This work is supported in part by the NIH/NCATS Clinical and Translational Science Awards to the University of Florida TL1 TR000066 and UL1 TR000064, a diversity supplement from Parent Grant R01AI056152 from the National Institute of Health, a BD Biosciences reagent grant, and the University of Florida.

All animal use was conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee.
1. Preparation of Mixes and Media
<ol>
	<li>Sterile PBS pH: 7.3 (1 L) 
	0.23 g NaH2PO4 
	1.15 g Na2HPO4 
	9.0 g NaCl 
	Take up volume with DI water. Sterilize with autoclave.</li>
	<li>Cell Culture Media (100 ml) 
	89 ml RPMI 
	10 ml 10% FBS 
	1 ml Antibiotic Antimycotic (ABAM) 100 &#956;g 50 mM 2-mercaptoethanol (3.5 &#956;g stock 2-mercaptoethanol into 96.5 &#956;g PBS)</li>
	<li>FACS Buffer (Fluorescent Activating Cell Sorting) (To be used during flow cytometry) 2% FBS in sterile PBS</li>
	<li>iTh17 Mix (Based on number of samples, determine the volume needed for all mixes and media) 
	1.5 &#956;g/ml anti-CD28 
	20 ng/ml IL-6 
	5 ng/ml TGF-&#946;
</li>
</ol>
2. Cells Will Be Plated in Triplicate under the Following Conditions
<ol>
	<li>Plate bound anti-CD3, anti-CD28 (this is the activation control mix).</li>
	<li>iTH17: plate bound anti-CD3, anti-CD28, IL-6, TGF-&#946;.</li>
</ol>
3. Plate-bound anti-CD3 (10 &#956;g/ml)
It is recommended that preparation of plate-bound anti-CD3 plates is done at least 4 hr prior to the time cells will be added to the plates.
<ol>
	<li>Add 30 &#956;l anti-CD3 to wells (anti-CD3 is diluted in sterile PBS) of a new 96 well U bottom Microtest tissue culture plate, and tap the sides of the plate to ensure uniform coverage of the wells. Incubate at 37 &#176;C for 4 hr, and then refrigerate until it is time to add the mixes and cells to the plate.</li>
</ol>
4. Mouse Dissection
<ol>
	<li>This protocol is based on the use of C57BL/6 mice, 3-8 months of age, and purchased from The Jackson Laboratory (Bar Harbor, ME).</li>
	<li>Sacrifice mouse using CO2 asphyxiation, and confirm death with subsequent cervical dislocation.</li>
	<li>Sterilize mice dissection tools and incision area with 70% ethanol and begin dissection.</li>
	<li>From the ventral view, grab the skin that is anterior to the urethral opening and begin cutting with scissors up the ventral midline until reaching the chin area. Take precautions not to tear or cut into the lining of the peritoneal wall.</li>
	<li>Pull back on the skin and pin down to allow comfortable access to lymph nodes during removal.</li>
	<li>The lymph nodes and spleens collected will all be removed with forceps, and placed in a sterile Petri dish containing 5 ml of autoMACS Running Buffer purchased from Miltenyi Biotec (refer to Figures 2 and 3 for lymph node and spleen removal diagrams).
	<ol>
		<li>Remove the axillary lymph nodes that are found near the axilla (armpit) behind the pectoral muscles of each mouse.</li>
		<li>Remove the brachial lymph nodes that are located in the connective tissues located near each axilla.</li>
		<li>Remove the superficial cervical lymph nodes found in the neck of each mouse.</li>
		<li>Remove the inguinal lymph nodes which are located in the hip region at the conjunction of the 3 blood vessels.</li>
		<li>To access the mesenteric lymph nodes, cut through the peritoneal lining up the ventral midline. Mesenteric lymph nodes are found in the connective tissue that holds the intestines together. They are generally found in a string of 4-8 nodes, and may appear as a "string of pearls". Make sure to pull out the entire string.</li>
		<li>The spleen is located in the abdominal area, behind the stomach and intestines. Remove it by pulling and detaching it from the pancreas.</li>
	</ol>
	</li>
	<li>Grind organs under a sterile hood using 2 frosted microscope slides. Place lymph nodes or spleen on the frosted side of one microscope slide, and rub with frosted side of the second slide until disintegrated. Repeat until all lymph nodes and spleen have been ground.</li>
</ol>
Note: It is recommended to start with the lymph nodes and finish with the spleen, as the blood will make it difficult to see the remaining lymph nodes in the autoMACs buffer.
<ol start="9">
	<li>To filter ground organs into a single cell suspension, begin by folding over a piece of 40 &mu;m nylon material several times, and placing in the top of a 15 ml centrifuge tube. The nylon material should be roughly 3 x 3 in.</li>
	<li>Use a 5 ml syringe and 21 G needle to aspirate the 5 ml of autoMACs Running buffer and ground organs. Dispense slowly into the folded nylon material. Take care to avoid puncturing the material with the needle.</li>
</ol>
Note: Once single cell suspension is achieved, the solution should appear as a pale, consistent solution with no visible pieces of solid tissue. If solids remain, re-aspirate and dispense through a new folded piece of nylon placed in a new 15 ml centrifuge tube.
<ol start="11">
	<li>Always keep cells on ice when not in use.</li>
</ol>
5. Cell Separation
<ol>
	<li>For optimal results, obtain at least an 80% pure CD4+CD25- cell population through autoMACS Pro Cell Separator using the MACS CD4+CD25+ regulatory T cell isolation kit, mouse. The protocol for the negative retention of the CD4+CD25- cell population is obtained through Miltenyi Biotec.</li>
</ol>
6. Th17 Differentiation
<ol>
	<li>Collect the CD4+CD25- cell population after separation with the autoMACS Pro Cell Separator.</li>
	<li>Count cells diluted in a 1:1000 ratio with trypan blue using a hemocytometer.</li>
	<li>Once concentration has been determined from cell counts, dilute cell suspension to 1 x 106 cells/ml in cell culture media.</li>
	<li>Take out 96 well plates with plate bound anti-CD3 after 4 hr.</li>
	<li>Wash anti-CD3-coated wells with 200 &#956;l of PBS. Repeat 2 times.
	<ol>
		<li>To wash add 200 &#956;l of sterile PBS to anti-CD3-coated wells and remove PBS into a waste container.</li>
	</ol>
	</li>
	<li>Add 100 &#956;l of the iTh17 mix or activation control mix (see point #2) to the wells in triplicate.</li>
	<li>Add 100 &#956;l of cells to each well in which either the Th17 mix or the activation control mix has been placed.</li>
	<li>Incubate cells for at least 72 hr or up to 5 days (Th17 differentiation can be obtained after 72 hr or after 5 days.)</li>
	<li>Th17 differentiation can be assessed by intracellular staining and flow cytometric analysis, ELISA, or qPCR</li>
</ol>
7. Cell Activation (Only Necessary for Intracellular Staining)
<ol>
	<li>Remove 96 well plates from incubators at the end of either 72 hr or 5 days
	<ol>
		<li>Recall that Th17 differentiation can be achieved after either 72 hr or 5 days.</li>
	</ol>
	</li>
	<li>For each condition (e.g. activation control or iTh17), transfer cells that are in triplicate into one well of a 24 well cell culture plate. The cells for one condition have now been pooled into one well of the 24 well culture plate, as opposed to being in triplicate in the 96 well U bottom culture plate.</li>
	<li>The total volume of each well in the 24 well cell culture plate is now 600 &#956;l. Raise the volume of each well to 1 ml with the cell culture media.</li>
	<li>Add PMA (phorbol myristate acetate) (50 ng/ml), ionomycin (1 &#956;M), and BFA (Brefeldin-A) (10 &#956;g/ml) to each well in the 24 well cell culture plate at the listed concentrations.</li>
	<li>Incubate at 37 &#176;C for 4 hr.</li>
</ol>
8. Intracellular Staining
<ol>
	<li>Stain cells with the desired extracellular and intracellular markers for flow cytometric analysis. To detect the presence of IL-17, intracellular staining is done with anti-IL-17A antibodies. Recommended extracellular surface markers include CD4, CD8, and CD25.
	<ol>
		<li>Use the Intracellular Cytokine Staining Starter Kit-Mouse from BD Bioscience for IL-17 Intracellular staining.</li>
		<li>For each sample, pellet cells, remove supernatant, and resuspend cell pellet in 200 &#956;l FACS buffer (2% FBS in PBS).</li>
		<li>Transfer resuspended cells to 96 cell flow cytometry plate.</li>
		<li>Spin cells down for 5 min at 1,200 rpm and discard supernatant.</li>
		<li>Add 200 &#956;l PBS FACS buffer, centrifuge for 5 min at 1,200 rpm, and discard supernatant.</li>
		<li>Resuspend cells in 100 &#956;l of FACS buffer and apply 100 &#956;l of extracellular antibody (Ab) mixture (extracellular Ab mixture is made in FACS buffer). Incubate for 15 min at RT, covered with foil.</li>
		<li>Repeat step 8.1.4.</li>
		<li>Repeat step 8.1.5 2x.</li>
		<li>Resuspend cells in 100 &#956;l of BD Cytofix/Cytoperm Buffer. Incubate for 20 min at RT, covered with foil.</li>
		<li>Add 100 &#956;l of 1x BD Perm/Wash buffer, centrifuge for 5 min at 1,200 rpm, and discard supernatant. Repeat.</li>
		<li>Add 50 &#956;l of intracellular Ab mixture. (Intracellular Ab mixture is made in 1x BD Perm/Wash) Incubate for 15 min at RT, covered with foil.</li>
		<li>Repeat step 8.1.10.</li>
		<li>Resuspend cells in 200 &#956;l of BD Staining Buffer.</li>
		<li>Place resuspended cells into flow cytometry tubes containing 200 &#956;l BD Staining Buffer (final volume is 400 &#956;l).</li>
		<li>Store at 4 &#176;C until samples are ready to be read.</li>
	</ol>
	</li>
</ol>
9. Flow Cytometric Analysis
<ol>
	<li>Gate live cell population.</li>
	<li>From the live cell population, gate on CD4+CD8- population.</li>
	<li>From the CD4+CD8- population, gate on IL-17A+ population.
	<ol>
		<li>Based on our previous experimental results, 100% of the IL-17A+ population will be CD25+.</li>
	</ol>
	</li>
</ol>
*Total absolute cell counts were obtained after pooling the sample triplicates 
**Absolute number of CD4+CD25+IL-17A+ cells was determined by multiplying the total number of cells by the live gate percentage and the percentage of total cells bearing the lineage-specific markers, CD4, CD25, and IL-17A, as determined by flow cytometry.
10. qPCR and ELISA
<ol>
	<li>Place cells not used for flow cytometric analysis in a 1.5 ml Eppendorf tube and centrifuge at 13,000 rpm for 5-10 min. The cells found in the cell pellet after centrifugation will be used for qPCR analysis. qPCR analysis was performed using PTC-200 Peltier Thermal cycler (Biorad).</li>
	<li>Collect supernatant from the centrifuged tubes for ELISAs. IL-17A ELISAs were performed using antibody pair TC11-18H10 (capture, catalog number 555068) and TC11-8H4 biotin (detection, catalog number 555067) purchased from BD Biosciences. IL-17A ELISA standards were obtained from eBioscience (catalog number 14-8171-80).</li>
	<li>After removing the supernatant, resuspend the remaining cells to be used for qPCR in 175 &#956;l RNA lysis buffer (use immediately for RNA extraction, cDNA synthesis, and qPCR, or store at -80 &#176;C for later use).
	<ol>
		<li>Refer to Table II for primer sequences for IL-17A and actin (house-keeping gene)</li>
	</ol>
	</li>
</ol>

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No disclosures to declare.

Here we have described the protocol to achieve in vitro Th17 differentiation. The study of Th17 differentiation is important, as differentiation of T lymphocytes into the Th17 subset is critical for the effective elimination of human pathogens13. Conversely, IL17 production has been associated with autoimmune disease progression13. The method of Th17 differentiation is applicable to many research settings as it can be applied to numerous distinct murine models of immune regulation and autoimmunity. This method helps to answer questions regarding the ability of T lymphocytes, to differentiate into a Th17 cell, denoted in this protocol by the production of IL-17A and expression of the transcription factor ROR&#947;T, to modulate immune responses. Moreover, it has been shown by others that Th17 cells can also be identified by the production of IL17F, Il22, and GM-CSF 8, 12. This protocol is significant with respect to other known T lymphocyte differentiation protocols, such as Th1 or Th2 differentiation, because it can be used to complement those protocols in order to better understand how T lymphocyte effector functions relate to immune system operation.
There are several critical steps to consider when conducting these experiments. Th17 differentiation can be achieved through an impure CD4+CD25- population, but for the most effective and reliable results, a CD4+CD25- population of at least 80 % purity is desired. Our method utilizes the protocol provided by Miltenyi Biotec for their autoMACS Pro Cell Separator. The autoMACS CD4+CD25+ Regulatory T Cell Isolation Kit, Mouse is used for negative selection of the CD4+CD25- cell population. Negative selection allows for the use of na&#239;ve cells that have not been potentially altered by antibody interactions.
Using certain cell surface markers in flow cytometric analysis can confirm the purity of a population. The recommended surface markers are CD4, CD8, and CD25 as they can readily identify the desired CD4+CD25- population. B220 and CD11b may be used to test for B cell and macrophage contamination, respectively. Samples consistently diluted to 1 x 106 cells/ml is also recommended as we have found that this concentration yields optimal differentiation. In the past we have found that cell concentrations that were too high or too low often had perturbed cellular expansion and biased results (such as skewed IL-17A production).
It is also important to remember to keep the samples on ice at all times when not in use. Cell viability is an important factor in achieving positive Th17 differentiation. This can be confirmed through cell counting using trypan blue dilutions. Because this protocol employs manual tissue homogenization by grinding the lymph nodes and spleen with frosted microscope slides, this can be a time consuming method. One design modification is to utilize automatic tissue dissociators as an alternative option.
In the event that the desired Th17 differentiation is not achieved, there are several trouble shooting tips that should be considered. First, controls must also be carefully considered so that the true extent of Th17 differentiation can be properly measured. We typically determine the extent of Th17 differentiation by comparing these results to the CD4+ T lymphocytes receiving only anti-CD3 and anti-CD28 stimulation. Also remember that CD4+ T lymphocytes need to be activated in order differentiate. One should also visually inspect cultured cells under the microscope to confirm the presence of blasted/activated T cells. Activated T cells visually appear significantly larger than cells that have not been stimulated. Clonal expansion can also be observed through visible clusters of blasting cells which have been derived from a single T cell clone. Cell activation can also be verified through the use of flow cytometric analysis using anti-CD4 and anti-CD25 antibodies to identify the CD4+CD25+ activated cells4
It is also important to mention that this protocol has been optimized for use with C57BL/6 mice, however published data shows that Th17 differentiation may be achieved in other mouse strains including NOD and BALB/c mice5, 11, 20 . It should be noted that Th17 differentiation, including overall frequency and absolute number of CD4+ T lymphocytes undergoing differentiation, is dependent upon genetic factors (such as mouse strain)20, and environmental factors (such as colony location)5 and may require optimization.
This Th17 differentiation protocol can serve as an invaluable means to further answer questions regarding the function of cells in the Th17 subset. Our method of Th17 differentiation has several significant advantages. As previously mentioned the use of a minimum 80% pure CD4+CD25- cell population provides a more consistent and effective differentiation result. Ease of isolation through use of the autoMACS Pro cell separator is an additional advantage. Furthermore the use of the optimal cytokine and activation signals provides the T cells with the appropriate environment in which to differentiate. Future implications for this protocol include determining the mechanistic means by which Th17 cells and the production of IL-17A are involved in the onset and progression of autoimmunity.

CD4+ T lymphocytes (T cells) play a critical role in immune system-mediated defense against infectious microorganisms. Conversely, T cells are also intimately associated with the onset and progression of autoimmune diseases such as type 1 diabetes, systemic lupus erythematosus, and rheumatoid arthritis. CD4+ T lymphocytes become activated through a combination of T cell receptor (TCR) interactions with cognate antigen/major histocompatibility complex II (MHCII) molecules, and CD28 receptor interactions with B7.1/B7.2 ligands15. In addition to the provision of TCR stimulation and CD28 co-stimulation, antigen-presenting cells also provide a cytokine environment, which determines the differentiation state of the T lymphocyte, thereby directing the T lymphocyte&#39;s response to the given antigen. Distinct pathogen/antigen-presenting cell interactions create distinct cytokine environments, which skew T lymphocytes down distinct pathways focused on the elimination of the initiating pathogen. Unfortunately, T lymphocyte effector pathways, originally designed to eradicate invading pathogens, can be erroneously directed against self-tissues15. Therefore, better understanding of each distinct CD4+ T cell subset&#39;s differentiation state is critical for our understanding of how to modulate the balance between elimination of pathogens and tolerance to self.
In addition to the Th1, Th2, and inducible regulatory T cell differentiation pathways, na&#239;ve T lymphocytes can also be driven by cytokines down the Th17 pathway. Whereas Th1 cells combat intracellular pathogens, Th2 cells eliminate extracellular pathogens, and regulatory T cells (Tregs) minimize inflammatory responses1, 16; Th17 cells play an important role in the elimination of extracellular bacteria and fungi. Th17 cells are generally denoted by expression of the lineage-specific transcription factor ROR&#947;T and production of IL-17A, which promotes the activation of macrophages and neutrophils1, 7.
Th17 cells have been implicated in several autoimmune disorders, and their associated rodent models. For example, it has been demonstrated that IL-23 (which is required to sustain the Th17 phenotype), but not IL-12, was the central culprit in experimental autoimmune encephalitis (EAE), the rodent disease model for MS. It has subsequently been shown that reductions in IL-17 production are correlated to EAE prevention2, 6, 17. Moreover, Th17 cells have been associated with other autoimmune disorders including arthritis and systemic lupus erythematosus (SLE)10, 16. IL-23 deficient p19-/- mice were shown to have very low numbers of Th17 cells, and are resistant to developing not only EAE, but also collagen-induced arthritis, a model for rheumatoid arthritis10, 18. In addition, mice treated with neutralizing IL-17A antibodies after the onset of collagen-induced arthritis were also found to have resolution of joint damage18. It should be noted that the role of Th17 cells in the progression of autoimmune disease remains to be characterized as recent research has also shown a protective role of Th17 cells in Type 1 diabetes9, 11 and intestinal inflammation14. These studies confirm the importance of Th17 differentiation in autoimmunity.
In vitro Th17 differentiation is a necessary method in T cell research because there are at least two perplexing questions that require further investigation: 1) How exactly does IL-6 regulate the balance between Treg and Th17 differentiation, and 2) what are the exact mechanisms behind IL-17-induced inflammatory disorders? Our method employs CD4+CD25- T cells from the spleens and lymph nodes of the C57BL/6 mouse. It is important to note that although it is possible to induce Th17 differentiation using an impure population, acquiring at least an 80% pure CD4+CD25- T cell population negates any worry of contamination and ensures more successful Th17 differentiation results. In order to achieve proper Th17 differentiation, CD4+CD25- T cells are incubated in the presence of anti-CD3 and anti-CD28, which provide activation signals, 1 and 2, respectively, and IL-6, and TGF-&#946;. Although it has been reported that IL-23 alone can be used to achieve Th17 differentiation, it was later demonstrated that IL-23 is necessary for the stability of the Th17 cell population, but IL-6 and TGF-&#946; are essential for Th17 differentiation3, 18, 19. Murine studies have shown that the IL-23 receptor is expressed on CD4+ T cells only after they have been stimulated with IL-6 and TGF-&#946;13, 18. Also, Th17 cells will successfully develop in the presence of IL-23-blocking antibodies as long as IL-6 and TGF-&#946; are present18, 19. As such, this Th17 differentiation protocol provides the appropriate conditions to successfully induce Th17 differentiation. Development of a better understanding of the mechanisms underlying Th17 differentiation and IL-17 production present the opportunity for the development of better therapeutics aimed at autoimmune disorders13.

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 <td></td>
 <td></td>
 <td>Reagent/Material</td>
 </tr>
 <tr>
 <td>Sterile Polyestrene Petri Dish</td>
 <td>Fisher Scientific</td>
 <td>0875713A</td>
 <td>60 mm x 15 mm</td>
 </tr>
 <tr>
 <td>autoMACS Running Buffer</td>
 <td>Miltenyi Biotec</td>
 <td>130-091-221</td>
 <td></td>
 </tr>
 <tr>
 <td>Premium Microscope Slides, Frosted</td>
 <td>Fisher Scientific</td>
 <td>12-544-3</td>
 <td>3" x 1"1 mm</td>
 </tr>
 <tr>
 <td>5 ml 21G1 Latex Free Syringe and Needle</td>
 <td>BD Biosciences</td>
 <td>309632</td>
 <td></td>
 </tr>
 <tr>
 <td>Corning 15 ml Centrifuge Tubes</td>
 <td>Sigma-Aldrich</td>
 <td>CLS430791</td>
 <td></td>
 </tr>
 <tr>
 <td>Nylon 40 microns</td>
 <td>Miami Aquaculture</td>
 <td>Nylon 40/26</td>
 <td></td>
 </tr>
 <tr>
 <td>Microtest tissue culture plate 96 well U bottom </td>
 <td>BD Biosciences</td>
 <td>35-3077</td>
 <td></td>
 </tr>
 <tr>
 <td>Corning Costar 24 well cell culture plates</td>
 <td>Sigma-Aldrich</td>
 <td>CL3524</td>
 <td></td>
 </tr>
 <tr>
 <td>Eppendorf Tubes 1.5 ml</td>
 <td>Fisher Scientific</td>
 <td>05-408-129</td>
 <td></td>
 </tr>
 <tr>
 <td>Purified NA/LE Hamster Anti-Mouse CD3e</td>
 <td>BD Biosciences</td>
 <td>553057</td>
 <td></td>
 </tr>
 <tr>
 <td>Purified NA/LE Hamster Anti-Mouse CD28</td>
 <td>BD Biosciences</td>
 <td>553294</td>
 <td></td>
 </tr>
 <tr>
 <td>Mouse IL-6 Recombinant Protein</td>
 <td>eBioscience</td>
 <td>14-8061-62</td>
 <td></td>
 </tr>
 <tr>
 <td>TGFbeta</td>
 <td>R&amp;D Systems</td>
 <td>240-B-002</td>
 <td></td>
 </tr>
 <tr>
 <td>Trypan blue solution 0.4 %</td>
 <td>Sigma-Aldrich</td>
 <td>66H2364</td>
 <td></td>
 </tr>
 <tr>
 <td>Pac Blue Rat Anti-Mouse CD4</td>
 <td>BD Biosciences</td>
 <td>558107</td>
 <td></td>
 </tr>
 <tr>
 <td>APC Rat Anti-Mouse CD8a</td>
 <td>BD Biosciences</td>
 <td>553035</td>
 <td></td>
 </tr>
 <tr>
 <td>PE Conjugated Anti-mouse CD25</td>
 <td>eBioscience</td>
 <td>E01155-516</td>
 <td></td>
 </tr>
 <tr>
 <td>Alexa Fluor 700 Rat Anti-mouse IL-17</td>
 <td>BD Biosciences</td>
 <td>560820</td>
 <td></td>
 </tr>
 <tr>
 <td>Intracellular Cytokine Staining Starter Kit-Mouse</td>
 <td>BD Biosciences</td>
 <td>51-2041AK (559311)</td>
 <td></td>
 </tr>
 <tr>
 <td>MACS CD4+CD25+ regulatory T cell isolation kit, mouse</td>
 <td>Miltenyi Biotec</td>
 <td>130-091-221</td>
 <td></td>
 </tr>
 <tr>
 <td>ABAM </td>
 <td>Cellgro</td>
 <td>30-004-CI</td>
 <td></td>
 </tr>
 <tr>
 <td>RPMI</td>
 <td>Corning, Cellgro</td>
 <td>10-040-CM</td>
 <td></td>
 </tr>
 <tr>
 <td>B 2-MercaptoEthanol</td>
 <td>MP Biomedical</td>
 <td>194834</td>
 <td>Hazardous</td>
 </tr>
 <tr>
 <td>Phorbol 12-Myristate 13-Acetate (PMA)</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Ionomycin Calcium Salt</td>
 <td>Sigma-Aldrich</td>
 <td>13909-1ML</td>
 <td>Hazardous</td>
 </tr>
 <tr>
 <td>Brefeldin A (BFA)</td>
 <td>MP Biomedicals</td>
 <td>159027</td>
 <td></td>
 </tr>
 <tr>
 <td>ELISA IL-17A Capture mAb </td>
 <td>BD Biosciences</td>
 <td>555068</td>
 <td></td>
 </tr>
 <tr>
 <td>ELISA IL-17A Detection mAb </td>
 <td>BD Biosciences</td>
 <td>555067</td>
 <td></td>
 </tr>
 <tr>
 <td>ELISA IL-17A Standard</td>
 <td>eBioscience</td>
 <td>14-8171-80</td>
 <td></td>
 </tr>
 <tr>
 <td>IL-2 ELISA Kit</td>
 <td>BD Biosciences</td>
 <td>555148</td>
 <td></td>
 </tr>
 <tr>
 <td>TMB Substrate Reagent Set </td>
 <td>BD Biosciences</td>
 <td>555214</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment </td>
 </tr>
 <tr>
 <td>autoMACS Pro Cell Separator</td>
 <td>Miltenyi Biotec</td>
 <td>130-092-545</td>
 <td></td>
 </tr>
 <tr>
 <td>Sorvall Legend RT+ Centrifuge</td>
 <td>ThermoScientific</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Napco series 8000 WJ CO2 Incubator</td>
 <td>ThermoScientific</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>PTC-200 Peltier Thermal Cycler</td>
 <td>Biorad</td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

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.

This Th17 differentiation protocol begins with the removal of the spleen and the axillary, brachial, mesenteric, cervical, and inguinal lymph nodes. A representation of the locations of each can be found in Figures 2 and 3. Figures 1 and 5 both provide a visual representation of the methods described in this protocol.
This Th17 protocol focuses on differentiation from the CD4+CD25- T lymphocyte population. It is important to note how efficiently the autoMACS Pro Cell separation enriches our desired CD4+CD25- population from the pooled total lymph node and spleen (Figure 4). The unfractionated, pooled lymph node and splenocytes, and the autoMACS-separated fractions were stained with antiCD4 and antiCD25 antibodies followed by flow cytometric analysis (Figure 4). Figure 4A shows a representative profile of the percentage of CD4+CD25+ and CD4+CD25- lymphocytes present in the unfractionated, pooled lymph nodes and spleen by flow cytometry. The isolation procedure also provides an enriched CD4+CD25+ Treg population from C57BL/6 mice which can be used in additional experiments (Figure 4B). Figure 4C shows our desired cell enrichment with a population that is 84% CD4+CD25- T cells, with the vast majority of CD4+CD25+ Tregs removed. We consistently have a small non-lymphoid cell population that is present in the enriched CD4+CD25-, but does it not interfere with the differentiation process (Figure 4C). Our enriched CD4+CD25- T cell population helps to assure a more successful Th17 differentiation.
Figure 5 shows a schematic of our Th17 differentiation procedure. Our enriched CD4+CD25- population is either incubated under Th17 differentiating conditions (anti-CD3, anti-CD28, IL-6, and TGF-&#946;) or control (anti-CD3, anti-CD28). It can be seen in Figure 6 that whereas incubation of CD4+CD25- T lymphocytes with anti-CD3, anti-CD28 for 5 days yielded CD25+ T lymphocytes, incubation under Th17 inducing conditions yielded a subset of IL17 producing CD4+CD25+ lymphocytes. Please refer to Table 1 for examples of CD4+CD25+IL-17A+ absolute cell number yields after 5 day incubation under iTh17 conditions.
Th17 differentiation status can further be assessed through quantitative PCR and ELISA. Figure 7 presents data from enriched CD4+CD25- T lymphocytes incubated under control or Th17-inducing conditions. CD4+CD25- T lymphocytes incubated under Th17 conditions up-regulate IL17A, which can be ascertained through qPCR (Figure 7A) and ELISA (Figure 7B). The Th17-specific transcription factor ROR&#947;T is also upregulated by CD4+CD25- T cells incubated under Th17-inducing conditions, and can be ascertained through qPCR (Figure 7A).
<img alt="Figure 1" src="/files/ftp_upload/50765/50765fig1.jpg" /> 
Figure 1. Th17 Differentiation schematic. 1. Coat wells of U-bottomed 96 well plate with anti-CD3 (10 &#956;g/ml) diluted in PBS. 2. Place plate in incubator for 4 hr at 37 &#176;C. While plate is incubating, dissect lymph nodes (LN) and spleen from mouse. Grind organs and filter to obtain a single cell suspension. Isolate CD4+CD25- T cells using the CD4+CD25+ Regulatory T cell Isolation Kit and Miltenyi autoMACS Pro Separator. CD4+CD25- cells should be diluted to 1 x 106 cells/ml. 3. Once 4 hr incubation of the 96 well plate is complete, wash wells with PBS (200 &#956;l/well) 3x. 4. Add 100 &#956;l CD4+CD25- T cells to plate-bound (PB) anti-CD3 coated wells. 5. Add 100 &#956;l anti-CD28 or 100 &#956;l iTh17 cocktail (antiCD28, TGF-&#946;, and IL-6). 6. Incubate plate for 3 or 5 days at 37 &#176;C. Following incubation assess differentiation by intracellular staining, qPCR, and/or ELISA.
<img alt="Figure 2" src="/files/ftp_upload/50765/50765fig2.jpg" /> 
Figure 2. LN Dissection Guide. A) 1 brachial lymph node can be found in the connective tissue located near each axilla (armpit) of the mouse. B) 1 axillary lymph node can be found in each axilla, behind the pectoral muscles. C) Mesenteric lymph nodes are located in the connective tissue of the intestines. They resemble a string of pearls. D) 4 superficial cervical lymph nodes are found in the neck of the mouse. E) 2 inguinal lymph nodes (1 on each side) can be located in the hip region at the location where 3 blood vessels meet.
<img alt="Figure 3" src="/files/ftp_upload/50765/50765fig3.jpg" /> 
Figure 3. Spleen dissection guide. Mouse spleens are found on the left side of the body behind the stomach and intestines. Simply remove by pulling and separating with the forceps.
<img alt="Figure 4" fo:content-width="6in" src="/files/ftp_upload/50765/50765fig4.jpg" /> 
Figure 4. Cell fractions from pooled LN and spleen cells before and after autoMACS Pro separation. A) LN and spleen cells were stained, ex vivo, to establish baseline cell populations prior to separation. Following separation, CD4+CD25+ (B) and CD4+CD25- (C) fractions were stained to assess purity of CD4+CD25+ and CD4+CD25- T lymphocyte populations, respectively. All samples were stained with CD4+ and CD25+ antibodies and analyzed by flow cytometry.
<img alt="Figure 5" fo:content-width="4.5in" src="/files/ftp_upload/50765/50765fig5.jpg" /> 
Figure 5. Th17 Differentiation. Incubate cells for up to 5 days. Harvest cells for intracellular staining and qPCR; harvest supernatants for ELISA.
<img alt="Figure 6" fo:content-width="6in" src="/files/ftp_upload/50765/50765fig6.jpg" /> 
Figure 6. Th17 differentiation assessed by flow cytometry. CD4+CD25- T lymphocytes were incubated in the presence of plate-bound anti-CD3, anti-CD28, IL-6, and TGF-&#946; (iTh17) or plate-bound anti-CD3 and anti-CD28 alone (control) for 5 days. Cells were then activated with PMA and Ionomycin for 4 hr at 37 &#176;C. Following activation, cells were stained with anti-CD4, anti-CD8, anti-CD25, and anti-IL-17A antibodies for flow cytometric analysis to assess Th17 differentiation. iTh17 = Th17 inducing conditions.
<img alt="Figure 7" fo:content-width="6in" src="/files/ftp_upload/50765/50765fig7.jpg" /> 
Figure 7. Th17 differentiation assessed by qPCR and ELISA. Lymphocytes from C57BL/6 mice were sorted and CD4+CD25- T cells were incubated in the presence of plate-bound anti-CD3 (10 &#956;g/ml), anti-CD28 (1.5 &#956;g/ml), IL-6 (20 ng/ml), and TGF-&#946; (5 ng/ml) or plate-bound anti-CD3 and anti-CD28 alone (control) for 5 days. A) Cells were then harvested to assess ROR&#947;T and IL-17A message expression via qPCR. B) Supernatants were harvested to assess IL-17A protein expression by ELISA.

Watch this Scientific Journal Video about Isolation and Th17 Differentiation of NaÃƒÂ¯ve CD4 T Lymphocytes at JoVE.com

Isolation and Th17 Differentiation of Na&#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.

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 ...

isolation-and-th17-differentiation-of-nave-cd4-t-lymphocytes

Universidad San Sebastian

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JoVE Journal

Immunology and Infection

34.3K Views.  The University of Florida. With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether naïve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

Video: Isolation and Th17 Differentiation of Naïve CD4 T Lymphocytes

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as 
insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during 
the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the 
radionuclide Chromium 51 (51Cr) to label target cells. These targets are then exposed to effector cells and the release of 51Cr from target 
cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of 51Cr.
 As effector cells, we used an activated autoreactive clonal population of CD8+ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with 51Cr labeled target NIT cells for 16 hours, release of 51Cr was recorded to calculate specific lysis 
Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37&deg;C, with 5% CO2, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.
Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.

Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

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  ...

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

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 ...

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.

Mouse Na&#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.

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

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

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&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#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 ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

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.

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.