This work was supported by a Biotechnology and Biological Sciences Research Council (BBSRC) grant (BB/H018573/1) and a BD Biosciences grant.

All mouse work performed in these protocols was undertaken according to the Animals Scientific Procedures Act, UK under the animal Project License 70/6965.
1. Retroviral Production
Prior to proceeding obtain all required approvals for producing genetically modified organisms and the use of retroviruses in mammalian cells.
<ol>
	<li>Growth of HEK 293T Cells
	<ol>
		<li>Grow HEK 293T cells on 10 cm tissue culture dishes in HEK 293T medium (Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) with 10% fetal bovine serum (FBS), 100 units/ml Penicillin 0.1 mg/ml Streptomycin, and 2 mM L-Glutamine). For general cell passage, split cells 1:10 when they reach confluence by removing medium and pipetting the cells off the plate with fresh HEK 293T medium. 
		NOTE: Cells should reach confluence in 2-3 days. 293T HEK cells attach loosely to the plate so trypsinization is not required.</li>
		<li>24 hr prior to transfection, split a confluent plate of cells 1:10 to a 10 cm plate (one plate per transfection) so that the next day the cells are ~50% confluent.</li>
	</ol>
	</li>
	<li>Transfection by Calcium Phosphate Precipitation
	<ol>
		<li>Approximately 1 hr prior to transfection, remove medium from cells and carefully add 9 ml of fresh HEK 293T medium to prevent cells detaching from the plate.</li>
		<li>Prepare 2x Hepes Buffered Saline (2x HBS) and a fresh 2.5 M CaCL2 solution as described in Table 1. Thaw DNA stocks. For the most efficient transfections use plasmid prep quality DNA.</li>
		<li>In a 6 ml round bottom or 15 ml conical tube add 5 &#181;g of retroviral DNA (pMIG)4, 5 &#181;g of Helper virus DNA (pCL-Eco)7, and H2O to 420 &#181;l. Add 80 &#181;l of 2.5 M CaCl2 bringing the final volume to 500 &#181;l. 
		Note: This along with the next step (addition of 2x HBS) can be done on a regular bench as long as general sterile technique is used.</li>
		<li>Slowly add 500 &#181;l of 2x HBS drop by drop to the above DNA mixture while gently vortexing so that the solution is continuously mixed and the CaPO4 precipitates in even-sized crystals.</li>
		<li>Transfer to a tissue culture hood and slowly add the CaPO4 DNA mixture (1 ml) dropwise to the 9 ml of medium covering the cells while constantly and gently swirling the plate. Once again be careful not to detach cells from the plate. Place the cells back into the incubator. 
		NOTE: At this point the CaPO4 precipitate will be readily visible under a microscope as small crystals about the size of bacteria. These are easily seen after 30-60 min when the crystals have had a chance to settle to the bottom of the plate.</li>
	</ol>
	</li>
	<li>Collection of Virus in Culture Supernatants.
	<ol>
		<li>The following day (anywhere from 12-24 hr after transfection) remove the medium and feed the cells with 10 ml of fresh HEK 293T medium once again being careful not to dislodge the cells from the plate. 
		NOTE: This dilutes out lymphocyte inhibitory substances that the HEK 293T cells secrete into the medium during the transfection.</li>
		<li>In the evening of the second day (~24 hr post transfection) feed cells with 3.5 ml of fresh HEK 293T medium. Be careful that the plate is placed exactly level in the incubator so that one side is not left dry.</li>
		<li>Collect medium ~12 hr later (store at 4 &#176;C) and feed with 3.5 ml of fresh HEK 293T medium. Repeat collection 2 more times at roughly 12 hr intervals so that about 10 ml of medium is collected. This is the virus stock.</li>
		<li>Spin out any residual HEK 293T cells and cellular debris by centrifuging at 600 x g for 5 min. Alternatively, filter using a 0.45 &#181;m, low protein binding, syringe filter. Store virus at 4 &#176;C, and for best titers, use within a day or two, as the titer will slowly diminish at 4 &#176;C. Do not freeze, as this significantly decreases the titer.</li>
	</ol>
	</li>
</ol>
2. Isolation of Primary Na&#239;ve CD4+ T Cells
<ol>
	<li>Isolation of Leukocyte Cells
	<ol>
		<li>Euthanize mice by cervical dislocation, CO2 asphyxiation, or other humane protocol appropriate to the animal handling rules of the institution.</li>
		<li>Dissect freshly euthanized mice and remove spleen and desired lymph nodes 15. Place the organs in wells of a 6 well tissue culture plate containing R10 medium (RPMI medium with 10% heat inactivated FBS, 100 units/ml Penicillin, 0.1 mg/ml Streptomycin, 2 mM L-Glutamine, and 0.1% &#946;-mercaptoethanol). Use a cell culture hood for dissection of mice and all subsequent manipulations to minimize chance of contamination in cultures. 
		NOTE: Keep the R10 medium cold and perform all the downstream purification steps at 4 &#176;C.</li>
		<li>Place a 70 &#181;m cell culture strainer into a 50 ml conical tube containing approximately 5 ml of R10 medium. Use one strainer for the spleen and lymph nodes of up to three mice.</li>
		<li>Add spleen and lymph nodes to the cell strainer and macerate using the end of a 5 ml syringe plunger. Rinse with 1-2 ml of R10 medium.</li>
		<li>Transfer cells to a 15 ml conical tube and bring the volume up to 14 ml with R10. Centrifuge at 600 x g for 5 min at 4 &#176;C. Discard supernatant by pouring it off with one clean movement to prevent disturbing the cell pellet.</li>
		<li>Add 2 ml of cold (4 &#176;C) red cell lysis buffer (Table 1) to each tube. Gently mix for 3.5 min, keeping the tube on ice. 
		NOTE: The timing of red cell lysis is critical to prevent death of lymphocytes. Therefore, the exact timing of each batch of Red cell lysis buffer will need to be optimized.</li>
		<li>Add &#8776;12-13 ml of R10 medium. Immediately centrifuge and discard supernatant as in step 2.1.5. Perform the washing steps 2x more with R10 medium to remove any residual red cell lysis buffer from the cells and avoid death of lymphocytes.</li>
		<li>Count cells in a hemocytometer diluting 1:20 in trypan blue to determine the yield of viable cells. Expect approximately 8-10 x 107 cells per mouse.</li>
	</ol>
	</li>
	<li>Isolation of CD4+ T Cells Using Magnetic Beads Kit to Remove CD8+, CD11b+, CD16/32+, CD45R+, MHC class II+ and Ter-119+ Cells.
	<ol>
		<li>Aliquot 8-10 x 107 cells from step 2.1.8 per 15 ml conical tube and centrifuge at 600 x g for 5 min at 4 &#176;C. Pour off the supernatant with one clean movement to prevent disturbing the cell pellet. Resuspend the cells in each tube in 100 &#181;l of heat inactivated FBS then add 100 &#181;l of antibody mix from the kit. Incubate for 30 min at 4 &#176;C with gentle mixing on a roller.</li>
		<li>While the above cells are incubating, prepare the beads.
		<ol>
			<li>Resuspend beads in the vial by vortexing for &#62;30 sec then transfer 1 ml of beads per 8-10 x 107 prepared cells to a 15 ml conical tube. Add 2 ml of R10 medium per ml of beads and gently mix.</li>
			<li>Place the tube in the magnet for 1 min then discard the supernatant. Remove tube from the magnet and resuspend beads in 4 ml of R10 medium. This amount of beads is sufficient for two rounds of incubation.</li>
		</ol>
		</li>
		<li>Wash the cells at the end of the antibody incubation (Step 2.2.1) by adding 10 ml of R10 medium per tube. Gently mix well with swirling of the tube and centrifuge cells at 600 x g for 5 min at 4 &#176;C. Pour off the supernatant with one clean movement to prevent disturbing the cell pellet. Resuspend cells in 1 ml of R10 medium.</li>
		<li>Add 2 ml of the washed beads from step 2.2.2.2 (&#189; the amount prepared for each tube) to each tube of antibody treated cells and incubate for 30 min at 4 &#176;C with gentle mixing on a roller.</li>
		<li>Resuspend the cell-bead mixture by gently pipetting 5 times with a pipet containing a narrow tip opening. Avoid foaming. Place the tube in the beads magnet for 2 min then transfer the supernatant containing the negatively selected cells to a new tube. Keep these cells, as they are the CD4+ population.</li>
		<li>Repeat negative selection one more time. Spin cells from step 2.2.5 at 600 x g for 5 min at 4 &#176;C. Pour off the supernatant as in step 2.2.3 and resuspend in 1 ml of R10 medium. Follow steps 2.2.4 and 2.2.5 again. After the final magnetic bead selection, count the cells to determine recovery (typical yields are 15-20 x 106 cells per 108 leukocytes).</li>
	</ol>
	</li>
	<li>Isolation of Na&#239;ve CD4+ T Cells (CD25- and CD62Lhigh) Using Cell Separation Columns
	<ol>
		<li>Centrifuge CD4+ T cells at 600 x g for 5 min at 4&#176; C and resuspend in 150-200 &#181;l of R10 medium per 108 cells. Add 2 &#181;l of stock biotinylated CD25 monoclonal antibody (clone 7D4). Incubate for 30 min at 4&#176; C with gentle mixing on a roller.</li>
		<li>Wash cells by adding 10 ml of cell separation column buffer (Table 1). Centrifuge at 600 x g for 5 min at 4 &#176;C. Remove as much supernatant as possible and estimate the volume of buffer/cells remaining. Resuspend to a final volume of approximately 90 &#181;l per 107 cells.</li>
		<li>Add 20 &#181;l of Streptavidin beads per 107 cells and mix with gentle flicks. Incubate for 15 min at 4 &#176;C.</li>
		<li>While cells are incubating, prepare the medium cell separation (MS) columns for the manual approach. Each MS column retains 107 cells, and since CD25+ cells typically represent about 10% of total CD4+ cells, use 1 column per 108 CD4+ T cells. Add 500 &#181;l of cell separation column buffer to the column and let flow through. Repeat 2 more times.</li>
		<li>At end of cell/bead incubation, wash cells by adding 10 ml of cell separation column buffer and centrifuging at 600 x g for 5 min at 4 &#176;C. Resuspend cells in 500 &#181;l of cell separation column buffer per 108 cells, but still use 500 &#181;l if there are fewer cells.</li>
		<li>Apply 500 &#181;l of cell/bead suspension to the column and collect the flow through. Pass this over the column 1 more time and collect the flow through once again. Rinse the column 3 times with 500 &#181;l of cell separation column buffer, adding each flow through from these rinses to the collected cells. These are the CD25- cells. Count cells to determine yield.</li>
		<li>If Tregs (CD25+ cells) are desired, isolate as follows.
		<ol>
			<li>Remove the column from the separator and hold it above an open universal tube taking care to maintain sterility. Quickly pipette 500 &#181;l of cell separation buffer onto the column and firmly flush out the positive fraction, using the plunger supplied with the column.</li>
			<li>Repeat the flushing procedure 2 more times. Pass the positive fraction over a fresh MS cell separation column and discard this flow-through. Firmly flush out the positive cells three times, as before and centrifuge cells at 600 x g for 5 min at 4&#176; C. Resuspend cells in 1 ml of R10 medium and count to determine the yield.</li>
		</ol>
		</li>
		<li>To obtain CD25- CD62Lhigh T cells, centrifuge the CD25- T cells isolated above in step 2.3.6 at 600 x g for 5 min at 4&#176; and resuspend in 150-200 &#181;l of R10 medium per 107 cells. Add 5 &#181;l of stock biotinylated CD62L monoclonal antibody (clone MEL-14) and incubate for 30 min at 4&#176; C with gentle mixing on a roller as before.</li>
		<li>Perform wash, Streptavidin bead binding, and cell separation column preparation as described for Treg isolation protocol (2.3.7). This time use the large cell separation (LS) column, which has a capacity of 108 cells.
		<ol>
			<li>Since CD62Lhigh T cells typically represent about 60-70% of CD25- cells, use 1 LS cell separation column per 108 cells. Add cell/bead mixture to LS cell separation column as described in step 2.3.6 &#8211; this time discarding the final flow through and column rinses. Collect the CD62Lhigh T cells as described in step 2.3.7 for the CD25+ cell collection.</li>
			<li>Centrifuge cells at 600 x g for 5 min at 4&#176; C. Resuspend cells in 1 ml of R10 and count to determine yield. Dilute to 0.75-1 X106 cells per ml in R10 medium.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Analyze Purity of Cells by Fluorescent-activated Cell Sorting (FACS).
	<ol>
		<li>FACS staining strategy
		<ol>
			<li>For cells pre and post all stages of isolation, stain with CD4-FITC, CD8a-PerCP-Cy5.5 (helper and cytotoxic T cells), and MHCII-PE (MHC class II expressing cells).</li>
			<li>For cells pre and post CD25 isolation, stain with CD4-FITC and CD25-PE (Tregs versus other helper T cells).</li>
			<li>For cells pre and post CD62L isolation stain with CD4-FITC, CD62L-PE and CD44-APC (na&#239;ve versus memory and effector helper T cells).</li>
		</ol>
		</li>
		<li>For each analysis place 105 cells in a well of a 96 well U-bottom plate. Centrifuge at 600 x g for 5 min at 4 &#176;C. Pour off the medium and wash the cells once with 200 &#181;l DPBS. Centrifuge as above and pour off DPBS.</li>
		<li>Add 50 &#181;l of DPBS per well and 0.5 &#181;l of each antibody (as indicated in 2.4.1) and incubate at RT for 30 min in the dark to avoid bleaching of fluorescent label.</li>
		<li>Wash cells 2x with DPBS as in 2.4.2 and immediately analyze on a flow cytometer using protocols and guidelines specific to the instrument at hand. 
		NOTE: After the final isolation step of a good preparation, 90-95% of cells will be CD4+ CD25- CD62Lhigh T cells.</li>
	</ol>
	</li>
</ol>
3. Retroviral Transduction of Activated CD4+ T Cells and their Differentiation into Specific T Helper Subsets
<ol>
	<li>Retroviral Transduction 
	NOTE: Retroviral integration and expression of genes requires cell division. Therefore, T cells must be activated O/N.
	<ol>
		<li>While isolating the na&#239;ve T cells, coat a 24 well tissue culture plate with anti-CD3 and anti-CD28 antibodies diluted in DPBS. Add 250 &#181;l per well. Use anti anti-CD3 at 1&#181;g/ml. Use anti-CD28 at 2 &#181;g/ml if cells will ultimately be differentiated under Th0, Th1, Th2 and iTregs conditions. Use anti-CD28 at 10 &#181;g/ml for Th9 and Th17 conditions. Incubate ~2 hr at 37 &#176;C in a tissue culture incubator.</li>
		<li>Just prior to culturing the cells, remove the antibody solution and wash the plate with ~250 &#181;l of DPBS per well to remove unbound antibody. Be careful not to let the plate dry out so process a maximum of 6 wells at a time. Remove DPBS wash and add 1 ml of na&#239;ve CD4+ T cells in R10 medium at 0.75-1 x 106 cells per ml. Activate cells O/N (14-16 hr).</li>
		<li>The following day, centrifuge cells in the plate at 900 x g for 5 min at 30&#176; C to attach cells to the bottom of the wells.</li>
		<li>Collect and save the medium being careful not to displace the cells, and replace with 1 ml virus culture supernatant prepared from the virus production protocol. Do not let the cells dry out so process a maximum of 4 wells at a time.
		<ol>
			<li>To each well add 1 &#181;l of 8 mg/ml polybrene and 10 &#181;l of 1M HEPES pH 7.5 to aid virus uptake and prevent significant alkalization in the ambient CO2 during the following centrifugation. Centrifuge cells in plate at 900 x g for 90 min at 30&#176; C.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Differentiation of Cells into Specific Subsets
	<ol>
		<li>Carefully remove the virus culture supernatant and replace with 1 ml of the medium collected above in step 3.1.4, as it contains IL-2 and other T cell growth factors. Add reagents indicated in Table 2 and culture cells for 3-4 days to differentiate cells into specific subsets.</li>
	</ol>
	</li>
	<li>Analysis of Cells
	<ol>
		<li>For intracellular staining of cytokines, treat cells with 1 &#181;g/ml each of Phorbol 12-myristate 13-acetate (PMA) and ionomycin for 4 hr. During the last 2 hr of stimulation, add 1 &#181;g/ml of brefeldin A.</li>
		<li>Resuspend cells from the bottom of each well by pipetting up and down several times. Transfer approximately 105 cells to a 96 well U bottom plate (usually 100 &#181;l from the 1 ml of culture of Th cells) for fixation and staining.</li>
		<li>For GFP staining, fix cells with 100 &#181;l of 2% paraformaldehyde at RT for exactly 5 min, as a longer incubation time will bleach the GFP and interfere with Foxp3 staining. Wash cells by adding 100 &#181;l of DPBS to each well and centrifuge at 600 x g at 22 &#176;C for 5 min. Pour off the supernatant. 
		Caution: Paraformaldehyde is toxic. Handle it in a fume hood with skin and eye protection.</li>
		<li>Fix the cells again using 100 &#181;l 1x Fixation buffer made from the Foxp3 staining kit (1 volume of Fixation buffer to 3 volumes of diluent). Incubate the cells for 45 min at RT or alternatively incubate at 4 &#176;C for 16 hr.</li>
		<li>After fixation, permeabilize cells by adding 100 &#181;l of permeabilization buffer from the same kit. Incubate at RT for 30-45 min then centrifuge at 600 x g at 22 &#176;C for 5 min.</li>
		<li>For antibody staining, remove fixation/permeabilization buffer and add 50 &#181;l of fresh permeabilization buffer. Add the required antibody diluted in permeabilization buffer at 0.5 &#181;l per 50 &#181;l buffer per sample. Incubate for 30-45 min at RT in dark to avoid bleaching of fluorescent label.</li>
		<li>Add 150 &#181;l permeabilization buffer and centrifuge at 600 x g at 22 &#176;C for 5 min. Discard the permeabilization buffer and add 200 &#181;l DPBS. Analyze on a flow cytometer using protocols and guidelines specific to the instrument at hand. 
		NOTE: Be sure to include proper controls for cytokine staining such as staining with isotype control antibodies, analyzing non-activated or Th0 activated cells, etc.</li>
	</ol>
	</li>
</ol>

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The authors have no conflicting interest in the publication of this work.

Retroviral mediated overexpression of genes is a powerful way to analyze function in helper T cells, as their development and function is often determined by the expression level of key regulators. However, cautious interpretation of the results is required because expression levels significantly above those of the endogenous gene can introduce many artifacts. Therefore, this technique should be combined with others to verify the relevance of function. For example, overexpression should be complemented by reduced expression using siRNAs or gene knockouts if available. With miRNAs, we complemented overexpression experiments with those of blocking by using viruses that overexpressed artificial miRNA targeting sites that acted as competitive inhibitors for a miRNA 15. Retroviral transduced cells can also be utilized in biochemical assays involving RNA and protein analysis. However, a major limitation of these experiments is efficiency of transduction resulting in a mixed population of transduced and untransduced cells. Therefore, these assays will most likely require sorting of the GFP+ population. Finally, in vitro differentiation assays should be combined with in vivo experiments, and one way this can be achieved is by adoptively transferring the transduced T cells into mice and following their differentiation and their effect on the immune response.
One of the key limitations to this system is the size of the RNA genome that can be packaged into the retroviral capsid. In our experience, the maximum insert size for MIG retroviral system that gives good virus production is 3-3.5 kb. Therefore, larger genes cannot be analyzed with this system, as they give poor virus titers. However, most genes are smaller than this size so this system is useful for a wide variety of gene studies.
With retroviral transduction, several alternatives within these protocols have been used. Many researchers have utilized packaging cell lines that stably express the retroviral genes (for example reference 16). However, we have obtained the highest titers using standard HEK 293T cells with co-transfection of the pCL-Eco helper virus vector. Isolation of na&#239;ve helper T cells can also be achieved through cell sorting rather than the magnetic bead and cell separation column protocol, but this requires access to a cell sorter, and the costs for sort time are typically higher than the bead reagents. Finally, there are variations on activation conditions used to differentiate helper T cells into the different subsets. For example, TCR stimulation of cells for too long before exposure to Treg inducing conditions can inhibit their induction 16. This can be a problem because retroviral expression requires cell division induced by stimulation of cells. Nevertheless, we have found efficient Treg induction using this protocol with O/N activation prior to retroviral transduction.
Within these protocols, successful application requires several factors. High titer retrovirus preparations need efficient transfection of the HEK 293T cells so high quality DNA and precisely prepared 2x HBS are important. In addition, the cell density of the HEK 293T cells needs to be roughly 50% at point of transfection because good expression of the transfected DNA requires that the cells are actively growing, and this will be inhibited if the cells are too sparse or dense. Cells at the optimal density during transfection should reach confluence at some point during the virus collection steps, but they will continue producing high titer virus stocks all the way through to the last collection. Efficient differentiation of the helper T cells requires good cell quality so ensure that isolated cells are to the purity illustrated in Figure 1. Likewise, the quality of the cells is dependent on the mice from which they were isolated. For these studies, we have used 6-8 week old C57BL/6 mice. Older mice can have less na&#239;ve cells, and other strains may differ in their differentiation. For example, BALB/c mice are more prone to Th2 responses than C57BL/6 mice 17 so as stated above, C57BL/6 T cells can be difficult to induce a Th2 response. In addition, any of the differentiation conditions may vary slightly from lab to lab, and the effect of gene overexpression may only become apparent in sub-optimal conditions so cytokine concentrations in the various polarization conditions may need to be titrated. Finally, effects of the overexpressed gene or the polarization conditions on cell proliferation can influence the transduction efficiency so measuring effects of the gene of interest may require optimizing the timing and concentration of polarizing reagents. Optimizing all these factors should lead to informative results with this system.

The immune response must be highly regulated to eliminate infections but prevent attacks on self-tissue that lead to autoimmunity. Helper T cells play an essential role in regulating the immune response, and a great deal of effort has been undertaken to understand their development and function (illustrated in several recent reviews 1-3). However, many questions remain, and many approaches have been utilized to study the mechanisms controlling helper T cell development and function. These have ranged from the use of in vitro cell culture systems to whole animals. Cell culture systems, especially those using cell lines, offer the benefit of ease of use and the ability to generate large amount of material to do sophisticated biochemical analyses. However, they suffer from their limited ability to reproduce the actual conditions occurring in an immune response. In contrast, whole animal experiments offer the benefit of relevance, but they can suffer from difficulties in manipulation and the ability to perform precise controls in addition to their large costs and ethical implications. Nevertheless, the vast majority of helper T cells studies today still require the use of whole animal experiments involving primary T cells because of the inability of cell lines to duplicate the exact steps occurring in the whole animal. Therefore, it is essential to utilize cost effective approaches that are highly informative. 
Genetics is one powerful tool to study helper T cell development and function, yet traditional methods involving gene knockouts or transgenes are time consuming and expensive so they are often out of reach of small labs. However, retroviral transduction offers a powerful, rapid and, cost effective genetic approach to study the mechanisms of specific gene products. Therefore, it is commonly used in papers studying helper T cell development and function. 
We have optimized a procedure for retroviral transduction of helper T cells. It utilizes the pMIG (Murine stem cell virus-Internal ribosomal entry site-Green fluorescent protein) retroviral expression vector, in which the gene of interest can be cloned and thereby expressed from the retrovirus long terminal repeat (LTR) 4. In addition, downstream of the inserted gene of interest is an internal ribosome entry sequence (IRES) followed by the green fluorescent protein (GFP) gene so transduced cells can easily be followed by their expression of GFP. The vector was originally derived from the Murine Stem Cell Virus (MSCV) vectors, which contain mutations in repressor binding sites in the LTRs making them resistant to silencing and thus, giving high expression in many cell types including helper T cells 5,6. Production of high titer retrovirus requires a simple transient transfection protocol of human embryonic kidney (HEK) 293T cells with the MIG vector and a helper virus vector that expresses the retroviral GAG, Pol, and Env genes. For this the pCL-Eco helper virus vector 7 works well in producing high titer replication incompetent retroviruses. 
Here these protocols for retroviral production and transduction of primary murine T cells are described in addition to some of our results using this approach to study miRNA regulation of gene expression controlling helper T cell differentiation. miRNAs are small RNAs of approximately 22 nucleotides in length that post-transcriptionally regulate gene expression by targeting homologous sequences in protein encoding messenger RNAs and suppressing translation and inducing message instability 8,9. miRNAs play critical roles in developmental gene regulation. They are essential in the earliest stages of development, as embryos that cannot produce miRNAs die at a very early stage 10. In addition miRNAs are important later on in the development of many tissues. They are thought to function by fine-tuning the expression of genes required for developmental programs 1. In helper T cells miRNAs play multiple roles and are required for regulatory T cell (Treg) development 11-14. We used retroviral transduction as a means to dissect the mechanisms of miRNA regulation of Treg differentiation 15. Through such studies important individual miRNAs were determined by retroviral-mediated overexpression. Subsequently, relevant genes regulated by these miRNAs were identified in order to understand the molecular pathways regulated by miRNAs in helper T cell differentiation.

<table><tbody><tr><td>RPMI</td><td>Sigma</td><td>R8758</td><td></td></tr><tr><td>DMEM</td><td>Sigma</td><td>D5671</td><td></td></tr><tr><td>Penicillin Streptomycin solution</td><td>Sigma</td><td>P4333</td><td></td></tr><tr><td>L-Glutamine</td><td>Sigma</td><td>G7513</td><td></td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Sigma</td><td>M3148</td><td></td></tr><tr><td>DPBS</td><td>Sigma</td><td>D8537</td><td></td></tr><tr><td>MIG vector</td><td>Addgene</td><td>Plasmid 9094</td><td></td></tr><tr><td>pCL-Eco vector</td><td>Addgene</td><td>Plasmid 12371</td><td></td></tr><tr><td>Cell strainer</td><td>BD Falcon</td><td>352350</td><td></td></tr><tr><td>Magnetic beads mouse CD4 cell kit&amp;nbsp;</td><td>Invitrogen (Dynabeads)</td><td>11415D</td><td></td></tr><tr><td>Streptavidin Beads&amp;nbsp;</td><td>Miltenyi Biotech</td><td>130-048-102</td><td></td></tr><tr><td>MS cell separation columns</td><td>Miltenyi Biotech</td><td>130-042-201</td><td></td></tr><tr><td>LS cell separation columns</td><td>Miltenyi Biotech</td><td>130-042-401</td><td></td></tr><tr><td>CD25 Biotenylated MAb&amp;nbsp;</td><td>BD Biosciences</td><td>85059</td><td>clone 7D4</td></tr><tr><td>CD62L Biotenylated MAb&amp;nbsp;</td><td>BD Biosciences</td><td>553149</td><td>clone MEL-14</td></tr><tr><td>Polybrene (Hexadimethrine Bromide)</td><td>Sigma</td><td>107689</td><td></td></tr><tr><td>Anti-CD3</td><td>eBiosciences</td><td>16-0031-85</td><td>clone 145-2C11&amp;nbsp;</td></tr><tr><td>Anti-CD28</td><td>eBiosciences</td><td>16-0281-85</td><td>clone 37.51&amp;nbsp;</td></tr><tr><td>Anti-IL-4</td><td>BD Biosciences</td><td>559062</td><td>clone 11B11</td></tr><tr><td>Anti-IFN-gamma</td><td>BD Biosciences</td><td>559065</td><td>clone XMG1.2</td></tr><tr><td>Anti-IL-2</td><td>BD Biosciences</td><td>554425</td><td>cloneJES6-5H4</td></tr><tr><td>Recombinant IL-12 p70</td><td>eBiosciences</td><td>14-8121</td><td></td></tr><tr><td>Recombinant IL-4</td><td>BD Biosciences</td><td>550067</td><td></td></tr><tr><td>Recombinant TGF-beta</td><td>eBiosciences</td><td>14-8342-62</td><td></td></tr><tr><td>Recombinant IL-6</td><td>eBiosciences</td><td>14-8061</td><td></td></tr><tr><td>Recombinant IL-2</td><td>eBiosciences</td><td>14-8021</td><td></td></tr><tr><td>PMA&amp;nbsp;</td><td>Sigma</td><td>P8139</td><td></td></tr><tr><td>Ionomycin</td><td>Sigma</td><td>I0634</td><td></td></tr><tr><td>Brefeldin A</td><td>eBiosciences</td><td>00-4506</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>16005</td><td>Paraformaldehyde is toxic so use appropriate caution when handling&amp;nbsp;</td></tr><tr><td>Foxp3 staining buffer set</td><td>eBiosciences</td><td>00-5523</td><td></td></tr><tr><td>Anti-CD4&amp;nbsp; FITC</td><td>eBiosciences</td><td>11-0041</td><td>clone GK1.5&amp;nbsp;</td></tr><tr><td>Anti-CD8a perCP-cy5.5</td><td>eBiosciences</td><td>45-0081-80</td><td>clone 53-6.7&amp;nbsp;</td></tr><tr><td>Anti-MHCII PE</td><td>eBiosciences</td><td>12-0920</td><td>&amp;nbsp;clone HIS19</td></tr><tr><td>Anti-CD25 PE</td><td>eBiosciences</td><td>12-0251-82</td><td>&amp;nbsp;clone PC61.5&amp;nbsp;</td></tr><tr><td>Anti-CD62L PE</td><td>eBiosciences</td><td>12-0621-82</td><td>clone MEL-14&amp;nbsp;</td></tr><tr><td>Anti-CD44 APC</td><td>eBiosciences</td><td>17-0441</td><td>clone IM7&amp;nbsp;</td></tr><tr><td>Anti-IFN-gamma FITC&amp;nbsp;</td><td>eBiosciences</td><td>11-7311-81</td><td>clone XMG1.2</td></tr><tr><td>Anti-IL-4 PE</td><td>BD Biosciences</td><td>554435</td><td>clone 11B11</td></tr><tr><td>Anti-IL-9 PE or APC</td><td>eBiosciences/Biolegend</td><td>50-8091-82/514104</td><td>clone RM9A4</td></tr><tr><td>Anti-IL-17a PE</td><td>BD Biosciences</td><td>559502</td><td>clone TC11-18H10</td></tr><tr><td>Anti-Foxp3&amp;nbsp; APC or PE</td><td>eBiosciences</td><td>17-5773-82/12-5773-80&amp;nbsp;</td><td>clone FJK-16s</td></tr><tr><td>NaCl</td><td>Sigma</td><td>S7653</td><td></td></tr><tr><td>KCl</td><td>Sigma</td><td>P9333</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;-2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>71643</td><td></td></tr><tr><td>Dextrose/Glucose</td><td>Sigma</td><td>G7021</td><td></td></tr><tr><td>HEPES, free acid</td><td>Sigma</td><td>H3375</td><td></td></tr><tr><td>NH&lt;sub&gt;4&lt;/sub&gt;Cl</td><td>Sigma</td><td>A9434</td><td></td></tr><tr><td>Disodium EDTA</td><td>Sigma</td><td>D2900000</td><td></td></tr><tr><td>KHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>237205</td><td></td></tr><tr><td>&amp;nbsp;CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;nbsp;</td><td>Sigma</td><td>C5670</td><td></td></tr></tbody></table>

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.

The success of this experimental system requires highly pure populations of T cells and high titer retrovirus preparations. Representative results are shown here as examples of successful experiments. Figure 1 shows the typical purity of pre- and post-selected populations at each stage of the na&#239;ve helper T cell isolation protocol. Figure 2 and 3 illustrate the analysis of retrovirus production through GFP expression in the transfected HEK 293T cells (Figure 2) and transduced T cells (Figure 3). Transfection efficiencies of the HEK 293T cells can vary significantly with different retroviral constructs, but this often doesn&#39;t correlate with the level of retrovirus production observed with the number of GFP+ T cells. Also, the number of GFP+ T cells can vary depending on the polarization conditions. Furthermore, the mean expression level of GFP and the inserted gene can vary depending on the number of virus copies integrated, the effect of the integration site on transcription, and post-transcriptional regulatory mechanisms affecting the viral transcript.
Finally, Figure 4 shows some typical results we have observed with helper T cell differentiation when the miRNAs miR-15b/16 are overexpressed. These results show some of the variability that can occur within an individual experiment so true effects must be substantiated by statistical analysis of multiple repeat experiments using different preparations of helper T cells. In these experiments Th2 responses can be difficult to observe in the C57BL/6 line used here because they are prone to Th1 responses. Likewise, IL-9 staining can be difficult to detect above background. Therefore, it is imperative to do isotype controls and set up proper compensation to ensure correct gating of cytokine expression. In our results we have found that miR-15b/16 enhances iTreg induction by inhibiting the mTOR signaling pathway through suppressing the expression of the components Rictor and mTOR 15. miR-15b/16 can sometimes influence Th0, Th1, and Th17 differentiation in individual experiments, but there is no significant effect when examined in multiple repeat experiments. In contrast miR-15b/16 overexpression does significantly suppress Th9 differentiation (see reference 18).
<img alt="Figure 1" src="/files/ftp_upload/54698/54698fig1.jpg" /> 
Figure 1. Typical purity of helper T cells at each stage of isolation. Representative flow cytometry results of the indicated antigens are shown from the gate of live cells designated in the Forward Scatter (FSC) and Side Scatter (SSC) plots. (A) Pre- and post-CD4 negative selection. Expression profiles of CD4, CD8a, and MHCII are shown. These illustrate the enrichment of helper T cells and the loss of cytotoxic T cells and MHC class II expressing cells. A good purification should result in ~90% CD4+ T cells at this stage. (B) CD25 selection. On the left are the expression profiles of CD4, CD8a, and MHCII, and on the right are the CD4 and CD25 expression profiles pre and post selection. At this point &#62;95% of CD25 negatively selected cells should be CD4+ CD25-. (C) CD62L selection. CD4, CD8a, and MHCII expression profiles are shown on the left. On the right the expression profiles for CD62L and CD44 are shown for pre- and post-CD62L selected cells along with CD4 and CD62L expression profile of post selected cells. After CD62L selection virtually all memory cells (CD44+) are removed leaving a highly enriched population of na&#239;ve helper T cells that contains 10-15% effector cells (CD62Llow). For all FACS profiles, equivalent settings and scales for a specific parameter were maintained throughout. Numbers represent the percentage of cells within a gated population. The slight decrease in size of the cells after the initial selection is presumably due to mechanical stress during the protocol. <a href="http://cloudflare.jove.com/files/ftp_upload/54698/54698fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54698/54698fig2.jpg" /> 
Figure 2. Analysis of retrovirus-transfected HEK 293T cells. GFP expression is shown in HEK 293T cells that were either untransfected or transfected and analyzed after collection of viral culture supernatants. GFP analysis was done on live cells from the gate on the FSC and SSC plot in the first panel. Numbers represent the percentage of GFP+ cells within the gated region. Typical transfection efficiencies range between 30-90%. <a href="http://cloudflare.jove.com/files/ftp_upload/54698/54698fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54698/54698fig3.jpg" /> 
Figure 3. Analysis of retrovirus transduced helper T cells. GFP expression is shown in retroviral-transduced helper T cells after differentiation in Th0, Th1, Th2, Th9, Th17, and Treg polarization conditions for three days. Analysis was gated on live and activated cells indicated in the FSC/SSC panel. Transduction efficiencies can vary between 10-75% depending on the construct and the polarization conditions. Likewise, the mean fluorescent intensity of GFP expression can vary. <a href="http://cloudflare.jove.com/files/ftp_upload/54698/54698fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54698/54698fig4.jpg" /> 
Figure 4. Effect of miR-15b/16 overexpression on helper T cell differentiation in different polarization conditions. Representative cytokine profiles are shown on the GFP+ population of cells from Figure 3. <a href="http://cloudflare.jove.com/files/ftp_upload/54698/54698fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/54698/54698table1.jpg" /> 
Table 1: Buffers used in these protocols. <a href="http://cloudflare.jove.com/files/ftp_upload/54698/54986table1.xlsx" target="_blank">Please click here to download this table as an Excel spreadsheet.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td colspan="3">Helper T cell polarization conditions</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Th0</td>
			<td>anti-IL-4&#160;</td>
			<td>5 &#181;g/ml</td>
		</tr>
		<tr>
			<td></td>
			<td>anti-IFN-&#947;&#160;</td>
			<td>5 &#181;g/ml</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Th1</td>
			<td>recombinant-IL-12&#160;</td>
			<td>20 ng/ml</td>
		</tr>
		<tr>
			<td></td>
			<td>anti-IL-4&#160;</td>
			<td>5 &#181;g/ml</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Th2</td>
			<td>recombinant IL-4&#160;</td>
			<td>40 ng/ml&#160;</td>
		</tr>
		<tr>
			<td></td>
			<td>anti-IFN-&#947;&#160;</td>
			<td>5 &#181;g/ml</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Th9</td>
			<td>recombinant TGF-&#946;&#160;&#160;&#160;</td>
			<td>2.5 ng/ml</td>
		</tr>
		<tr>
			<td></td>
			<td>recombinant IL-4</td>
			<td>40 ng/ml&#160;</td>
		</tr>
		<tr>
			<td></td>
			<td>anti-IFN-&#947;&#160;</td>
			<td>10 &#181;g/ml</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Th17</td>
			<td>recombinant TGF-&#946;</td>
			<td>2.5 ng/ml&#160;</td>
		</tr>
		<tr>
			<td></td>
			<td>recombinant IL-6</td>
			<td>50 ng/ml&#160;</td>
		</tr>
		<tr>
			<td></td>
			<td>anti-IFN-&#947;</td>
			<td>5 &#181;g/ml</td>
		</tr>
		<tr>
			<td></td>
			<td>anti-IL-4&#160;</td>
			<td>5 &#181;g/ml</td>
		</tr>
		<tr>
			<td></td>
			<td>anti-IL-2&#160;</td>
			<td>5 &#181;g/ml</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tregs</td>
			<td>recombinant TGF-&#946;</td>
			<td>2.5 ng/ml&#160;</td>
		</tr>
		<tr>
			<td></td>
			<td>recombinant IL-2</td>
			<td>5 ng/ml&#160;</td>
		</tr>
	</tbody>
</table>
Table 2: Helper T cell subset polarization conditions.

Watch this Scientific Journal Video about Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function at JoVE.com

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

retroviral-transduction-helper-t-cells-as-genetic-approach-to-study

Research

JoVE Journal

Immunology and Infection

11.0K Views.  The Royal Veterinary College. 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.

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

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 ...

Retroviral Scanning: Mapping MLV Integration Sites to Define Cell-specific Regulatory Regions

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV integration sites can be used as functional markers of active regulatory elements. Here, we present a retroviral scanning tool, which allows the genome-wide identification of cell-specific enhancers and promoters. Briefly, the target cell population is transduced with an mLV-derived vector and genomic DNA is digested with a frequently cutting restriction enzyme. After ligation of genomic fragments with a compatible DNA linker, linker-mediated polymerase chain reaction (LM-PCR) allows the amplification of the virus-host genome junctions. Massive sequencing of the amplicons is used to define the mLV integration profile genome-wide. Finally, clusters of recurrent integrations are defined to identify cell-specific regulatory regions, responsible for the activation of cell-type specific transcriptional programs.
The retroviral scanning tool allows the genome-wide identification of cell-specific promoters and enhancers in prospectively isolated target cell populations. Notably, retroviral scanning represents an instrumental technique for the retrospective identification of rare populations (e.g. somatic stem cells) that lack robust markers for prospective isolation.

Here, we describe a protocol for genome-wide mapping of the integration sites of Moloney murine leukemia virus-based retroviral vectors in human cells.

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV ...

Genetics

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies 1. CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines 2-7. However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies.
TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases 8-10. However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic 11-13, HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture 14-16. Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs.
Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.

Generation of T lymphocytes from induced pluripotent stem (iPS) cells gives an alternative approach of using embryonic stem cells for T cell-based immunotherapy. The method shows that by utilizing either in vitro or in vivo induction system, iPS cells are able to differentiate into both conventional and antigen-specific T lymphocytes.

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

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.

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 ...

Co-Culture and Transduction of Murine Thymocytes on Delta-Like 4-Expressing Stromal Cells to Study Oncogenes in T-Cell Leukemia

Transduced mouse immature thymocytes can be differentiated into T cells&#160;in vitro&#160;using the delta-like 4-expressing bone marrow stromal cell line co-culture system (OP9-DL4). As retroviral transduction requires dividing cells for transgene integration, OP9-DL4 provides a suitable in vitro environment for cultivating hematopoietic progenitor cells. This is particularly advantageous when studying the effects of the expression of a specific gene during normal T cell development and leukemogenesis, as it allows researchers to circumvent the time-consuming process of generating transgenic mice. To achieve successful outcomes, a series of coordinated steps involving the simultaneous manipulation of different types of cells must be carefully performed. Although these are very well-established procedures, the lack of a common source in the literature often means a series of optimizations are required, which can be time-consuming. This protocol has been shown to be efficient in transducing primary thymocytes followed by differentiation on OP9-DL4 cells. Detailed here is a protocol that can serve as a quick and optimized guide for the co-culture of retrovirally transduced thymocytes on OP9-DL4 stromal cells.

This protocol describes the isolation of double-negative thymocytes from the mouse thymus followed by retroviral transduction and co-culture on the delta-like 4-expressing bone marrow stromal cell line co-culture system (OP9-DL4) for further functional analysis.

Transduced mouse immature thymocytes can be differentiated into T cells&#160;in vitro&#160;using the delta-like 4-expressing bone marrow stromal cell line ...

Cancer Research

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

Deciphering Early Follicular Helper T-Cell Commitment in Acute LCMV Infection

Accessing Early Differentiation of Virus-Specific Follicular Helper CD4+ T Cell in Acute LCMV-Infected Mice

Follicular Helper T (TFH) cells are perceived as an independent CD4+ T cell lineage that assists cognate B cells in producing high-affinity antibodies, thus establishing long-term humoral immunity. During acute viral infection, the fate commitment of virus-specific TFH cells is determined in the early infection phase, and investigations of the early-differentiated TFH cells are crucial in understanding T cell-dependent humoral immunity and optimizing vaccine design. In the study, using a mouse model of acute lymphocytic choriomeningitis virus (LCMV) infection and the TCR-transgenic SMARTA (SM) mouse with CD4+ T cells specifically recognizing LCMV glycoprotein epitope I-AbGP66-77, we described procedures to access the early fate commitment of virus-specific TFH cells based on flow cytometry stainings. Furthermore, by exploiting retroviral transduction of SM CD4+ T cells, methods to manipulate gene expression in early-differentiated virus-specific TFH cells are also provided. Hence, these methods will help in studies exploring the mechanism(s) underlying the early commitment of virus-specific TFH cells.

Author Spotlight: Elucidating the Pathways of TFH Cell Differentiation in Acute LCMV Challenges

The current study showcases protocols for assessing the early fate commitment of virus-specific TFH cells and manipulating gene expression in these cells.

Follicular Helper T (TFH) cells are perceived as an independent CD4+ T cell lineage that assists cognate B cells in producing high-affinity antibodies, ...

Retroviral Transduction of Guide RNA for Gene Editing in Th17 Differentiation

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

T helper cells that produce IL-17A, known as Th17 cells, play a critical role in immune defense and are implicated in autoimmune disorders. CD4 T cells can be stimulated with antigens and well-defined cytokine cocktails in vitro to mimic Th17 cell differentiation in vivo. Research has been conducted extensively on the Th17 differentiation regulation mechanisms using the in vitro Th17 polarization assay.
Conventional Th17 polarization methods typically involve obtaining na&#239;ve CD4 T cells from genetically modified mice to study the effects of specific genes on Th17 differentiation and function. These methods can be time-consuming and costly and may be influenced by cell-extrinsic factors from the knockout animals. Thus, a protocol using retroviral transduction of guide RNA to introduce gene knockout in CRISPR/Cas9 knockin primary mouse T cells serves as a very useful alternative approach. This paper presents a protocol to differentiate na&#239;ve primary T cells into Th17 cells following retroviral-mediated gene targeting, as well as the subsequent flow cytometry analysis methods for assaying infection and differentiation efficiency.

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.

T helper cells that produce IL-17A, known as Th17 cells, play a critical role in immune defense and are implicated in autoimmune disorders. CD4 T cells ...

Efficient Retroviral Transduction and Competitive Homing for Investigating GPCR-Mediated T-Cell Localization in Diverse Tissue Microenvironments

Understanding how G-protein coupled receptor (GPCR) expression affects cell positioning within diverse tissue microenvironments is essential for elucidating immune cell trafficking mechanisms. We present a competitive homing assay designed to study GPCR-mediated T-cell localization to organs expressing their cognate chemoattractant ligands, applicable for both short-term and long-term studies. The approach involves an improved protocol for recombinant murine stem cell virus (MSCV) transduction of T cells to express the GPCR of interest or a control construct, followed by competitive homing in recipient mice. Cell distribution across different organs is analyzed using flow cytometry and/or confocal microscopy. In short-term experiments (10-12 h), confocal microscopy revealed distinct cell localization patterns, including to alveoli, bronchi submucosa, venous sites, and interstitium in the lung, as well as the epithelium lining the trachea, stomach, and uterine horn. In long-term studies (1-7 weeks), flow cytometry provided insights into preferential cell accumulation, revealing dynamic changes and potential maturation or repositioning within tissues over time. This competitive homing assay is a robust tool for studying GPCR-mediated cell positioning, offering valuable insights into tissue-specific distribution and potential applications in immunology and therapeutic research.

We present improved protocols for retroviral transduction of trafficking receptors and competitive homing to study receptor-mediated organ- and microenvironment-specific lymphocyte positioning. This method offers valuable insights into immune cell trafficking mechanisms and has potential applications in future basic and therapeutic research.