The authors would like to thank all members of the Reynolds lab at Rosalind Franklin University of Medicine and Science, and the Chen Dong lab at the University of Texas MD Anderson Cancer Center for optimization of this protocol. This work was supported by a grant to J.M.R. from the National Institutes of Health (K22AI104941).

All experimental procedures are performed using protocols approved by the office of Environmental Health and Safety at the Rosalind Franklin University of Medicine and Science. C57BL/6 mice (purchased from NCI) used for this protocol were housed under specific pathogen-free conditions, and all animal experiments were performed using protocols approved by the Institutional Animal Care and Use Committee (IACUC) at the Rosalind Franklin University of Medicine and Science.
1. Preparation of Instruments, Supplies, and Reagents
<ol>
	<li>Coat 48-well or 24-well plates with anti-CD3 and anti-CD28 (Table 1) in sterile PBS, cover and wrap in parafilm to prevent evaporation and contamination, and then incubate O/N at 4 oC the day before T cell isolation. Alternatively, coat wells 1 h at 37 oC. Perform Th0, Th1, Th2, and iTreg conditions by coating both anti-CD3 and anti-CD28 at 1&#956;g/ml in 0.5-1 ml total volume (48-well plate). For Th17, we typically coat anti-CD3 at 2 &#956;g/ml and anti-CD28 at 1 &#956;g/ml. 
	NOTE: We have found that a lower anti-CD3 concentration is optimal for Th1, Th2, and iTreg generation while a higher anti-CD3 concentration is optimal for Th17 generation. Thus, anti-CD3 and anti-CD28 concentrations should be titrated and optimized for individual laboratories (Table 2).</li>
	<li>Sterilize a set of dissection tools (scissors and forceps) by autoclave or disinfectant solution. A tube or small beaker containing 70% ethanol will be needed for quick sterilization of tools between dissections. Finally, a spray bottle containing 70% ethanol will be needed for sterilizing the surface of the mouse before dissection.</li>
	<li>Prepare a dissection surface by wrapping a flat piece of Styrofoam or corkboard in aluminum foil. Dissecting pins or needles are used to fix the animal in place.</li>
	<li>Prepare dishes or plates for the collection of tissues. If pooling tissues, separate 60mm dishes containing 3ml of sterile PBS + 1% FBS (PBS+) for spleen and lymph node tissues is sufficient. If keeping tissues from individual mice separate, prepare a 24-well plate containing 1ml of PBS+ in the desired number of wells.</li>
	<li>Prepare complete RPMI media for cell culture and pre-warm before use (Table 1). 
	NOTE: We routinely use RPMI but other media such as IMDM and DMEM may be equally effective or superior depending on the lab and the experiment.</li>
	<li>Cut 2x2 cm squares of nylon mesh with a 120 &#956;m pore size and autoclave to sterilize (Table 1).</li>
	<li>Optional: if using the high-speed magnetic cell sorting system such as autoMACS for CD4+ cell enrichment, pre-warm running and rinsing buffers in a 37 oC water bath to prevent damage to the machine.</li>
	<li>NOTE: Enrichment is recommended to increase the sorting yield and decrease sorting time.</li>
	<li>All preparation steps, except for dissection, should be performed in a sterile tissue culture hood.</li>
</ol>
2. Isolation of Lymph Nodes and Spleen from Mice
<ol>
	<li>Euthanize the required number of animals using an institutionally-approved technique. Use C57BL/6 female mice, age 5-10 wks, due to their high percentage of na&#239;ve T cells and lower body fat in comparison to males or to older mice. 
	NOTE: Cells derived from male mice will perform similarly in vitro and the protocol steps remain the same. Female mice, 5-10 wks old, will typically yield 3-6 x 106 na&#239;ve CD4+ T cells per mouse. However, using different strains of mice may affect yield and performance. For example, cells from C57BL/6 mice are better for generating Th1 cells while cells from Balb.c mice are better for generating Th2 cells.</li>
	<li>Spread the limbs of the animal and pin them in place as shown in Figure 1. Cut the skin longitudinally from the anus to the chin while being careful not to puncture deeper tissue.</li>
	<li>Spread open the skin with forceps and pin the skin in place to allow easy access to lymph nodes as shown in Figure 1.</li>
	<li>Harvest the accessible lymph nodes from the locations shown in Figure 1. Grabbing the lymph node with a pair of forceps while separating tissue with another pair of forceps will allow for easy removal. Place the tissue in the collection dish.</li>
	<li>Harvest the spleen from the location shown in Figure 1. In this case, careful cutting into the peritoneum is needed to gain access to the spleen. Place the tissue in a collection dish.</li>
	<li>Repeat the procedure if more tissues will be harvested. Otherwise proceed to the next steps. At this point, perform all remaining steps on ice until the plating of the T cells after sorting.</li>
</ol>
3. Tissue Processing and CD4+ Enrichment
<ol>
	<li>Process the lymph nodes and spleen in different dishes to increase the yield as lymph node cell preparations do not require erythrocyte lysis, which may result in minor leukocyte death. Therefore, the following steps describe a method for processing splenic and lymph node populations separately followed by combining prior to labeling for sorting.</li>
	<li>Remove the tissue (spleen or pooled lymph nodes) from the collection dish and place in a new 60 mm dish containing 3 ml of PBS+. Place a square of sterile nylon mesh over the tissue using sterilized forceps.</li>
	<li>Take the thumb side of a syringe plunger (3-10 ml) and gently smash the tissue against the mesh. Almost all of the tissue should go into suspension. Alternatively, place the tissue between two autoclaved frosted slides and grinded into suspension.</li>
	<li>Pipette the suspension up and down a few times to break up the remaining soluble clumps. Place a new square piece of nylon mesh over the opening of a 15 ml conical tube and filter the suspension through to remove the remaining debris.</li>
	<li>Repeat steps 2-4 for additional lymph node and splenic tissue.</li>
	<li>Once the suspension has been filtered into a 15ml tube fill the remainder of the tube with PBS+ and invert a few times. Centrifuge the cells at 475 x g for 5 min at 4 oC.</li>
	<li>For the splenic cells, aspirate the supernatant following centrifugation and resuspend the cell pellet in 1 ml of ice-cold 1X ACK solution per spleen for 1 min to lyse erythrocytes. Then add 10 ml of PBS+ on top of the ACK, invert the tube, and centrifuge 475 x g for 5 min at 4 oC.</li>
	<li>For lymph node cells, aspirate the supernatant and resuspend in 2ml of PBS+. If desired these can be combined with the splenic cells after their ACK lysis and washing step.</li>
	<li>Aspirate the supernatant after centrifugation of the splenocytes and resuspend in another 10 ml of PBS+. At this point the lymph node suspension can be added on top of the spleen suspension if tissue pooling is required for sorting. Centrifuge the tube again at 475 x g for 5 min at 4 oC.</li>
	<li>The cell pellet can now be prepared for CD4 enrichment. If this step will not be performed, proceed to the sorting preparation steps.</li>
	<li>For CD4 enrichment using the high-speed magnetic cell sorting system, resuspend the cellular pellet with CD4 beads. Typically 15 &#956;l of beads diluted in 85 &#956;l of PBS+ is used to label the tissues from 1 mouse. Ramp up the volume of bead mixture as needed, resuspend the pellet, and incubate for 15-30 min at 4 oC.</li>
	<li>Wash the cells with 10 ml of PBS+, pass the suspension through nylon into a new 15 ml tube to remove debris, and centrifuge again at 475 x g for 5 min at 4 oC.</li>
	<li>Resuspend the pellet in 100 &#956;l of PBS+ per mouse and proceed to positive selection on the high-speed magnetic cell sorting system. If using a manual separation system, follow the manufacturer&#8217;s instructions for enrichment. Alternatively, resuspend the samples in FACS buffer: PBS with 1mM EDTA (pH = 8) and 1% BSA, sterile-filtered.</li>
	<li>Remove the positive fraction and wash again with 10ml of PBS+. The enriched CD4+ fraction is now ready to be labeled for sorting.</li>
</ol>
4. Sorting Na&#239;ve CD4+ T cells
<ol>
	<li>Label the cell pellet with PBS+ containing fluorescent antibodies directed against CD62L, CD44, CD25, and CD4. If using the antibodies listed in Table 1, dilutions for each are provided. For staining, a 100 &#956;l volume of the antibody cocktail is used per tissues from one mouse.</li>
	<li>Resuspend the pellet in antibody cocktail and incubate for 15-30 min at 4 oC in the dark.</li>
	<li>Wash the cells with 10ml of PBS+ and centrifuge 475 x g for 5 min at 4 oC.</li>
	<li>Pass the mixture over another nylon filter or cell strainer cap to remove any leftover debris. Pipette the labeled cells into a tube that is compatible for the cell sorter. Keep cells on ice and protected from the light until the sort.</li>
	<li>Prepare collection tubes by pipetting 1-2ml of complete RPMI media or FBS into the bottom of tubes compatible for collection on a cell sorter.</li>
	<li>Sort the na&#239;ve CD4+ T cells as a CD4+CD25-CD62L+CD44- population (Figure 2). Wash the collected cells with complete RPMI media and centrifuge as described above.</li>
	<li>Resuspend the pellet in complete RPMI, count the na&#239;ve cells, and adjust the concentration to 1 x 106 cells/ml.</li>
</ol>
5. Setting Up the In vitro&#160;Differentiation
<ol>
	<li>Aspirate the wells of the anti-CD3 and anti-CD28 coated plates and wash each well with 1ml of sterile PBS (no FBS). For 48-well plates, plate 0.5 x 106 cells/well in 0.5 ml of RPMI. For 24-well plates, culture 1 x 106 cells/well in 1.0 ml of RPMI.</li>
	<li>If testing the influence of a factor such as a drug add the substance to the appropriate wells either before, during, or after the differentiation process depending on the experiment. Next, supplement the culture media with cytokines and blocking antibodies accordingly. Th0: 30 U/ml hIL-2. Th1: 15 ng/ml rmIL-12, 30 U/ml hIL-2, and 5,000 ng/ml 11B11.Th2: 10 ng/ml rmIL-4, 30 U/ml hIL-2, 2,000 ng/ml soluble 37.51, and 5,000 ng/ml XMG1.2. iTreg: 15 ng/ml hTGF&#946;, 30 U/ml hIL-2, 5,000 ng/ml XMG1.2, and 5,000 ng/ml 11B11. Th17: 20 ng/ml rmIL-6, 3 ng/ml hTGF&#946;, 5,000 ng/ml XMG1.2, and 5,000 ng/ml 11B11. 
	NOTE: The conditions presented above were found to be optimal for maximizing cytokine production as measured at the end of the differentiation experiment (Representative Results section). However, each condition should be optimized for individual laboratories (Table 2). Furthermore, the addition of soluble anti-CD28 in addition to the plate-bound anti-CD28 has been found to enhance Th2 cytokine production in our laboratory, but has no effect on other T cell subsets:</li>
	<li>Incubate the plate at 37 oC with 5% CO2 for 4-5 d prior to analysis of gene expression and cytokine production. Alternatively, remove the cells from the TCR stimuli after 2 d, adjusted to 1 x 106 cells/ml with fresh media, and plate in new wells (non-coated) to enhance proliferation and final yield. In this case, fresh polarizing cytokines and antibodies are not needed but 10 U/ml of IL-2 should be added to new media for Th0, Th1, Th2, and iTreg cultures.</li>
</ol>
6. Analysis of Differentiation
<ol>
	<li>For cytokine analysis by flow cytometry, restimulate each well with 10ng/ml PMA and 1,000 ng/ml ionomycin or 1 &#956;g/ml anti-CD3 for 4-6 hr in the presence of a golgi inhibitor such as brefeldin A (Table 1). Perform intracellular cytokine staining depending on the experiment (Figure 3). 
	NOTE: Stain non-lineage-specific cytokines or transcription factors, such as IL-4 (not shown) and IFN&#947; (Figure 3) for Th17 cultures, for each condition as negative controls and as an indicator of differentiation efficiency. Furthermore, stain Foxp3 for Th17 cultures as the reciprocal development of Tregs may occur with the addition of TGF&#946;.</li>
	<li>For protein quantification, collect the supernatants from each well and assay by cytokine-specific ELISA (Figure 3). If there is concern that the factor being tested has influenced proliferation compared to control samples, the cells can be washed, counted, normalized, and then restimulated with 1 &#956;g/ml of anti-CD3 for 24 h before collecting supernatants for ELISA assays. If assaying IL-4 from Th2 conditions, washing and restimulating is necessary to remove IL-4 that may be leftover from the initial differentiation culture.</li>
	<li>For gene expression analysis by real time PCR, harvest cells after the incubation period, wash, normalize, and re-stimulate for 2-6 h with 1 &#956;g/ml of anti-CD3 prior to harvesting mRNA (Figure 3).</li>
</ol>

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The authors declare no competing financial interests.

While the spleen contains na&#239;ve Th cells, the proportion of this population in lymph nodes is much higher. Failure to properly identify and remove lymph nodes in this protocol will result in a poor yield of na&#239;ve cells. This can be especially difficult in older mice or male mice that have more fat tissue. As shown in Figure 1, proper fixing and pinning of the animal limbs and skin will allow for easier visualization of the accessible exterior lymph nodes. Once lymph nodes and spleens are proces...

The concept of distinct lineages or subsets of CD4+ T helper (Th) cells has been around since the latter part of the 20th century1. Recognition of cognate antigen in the presence of costimulatory signals results in several rounds of cellular proliferation and the eventual differentiation into effector Th cells. The type of Th cell generated during this process is dependent on the cytokine environment present during activation2. Initially, na&#239;ve Th cells were thought to polarize into 2 distinct lineages following T cell receptor (TCR) activation, costimulatory CD28 ligation, and cytokine signaling. Type 1 helper cells (Th1) are characterized by their effector production of the IFN&#947; cytokine as well as their requirement for IL-12 signaling during the differentiation process3,4. Eventually it was discovered that differentiated Th1 cells have a genetic profile that is most distinctively characterized by the expression of the T box family transcription factor, Tbx21 (T-bet), which is considered the master regulator of the Th1 genetic program5. Furthermore, IL-12 as well as IFN&#947; can promote T-bet expression6,7. In the immune response, Th1 cells are important for the host defense against intracellular pathogens as well as strong promoters of autoimmune inflammation. In contrast, type 2 helper cells (Th2) require IL-4 for their development and their effector cytokines, including IL-4, IL-5, and IL-13, are important for driving B cell responses and are pathogenic in allergy8,9. Similar to Th1 cells, Th2 cells were found to express their own master transcriptional regulator, termed GATA-310,11. Interestingly, the presence of polarizing cytokines and the generation of a specific Th lineage are antagonistic to the development of others2,12, suggesting that only a particular Th subset may become dominant during an immune response.
Since the identification of the Th1 and Th2 lineages, further work has demonstrated even more unique subsets of T helper cells, including follicular helper (TFH), IL-9-producing (Th9), and IL-22-producing (Th22)(recently reviewed in13). For the purposes of in vitro differentiation experiments, this protocol will focus only on two additional Th subsets, termed regulatory T cells (Treg) and IL-17-producing CD4+ T cells (Th17). CD25+ regulatory T cells can occur naturally (nTreg) in the thymus; na&#239;ve Th cells may also be induced (iTreg) to become regulatory in the periphery (reviewed in14,15). Both types of Tregs express a characteristic transcription factor, termed forkhead box P3 (Foxp3), which is critical for their effector suppression mechanisms that include soluble anti-inflammatory mediator production, IL-2 consumption, and cell contact-dependent mechanisms14,15. The lack of Foxp3 expression results in a severe, multi-organ autoimmune disorder termed immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX), demonstrating the critical role of this Th subset in resolving inflammation and regulating peripheral tolerance to self antigens16. In vitro, na&#239;ve CD4+ T helper cells up-regulate Foxp3 and become committed to the Treg program upon stimulation with IL-2 and TGF-&#946;14,15. There may be moderate to considerable plasticity in CD4+ T cell lineages, especially when considering only cytokine production (reviewed in 17,18). However, for the purposes of in vitro differentiation protocols, we will be discussing each subset as a unique lineage.
Recently, a subset of Th17 cells that produces the IL-17 cytokine was identified as a unique lineage with pro-inflammatory functions that are particularly pathogenic during autoimmune inflammation19-21. Th17 cells express a unique transcription factor, termed retinoid-related orphan receptor gamma t (ROR&#947;t) that coordinates the Th17 genetic program22. TGF&#946; is important for the generation of Th17 lineage through the induction of ROR&#947;t. However, the effect of TGF&#946; signaling is believed to only induce Th17 commitment upon synergizing with IL-6 (reviewed in12). Further studies have shown that a variety of other signals that can positively regulate Th17 commitment, including IL-1&#946;, increased sodium, and TLR signaling23-26. Other reports have suggested that the pathogenic Th17 cells in vivo are the ones that actually bypass TGF&#946; signaling and instead rely on a combination of IL-1, IL-6, and IL-23 for their differentiation27. Thus, Th17 cells may be derived from a variety of signaling pathways; for the purposes of this protocol, the commonly-used (TGF&#946; and IL-6) pathway for Th17 lineage commitment will be presented.
The differentiation protocols described below for all effector lineages relies on fixed antibody as stimuli for the TCR and CD28 throughout the entire course of the experiment. However, others have demonstrated that TCR activation with antigen-presenting cells28 or cross-linking anti-CD3 and anti-CD28 antibodies with hamster antibody for 2 days29 are also highly effective means of inducing the generation of various Th subsets. The protocol presented here builds on previously reported methods for isolating murine CD4+ T cells from secondary lymphoid organs30 and generating Th17 cells31. One major difference is that this protocol relies on the use of a cell sorter to isolate na&#239;ve CD4+ T cells from lymphoid tissues. However, many companies now offer rapid separation kits that can enrich for na&#239;ve CD4+ T cells, which may be able to bypass the requirement for sorting depending on the experiment. The methods and reagents presented in this protocol are what we routinely use and find to be the most effective. However, keep in mind that alternative reagents and methodologies exist for many of the steps presented below and it is up to the individual lab to determine what will work best for their purposes.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Complete RPMI:</td>
			<td></td>
			<td></td>
			<td>Warm in a 37 oC water bath before use</td>
		</tr>
		<tr>
			<td>RPMI 1640 Media</td>
			<td>Life Technologies</td>
			<td>11875119</td>
			<td></td>
		</tr>
		<tr>
			<td>10 % FBS</td>
			<td>Life Technologies</td>
			<td>26140-079</td>
			<td></td>
		</tr>
		<tr>
			<td>1000X 2-mercaptoethanol</td>
			<td>Life Technologies</td>
			<td>21985023</td>
			<td></td>
		</tr>
		<tr>
			<td>100X Pen/Strep</td>
			<td>Life Technologies</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>100X L-glutamine</td>
			<td>Life Technologies</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>120 micron nylon mesh</td>
			<td>Amazon</td>
			<td>CMN-0120-10YD</td>
			<td>Cut into 2 cm2 squares and autoclave</td>
		</tr>
		<tr>
			<td>Alternative: 100 micron cell strainers</td>
			<td>Fisher</td>
			<td>08-771-19</td>
			<td>Alternative to cutting nylon mesh</td>
		</tr>
		<tr>
			<td>autoMACS running buffer</td>
			<td>Miltenyi</td>
			<td>130-091-221</td>
			<td>Warm in a 37 oC water bath before use</td>
		</tr>
		<tr>
			<td>autoMACS rinsing solution</td>
			<td>Miltenyi</td>
			<td>130-091-222</td>
			<td>Warm in a 37 oC water bath before use</td>
		</tr>
		<tr>
			<td>CD4 beads</td>
			<td>Miltenyi</td>
			<td>130-049-201</td>
			<td></td>
		</tr>
		<tr>
			<td>ACK lysis buffer</td>
			<td>Life Technologies</td>
			<td>A10492-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytokines:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Human (h) IL-2</td>
			<td>Peprotech</td>
			<td>200-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant mouse (rm) IL-4</td>
			<td>Peprotech</td>
			<td>214-14</td>
			<td></td>
		</tr>
		<tr>
			<td>rmIL-6</td>
			<td>R &#38; D Systems</td>
			<td>406-ML-025</td>
			<td></td>
		</tr>
		<tr>
			<td>rmIL-12</td>
			<td>Peprotech</td>
			<td>210-12</td>
			<td></td>
		</tr>
		<tr>
			<td>hTGFb</td>
			<td>R &#38; D Systems</td>
			<td>240-B-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2C11 (anti-CD3)</td>
			<td>BioXcell</td>
			<td>BE0001-1</td>
			<td></td>
		</tr>
		<tr>
			<td>37.51 (anti-CD28)</td>
			<td>BioXcell</td>
			<td>BE0015-1</td>
			<td></td>
		</tr>
		<tr>
			<td>11B11 (anti-IL-4)</td>
			<td>BioXcell</td>
			<td>BE0045</td>
			<td></td>
		</tr>
		<tr>
			<td>XMG1.2 (anti-IFNg)</td>
			<td>BioXcell</td>
			<td>BE0055</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CD62L-FITC</td>
			<td>BioLegend</td>
			<td>104406</td>
			<td>Use at 1:100</td>
		</tr>
		<tr>
			<td>anti-CD25-PE</td>
			<td>BioLegend</td>
			<td>102008</td>
			<td>Use at 1:400</td>
		</tr>
		<tr>
			<td>anti-CD4-PerCP</td>
			<td>BioLegend</td>
			<td>100434</td>
			<td>Use at 1:1000</td>
		</tr>
		<tr>
			<td>anti-CD44-APC</td>
			<td>BioLegend</td>
			<td>103012</td>
			<td>Use at 1:500</td>
		</tr>
		<tr>
			<td>Phorbol&#160; 12-myristate 13 acetate (PMA)</td>
			<td>Sigma-Aldrich</td>
			<td>P-8139</td>
			<td>Prepare a stock at 0.1 mg/ml in DMSO and freeze aliquots at -20 oC</td>
		</tr>
		<tr>
			<td>Ionomycin</td>
			<td>Sigma-Aldrich</td>
			<td>I-0634</td>
			<td>Prepare a stock at 0.5 mg/ml in DMSO and freeze aliquots at -20 oC</td>
		</tr>
		<tr>
			<td>Brefeldin A</td>
			<td>eBioscience</td>
			<td>00-4506-51</td>
			<td>Use at 1:1000</td>
		</tr>
	</tbody>
</table>

mouse na&#239;ve cd4+ t cell isolation and in vitro differentiation into t cell subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

The time point for the analysis of differentiation can vary depending on the Th condition being tested as well as the strength of T cell receptor activation. After 2-3 days of differentiation, cells can be visualized by light microscopy to determine the extent of T cell proliferation. Wells exhibiting extensive proliferation and clumping of cells will most likely be ready for analysis at day 4. Differentiation conditions relying on the addition of exogenous IL-2, such as Th1 and Th2, will likely exhaust the media after 4...

Watch this Scientific Journal Video about Mouse NaÃ¯ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets at JoVE.com

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

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve 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

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

mouse-nave-cd4-t-cell-isolation-vitro-differentiation-into-t-cell

Research

JoVE Journal

Immunology and Infection

51.2K Views.  Rosalind Franklin University of Medicine and Science. 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. 

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

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 ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

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 ...

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

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&amp;#239;ve CD4 T Lymphocytes

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 na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10R&#945; and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 &#176;C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

We developed an in vitro assay to investigate the role of immunoregulatory pathways in the regulation of cytokine secretion by HIV-specific CD4 T cells.

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been considered as the only source of &#945;&#946;T cells. Yet, thymic involution begins early in life leading to a drastically reduced output of na&#239;ve &#945;&#946;T cells into the periphery. Nevertheless, even centenarians can build immunity against newly acquired pathogens. Recent research suggests extrathymic &#945;&#946;T cell development, however our understanding of pathways that may compensate for thymic loss of function are still rudimental. &#947;&#948; T cells are innate lymphocytes that constitute the main T-cell subset in the tissues. We recently ascribed a so far unappreciated outstanding function to a &#947;&#948; T cell subset by showing that the scarce entity of CD4+ V&#948;1+&#947;&#948; T cells can transdifferentiate into &#945;&#946;T cells in inflammatory conditions. Here, we provide the protocol for the isolation of this progenitor from peripheral blood and its subsequent cultivation. V&#948;1 cells are positively enriched from PBMCs of healthy human donors using magnetic beads, followed by a second step wherein we target the scarce fraction of CD4+ cells with a further magnetic labeling technique. The magnetic force of the second labeling exceeds the one of the first magnetic label, and thus allows the efficient, quantitative and specific positive isolation of the population of interest. We then introduce the technique and culture condition required for cloning and efficiently expanding the cells and for identification of the generated clones by FACS analysis. Thus, we provide a detailed protocol for the purification, culture and ex vivo expansion of CD4+ V&#948;1+&#947;&#948; T cells. This knowledge is prerequisite for studies that relate to this &#945;&#946;T cell progenitor`s biology and for those who aim to identify the molecular triggers that are involved in its transdifferentiation.

Isolation and Ex Vivo Culture of V&amp;#948;1+CD4+&amp;#947;&amp;#948; T Cells, an Extrathymic &amp;#945;&amp;#946;T-cell Progenitor

Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral V&#948;1+CD4+ T cells that is, as we showed recently, an extrathymic &#945;&#946; T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been ...

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

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

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing autoimmune diseases. Clinical approaches to adoptively transfer Tregs, or to deplete Tregs in cancer, are underway with promising first outcomes. 
Because the number of naturally occurring Tregs (nTregs) is very limited, studying certain Treg features using in vitro induced Tregs (iTregs) can be advantageous. To date, the best although not absolutely specific protein marker to delineate Tregs is the transcription factor FOXP3. Despite the importance of Tregs including non-redundant roles of peripherally induced Tregs, the protocols to generate iTregs are currently controversial, particularly for human cells. This protocol therefore describes the in vitro differentiation of human CD4+FOXP3+ iTregs from human na&#239;ve T cells using a range of Treg-inducing factors (TGF-&#946; plus IL-2 only, or their combination with retinoic acid, rapamycin or butyrate) in parallel. It also describes the phenotyping of these cells by flow cytometry and qRT-PCR. 
These protocols result in reproducible expression of FOXP3 and other Treg signature genes and enable the study of general FOXP3-regulatory mechanisms as well as protocol-specific effects to delineate the impact of certain factors. iTregs can be utilized to study various phenotypic aspects as well as molecular mechanisms of Treg induction. Detailed molecular studies are facilitated by relatively large cell numbers that can be obtained. 
A limitation for the application of iTregs is the relative instability of FOXP3 expression in these cells compared to nTregs. iTregs generated by these protocols can also be used for functional assays such as studying their suppressive function, in which iTregs induced by TGF-&#946; plus retinoic acid and rapamycin display superior suppressive activity. However, the suppressive capacity of iTregs can differ from nTregs and the use of appropriate controls is crucial.

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

This protocol describes the reproducible generation and phenotyping of human induced regulatory T cells (iTregs) from na&#239;ve CD4+ T cells in vitro. Different protocols for FOXP3 induction allow for the study of specific iTreg phenotypes obtained with respective protocols.

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing ...

An In Vivo Mouse Model to Measure Na&#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played by T cells in an immune response. This protocol describes the in vitro differentiation of bone marrow (BM) progenitors to obtain granulocyte macrophage colony-stimulating factor (GM-CSF) derived-dendritic cells (DCs). The protocol also describes the adoptive transfer of ovalbumin peptide (OVAp)-loaded GM-CSF-derived DCs and na&#239;ve CD4 T cells from OTII transgenic mice in order to analyze the in vivo activation, proliferation, and Th1 differentiation of the transferred CD4 T cells. This protocol circumvents the limitation of purely in vivo methods imposed by the inability to specifically manipulate or select the studied cell population. Moreover, this protocol allows studies in an in vivo environment, thus avoiding alterations to functional factors that may occur in vitro and including the influence of cell types and other factors only found in intact organs. The protocol is a useful tool for generating changes in DCs and T cells that modify adaptive immune responses, potentially providing important results to understand the origin or development of numerous immune associated diseases.

An &lt;em&gt;In Vivo&lt;/em&gt; Mouse Model to Measure Na&amp;#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Here, we present a protocol for the in vivo determination of na&#239;ve CD4 T cell (T cell) activation, proliferation, and Th1 differentiation induced by GM-CSF bone marrow (BM)-derived dendritic cells (DCs). In addition, this protocol describes BM and T-cell isolation, DC generation, and DC and T-cell adoptive transfer.

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played ...

Determination of Regulatory T Cell Subsets in Murine Thymus, Pancreatic Draining Lymph Node and Spleen Using Flow Cytometry

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, thymic derived Treg (tTreg) cells and peripherally induced Treg (pTreg) cells. They are present in different organs of our body and can be distinguished by specific markers, such as Helios and Neuropilin 1. It has been reported that tTreg cells are functionally more suppressive than pTreg cells. Therefore, it is important to determine the proportion of both tTreg and pTreg cells when investigating heterogeneous cell populations. Herein, we collected thymic glands, pancreatic draining lymph nodes and spleens from normoglycemic non-obese diabetic mice to distinguish tTreg cells from pTreg cells using flow cytometry. We manually prepared single cell suspensions from these organs. Fluorochrome conjugated surface CD4, CD8, CD25, and Neuropilin 1 antibodies were used to stain the cells. They were kept in the fridge overnight. On the next day, the cells were stained with fluorochrome conjugated intracellular Foxp3 and Helios antibodies. These markers were used to characterize the two subsets of Treg cells. This protocol demonstrates a simple but practical way to prepare single cells from murine thymus, pancreatic draining lymph node and spleen and use them for subsequent flow cytometric analysis.

Herein, we present a protocol to prepare single cells from murine thymus, pancreatic draining lymph node and spleen to further study these cells using flow cytometry. In addition, this protocol was used for determining the subsets of regulatory T cells using flow cytometry.

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, ...