Name: Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation (Video) | JoVE Journal
Uploaded: 2013-08-13T13:00:00.000+00:00
Duration: 15 min 33 s
Description: Learn adenoviral transduction of naive CD4 T cells to study Treg differentiation using high-efficiency gene overexpression. This method enables precise manipulation of early molecular events without affecting cell viability.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

The authors would like to thank Lirui Du for constructing the pCAGAdDU vector and Oliver Gorka for provision of the fixation protocol.

1. Cloning of a Gene of Interest into an Entry Vector
<ol>
	<li>Clone the gene of interest into an entry vector. PCR amplification of the gene followed by blunt-end ligation into a topoisomerase-coupled vector (e.g. pENTR/D-TOPO) or restriction enzyme-mediated cloning may be used for this procedure.</li>
</ol>
2. Transferring the Gene of Interest into the pCAGAdDu Destination Vector
<ol>
	<li>Transfer the gene of interest from the entry vector into the destination vector by LR recombination (e.g. Gateway LR Clonase II Enzyme Mix). This will create the adenoviral expression vector (Figure 1).</li>
	<li>Linearize 10 &mu;g of the adenoviral expression vector in a PacI restriction digest, precipitate the DNA and resuspend it in water at a concentration of 3 &mu;g per 100 &mu;l. Linearization liberates the viral inverted repeats (ITR), which are required for replication and encapsidation of the viral DNA into virus particles.</li>
</ol>
3. Generation of the Primary Virus Lysate
<ol>
	<li>Seed 1 x 105 HEK293A cells in 2 ml cell culture media (DMEM, 10% FBS, 5% PenStrep) in one well of a 6-well plate and incubate the cells for 6 - 14 hr at 37 &deg;C in a 10% CO2 incubator to allow them to adhere. Cells should then be at approximately 50% confluency.</li>
	<li>Lipofection: Transfer 6 &mu;l of jetPEI reagent into 94 &mu;l of 50 &mu;M NaCl, vortex briefly. Add the mixed solution to 100 &mu;l linearized adenovirus vector while vortexing and incubate this transfection mix for 15 - 30 min at room temperature.</li>
</ol>
Perform all following steps under the appropriate biosafety conditions for adenovirus infection!
<ol start="3">
	<li>Dispense the solution dropwise on the HEK293A cell-containing well and incubate the cells at 37 &deg;C and 10% CO2. When using a fluorescence marker, evaluate the transfection efficiency visually after 12 - 36 hr using an inverted fluorescence microscope. Add 0.5 ml of fresh medium every 3 days.</li>
	<li>Check every 2 - 3 days with a light or fluorescence microscope for cytopathic effects (CPE), which are areas with enlarged and rounded cells that start to detach. This is indicative of efficient virus generation. Upon occurrence of broader zones of CPE (Figure 3), it will take 24 - 72 hr before all cells are infected.</li>
	<li>When all cells show signs of CPE, but before an overall detachment of cells occurs, detach the cells by gentle pipetting and transfer cells with supernatant (SN, 3 - 5 ml) to a 15 ml polystyrene tube.</li>
	<li>Freeze the cells in the SN on dry ice for 15 - 20 min and thaw them quickly at 37 &deg;C afterwards to rupture the cells. Repeat this freeze-and-thaw-cycle (F/TC) two more times. Keep the primary virus lysate on ice for usage within a day or freeze it at -80 &deg;C for long-term storage. Any additional F/TC will reduce the virus titer by 30 - 50%.</li>
</ol>
4. Virus Amplification
<ol>
	<li>Seed and grow HEK293A cells to 90% confluence on a 14 cm tissue culture dish.</li>
	<li>Infect the cells with half of the primary virus lysate (1.5 - 2.5 ml) and incubate the cells for at least 36 hr. Nearly all cells should be infected then, which can be determined by fluorescence microscopy. For the re-amplification of an already amplified (i.e. more concentrated) virus stock, infect HEK293A at a multiplicity of infection (MOI) of 50 (see 6.3).</li>
	<li>Cells should be harvested when all of them show CPE but before detachment. With efficient virus production this state is reached within 48 hr after infection. (If it takes up to one week, consider another round of amplification by increasing the amount of adenovirus stock used for the infection described in 4.2.)</li>
	<li>Detach the cells by gentle pipetting and transfer cells and SN to a 50 ml polystyrene tube. Spin cells down at 300 x g for 10 min at 4 &deg;C.</li>
	<li>Remove the SN and resuspend the pellet in a suitable volume of medium or SN (ca. 1 ml).</li>
	<li>Perform 3 F/TC to disrupt the cells and centrifuge at 800 x g for 15 min at 4 &deg;C. Take off the SN that contains the virus particles (i.e. the concentrated virus lysate), and aliquot the virus lysate to store it at -80 &deg;C.</li>
</ol>
5. Virus Titer Determination
<ol>
	<li>Seed 105 A549 cells per well into 5 wells of a 12-well plate in 1 ml medium and let cells adhere for 6 hr.</li>
	<li>Use 1 &mu;l of concentrated adenovirus (thawed on ice) to perform a serial dilution in medium (1:5,000, 1:10,000, 1:50,000, 1:100,000) and add 10 &mu;l per well. Leave one well uninfected to adjust the gating in flow cytometry.</li>
	<li>After 36 hr, take off the SN, wash with PBS and detach cells (e.g. by trypsinization). For biohazard precautions, it is recommended to fix cells in 100 &mu;l 4% paraformaldehyde in PBS for 10 min at room temperature and wash with PBS one time.</li>
	<li>Perform a FACS analysis of infection marker expression. Plot '&mu;l viral lysate applied' against the absolute number of infected cells (Figure 4). Determine the linear range of infection and calculate the titer per ml of undiluted virus from the standard curve over the linear range using x = 1,000 &mu;l.</li>
</ol>
6. T Cell Infection
<ol>
	<li>Isolate naive/resting CD4 T cells from DO11.10 tg; CAR&Delta;1 tg mice using MACS (Naive CD4+ T Cell Isolation Kit II) or FACS sorting (CD4+ CD25- CD62L+ CD44-).</li>
	<li>For small-scale experiments, pipette an appropriate volume of viral lysate to achieve an MOI of 50 into one well of a 96-well round bottom plate.</li>
	<li>Add up to 4 x 105 T cells in a final infection volume of 50 &mu;l in T cell medium (RPMI1640, 10% FBS, 5% PenStrep, 5% NaPyruvate, 1x NEAA, 1x MEM Essential vitamin, 1x L-Glutamine, 1:250,000 Mercaptoethanol, 10 mM HEPES). 
	Example: 
	An MOI of 50 shall be used to infect 3 x 105 T cells; the viral titer is 3 x 109 ml-1 
	Virus volume = MOI x T cell number / viral titer = 50 x (3 x 105) / (3 x 109 ml-1) = 0.005 ml</li>
</ol>
Note: for infection of larger cell numbers, scale up using an MOI of 50 in an infection volume of 165 &mu;l per 106 naive T cells in a polystyrene tube with loose cup (up tp 3 ml per tube).
<ol start="4">
	<li>Incubate cells for 90 min at 37 &deg;C in a 5% CO2 incubator.</li>
	<li>Spin down cells at 300 x g for 5 min at room temperature, take off SN, resuspend in 200 &mu;l PBS. Centrifuge again and take off SN.</li>
</ol>
(Optional: the cells may be washed again to remove the virus more efficiently)
<ol start="6">
	<li>Resuspend cells in 200 &mu;l T cell medium without stimulating antibodies and without IL-2 or other cytokines and rest them for 40 hr at 37 &deg;C in a 5% CO2 incubator to allow expression of the gene of interest before activation.</li>
</ol>
7. T Cell Activation and Polarization
<ol>
	<li>Pipette a volume of anti-CD3- and anti-CD28-coupled beads that equals the cell number (e.g. 4 x 105) into a small reagent cup, add the 10-fold volume of PBS and put them on a magnet for 2 min. Take off the supernatant and resuspend the beads in 200 &mu;l polarizing medium (for Tregs: T cell medium + 1 ng/ml TGF&beta;, 100 U/ml IL-2).</li>
 </ol>
	Note: Cells can also be activated using tissue culture dishes coated with anti-CD28 and anti-CD3 antibodies, or in a DO11.10 T cell receptor-specific manner using irradiated BALB/c splenocytes pulsed with ovalbumin 323-339 peptide antigen.
 <ol start="2">
	<li>Centrifuge the rested cells as before, take off the SN and resuspend cells in 200 &mu;l polarizing medium containing anti-CD3- and anti-CD28-antibody-coupled beads. Incubate for 72 hr at 37 &deg;C in a 5% CO2 incubator without changing medium.</li>
</ol>
8. T Cell Fixation and Staining for Flow Cytometry
<ol>
	<li>Wash cells: Spin down cells at 300 x g for 5 min at room temperature, take off SN, resuspend in 200 &mu;l PBS. Centrifuge again and take off SN. Perform all following washing steps accordingly.</li>
	<li>Resuspend the cells in 100 &mu;l fixable dead cell staining solution and incubate for 30 min at 4 &deg;C.</li>
	<li>Wash cells, resuspend in 100 &mu;l PBS, add 100 &mu;l 4% paraformaldehyde in PBS, incubate 15 min at RT.</li>
	<li>Wash cells, resuspend them in 200 &mu;l ice-cold 70% methanol in PBS and incubate for 30 min on ice.</li>
</ol>
Note: Cells can be treated from now on without biohazard precaution!
<ol start="5">	
 <li>Prepare 60 &mu;l master-mix of 60 &mu;l PBS + 10 &mu;g/ml Fc-block (anti-FCR3 to block unspecific binding). Wash cells, resuspend them in 40 &mu;l of PBS + anti-FCR3 and incubate 15 min at RT.</li>
	<li>Add 20 &mu;l of PBS + anti-FCR3 containing 1 &mu;g PE-coupled anti-Foxp3 antibody, mix well and incubate at 4 &deg;C over night.</li>
	<li>Wash cells twice in PBS and analyze cells on a flow cytometer.</li>
</ol>

<ol><li>Lahl, K., Loddenkemper, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Selective+depletion+of+Foxp3%2B+regulatory+T+cells+induces+a+scurfy-like+disease%5BTitle%5D)+AND+%22The+Journal+of+Experimental+Medicine%22%5BJournal%5D)">Selective depletion of Foxp3+ regulatory T cells induces a scurfy-like disease.</a> The Journal of Experimental Medicine. 204 (1), 57-63 (2007).</li>
<li>Kretschmer, K., Apostolou, I., Hawiger, D., Khazaie, K., Nussenzweig, M. C., von Boehmer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inducing+and+expanding+regulatory+T+cell+populations+by+foreign+antigen%5BTitle%5D)+AND+%22Nature+Immunology%22%5BJournal%5D)">Inducing and expanding regulatory T cell populations by foreign antigen.</a> Nature Immunology. 6 (12), 1219-1227 (2005).</li>
<li>Zhang, X., Izikson, L., Liu, L., Weiner, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Activation+of+CD25%28%2B%29CD4%28%2B%29+regulatory+T+cells+by+oral+antigen+administration%5BTitle%5D)+AND+%22Journal+of+Immunology+%28Baltimore%2C+Md%29%22%5BJournal%5D)">Activation of CD25(+)CD4(+) regulatory T cells by oral antigen administration.</a> Journal of Immunology (Baltimore, Md). 167 (8), 4245-4253 (2001).</li>
<li>Josefowicz, S. Z., Niec, R. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extrathymically+generated+regulatory+T+cells+control+mucosal+TH2+inflammation%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Extrathymically generated regulatory T cells control mucosal TH2 inflammation.</a> Nature. 482 (7385), 395-399 (2012).</li>
<li>Samstein, R. M., Josefowicz, S. Z., Arvey, A., Treuting, P. M., Rudensky, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extrathymic+generation+of+regulatory+T+cells+in+placental+mammals+mitigates+maternal-fetal+conflict%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Extrathymic generation of regulatory T cells in placental mammals mitigates maternal-fetal conflict.</a> Cell. 150 (1), 29-38 (2012).</li>
<li>Laurence, A., Amarnath, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((STAT3+Transcription+Factor+Promotes+Instability+of+nTreg+Cells+and+Limits+Generation+of+iTreg+Cells+during+Acute+Murine+Graft-versus-Host+Disease%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">STAT3 Transcription Factor Promotes Instability of nTreg Cells and Limits Generation of iTreg Cells during Acute Murine Graft-versus-Host Disease.</a> Immunity. 37 (2), 209-222 (2012).</li>
<li>Terabe, M., Berzofsky, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunoregulatory+T+cells+in+tumor+immunity%5BTitle%5D)+AND+%22Current+Opinion+in+Immunology%22%5BJournal%5D)">Immunoregulatory T cells in tumor immunity.</a> Current Opinion in Immunology. 16 (2), 157-162 (2004).</li>
<li>Li, X., Kostareli, E., Suffner, J., Garbi, N., Hämmerling, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+Treg+depletion+induces+T-cell+infiltration+and+rejection+of+large+tumors%5BTitle%5D)+AND+%22European+Journal+of+Immunology%22%5BJournal%5D)">Efficient Treg depletion induces T-cell infiltration and rejection of large tumors.</a> European Journal of Immunology. 40 (12), 3325-3335 (2010).</li>
<li>Fontenot, J. D., Gavin, M. A., Rudensky, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Foxp3+programs+the+development+and+function+of+CD4%2BCD25%2B+regulatory+T+cells%5BTitle%5D)+AND+%22Nature+Immunology%22%5BJournal%5D)">Foxp3 programs the development and function of CD4+CD25+ regulatory T cells.</a> Nature Immunology. 4 (4), 330-336 (2003).</li>
<li>Hori, S., Nomura, T., Sakaguchi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Control+of+regulatory+T+cell+development+by+the+transcription+factor+Foxp3%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Control of regulatory T cell development by the transcription factor Foxp3.</a> Science. 299 (5609), 1057-1061 (2003).</li>
<li>Chen, W., Jin, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Conversion+of+Peripheral+CD4%2BCD25-+Naive+T+Cells+to+CD4%2BCD25%2B+Regulatory+T+Cells+by+TGF-%CE%B2+Induction+of+Transcription+Factor+Foxp3%5BTitle%5D)+AND+%22The+Journal+of+Experimental+Medicine%22%5BJournal%5D)">Conversion of Peripheral CD4+CD25- Naive T Cells to CD4+CD25+ Regulatory T Cells by TGF-β Induction of Transcription Factor Foxp3.</a> The Journal of Experimental Medicine. 198 (12), 1875-1886 (2003).</li>
<li>Xu, W., Lan, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adoptive+transfer+of+induced-treg+cells+effectively+attenuates+murine+airway+allergic+inflammation%5BTitle%5D)+AND+%22PloS+ONE%22%5BJournal%5D)">Adoptive transfer of induced-treg cells effectively attenuates murine airway allergic inflammation.</a> PloS ONE. 7 (7), e40314(2012).</li>
<li>Circosta, P., Granziero, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((T+cell+receptor+%28TCR%29+gene+transfer+with+lentiviral+vectors+allows+efficient+redirection+of+tumor+specificity+in+naive+and+memory+T+cells+without+prior+stimulation+of+endogenous+TCR%5BTitle%5D)+AND+%22Human+Gene+Therapy%22%5BJournal%5D)">T cell receptor (TCR) gene transfer with lentiviral vectors allows efficient redirection of tumor specificity in naive and memory T cells without prior stimulation of endogenous TCR.</a> Human Gene Therapy. 20 (12), 1576-1588 (2009).</li>
<li>Patel, C., Muthuswamy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High+efficiency%2C+Site-specific+Transfection+of+Adherent+Cells+with+siRNA+Using+Microelectrode+Arrays+%28MEA%29%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays (MEA).</a> J. Vis. Exp. (67), e4415(2012).</li>
<li>Zhong, S., Malecek, K., Perez-Garcia, A., Krogsgaard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Retroviral+transduction+of+T-cell+receptors+in+mouse+T-cells%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Retroviral transduction of T-cell receptors in mouse T-cells.</a> J. Vis. Exp. (44), e2307(2010).</li>
<li>Leon, R. P., Hedlund, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adenoviral-mediated+gene+transfer+in+lymphocytes%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Adenoviral-mediated gene transfer in lymphocytes.</a> Proceedings of the National Academy of Sciences of the United States of America. 95 (22), 13159-13164 (1998).</li>
<li>Wan, Y. Y., Leon, R. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transgenic+expression+of+the+coxsackie%2Fadenovirus+receptor+enables+adenoviral-mediated+gene+delivery+in+naive+T+cells%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Transgenic expression of the coxsackie/adenovirus receptor enables adenoviral-mediated gene delivery in naive T cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 97 (25), 13784-13789 (2000).</li>
<li>Hurez, V., Dzialo-Hatton, R., Oliver, J., Matthews, R. J., Weaver, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+adenovirus-mediated+gene+transfer+into+primary+T+cells+and+thymocytes+in+a+new+coxsackie%2Fadenovirus+receptor+transgenic+model%5BTitle%5D)+AND+%22BMC+Immunology%22%5BJournal%5D)">Efficient adenovirus-mediated gene transfer into primary T cells and thymocytes in a new coxsackie/adenovirus receptor transgenic model.</a> BMC Immunology. 3, 4(2002).</li>
<li>Eitelhuber, A. C., Warth, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dephosphorylation+of+Carma1+by+PP2A+negatively+regulates+T-cell+activation%5BTitle%5D)+AND+%22The+EMBO+Journal%22%5BJournal%5D)">Dephosphorylation of Carma1 by PP2A negatively regulates T-cell activation.</a> The EMBO Journal. 30 (3), 594-605 (2011).</li>
<li>Glasmacher, E., Hoefig, K. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Roquin+binds+inducible+costimulator+mRNA+and+effectors+of+mRNA+decay+to+induce+microRNA-independent+post-transcriptional+repression%5BTitle%5D)+AND+%22Nature+Immunology%22%5BJournal%5D)">Roquin binds inducible costimulator mRNA and effectors of mRNA decay to induce microRNA-independent post-transcriptional repression.</a> Nature Immunology. 11 (8), 725-733 (2010).</li>
<li>Russell, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Update+on+adenovirus+and+its+vectors%5BTitle%5D)+AND+%22The+Journal+of+General+Virology%22%5BJournal%5D)">Update on adenovirus and its vectors.</a> The Journal of General Virology. 81 (Pt. 11), 2573-2604 (2000).</li>
<li>Graham, F. L., Smiley, J., Russell, W. C., Nairn, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characteristics+of+a+Human+Cell+Line+Transformed+by+DNA+from+Human+Adenovirus+Type+5%5BTitle%5D)+AND+%22Journal+of+General+Virology%22%5BJournal%5D)">Characteristics of a Human Cell Line Transformed by DNA from Human Adenovirus Type 5.</a> Journal of General Virology. 36 (1), 59-72 (1977).</li>
<li>Landy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamic%2C+structural%2C+and+regulatory+aspects+of+lambda+site-specific+recombination%5BTitle%5D)+AND+%22Annual+Review+of+Biochemistry%22%5BJournal%5D)">Dynamic, structural, and regulatory aspects of lambda site-specific recombination.</a> Annual Review of Biochemistry. 58, 913-949 (1989).</li>
<li>Bernard, P., Couturier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+killing+by+the+F+plasmid+CcdB+protein+involves+poisoning+of+DNA-topoisomerase+II+complexes%5BTitle%5D)+AND+%22Journal+of+Molecular+Biology%22%5BJournal%5D)">Cell killing by the F plasmid CcdB protein involves poisoning of DNA-topoisomerase II complexes.</a> Journal of Molecular Biology. 226 (3), 735-745 (1992).</li>
<li>Thai, T. -H., Calado, D. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulation+of+the+Germinal+Center+Response+by+MicroRNA-155%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Regulation of the Germinal Center Response by MicroRNA-155.</a> Science. 316 (5824), 604-608 (2007).</li>
<li>Rodriguez, A., Vigorito, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Requirement+of+bic%2FmicroRNA-155+for+Normal+Immune+Function%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Requirement of bic/microRNA-155 for Normal Immune Function.</a> Science. 316 (5824), 608-611 (2007).</li>
<li>Cobb, B. S., Hertweck, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+role+for+Dicer+in+immune+regulation%5BTitle%5D)+AND+%22The+Journal+of+Experimental+Medicine%22%5BJournal%5D)">A role for Dicer in immune regulation.</a> The Journal of Experimental Medicine. 203 (11), 2519-2527 (2006).</li>
<li>Steiner, D. F., Thomas, M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MicroRNA-29+Regulates+T-Box+Transcription+Factors+and+Interferon-%CE%B3+Production+in+Helper+T+Cells%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">MicroRNA-29 Regulates T-Box Transcription Factors and Interferon-γ Production in Helper T Cells.</a> Immunity. , (2011).</li>
</ol>

The authors declare no conflict of interest.

Virus Generation and Titration
For optimal transfection results, the quality and amount of linearized vector appear most important. We did not observe negative effects on primary lysate production from an initial overgrowth of the culture since the infection will quickly proceed once efficient virus production occurs. However, virus production by HEK293A cells can be affected by long inserts that decrease the efficiency. Some open reading frames were actually found to interfere with virus product...

Tregs are crucial to maintain immune tolerance and to dampen overshooting immune responses. Tregs suppress bystander T cell activation. Consequently, ablation of Tregs leads to fatal autoimmunity and self-destruction driven by activated T cells1. Tregs develop in the thymus during negative selection of CD4 single-positive precursors, but they can also differentiate in the periphery from naive CD4 T cells upon low-dose antigen stimulation with suboptimal co-stimulation 1,2. Thymic Tregs seem to suppress tissue autoimmunity against self-antigens, whereas peripheral Tregs have been implicated in providing tolerance in the gut or lung. These induced Tregs potently prevent T cell activation after recognition of foreign antigens in the mucosa, including environmental antigens from food and air, commensal bacteria, and allergens 3,4. In addition, Tregs are crucial to establish maternal tolerance to fetal peptides 5 and to prevent graft-versus-host disease 6. At the same time, Tregs also mediate unwanted effects by attenuating immune surveillance of tumor cells 7,8. The hallmark feature of Tregs is the expression of the subset-specifying transcription factor Foxp3, a fork-head domain-containing transcription factor that is necessary and sufficient to confer Treg function 9,10. Some signaling pathways that can induce Foxp3 expression are known. However, the molecular processes that control, regulate, or modulate Treg differentiation in response to T cell receptor triggering are less well understood.
Tregs can very effectively be induced in vitro through the stimulation of naive CD4 T cells with anti-CD3 and anti-CD28 antibodies in the presence of TGF&beta; and IL-2 11. As the emerging Tregs are functional in vivo, the manipulation of molecules that promote Treg differentiation bears enormous potential for future therapies, for example, the treatment of asthma, Crohns disease, and transplantation 11,12. Conversely, therapeutic modulation of molecules to block Treg differentiation may provide benefit in future approaches of combined treatment of tumor patients.
In vitro differentiation assays have been instrumental for the description of molecular changes that are associated with T cell subset differentiation. At the moment, experimental attempts to search or screen for gene products that control T cell differentiation are hampered by the fact that the most common methods of ectopic gene expression fail in naive T cells. For example, electroporation and retroviral transduction are only effective in activated T cells. In contrast to initial expectations, lentiviral transduction, which is typically effective in resting cells, requires pre-activation of naive T cells by cytokines13. Furthermore, the transfer of cDNA or mRNA during electroporation involves depolarization of the plasma membrane, which itself confers features of T cell activation and may even mobilize Ca2+ signaling and activate NFAT proteins (unpublished observation and ref. 14). Similarly, for retroviral transduction, the naive T cells have to be activated for 18 - 40 hr. During this time, the breakdown of the nuclear membrane in the course of cell division occurs and allows for the subsequent genomic integration of the retroviral vector 15. These methods are therefore not able to address the early molecular regulation of initial T cell encounter with antigen, which is the decisive phase of helper T cell differentiation.
Adenoviral transduction is known to confer transient ectopic gene expression in a number of human cell types that express the human Coxsackie adenovirus receptor (CAR). It proceeds without requirement for cell activation or cell-cycle progression. The surface expression of CAR is essential for efficient virus attachment and internalization, and transgenic expression of the truncated version CAR&Delta;1 under a T cell-specific promoter was found to render mouse thymocytes and T cells susceptible to adenoviral infection 16. Importantly, the transgene does not alter thymocyte development or in vitro differentiation of naive CD4 T cells into different subsets (data not shown; ref. 17). Adenovirus-mediated transduction of T cells was previously used for overexpression 17,18 and knock-down approaches 19,20. The transgenic T cells can be purified from commercially available DO11.10 tg; CAR&Delta;1 tg (Taconic, Inc. and ref. 17). Importantly, adenoviral transduction allows high expression of a gene of interest in naive T cells without inducing obvious signs of activation. The T cells remain naive (CD44low, CD62Lhigh) and resting (CD25-, CD69-) after infection and can be activated and differentiated into Treg similar to non-infected cells.
 
Production of recombinant adenoviruses can be achieved after transfection of HEK293A cells with adenoviral plasmids (Figure 1). These plasmids typically contain the human type 5 adenovirus genome with E1 and E3 genes deleted to render recombinant adenoviruses replication-incompetent 21. HEK293A cells complement replication deficiency as they have been immortalized through stable integration of sheared adenovirus 22. Since adenoviral vectors are large (~40 kb) and consequently not well suited for traditional restriction enzyme-mediated cloning, we employed the Gateway system. The gene of interest is initially cloned into a smaller entry vector, from which it can be easily transferred into the adenoviral destination vector via lambda recombination reaction (LR) 23. We constructed the pCAGAdDu vector by combining the CAG promoter (chicken actin promoter and CMV enhancer) with an expression cassette containing LR sites flanking the procaryotic ccdB selection marker 24 . This expression cassette is fused to an internal ribosome entry site (IRES) element that allows coexpression of the eukaryotic infection marker enhanced green fluorescent protein (eGFP), which is fused to a sequence containing the bovine growth hormone poly(A)-signal. We chose the CAG cis-regulatory sequences, since the prototypic CMV promoter was found to be highly activation-dependent and therefore unfavorable for gene expression in naive T cells.
Here, we provide a protocol for efficient in vitro Treg differentiation and a method to transduce naive CD4 T cells without activation (Figure 2). The method enables ectopic gene expression or knock down preceding CD4 T cell differentiation at the naive state. It allows testing the effect of an overexpressed gene of interest during early signaling events upon initial TCR stimulation until T cell subset commitment. Our validation experiments also provide the basis to establish similar adenovirus application in the differentiation of other T cell subsets such as Th1, Th2, Th9, Th17, Th22, or Tfh cells.

<table><tbody><tr><td>pENTR/D-TOPO Cloning Kit</td><td>Invitrogen</td><td>K240020</td><td></td></tr><tr><td>Gateway LR Clonase II Enzyme mix</td><td>Invitrogen</td><td>11791020</td><td></td></tr><tr><td>PacI</td><td>New England Biolabs</td><td>R057S</td><td></td></tr><tr><td>jetPEI</td><td>Polyplus-transfection</td><td>101-10N</td><td></td></tr><tr><td>HEK293A Cell Line</td><td>Invitrogen</td><td>R705-07</td><td></td></tr><tr><td>A549</td><td>ATCC</td><td>CCL-185</td><td></td></tr><tr><td>6-well plates</td><td>BD Falcon</td><td>353046</td><td></td></tr><tr><td>14 cm tissue culture dish</td><td>Nunc</td><td>168381</td><td></td></tr><tr><td>BALB/cJ-Tg(DO11.10)10Dlo Tg(CAR&amp;Delta;1)1Jdgr</td><td>Taconic Farms</td><td>Model Nr. 4285</td><td></td></tr><tr><td>Naive CD4+ T Cell Isolation Kit II</td><td>Miltenyi Biotech</td><td>130-094-131</td><td></td></tr><tr><td>DMEM</td><td>Invitrogen</td><td>41966-052</td><td></td></tr><tr><td>FBS</td><td>PAN Biotech</td><td>1502-P110704</td><td></td></tr><tr><td>PenStrep</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>RPMI 1640</td><td>Lonza</td><td>BE12-167F</td><td></td></tr><tr><td>NaPyruvate</td><td>Lonza</td><td>BE13-115E</td><td></td></tr><tr><td>NEAA 100x</td><td>Lonza</td><td>BE13-114E</td><td></td></tr><tr><td>L-Glutamine</td><td>Invitrogen</td><td>25030</td><td></td></tr><tr><td>HEPES Buffer Solution (1 M)</td><td>Invitrogen</td><td>15630-056</td><td></td></tr><tr><td>&amp;beta;-Mercaptoethanol</td><td>Sigma-Aldrich</td><td>M-7522</td><td></td></tr><tr><td>MEM Essential vitamin mixture (100x)</td><td>Lonza</td><td>13-607C</td><td></td></tr><tr><td>Dynabeads Mouse T-Activator CD3/CD28 for Cell Expansion and Activation</td><td>Invitrogen</td><td>114-56D</td><td></td></tr><tr><td>Recombinant Human TGF-beta 1</td><td>R&amp;amp;D Systems</td><td>240B</td><td></td></tr><tr><td>Proleukin S (18 x 106IE)</td><td>Novartis</td><td></td><td></td></tr><tr><td>LIVE/DEAD Fixable Blue Dead Cell Stain Kit</td><td>Invitrogen</td><td>L-23105</td><td></td></tr><tr><td>Mouse BD Fc BlockT</td><td>BD Pharmingen</td><td>553141</td><td></td></tr><tr><td>Anti-Mouse/Rat Foxp3 PE</td><td>eBioscience</td><td>12-5773-82</td><td></td></tr><tr><td>Mmu-miR-155 TaqMan MicroRNA Assay</td><td>Roche Applied Biosystems</td><td>4427975</td><td></td></tr><tr><td>LightCycler 480 Probes Master</td><td>Roche Applied Biosystems</td><td>04902343001</td><td></td></tr><tr><td>TaqMan MicroRNA Reverse Transcription Kit</td><td>Roche Applied Biosystems</td><td>4366596</td><td></td></tr></tbody></table>

adenoviral transduction of naive cd4 t cells to study treg differentiation

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGF&beta; and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown.
Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44low, CD62Lhigh) and resting (CD25-, CD69-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.

Virus Production
For generation of high virus titers, the timing of HEK293A cell harvest in primary virus production or virus amplification is crucial. Representative fluorescent and phase contrast images with visual signs of virus production are shown in Figure 3. The CPE were observed 10 days after transfection of HEK293A cells with a control pCAGAdDu vector without insert. CPE are characterized by the appearance of areas with enlarged and round-up cells that start to detach an...

Adenoviral gene transfer into naive CD4 T cells with transgenic expression of the Coxsackie adenovirus receptor enables the molecular analysis of regulatory T cell differentiation in vitro.

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

This protocol provides a workflow for the production of infectious adenoviral particles that are used to transduce a gene of interest into naive T cells, which upon atopic expression of the transgene or differentiated into regulatory T cells or tregs. First the gene of interest is cloned into the adenoviral destination vector. The vector is then transferred into the adenovirus producer cell line, HC 2 93 A and infectious adenoviral particles are generated.The resulting viruses are used to infect naive T cells from mice with an adenovirus receptor transgene after infection. The cells are then rested to ensure expression of the gene of interest. In naive T cells, the T cells are activated in polarizing conditions to induce treg differentiation.Then the cells are fixed and stained for flow cytometric analysis. Ultimately, results can be obtained that show expression of the treg differentiation marker, Fox P three and the infection marker GFP, which may be influenced by the overexpressed gene of interest. The main advantage of this technique over existing methods like retroviral gene transduction is that it allows overexpression of a gene of interest in naive T-cells.This results in a high level of overexpression present at the moment of T-cell activation. This method can help answer key questions in the regulatory T-cell field. For example, about the post transcription gene regulation that underlies the establishment of T-Rex cell identity.Begin this protocol by cloning the gene of interest into an entry vector. The PCR amplified gene of interest is blunt and ligated into the topoisomerase coupled vector P enter D topo. The gene of interest is then transferred by LR Recombination into the P kakadu destination vector.It contains the adenoviral genes and an internal ribosomal entry site or iris, followed by the open reading frame of GFP to create an adenoviral expression vector with an infection marker. Next 10 micrograms of the adenoviral expression vector is linearized. In a PAC one restriction digest, the DNA is then precipitated and resuspended in water at a concentration of three micrograms per 100 microliters.Linearization liberates the viral inverted repeats or ITR, which are required for replication and encapsulation of the viral DNA into virus particles. All of the following steps should be performed under the appropriate biosafety conditions for adenovirus infection. To prepare linearized vector for cot transfection transfer six microliters of jet PEI reagent into 94 microliters of 50 micromolar sodium chloride vortex.Briefly then, while vortexing add the mixed solution to 100 microliters of linearized adenovirus vector. Incubate this transfection mix for 15 to 30 minutes at room temperature. Next, remove HEC 2 9 3 A cells grown to 50%co fluency on six well plates from the incubator.Dispense the solution dropwise into the wells containing HEC 2 9 3 A cells and incubate them at 37 degrees Celsius and 10%carbon dioxide. Add 0.5 milliliters of fresh medium every three days using GFP expression as an infection marker. Evaluate the transfection efficiency visually with an inverted fluorescence microscope every two to three days check for cytopathic effects evidenced by areas with enlarged and rounded cells that start to detach.This is indicative of efficient virus generation. Once broader zones of cytopathic effects appear, all cells will be infected within 24 to 72 hours. When all of the cells show signs of cytopathic effects, but before an overall detachment of cells occurs, harvest the cells by gentle pipetting and transfer them with the supernatant to a 15 milliliter polystyrene tube.Next to rupture the cells freeze the suspensions on dry ice for 15 to 20 minutes, then quickly thaw them at 37 degrees Celsius. Repeat this, freeze and thaw cycle two more times. The ruptured cell suspension is now referred to as the primary virus.Lysate.Keep all virus lysates on ice. If they'll be used within a day for long-term storage, freeze them at negative 80 degrees Celsius. Keep in mind that any additional freeze thaw cycles will reduce the virus tighter by 30 to 50%To amplify the primary virus lysate infect HC 2 93 A cells grown to 90%confluence on 15 tissue culture dishes with half of the primary virus, lysate and incubate for at least 36 hours.After 36 hours check for infection by fluorescence microscopy, nearly all cells should be infected. Once all of the cells show cytopathic effects but not detachment, harvest the cells by gentle pipetting with efficient virus production. This state is reached within 48 hours of infection, transfer the cells and supernatant to a 50 milliliter polystyrene tube.Spin cells down at 300 G for 10 minutes at four degrees Celsius. Following the spin, remove the supernatant and resuspend the pellet in a suitable volume of medium. One milliliter should result in a titer in the range of 10 to the 10th per milliliter.Perform three freeze thaw cycles as before to disrupt the cells. Then centrifuge at 900 G for 20 minutes at four degrees Celsius. After the spin, carefully transfer the supernatant, which is now referred to as the concentrated virus, lysate to a cryo tube.Make aliquots for multiple infections and store them at negative 80 degrees Celsius. Finally, determine the virus titer by fax analysis of infection marker expression in a 5 4 9 cells. According to the instructions in the accompanying document, this part of the protocol is performed using naive rusting CD four T cells isolated from D 1110 transgenic car delta one transgenic mice for small scale experiments.Peppe an appropriate volume of viral lysate to achieve an MOI of 50 into one well of a 96 well round bottom plate. Add up to four times 10 to the fifth T cells in a final infection. Volume of 50 microliters in T-cell.Medium medium. This setup allows parallel experimental approaches. Incubate cells for 90 minutes at 37 degrees Celsius in a 5%carbon dioxide incubator.Following the incubation, spin the cells down at 300 G for five minutes at room temperature after the spin resus, suspend them in 200 microliters of PBS centrifuge again and take off the supernatant Resus suspend cells in 200 microliters of T-cell medium without stimulating antibodies and without IL two or other cytokines. Then rest the cells for 40 hours at 37 degrees Celsius in a 5%carbon dioxide incubator to allow expression of the gene of interest before activation to activate and polarize the T cells pipette anti CD three and anti CD 28 coupled beads into a small reagent cup. The number of beads should be equal to the number of cells, then tenfold the volume of PBS and place them on a magnet for two minutes.Next, take off the supernatant and resus. Suspend the beads in 200 microliters of polarizing medium centrifuge the rested cells at 300 G for five minutes at room temperature following the spin take off the super datin and resuspend cells in 200 microliters of polarizing medium containing antibody coupled beads incubate for 72 hours at 37 degrees Celsius in a 5%carbon dioxide incubator without changing medium. The protocol for staining and fixation is described in brief here.First, the cells are washed in PBS and incubated with fixable dead cell staining solution. After another wash step, the cells are fixed in para formaldehyde following which the cells can be treated without adenovirus biosafety precautions after fixation, the cells are washed again and permeable with methanol. After another wash step, the cells incubated with anti FCR three to block non-specific binding.Finally, the cells are incubated with anti FCR three and PE coupled anti fox P three antibodies. There is the risk of losing a lot of cells during the fixation and staining procedure due to the multiple washing steps to avoid loss. Make sure to take off the supinate right after centrifugation without disturbing the palette with a piper tip After the cells have been stained, wash the cells twice in PBS and analyze them on a flow cytometer.A 96 well reader on the flow cytometer allows performing T-cell infection, differentiation, fixation, and staining in the same 96 well plate providing an efficient workflow for parallel analyses of multiple conditions, but the analysis can also be performed in standard fax tubes to assess the effect of adenovirus infection on T-cell viability and treg differentiation. Naive T cells purified from DO 1110 transgenic car delta one transgenic mice were infected with control adenovirus at different MOIs and activated in treg polarizing conditions. Fixed and stained cells were analyzed by flow cytometry for the expression of the infection marker.GFP incorporation of dead cell die and for the differentiation marker. FOX P three as shown here. Increasing the MOI from one to 50 results in a higher percentage of infected cells.While neither viability nor treg differentiation is affected to determine whether adenovirus infection affects the activation status of T cells. Infected and non-infected cells were analyzed for expression of activation and resting markers by facts as seen here. Expression of these markers was indistinguishable in infected and non-infected cells when cells were then activated in treg polarizing conditions.Upregulation of activation markers and downregulation of naive T-cell markers was very similar in infected and non-infected cells taken together. These data indicate that an infection of T cells at a naive state does not interfere with subsequent T-cell activation to test the level of overexpression achieved with the described adenoviral transduction protocol, naive T cells were infected with microRNA 1 55 encoding or control adenovirus at an MOI of 50 cells were either rested for 40 hours, activated for 40 hours, or were rested for 40 hours, followed by an activation of 40 hours. CDNA was generated and micro RNA 1 55 expression was determined relative to snow RNA 202.By quantitative PCR microRNA 1 55 overexpression levels in naive T cells rested for 40 hours matches endogenous microRNA. 1 55 expression levels. After 40 hours of T-cell activation, activation of those cells still induces activation dependent microRNA 1 55 upregulation.This demonstrates the high level of overexpression in naive T cells by adenovirus on top of the endogenous activation induced micro RNA expression. Once mastered the procedure of cloning virus generation and T cell infection can be completed within four weeks. The virus, once it's generated, can be stored long term or re amplified within three days.While attempting this procedure, it is important to remember to carefully control all experiments with an empty vector and to first establish the effect of adenoviral infection itself on the studied process. Please keep in mind that adenoviral infection and overexpression is transient and lasts for only about five to seven days.

adenoviral-transduction-naive-cd4-t-cells-to-study-treg

Research

JoVE Journal

Immunology and Infection

We describe a method for generating regulatory, memory and naïve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

This video demonstrates a method of converting dendritic cells (DCs) to tolerogenic DCs (TolDCs) using an immunomodulatory pharmacological agent. This conversion is confirmed by evaluating the TolDC function through their immunosuppressive effect on CD4+ T cells.

Effect of an Immunomodulatory Pharmacological Agent on the Interaction Between Dendritic Cells and CD4+ T Cells

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether naïve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Naïve CD4+ T cells polarize to various subsets depending on the environment at the time of activation.  The differentiation of naïve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

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.

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

This protocol describes the reproducible generation and phenotyping of human induced regulatory T cells (iTregs) from naïve CD4+ T cells in vitro. Different protocols for FOXP3 induction allow for the study of specific iTreg phenotypes obtained with respective protocols.

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Na&#239;ve CD4+ T Cells Using a TGF-&#946;-containing Protocol

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.

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

This protocol describes the differentiation of naïve CD4+ T cells into pathogenic Th17 cells in vitro. Specifically, when combined with a multi-parameter flow cytometry-based approach, 90% purity of pathogenic Th17 cells can be obtained from naïve CD4+ T cells using this differentiation method.

In Vitro Differentiation of Naive CD4+ T Cells into Pathogenic Th17 Cells in Mouse

We present a protocol for retroviral transduction of guide RNA into primary T cells from Cas9 transgenic mice, providing an efficient alternative for gene editing in studying Th17 differentiation.

Retroviral CRISPR/Cas9-Mediated Gene Targeting for the Study of Th17 Differentiation in Vitro

We describe a procedure to assess the capacity for pharmacologic agents to generate tolerogenic dendritic cells from naïve monocyte-derived dendritic cells in vitro and validate their potency by autologous regulatory T cell generation.

A Simple and Efficient Method for Testing Immunomodulatory Agents for Generation of Tolerogenic Dendritic Cells from Human CD14+ Monocytes

Learn adenoviral transduction of naive CD4 T cells to study Treg differentiation using high-efficiency gene overexpression. This method enables precise manipulation of early molecular events without affecting cell viability.

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Explore More Videos