remove supernatant from Phoenix cells 48hr post-transfection and pass the supernatant through a 0.2 micron filter to remove any cell debris. </li>
	<li>Mix 3 mL of virus-containing supernatant with 3 mL of pre-activated B cells (as in step 1). In order to enhance viral adsorption to the cells, add polybrene at a final concentration of 16 &mu;g per mL.</li>
	<li>Infect the cells by centrifugation at 2500 rpm for 90 minutes at 30&deg;C.</li>
	<li>Immediately after the spin, incubate for a further 48-72 hrs at 37&deg;C to allow for cell growth and proliferation. </li>
</ol>
4. Flow Cytometry Analysis
<ol>
	<li>Using a flow cytometer, analyze the cells for surface expression of B220 and surface IgG1. The presence of IgG1 at the surface of the cells indicates that class switch recombination has occurred as a result of successful rescue via the retroviral complementation of the AID gene.</li>
</ol>
5. Representative Results
We have successfully rescued the deficiency of the protein factor Activation Induced Cytidine Deaminase (AID) from AID-deficient (AID-/-) B-lymphocytes using retroviral transduction. Briefly, we generated recombinant retroviruses expressing mouse AID (mAID) protein by transfecting Phoenix cells with pMX-mAID-GFP 15-18. The harvested viruses were infected into AID-deficient B cells obtained from AID-deficient mice in ex vivo class switch recombination (CSR) stimulation conditions 19. The infected B cells were then analyzed for CSR to IgG1 after 72hrs in culture using flow cytometry analysis. Figure 2 shows the detection of IgG1 at the surface of B cells with AID but not the empty construct, indicating that CSR has occurred as a result of AID expression rescue. 
<img src="/files/ftp_upload/2371/2371fig1.jpg" alt="Figure 1" /> Figure 1: Scheme of retroviral packaging within producer cells: Gene of interest, represented by &Psi;, was subcloned into pMX-IRES-GFP, as previously described 15. The plasmid was transfected into an ecotropic viral packaging cell line capable of producing gag-pol and envelope protein (Phoenix, Orbigen), using calcium phosphate method 13, 14. Forty-eight hours after transfection, supernatant containing viral particles was harvested for infection into target primary B cells obtained from mice. 
<img src="/files/ftp_upload/2371/2371fig2.jpg" alt="Figure 2" /> Figure 2: AID deficient B cells, stimulated with anti-CD40 and IL-4 were infected with a retrovirus containing pMX-mAID-GFP or a retrovirus expressing GFP alone. The level of CSR to IgG1 was evaluated by FACS analysis.

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+sequencing+and+comparative+analysis+of+the+mouse+genome.">Initial sequencing and comparative analysis of the mouse genome.</a> Nature. 420, 520-562 (2002).</li><li>Griffiths, A. J. F., Miller, J. H., Suzuki, D. T., Lewontin, R. C., Gelbart, W. 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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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AID+from+bony+fish+catalyzes+class+switch+recombination.">AID from bony fish catalyzes class switch recombination.</a> J Exp Med. 202, 733-738 (2005).</li><li>Delphin, S., Stavnezer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+antibody+class+switching+to+IgE:+characterization+of+an+IL-4-responsive+region+in+the+immunoglobulin+heavy-chain+germline+epsilon+promoter.">Regulation of antibody class switching to IgE: characterization of an IL-4-responsive region in the immunoglobulin heavy-chain germline epsilon promoter.</a> Ann N Y Acad Sci. 764, 123-135 (1995).</li><li>Castigli, 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+expression+and+function+in+murine+B+cell+ontogeny.">CD40 expression and function in murine B cell ontogeny.</a> Int Immunol. 8, 405-411 (1996).</li><li>Ballantyne, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+recombination+of+a+switch+substrate+retrovector+in+CD40-+activated+B+lymphocytes:+implications+for+the+control+of+CH+gene+switch+recombination.">Efficient recombination of a switch substrate retrovector in CD40- activated B lymphocytes: implications for the control of CH gene switch recombination.</a> J Immunol. 161, 1336-1347 (1998).</li></ol>

No conflicts of interest declared.

Retroviral transduction of splenic B cells as described here and as depicted in Figure 1 is a genetic approach that is useful in the study of B-lymphocytes because many of the developmental events in lymphopoesis are controlled by transcriptional regulation 1, 2. In the later stages of B cell maturation, triggering via CD40L is essential for the induction of B cell growth, entry into the cell cycle, and proliferation 11, 20. As shown in Figure 2, B cells can be stim...

<table><tbody><tr><td>FCS</td><td>Atlanta Biologicals</td><td>S11550</td><td></td></tr><tr><td>RPMI</td><td>Invitrogen</td><td>22400</td><td></td></tr><tr><td>PBS</td><td>Invitrogen</td><td>20012</td><td></td></tr><tr><td>Red Blood Cell (RBC) lysis buffer</td><td>Sigma-Aldrich</td><td>R7757</td><td></td></tr><tr><td>CD43 beads</td><td>Miltenyi Biotec</td><td></td><td></td></tr><tr><td>B Cell Complete Media</td><td>Various</td><td>Various Components</td><td>RPMI, 15% FCS, 1% Non-Essential Amino Acids, 1% Sodium Pyruvate, 1% HEPES, 1% Pen-Strep, 50&amp;#x03BC;M &amp;#946;-Mercapt&amp;#8211;thanol</td></tr><tr><td>IL-4</td><td></td><td></td><td></td></tr><tr><td>Anti-CD40</td><td>BD Biosciences</td><td>553787</td><td></td></tr><tr><td>polybrene</td><td>Sigma-Aldrich</td><td>107689 &amp;#8232;</td><td></td></tr><tr><td>Chloroquine diphosphate salt</td><td>Sigma-Aldrich</td><td>C6628</td><td>Used at 100mM</td></tr><tr><td>Ph&amp;#8211;nix Eco cells (Murine)</td><td>Orbigen</td><td>RVC-10002</td><td></td></tr><tr><td>PE-Cy5-&amp;alpha;-mouse-CD45R (B220)</td><td>eBioscience</td><td>15-0452-81</td><td></td></tr><tr><td>PE-&amp;alpha;-mouse-IgG1</td><td>BD Biosciences</td><td>A85-1</td><td></td></tr></tbody></table>

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.

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

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

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

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

14.1K Views.  Columbia University College of Physicians and Surgeons. 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.

Video: Recombinant Retroviral Production and Infection of B Cells

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

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 ...

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

Production of Replication-Defective Retrovirus by Transient Transfection of 293T cells

Our lab studies human myeloproliferative diseases induced by such oncogenes as Bcr-Abl or growth factor receptor-derived oncogenes (ZNF198-FGFR1, Bcr-PDGFR&#945;, etc.). We are able to model and study a human-like disease in our mouse model, by transplanting bone marrow cells previously infected with a retrovirus expressing the oncogene of interest. Replication-defective retrovirus encoding a human oncogene and a marker (GFP, RFP, antibiotic resistance gene, etc.) is produced by a transient transfection protocol using 293T cells, a human renal epithelial cell line transformed by the adenovirus E1A gene product. 293 cells have the unusual property of being highly transfectable by calcium phosphate (CaPO4), with up to 50-80% transfection efficiency readily attainable. Here, we co-transfect 293 cells with a retroviral vector expressing the oncogene of interest and a plasmid that expresses the gag-pol-env packaging functions, such as the single-genome packaging constructs kat or pCL, in this case the EcoPak plasmid. The initial transfection is further improved by use of chloroquine. Stocks of ecotropic virus, collected as culture supernatant 48 hrs. post-transfection, can be stored at -80&deg;C and used for infection of cell-lines in view of transformation and in vitro studies, or primary cells such as mouse bone marrow cells, that can then be used for transplant in our mouse model.

This technique demonstrates an efficient way to prepare replication-defective retroviral stocks encoding a human oncogene, and subsequently used for induction of myeloproliferative disease in the mouse model.

Our lab studies human myeloproliferative diseases induced by such oncogenes as Bcr-Abl or growth factor receptor-derived oncogenes (ZNF198-FGFR1, ...

Biology

A Method For Production of Recombinant mCD1d Protein in Insect Cells.

CD1 proteins constitute a third class of antigen-presenting molecules. They are cell surface glycoproteins, expressed as approximately 50-kDa glycosylated heavy chains that are noncovalently associated with beta2-microglobulin. They bind lipids rather than peptides. Although their structure confirms the similarity of CD1 proteins to MHC class I and class II antigen presenting molecules, the mCD1d groove is relatively narrow, deep, and highly hydrophobic and it has two binding pockets instead of the several shallow pockets described for the classical MHC-encoded antigen-presenting molecules. Based upon their amino acid sequences, such a hydrobphobic groove provides an ideal environment for the binding of lipid antigens. The Natural Killer T (NKT) cells use their TCR to recognize glycolipids bound to or presented by CD1d. T cells reactive to lipids presented by CD1 have been involved in the protection against autoimmune and infectious diseases and in tumor rejection. Thus, the ability to identify, purify , and track the response of CD1-reactive NKT cell is of great importance . The generation of tetramers of alpha Galactosyl ceramide (a-Galcer) with CD1d has significant insight into the biology of NKT cells. Tetramers constructed from other CD1 molecules have also been generated and these new reagents have greatly expanded the knowledge of the functions of lipid-reactive T cells, with potential use in monitoring the response to lipid-based vaccines and in the diagnosis of autoimmune diseases and other treatments.

A Method to prepare Insect cells and infect them with baculovirus for the the purpose of production of recombinant mCD1d proteinand generating mCD1d tetramers.

CD1 proteins constitute a third class of antigen-presenting molecules. They are cell surface glycoproteins, expressed as approximately 50-kDa glycosylated ...

A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells

Lentiviral vectors (LVs) are a powerful means of delivering genetic material to many types of cells. Because of safety concerns associated with these HIV-1 derived vectors, producing large quantities of LVs is challenging. In this paper, we report a method for producing high titers of self-inactivating LVs. We retrovirally transduce the tet-off stable producer cell line GPR to generate a cell line, GPRS, which can express all the viral components, including a dendritic cell-specific glycoprotein, SVGmu. Then, we use concatemeric DNA transfection to transfect the LV transfer plasmid encoding a reporter gene GFP in combination with a selectable marker. Several of the resulting clones can produce LV at a titer 10-fold greater than what we achieve with transient transfection. Plus, these viruses efficiently transduce dendritic cells in vitro and generate a strong T cell immune response to our reporter antigen. This method may be a good option for producing strong LV-based vaccines for clinical studies of cancer or infectious diseases.

Here, we use retroviral transduction and concatemeric transfection to create a cell line that can express the components of a lentiviral vector (LV) in the absence of tetracycline. This LV encodes GFP and is pseudotyped with a glycoprotein, SVGmu, which is specific for a receptor on dendritic cells.

Lentiviral  vectors (LVs) are a powerful means of delivering genetic material to many types  of cells. Because of safety concerns associated with these ...

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

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

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

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

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, cloning TCRs specific for various tumor-associated antigens has been the goal of many studies. To elicit an effective T cell response, the TCR must recognize the target antigen with optimal affinity. However, cloning such TCRs has been a challenge and many available TCRs possess sub-optimal affinity for the cognate antigen. In this protocol, we describe a method of cloning de novo high affinity antigen-specific TCRs using existing TCRs by exploiting hemichain centricity. It is known that for some TCRs, each TCR&#945; or TCR&#946; hemichain do not contribute equally to antigen recognition, and the dominant hemichain is referred to as the centric hemichain. We have shown that by pairing the centric hemichain with counter-chains differing from the original counter-chain, we are able to maintain the antigen specificity, while modulating its interaction strength for the cognate antigen. Thus, the therapeutic potential of a given TCR can be improved by optimizing the pairing between the centric and counter hemichains.

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Herein we describe a novel method to generate antigen-specific T cell receptors (TCRs) by pairing the TCR&#945; or TCR&#946; of an existing TCR, possessing the antigen-specificity of interest, with complementary hemichain of the peripheral T cell receptor repertoire. The de novo generated TCRs retain antigen-specificity with varying affinity.

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity. Many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper T cell development and function. However, studies using mouse models have proven to be highly informative due to the availability of genetic, cellular, and biochemical systems. One genetic approach in mice used by many labs involves retroviral transduction of primary helper T cells. This is a powerful approach due to its relative ease, making it accessible to almost any laboratory with basic skills in molecular biology and immunology. Therefore, multiple genes in wild type or mutant forms can readily be tested for function in helper T cells to understand their importance and mechanisms of action. We have optimized this approach and describe here the protocols for production of high titer retroviruses, isolation of primary murine helper T cells, and their transduction by retroviruses and differentiation toward the different helper subsets. Finally, the use of this approach is described in uncovering mechanisms utilized by microRNAs (miRNAs) to regulate pathways controlling helper T cell development and function.

Many experimental systems have been utilized to understand the mechanisms regulating T cell development and function in an immune response. Here a genetic approach using retroviral transduction is described, which is economic, time efficient, and most importantly, highly informative in identifying regulatory pathways.

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...

Transduction and Expansion of Primary T Cells in Nine Days with Maintenance of Central Memory Phenotype

Emerging immunotherapies to treat infectious diseases and cancers often involve transduction of cellular populations with genes encoding disease-targeting proteins. For example, chimeric antigen receptor (CAR)-T cells to treat cancers and viral infections involve the transduction of T cells with synthetic genes encoding CAR molecules. The CAR molecules make the T cells specifically recognize and kill cancer or virally infected cells. Cells can also be co-transduced with other genes of interest. For example, cells can be co-transduced with genes encoding proteins that target cells to specific locations. Here, we present a protocol to transduce primary peripheral blood mononuclear cells (PBMCs) with genes encoding a virus-specific CAR and the B cell follicle homing molecule chemokine receptor type 5 (CXCR5). This procedure takes nine days and results in transduced T cell populations that maintain a central memory phenotype. Maintenance of a central memory or less differentiated phenotype has been shown to associate with persistence of cells post-infusion. Furthermore, cells produced with this method show high levels of viability, high levels of co-expression of the two transduced genes, and large enough quantities of cells for immunotherapeutic infusion. This nine-day protocol may be broadly used for CAR-T cell and other T cell immunotherapy approaches. The methods described here are based on studies presented in our previous publications.

We outline a 9-day protocol for the transduction and expansion of rhesus macaque peripheral blood mononuclear cells which yields cells with excellent co-expression of the genes of interest in sufficient number for infusion studies of cell efficacy.

Emerging immunotherapies to treat infectious diseases and cancers often involve transduction of cellular populations with genes encoding disease-targeting ...

Bioengineering

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.

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

Recombinant Retroviral Production and Infection of B Cells

Summary

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Chapters in this video

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

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Production of Replication-Defective Retrovirus by Transient Transfection of 293T cells

A Method For Production of Recombinant mCD1d Protein in Insect Cells.

A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells

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

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

Transduction and Expansion of Primary T Cells in Nine Days with Maintenance of Central Memory Phenotype

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