The work was supported by the National Key R&#38;D Program of China (No.2022YFC2304604), National Natural Science Foundation of China (No.81971812), National Natural Science Foundation of China (No.82272235), Science Foundation of the Commission of Health of Jiangsu Province (No. ZDB2020009), Jiangsu Province Key research, development Program (Social Development) Special Project (No.BE2021734), National Key R &#38; D Program of Ministry of Science and Technology (No.2020YFC083700), Jiangsu Provincial Key Laboratory of Critical Care Medicine (BM2020004), Key project of National Natural Science Foundation of China (81930058), National Natural Science Foundation of China (82171717), Central Universities Basic Research Funds (2242022K4007), and Jiangsu Provincial Natural Science Foundation General Project (BK20211170).

The Institutional Review Committee for Animal Studies at Southeast University approved all the animal studies that are detailed in this study, which were carried out in compliance with both local and institutional office standards. Spleen samples were taken from C57BL6/J mice. Both female and male mice, aged between 5 and 8 weeks, were included in this study. The culture medium and buffer were stored at 4 &#176;C for up to 1 month. Surgical instruments were autoclaved before use. Wear latex gloves and masks to avoid contamination of skin, eyes, and clothing with reagents; use a lot of water or saline rinse for the skin and eyes.
1. Precoating the 24-well tissue culture plate with anti-CD3
<ol>
	<li>Dilute anti-CD3 to a final concentration of 1 &#181;g/mL in sterile 1x phosphate-buffered saline (PBS) or RPMI 1640 medium.</li>
	<li>Fill a 24-well tissue culture plate well with 1 mL of anti-CD3 solution (1 &#181;g/mL); then, cover the well with parafilm.</li>
	<li>Place the anti-CD3 coated plate in the refrigerator (2 &#176;C-8 &#176;C) for 16 h or keep it at 37 &#176;C in a cell incubator for 2-3 h.</li>
</ol>
2. Mouse spleen isolation and preparation of spleen single-cell suspension
<ol>
	<li>Induce complete anesthesia in C57BL/6J with 3% isoflurane and euthanize by inhaling 100% carbon dioxide for 2 min. Immediately after the mice&#39;s death, soak them in 75% ethanol for disinfection to prevent contamination.</li>
	<li>Place the mouse in a supine position on the clean bench, cut it open along the midline of the abdomen, and separate the abdominal structures layer by layer. Gently open the greater omentum and stomach with tweezers, gently pull out the spleen with the gastrosplenic ligament, and bluntly separate the spleen from surrounding tissues and ligaments to obtain an intact spleen (be careful to avoid spleen crushing or rupture). 
	NOTE: To maintain a sterile state, the following operation is carried out in a sterile super clean bench.</li>
	<li>Place a 70 &#181;m cell filter in a 100 mm sterile culture dish, add the spleen to the cell strainer, and crush it with a syringe plunger. At the same time, add 5-8 mL of homogenate flushing fluid to push all the cells through the filter into the culture dish. 
	NOTE: The composition of the lymphocyte separation liquid kit can be seen in the Table of Materials.</li>
	<li>Centrifuge the filtrate at 450 &#215; g for 5 min at room temperature. Discard the supernatant.</li>
	<li>Resuspend the cell pellet with the sample diluent provided with the lymphocyte separation kit or with PBS or RPMI 1640 medium. Adjust the cell concentration of the cell suspension to 2 &#215; 108-1 &#215; 109 cells/mL. 
	NOTE: Typically, 4-6 mL of sample diluent is required for one spleen.</li>
	<li>In a centrifuge tube, take the same amount of lymphocyte separation solution as the tissue single-cell suspension. Carefully pipette the single-cell suspension onto the surface of the lymphocyte separation solution and centrifuge at 800 &#215; g, 25 &#176;C for 30 min. Set the Acceleration and Deceleration of the centrifuge to an appropriate range (e.g., set at 3 if there are the usual 1 to 9 gears). 
	NOTE: The lymphocyte separation solution should not be less than 3 mL.</li>
	<li>After centrifugation, observe the four layers in the centrifuge tube from the top to the bottom. The first layer corresponds to the sample diluent, followed by the annular milky white lymphocyte layer. The third layer is the separation liquid, and the fourth layer consists of red blood cells.</li>
	<li>Use a pipette to carefully draw the second layer of annular milky white lymphocyte layer into another centrifuge tube and add 10 mL of cleaning solution to the centrifuge tube to mix the cells.</li>
	<li>Centrifuge at 250 &#215; g for 5 min at room temperature. Discard the supernatant. Use 10 mL of RPMI 1640 medium to resuspend the cell pellet for cell counting.</li>
</ol>
3. Purification of na&#239;ve CD4+ T cells based on negative selection of magnetic beads
NOTE: Isolate untouched and highly purified na&#239;ve CD4+ T cells (CD4+CD44lowCD62Lhigh) from mouse splenocytes by immunomagnetic negative selection.
<ol>
	<li>Prepare the sample to have 1 &#215; 108 cells/mL within the volume range of 0.1-2 mL.</li>
	<li>Add 50 &#181;L/mL of rat serum and transfer the sample to a 5 mL polystyrene round-bottom tube. 
	NOTE: The composition of the naive CD4 T cell sorting kit can be seen in the Table of Materials.</li>
	<li>Add 50 &#181;L/mL of Isolation Cocktail to the sample, mix well, and incubate for 7.5 min at room temperature.</li>
	<li>Add 50 &#181;L/mL of Depletion Cocktail to the sample, mix well, and incubate for 2.5 min at room temperature.</li>
	<li>Vortex the magnetic beads to ensure even dispersion. Add 75 &#181;L/mL of the magnetic beads to the sample, mix well, and incubate for 2.5 min at room temperature. 
	NOTE: The particles should appear evenly dispersed; vortexing time should not be less than 30 s.</li>
	<li>Top up the sample with RPMI 1640 medium to a final volume of 2.5 mL. Mix several times by gently pipetting up and down.</li>
	<li>Place the tube (without the lid) into the magnet and incubate for 2.5 min at room temperature.</li>
	<li>Picking up the magnet, invert the magnet and tube in one continuous motion and pour theenriched cell suspension into a new tube. 
	NOTE: Keep the tube and magnet inverted for 2-3 s before turning them upright. Do not disturb any drops that may remain on the mouth of the tube.</li>
	<li>Determine the cell number using a hemocytometer.</li>
	<li>Perform flow cytometric analysis for na&#239;ve CD4+ T cells before and after isolation (Figure 1).</li>
	<li>Transfer the cells to a new centrifuge tube, centrifuge at 400 &#215; g for 5 min at room temperature, and discard the supernatant.</li>
	<li>Resuspend the cells in 200-500 &#181;L of RPMI 1640 medium (supplemented with 10% fetal bovine serum). For each 1 mL of cell culture (e.g., ~106 cells/mL), add 2 &#181;L of leukocyte activation cocktail and mix it. Culture the cells in a CO2 incubator at 37 &#176;C and saturated humidity for 4-6 h. 
	NOTE: The cells were vortexed once every 1-2 h.</li>
	<li>Centrifuge the cells at 400 &#215; g, 4 &#176;C for 5 min. Carefully discard the supernatant and resuspend the cells in 200 &#181;L of PBS. Add 1 &#181;L of Fc blocker to the cells (e.g., ~106 cells/mL). Incubate the cells for 10 min at 4 &#176;C.</li>
	<li>Wash the cells once with 1 mL of PBS by centrifuging at 400 &#215; g for 5 min at 4 &#176;C. Discard the supernatant and resuspend the cells in 200 &#181;L of PBS.</li>
	<li>Add antibody cocktail (Fixable Viability Dye, 1:1,000; anti-CD4, 1:200; anti-CD44, 1:200; anti-CD62L, 1:200) and incubate the cells for 15-20 min in the dark at 4 &#176;C.</li>
	<li>Wash the cells with 2 x 1 mL of PBS and then, centrifuge at 400 &#215; g for 5 min at 4 &#176;C.</li>
	<li>Discard the supernatant, resuspend the cells in 250 &#181;L of fixed buffer, and incubate them for 40-60 min in the dark at 4 &#176;C. 
	NOTE: The fix buffer is provided with the Foxp3/transcription factor staining buffer, which is a 4x stock solution that must be diluted with permeabilization diluent.</li>
	<li>Add 1 mL of 1x permeabilization buffer to wash the cells 2x by centrifuging the cells at 300 &#215; g, 4 &#176;C for 5 min.</li>
	<li>Discard the supernatant, resuspend the cells in 200 &#181;L of permeabilization buffer, and add the antibody (Foxp3 [nuclear], 1:200) to the cells. After fixation and membrane rupture, incubate the cell-antibody mixtures in the dark for 40-60 min with shaking occasionally for short periods at 4 &#176;C. 
	NOTE: The Permeabilization/Wash solution is a 10x stock solution that must be diluted before use with PBS.</li>
</ol>
4. Induction of pathogenic Th17 cells in vitro
<ol>
	<li>Remove sterile PBS from the precoated 24-well plates prior to use.</li>
	<li>Use Th17 cell culture medium (Table 1) and contrast medium for culture, including Th0 cell culture medium (Table 1), classical non-pathogenic Th17 culture medium (Table 1), and classical pathogenic Th17 culture medium (Table 1). Resuspend the enriched naive CD4 + T cells and dispense into different wells in the same 24-well plate (precoated with anti-CD3), adjusting the concentration to 4 &#215; 105 cells/mL, with 1 mL of medium in each well.</li>
	<li>Add 1 &#181;L/mL of anti-CD28 solution (final concentration: diluted with medium to 2 &#181;g/mL) to each well. Culture the cells in a 5% CO2 incubator at 37 &#176;C for 5 days.</li>
	<li>Replace the cell culture supernatant medium with culture medium (Th0, non-pathogenic Th17, classical pathogenic Th17, and objective Th17) by carefully pipetting to distribute the cells evenly, discard half of the medium containing the cells, and then add new medium equal to the discard volume on day 2. Repeat on day 4.</li>
	<li>Observe the cells under an optical microscope on day 5 (Figure 2). Collect the cell supernatants from each group and cryopreserve them at -80 &#176;C.</li>
</ol>
5. Flow cytometric analysis for pathogenic Th17 and Th0 cell differentiation
<ol>
	<li>Harvest all cells (per well) after 5 days of initiation of culture. Perform steps 3.12-3.15.</li>
	<li>Add an antibody cocktail (Fixable Viability Dye, 1:1,000; anti-CD4, 1:200) and incubate the cells for 15-20 min in the dark at 4 &#176;C.</li>
	<li>Perform steps 3.17-3.19.</li>
	<li>Discard the supernatant, resuspend the cells in 200 &#181;L of permeabilization buffer, and add the antibody cocktail (IL-17A [intracellular], 1:200; ROR&#947;T [nuclear], 1:200) to the cells. After fixation and membrane rupture, incubate the cell-antibody mixtures in the dark for 40-60 min with shaking occasionally for short periods at 4 &#176;C.</li>
	<li>Add 1 mL of 1x Permeabilization/Wash solution to wash the cells 2x, centrifuging the cells at 400 &#215; g for 5 min at 4 &#176;C; then, discard the supernatant.</li>
	<li>Examine the cells on a flow cytometer and analyze the data using the software (Figure 3).
	<ol>
		<li>Turn on the flow cytometer, and turn on the self-starting cleaning and calibration process.</li>
		<li>Select the laser channel used in this experiment, and set up a blank tube, single-stained tubes, and sample tubes.</li>
		<li>Click on the blank tube option and test on the machine; adjust the voltage so that the cell event is, as far as possible, in the center of the collection box.</li>
		<li>Collect single-stained tube cells and perform spectral detection of the corresponding channels to confirm that the fluorescent antibody is added correctly.</li>
		<li>Click the unmix button and let the machine automatically adjust the spectral compensation. With other flow cytometry instruments, adjust the fluorescence compensation normally.</li>
		<li>Collect each sample tube cell, save the data format as FCS files, and export. Analyze and plot in the relevant flow cytometry data analysis software.
		<ol>
			<li>Circle the main cell population and remove possible cell adhesions through the diagonal circle gates of FSC-H and FSC-A.</li>
			<li>Circle live cells according to the FVS negative circle area; next, circle the CD4+ cell population.</li>
			<li>Construct the cross gate with the horizontal axis as IL-17 A and the vertical axis as ROR&#947;T; the double positive selection in the Q2 area is the Th17 cell population.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
6. Enzyme-linked immunosorbent assay (ELISA) for detection of IL-17A secretion induced by different media
<ol>
	<li>Thaw the frozen cell supernatants collected in Section 4.5.</li>
	<li>Add 100 &#181;L of the diluted standard, blank, and sample into the designated wells. Use the sealer to cover the plate and incubate for 90 min at 37 &#176;C. 
	NOTE: Add the solutions to the bottom of the micro-ELISA plate well without touching the inside wall or causing any foaming.</li>
	<li>Decant the liquid from each well and immediately add 100 &#181;L of Biotinylated Detection Antibody working solution. Use a new sealer to cover the plate and incubate for 1 h at 37 &#176;C.</li>
	<li>Decant the solution and add 350 &#181;L of wash buffer to each well. After 1 min, aspirate or decant the solution from each well and pat it dry against clean absorbent paper. Repeat this wash step 3x.</li>
	<li>Add 100 &#181;L of the horseradish peroxidase Conjugate working solution to each well. Use a new sealer to cover the plate and incubate for 30 min at 37 &#176;C.</li>
	<li>Decant the solution from each well, and repeat the wash process 5x as described in step 6.4.</li>
	<li>Add 90 &#181;L of the Substrate Reagent to each well, and incubate for about 15 min at 37 &#176;C with a new sealer, protecting the plate from light. 
	NOTE: The reaction time can be altered based on the actual color change but should not exceed 30 min. Let the microplate reader warm up for ~15 min before OD measurement.</li>
	<li>Add 50 &#181;L/well of Stop Solution in the same order as the substrate solution.</li>
	<li>Determine the optical density (OD value) of each well at once with a microplate reader set to 450 nm.</li>
	<li>Obtain the OD values for the standard samples and replicate wells of the samples, and subtract the OD values of the blank wells to obtain the corrected values. Then, perform linear fitting with concentration as the abscissa and OD value as the ordinate. Based on the fitted equation, calculate the IL-17A concentration values for the sample wells. 
	NOTE: After 4 days of differentiation, the content of IL-17A in the supernatant of each group is shown in Figure 4. The fitting method can refer to the Orgin21 software. The standard curve obtained from this experiment has an R2 value of 0.9946.</li>
</ol>
7. T-cell differentiation by signature gene expression assays via quantitative PCR (qPCR)
NOTE: To rule out the possibility of flow cytometry instability due to the effects of dyes and fixation/rupture, we detected the expression of characteristic genes by qPCR to elucidate the differentiation effect of pathogenic Th17 at the transcriptional level.
<ol>
	<li>Measure the number of cells at the end of T cell culture. Collect the cells in 1.5 mL centrifuge tubes and centrifuge at 400 &#215; g for 5 min. Carefully discard all the supernatants.</li>
	<li>RNA extraction 
	NOTE: The extraction process must be carried out in a fume hood or an ultra-clean bench to avoid RNA enzyme contamination.
	<ol>
		<li>Add 500 &#181;L of Lysis Buffer (e.g., to 1-2 &#215; 106 cells) to the sample tube (step 7.1), immediately shake and mix until there is no cell mass, and let it stand for 1 min.</li>
		<li>Add the mixture to the adsorption column placed in a collection tube, centrifuge at 13,400 &#215; g for 30 s, and discard the filtrate.</li>
		<li>Add the specified amount of absolute ethanol to the Washing Buffer bottle before using it for the first time. Add 500 &#181;L of washing buffer to the adsorption column, centrifuge at 13,400 &#215; g for 30 s, and discard the filtrate.</li>
		<li>Repeat the washing in step 7.2.3.</li>
		<li>Place the adsorption column RA into the empty collection tube and centrifuge at 13,400 &#215; g for 2 min to remove the rinse solution. 
		NOTE: The rinse solution must be removed to prevent the residual ethanol in the rinse solution from inhibiting the downstream reaction.</li>
		<li>Remove the adsorption column RA and place it in a clean RNase-free centrifuge tube. Add 50 &#181;L of RNase-free H2O to the middle of the adsorption membrane, let it stand for 1 min at room temperature, and centrifuge at 9,600 &#215; g for 1 min.</li>
	</ol>
	</li>
	<li>Measure the purity and concentration of RNA.</li>
	<li>Synthesize the first strand of cDNA by following the kit manufacturer&#39;s instructions.
	<ol>
		<li>Remove genomic DNA. Take 500 ng of the extracted template RNA in an RNase-free centrifuge tube; add RNase-free ddH2O and 4x gDNA mix to form a mixture. Blend gently with a pipette, and place in a 42 &#176;C water bath for 2 min. 
		NOTE: The amount of the reaction mixture and the amount added are related to the RNA concentration; refer to the instructions in detail. The 20 &#181;L reaction system was used in this experiment.</li>
		<li>Directly add a 5x reverse transcription reaction system in the reaction tube of the previous step. Take 4 &#181;L of the reverse transcription reaction system and 16 &#181;L of the mixture from the previous step and gently mix with a pipette. 
		NOTE: The reverse transcription system includes all the required reverse transcriptase, which is a directly configured reverse transcription system.</li>
		<li>Set the reverse transcription reaction: 37 &#176;C 15 min, 85 &#176;C, 5 s, and finally cool to 4 &#176;C.</li>
	</ol>
	</li>
	<li>Set up the PCR reaction following the manufacturer&#39;s instructions.
	<ol>
		<li>Add 10 &#181;L of 2x PCR reaction enzyme mixture in the qPCR tube, 0.4 &#181;L of primer 1, 0.4 &#181;L of primer 2, 0.4 &#181;L of 50x of ROX Reference Dye 1, 2 &#181;L of template cDNA, and 6.8 &#181;L of ddH2O to prepare a 20 &#181;L mixture.</li>
		<li>Perform PCR in the real-time fluorescence quantitative PCR instrument using the following settings: stage 1, 95 &#176;C, 30 s, Rep x 1; stage 2, first 95 &#176;C, 10 s, then 60 &#176;C, 30 s, Rep x 40; stage 3, first 95 &#176;C, 15 s, next 60 &#176;C, 60 s, finally 95 &#176;C, 15 s, Rep x 1. 
		NOTE: After 4 days of differentiation, the relative expression levels of Il17a, Il17f, Rora, and Il23r in na&#239;ve CD4+ T cells are shown in Figure 5.</li>
	</ol>
	</li>
</ol>

Th17 Cell Differentiation from Naive CD4+ T-Cells Using Flow Cytometry

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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unexpected+targets+and+triggers+of+autoimmunity.">Unexpected targets and triggers of autoimmunity.</a> J Clin Immunol. 34, S56-S60 (2014).</li><li>Chang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+conserved+non-coding+aequences+CNS6+and+CNS9+control+cytokine-induced+Rorc+transcription+during+T+helper+17+cell+differentiation.">The conserved non-coding aequences CNS6 and CNS9 control cytokine-induced Rorc transcription during T helper 17 cell differentiation.</a> Immunity. 53 (3), 614-626 (2020).</li><li>Bunte, K., Beikler, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Th17+cells+and+the+IL-23/IL-17+axis+in+the+pathogenesis+of+periodontitis+and+immune-mediated+inflammatory+diseases.">Th17 cells and the IL-23/IL-17 axis in the pathogenesis of periodontitis and immune-mediated inflammatory diseases.</a> Int J Mol Sci. 20 (14), 3394 (2019).</li><li>Zhao, Y., Liu, Z., Qin, L., Wang, T., Bai, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+the+mechanisms+of+Th17+differentiation+and+the+Yin-Yang+of+Th17+cells+in+human+diseases.">Insights into the mechanisms of Th17 differentiation and the Yin-Yang of Th17 cells in human diseases.</a> Mol Immunol. 134, 109-117 (2021).</li><li>Berghmans, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interferon-&#947;+orchestrates+the+number+and+function+of+Th17+cells+in+experimental+autoimmune+encephalomyelitis.">Interferon-&#947; orchestrates the number and function of Th17 cells in experimental autoimmune encephalomyelitis.</a> J Interferon Cytokine Res. 31 (7), 575-587 (2011).</li><li>Wu, B., Wan, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+control+of+pathogenic+Th17+cells+in+autoimmune+diseases.">Molecular control of pathogenic Th17 cells in autoimmune diseases.</a> Int Immunopharmacol. 80, 106187 (2020).</li><li>Du, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+hormone+releasing+hormone+signaling+promotes+Th17+cell+differentiation+and+autoimmune+inflammation.">Growth hormone releasing hormone signaling promotes Th17 cell differentiation and autoimmune inflammation.</a> Nat Commun. 14 (1), 3298 (2023).</li><li>Ernst, D. N., Weigle, W. O., Noonan, D. J., McQuitty, D. N., Hobbs, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+age-associated+increase+in+IFN-gamma+synthesis+by+mouse+CD8++T+cells+correlates+with+shifts+in+the+frequencies+of+cell+subsets+defined+by+membrane+CD44,+CD45RB,+3G11,+and+MEL-14+expression.">The age-associated increase in IFN-gamma synthesis by mouse CD8+ T cells correlates with shifts in the frequencies of cell subsets defined by membrane CD44, CD45RB, 3G11, and MEL-14 expression.</a> J Immunol. 151 (2), 575-587 (1993).</li><li>Radtke, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IBEX:+an+iterative+immunolabeling+and+chemical+bleaching+method+for+high-content+imaging+of+diverse+tissues.">IBEX: an iterative immunolabeling and chemical bleaching method for high-content imaging of diverse tissues.</a> Nat Protoc. 17 (2), 378-401 (2022).</li><li>El-Hindi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-cell-specific+CerS4+depletion+prolonged+inflammation+and+enhanced+tumor+burden+in+the+AOM/DSS-induced+CAC+model.">T-cell-specific CerS4 depletion prolonged inflammation and enhanced tumor burden in the AOM/DSS-induced CAC model.</a> Int J Mol Sci. 23 (3), 1866 (2022).</li><li>Cooney, L. A., Towery, K., Endres, J., Fox, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitivity+and+resistance+to+regulation+by+IL-4+during+Th17+maturation.">Sensitivity and resistance to regulation by IL-4 during Th17 maturation.</a> J Immunol. 187 (9), 4440-4450 (2011).</li><li>Zhu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+interleukin-2-based+fusion+molecule,+HCW9302,+differentially+promotes+regulatory+T+cell+expansion+to+treat+atherosclerosis+in+mice.">A novel interleukin-2-based fusion molecule, HCW9302, differentially promotes regulatory T cell expansion to treat atherosclerosis in mice.</a> Front Immunol. 14, 1114802 (2023).</li></ol>

The authors declare that there are no conflicts of interest regarding the publication of this paper.

This procedure offered a productive way to increase the number of mice splenic na&#239;ve CD4+ T cells for the in vitro production of pathogenic Th17 cells. Although we use more cytokines than other reported Th17 cell culture media, we are committed to optimizing the growth conditions of pathogenic Th17 cells. We are considering further optimization of our induced differentiation protocol.
Here, we simply used flow cytometry and qPCR to examine the production of hallmark cytokines. With a few minor modifications, this approach can also be used for other function tests, such as cell proliferation.
We used a Chinese-produced lymphocyte isolation kit to isolate mouse spleen lymphocytes because it is effective and time-saving. Lymphocyte separation solutions based on other brands can also achieve the purpose of separating mouse spleen lymphocytes through different steps. Another method is to directly lyse the red blood cells of the obtained spleen cell suspension; however, we found that spleen red blood cells often cannot be lysed at one time.
Some problems can arise during the execution of this protocol. First, the number of na&#239;ve CD4+ T cells obtained by magnetic bead sorting may be very low (protocol step 3). This could be attributed to the process of crushing organs being insufficient. It is important to ensure that the organ is properly homogenized. Increasing the frequency of rinsing during the homogenization process will improve the recovery rate. To obtain a higher number of splenic na&#239;ve CD4+ T cells, we suggest using younger mice (6-10 weeks old). There are various methods available for separating spleen lymphocytes, and the yield may vary depending on the separation liquid used. It is recommended to use a universally certified separation liquid and try to extract the lymphocyte layer as much as possible.
Second, the proportion of CD4+ T cells in flow cytometry may be &#60;80% (protocol step 5). One possible cause of this issue could be an inaccurate splenocyte count, resulting in a cell count greater than that of the additional antibody cocktail and magnetic beads. To increase the effectiveness of na&#239;ve CD4+ T cell purification, cell counting must be precise. Additionally, 10% more antibody cocktail and magnetic beads can be used over what this protocol recommends. Lastly, flow cytometry can be performed immediately after sorting na&#239;ve CD4+ T cells.
Third, there may not be many T cell clusters formed during the culture, and most of the cells may have died during the differentiation of T cells (protocol step 4). The potential reason for this problem may be the inaccurate determination of cell numbers for na&#239;ve CD4+ T cells before seeding, resulting in a low cell density. It is recommended to adopt a more accurate counting method to achieve the desired cell density of approximately 4 &#215; 105 cells/mL for each well in a 48-well plate. Another possible cause could be technical problems with the CO2 incubator, such as incorrect temperature or CO2 concentration. Lastly, excessive force while changing the cell culture medium could potentially cause cell death.
Fourth, the relative expression levels of the signature cytokine genes may be low (protocol step 7). To ensure the authenticity of the extracted RNA, it is recommended to use a nanodroplet detection concentration of more than 100 ng/mL. The potential reason for the decrease in concentration may be the unhealthy nature of cultured cells, such as a large part of the collected cells are dead or in the process of death. To obtain the true RNA concentration, it is necessary to resolve the situation that may lead to poor growth during cell culture. An alternative reason behind this concern might be the excessively low final cell count achieved during RNA extraction, possibly due to inadvertent cell loss during the supernatant disposal. Employing advanced RNA extraction techniques such as one-step RNA extraction kits could prove advantageous. The ideal OD260/OD280 ratio, as measured by Nanodrop, should fall within the range of 1.9-2.1. In the event of an excessively low ratio, protein contamination becomes a possibility. Increasing the frequency of RNase-free buffer washing may aid in mitigating this issue. Conversely, an uncharacteristically low ratio implies potential RNA degradation. To counteract this issue, it is recommended to employ RNase-free water and utilize 1.5 mL tubes for RNase decontamination purposes.
In conclusion, the current protocol describes the use of new cell culture medium to directly induce naive CD4+ T cells to differentiate into pathogenic Th17 cells in vitro. Compared with direct separation, there is no doubt that this method is more direct, inexpensive, and more efficient. The configuration of the medium is also very simple so that the constructed Th17 cells can be more intuitively used for subsequent experiments, providing a very good cell model for the study of many diseases.

After leaving the thymus, naive CD4+ T lymphocytes pass through secondary lymphoid organs. Antigen-presenting cells that transmit homologous antigens to na&#239;ve CD4+ T cells activate them, starting a series of differentiation programs that eventually result in the production of highly specialized T helper (Th) cell lineages1. Interleukin 17 (IL-17) production characterizes Th17 cells, a subpopulation of pro-inflammatory Th cells2. Th17 cells play a role in the host&#39;s defense against extracellular pathogens and in the pathogenesis of many autoimmune diseases, such as autoimmune uveitis and multiple sclerosis. Signals from T-cell receptors and cytokines IL-6 and transforming growth factor-&#946; (TGF-&#946;) induce the differentiation of naive T cells into Th17 cells through the phosphorylation of signal transducer and activator of transcription (STAT)33. STAT3 is further amplified in a positive feedback loop through signaling mediated by IL-23 and IL-214,5. Phosphorylation of STAT3 can induce the expression of the transcription factors ROR&#947;t and ROR&#945;, which act as master switches regulating the cytokine profile of IL-17A, IL-17F, IL-21, and IL-22 in Th17 cells6. However, it has been reported that IL-6- and TGF-&#946;-induced Th17 cells are insufficient to trigger autoimmune diseases, which require co-stimulation by IL-23 or separate co-stimulation of IL-6, IL-1&#946;, and IL-23 in the absence of TGF-&#946;7,8.
Th17 subsets that cannot effectively induce experimental autoimmune encephalomyelitis (EAE) are sometimes referred to as non-pathogenic Th17 while Th17 subsets that can induce EAE are known as pathogenic Th179. Current studies have shown that although pathogenic Th17 and non-pathogenic Th17 co-express the core transcription factor ROR&#947;t, there are great differences in the ability to produce IL-17A and pro-inflammatory and anti-inflammatory properties10. In addition to the high expression of ROR&#947;t, CCR6, STAT4, and RUNX4, which are common characteristic transcription factors of Th17, pathogenic Th17 cells also show additional gene signal expression characteristics related to the disease, such as TBX21, IFN-&#947;, and CXCR3, which have the characteristics of Th1 cell subsets. Pathogenic Th17 cells can secrete high levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), IFN-&#947;, TNF-&#945;, and other cytokines11,12. The phenotype of non-pathogenic Th17 cells is unstable, and only under the stimulation of CD3 and cytokine IL-2 can some of these cells differentiate into pathogenic Th17 cells. Therefore, in common clinical disease models such as rheumatoid arthritis, multiple sclerosis, and acute respiratory distress syndrome, pathogenic Th17 cells primarily exert pathogenic effects.
Pathogenic and non-pathogenic Th17 cells can differentiate in vitro under the influence of different cytokines. In recent years, several studies have proposed methods for inducing the differentiation of Th17 cells using different types and concentrations of cytokines. Th17 cells are stimulated by a combination of IL-6, IL-1&#946;, and IL-2313,14,15,16. It has been proved that IL-6 and TGF-&#946;, two cytokines necessary for Th17 cell differentiation, synergistically regulate the expression of ROR&#947;t and Th17 cell differentiation by interacting with two different conserved non-coding DNA sequences at the Rorc gene locus17. The stable phase of pathogenic Th17 cells is mainly maintained by IL-2318,19. IL-23 binds to its receptor and activates the JAK-STAT signaling pathway20, thereby causing phosphorylation of Jak2 and Tyk2 and promoting phosphorylation of STAT1, STAT3, STAT4, and STAT5. IL-4 and IFN-&#947; are negative regulators of this pathway. However, studies have shown that IL-1&#946; may positively regulate the transcription of Ror&#945; and Ror&#947;t through the mTOR pathway to maintain the stability of the Th17 cell phenotype21.
Due to the heterogeneity of numerous studies, we chose the induction protocols for pathogenic and non-pathogenic Th17 cells from the latest research as controls22. The results indicate that, assuming that everything is carried out according to this protocol, after 5 days of culture under the condition of generating pathogenic Th17, more than 90% of the surviving cells can be pathogenic Th17 cells.

<table><tbody><tr><td>&lt;strong&gt;Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Rat anti-mouse CD62L, BV650, clone MEL-14, 1:200 dilution</td><td>BD</td><td>Cat# 564108; RRID: AB_2738597</td><td></td></tr><tr><td>Rat monoclonal anti-CD4, FITC, clone RM4-5, 1:200 dilution</td><td>BioLegend</td><td>Cat#100509; RRID: AB_312712</td><td></td></tr><tr><td>Rat monoclonal anti-IL-17A, PE, clone TC11-18H10.1, 1:200 dilution</td><td>BioLegend</td><td>Cat#506903; RRID: AB_ 315463</td><td></td></tr><tr><td>Rat monoclonal anti mouse/human CD44, APC, clone IM&amp;amp;, 1:200 dilution</td><td>BioLegend</td><td>Cat#103012; RRID: AB_312963</td><td></td></tr><tr><td>Rat monoclonal anti-ROR&amp;gamma;T, APC, clone B2D, 1:200 dilution</td><td>Invitrogen</td><td>Cat#17-6981-80; RRID: AB_2573253</td><td></td></tr><tr><td>Rat monoclonal FOXP3 antibody, PE, clone FJK-16s, 1:200 dilution</td><td>Invitrogen</td><td>Cat#12-5773-82; RRID: AB_465936</td><td></td></tr><tr><td>&lt;strong&gt;Chemicals, peptides, and recombinant proteins&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Anti-Mouse CD3 SAFIRE purified</td><td>biogems</td><td>Cat#05112-25</td><td></td></tr><tr><td>Anti-Mouse CD28 SAFIRE purified</td><td>biogems</td><td>Cat#10312-25</td><td></td></tr><tr><td>Anti-Mouse IFN gamma</td><td>biogems</td><td>Cat#80822-25</td><td></td></tr><tr><td>Anti-Mouse IL-4</td><td>biogems</td><td>Cat#81112-25</td><td></td></tr><tr><td>Ethanol</td><td>Xilong scientific</td><td>Cat#64-17-5</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Gibco</td><td>Cat#10437-028</td><td></td></tr><tr><td>FcR Blocking reagent</td><td>Miltenyi Biotec</td><td>Cat#130-092-575</td><td></td></tr><tr><td>GlutaMAX supplement</td><td>gibco</td><td>Cat#35050079</td><td></td></tr><tr><td>Mouse rIL-1 beta</td><td>Sino Biological</td><td>Cat#50101-MNAE</td><td></td></tr><tr><td>Mouse rIL-6</td><td>Sino Biological</td><td>Cat#50136-MNAE</td><td></td></tr><tr><td>Mouse rIL-23</td><td>Sino Biological</td><td>Cat#CT028-M08H</td><td></td></tr><tr><td>Mouse TGF beta 1</td><td>Sino Biological</td><td>Cat#50698-M08H</td><td></td></tr><tr><td>PBS</td><td>Procell</td><td>Cat#PB180327</td><td></td></tr><tr><td>Recombinant Murine IL-2</td><td>peprotech</td><td>Cat#212-12</td><td></td></tr><tr><td>RPMI 1640 with L-glutamine</td><td>Gibco</td><td>Cat#11875-119</td><td></td></tr><tr><td>Penicillin-streptomycin solution</td><td>Gibco</td><td>Cat#15070063</td><td></td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Sigma-Aldrich</td><td>Cat#M6250-100ML</td><td></td></tr><tr><td>&lt;strong&gt;Critical commercial assays&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Animal Organ Lymphocyte Separation Solution Kit</td><td>Tbdscience</td><td>Cat#TBD0018SOP</td><td>Contains animal spleen tissue lymphocyte separation liquid, tissue sample diluent (cat no. 2010C1119), sample cleaning solution (cat no. 2010X1118), sample washing solution (cat no. TBTDM-W), tissue homogenate flushing liquid (F2013TBD)</td></tr><tr><td>ChamQ SYBR qPCR Master Mix (High ROX Premixed)</td><td>Vazyme</td><td>Cat#Q341-02</td><td>https://www.vazymebiotech.com/product-center/chamq-sybr-qpcr-master-mix-high-rox-premixed-q341.html.</td></tr><tr><td>Fixation/permeabilization Concentrate</td><td>invitrogen</td><td>Cat#00-5123-43</td><td></td></tr><tr><td>Fixation / Permeabilization Diluent</td><td>invitrogen</td><td>Cat#00-5223-56</td><td></td></tr><tr><td>Fixable Viability Dye EF506</td><td>invitrogen</td><td>Cat#65-0866</td><td></td></tr><tr><td>HiScript II Q RT SuperMix for qPCR (+gDNA wiper)</td><td>Vazyme</td><td>Cat#R223-01</td><td>https://www.vazymebiotech.com/product-center/hiscript-ii-q-rt-supermix-for-qpcr-gdna-wiper-r223.html.</td></tr><tr><td>Leukocyte Activation Cocktail</td><td>BD</td><td>Cat#550583</td><td></td></tr><tr><td>Mouse IL-17A (Interleukin 17A) ELISA Kit</td><td>Elabscience&amp;reg;</td><td>Cat#E-EL-M0047</td><td></td></tr><tr><td>Na&amp;iuml;ve CD4&lt;sup&gt;+&lt;/sup&gt; T cells isolation kit, mouse</td><td>STEMCELL</td><td>Cat#19765</td><td>EasySep kit contains mouse CD4+ T cell isolation cocktail [cat no. 19852C.1], mouse memory T cell depletion cocktail [cat no. 18766C], streptavidin RapidSphered 50001 [cat no. 50001], normal rat serum [cat no. 13551]); only store rat serum at -20 &amp;deg;C; other components to be stored at 2-8 &amp;deg;C.</td></tr><tr><td>Permeabilization Buffer</td><td>invitrogen</td><td>Cat#00-8333-56</td><td></td></tr><tr><td>SPARKeasy Cell RNA Kit</td><td>Sparkjade</td><td>Cat#AC0205-B</td><td>https://www.sparkjade.com/product/detail?id=85</td></tr><tr><td>&lt;strong&gt;Experimental models: Organisms/strains&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Mouse: C57BL/6</td><td>Gempharmatech</td><td>Cat#000013</td><td></td></tr><tr><td>&lt;strong&gt;Oligonucleotides&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Mouse Il17a TaqMan primers with probe</td><td>ribobio</td><td>NA</td><td></td></tr><tr><td>Mouse Il17f TaqMan primers with probe</td><td>ribobio</td><td>NA</td><td></td></tr><tr><td>Mouse Il23r TaqMan primers with probe</td><td>ribobio</td><td>NA</td><td></td></tr><tr><td>Mouse Rora TaqMan primers with probe</td><td>ribobio</td><td>NA</td><td></td></tr><tr><td>&amp;beta;-actin TaqMan primers with probe</td><td>ribobio</td><td>NA</td><td></td></tr><tr><td>&lt;strong&gt;Software and algorithms&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cytek Aurora</td><td>Cytek</td><td>https://spectrum.cytekbio.com/</td><td></td></tr><tr><td>FlowJo v10.8.1</td><td>Tree Star</td><td>https://www.flowjo.com</td><td></td></tr><tr><td>GraphPad prism 9</td><td>GraphPad Software</td><td>https://www.graphpad-prism.cn</td><td></td></tr><tr><td>&lt;strong&gt;Other&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1 mL syringe</td><td>Kindly</td><td>NA</td><td></td></tr><tr><td>1.5 mL Centrifuge tubes</td><td>Eppendorf</td><td>Cat#MCT-150-C</td><td></td></tr><tr><td>5 mL Round-bottom tubes</td><td>Corning</td><td>Cat#352235</td><td></td></tr><tr><td>15 mL Centrifuge tubes</td><td>NEST</td><td>Cat#601052</td><td></td></tr><tr><td>48-Well tissue culture plate (flatten bottom)</td><td>Corning</td><td>Cat#3548</td><td></td></tr><tr><td>50 mL Centrifuge tubes</td><td>NEST</td><td>Cat#602052</td><td></td></tr><tr><td>70 &amp;micro;m Cell strainer</td><td>Biosharp</td><td>Cat#BS-70-XBS</td><td></td></tr><tr><td>96-well Unskirted qPCR Plates</td><td>VIOX scientific</td><td>Cat#V4801-M</td><td></td></tr><tr><td>100 mm Petri dish</td><td>Corning</td><td>Cat#430167</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5425R</td><td></td></tr><tr><td>Cell culture CO2 incubator</td><td>Thermo Fisher</td><td>HEPA Class100</td><td></td></tr><tr><td>Cytek Aurora</td><td>Cytek</td><td>NA</td><td></td></tr><tr><td>dissecting scissors</td><td>RWD</td><td>S12003-09</td><td></td></tr><tr><td>Hemocytometer</td><td>AlphaCell</td><td>Cat#J633201</td><td></td></tr><tr><td>NanoDrop 2000 Spectrophotometer</td><td>Thermo Fisher</td><td>ND-2000</td><td></td></tr><tr><td>Real-time PCR System</td><td>Roche</td><td>LightCycler96</td><td></td></tr><tr><td>Surgical tweezers</td><td>RWD</td><td>F12005-10</td><td></td></tr><tr><td>Thermal cycler</td><td>Bio-Rad</td><td>C1000 Touch</td><td></td></tr></tbody></table>

in vitro differentiation of naive cd4+ t cells into pathogenic th17 cells in mouse

In vitro T cell differentiation techniques are essential for both functional and mechanistic investigations of CD4+ T cells. Pathogenic Th17 cells have been linked to a wide range of diseases in recent times, including multiple sclerosis (MS), rheumatoid arthritis, acute respiratory distress syndrome (ARDS), sepsis, and other autoimmune disorders. However, the currently known in vitro differentiation protocols have difficulty achieving high purity of pathogenic Th17 cells, with the induction efficiency often below 50%, which is a key challenge in in vitro experiments. In this protocol, we propose an enhanced in vitro culture and differentiation protocol for pathogenic Th17 cells, which is used to directly differentiate naive CD4+ T cells isolated from mouse spleens into pathogenic Th17 cells. This protocol provides detailed instructions on splenocyte isolation, purification of naive CD4+ T cells, and differentiation of pathogenic Th17 cells. Through this protocol, we can achieve a differentiation purity of approximately 90% for pathogenic Th17 cells, which meets the basic needs of many cellular experiments.

Our protocol was developed based on earlier research about pathogenic Th17 cell differentiation. The first step of the experiment is to detect the purity of na&#239;ve CD4+ T cells isolated from the spleen by magnetic bead sorting, which is the basis for the success of our subsequent pathogenic Th17 cell differentiation. The purity of na&#239;ve CD4+ T cells was detected using surface markers CD62L23 and CD4424 while FOXP325 was used as a marker of Treg cells. We found that the content of Treg cells was significantly reduced after sorting, and the purity of na&#239;ve CD4+ T cells could reach at least 95% (Figure 1). To compare the differentiation of pathogenic Th17 cells, na&#239;ve CD4+ T cells were cultured with Th0 medium (Table 1) and Th17 differentiation medium (Table 1) for a total of 5 days. It was found that T cells showed cluster growth in both Th0 and Th17 media (Figure 2).
Next, the cells were fixed, permeabilized, and labeled with antibodies against several cytokines in differentiated CD4+ T cells based on flow cytometry. We examined IL17A26 and ROR&#947;T27 as the hallmark cytokines of Th17 cell differentiation and found that 90% of na&#239;ve CD4+ T cells successfully differentiated into pathogenic Th17 cells under the stimulation of new Th17 cell culture medium (Figure 3). Figure 5 shows the signature genes of Th17 cells detected by PCR, which proved that the pathogenic Th17 cells we obtained by differentiation were stably expressing.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66718/66718fig01.jpg" /> 
Figure 1: Gating strategy for analysis of signature cytokines in C57BL/6J mouse before and after na&#239;ve CD4+ T cell isolation. Abbreviations: FSC-H = forward scatter-peak height; SSC-H = side scatter-peah height; FSC-A = forward scatter-peak area; FITC = fluorescein isothiocyanate; PE = phycoerythrin. <a href="https://www.jove.com/files/ftp_upload/66718/66718fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66718/66718fig02.jpg" /> 
Figure 2: Representative images of mouse na&#239;ve CD4+ T cells cultured under pathogenic Th17 and Th0 conditions for 5 days. (A) Th0 cells; (B) pathogenic Th17 cells. Scale bars = 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/66718/66718fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66718/66718fig03.jpg" /> 
Figure 3: Flow cytometry analysis after differentiation induced by Th0 cell culture medium and Th17 cell culture medium. Abbreviations: FSC-H = forward scatter-peak height; SSC-H = side scatter-peah height; FSC-A = forward scatter-peak area; FITC = fluorescein isothiocyanate; PE = phycoerythrin. <a href="https://www.jove.com/files/ftp_upload/66718/66718fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66718/66718fig04.jpg" /> 
Figure 4: The content of IL-17A in the supernatant of Th0 cell culture medium and Th17 cell culture medium after induction of differentiation. <a href="https://www.jove.com/files/ftp_upload/66718/66718fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66718/66718fig05.jpg" /> 
Figure 5: Representative results of the expression levels of signature cytokines in differentiated CD4+ T cells of C57BL/6J mouse. <a href="https://www.jove.com/files/ftp_upload/66718/66718fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3"></td>
			<td colspan="3">&#160;Target pathogenic Th17 cell culture medium</td>
			<td colspan="3">Th0 cell culture medium</td>
			<td colspan="3">Classical non-pathogenic Th17 cell culture medium</td>
			<td colspan="3">Classical pathogenic Th17 cell culture medium.</td>
		</tr>
		<tr>
			<td colspan="3">Reagent</td>
			<td colspan="2">Final concentration&#160;</td>
			<td>Amount</td>
			<td colspan="2">Final concentration&#160;</td>
			<td>Amount</td>
			<td colspan="2">Final concentration&#160;</td>
			<td>Amount</td>
			<td colspan="2">Final concentration&#160;</td>
			<td>Amount</td>
		</tr>
		<tr>
			<td colspan="3">Penicillin-Streptomycin (100x)</td>
			<td colspan="2">1x</td>
			<td>500 &#956;L</td>
			<td colspan="2">1x</td>
			<td>500 &#956;L</td>
			<td colspan="2">1x</td>
			<td>500 &#956;L</td>
			<td colspan="2">1x</td>
			<td>500 &#956;L</td>
		</tr>
		<tr>
			<td colspan="3">Fetal Bovine Serum</td>
			<td colspan="2">10%</td>
			<td>5 mL</td>
			<td colspan="2">10%</td>
			<td>5 mL</td>
			<td colspan="2">10%</td>
			<td>5 mL</td>
			<td colspan="2">10%</td>
			<td>5 mL</td>
		</tr>
		<tr>
			<td colspan="3">&#946;-mercaptoethanol</td>
			<td colspan="2">50 &#956;M</td>
			<td>50 &#956;L</td>
			<td colspan="2">50 &#956;M</td>
			<td>50 &#956;L</td>
			<td colspan="2">50 &#956;M</td>
			<td>50 &#956;L</td>
			<td colspan="2">50 &#956;M</td>
			<td>50 &#956;L</td>
		</tr>
		<tr>
			<td colspan="3">GlutaMAXTM supplement (100x)</td>
			<td colspan="2">1x</td>
			<td>500 &#956;L</td>
			<td colspan="2">1x</td>
			<td>500 &#956;L</td>
			<td colspan="2">1x</td>
			<td>500 &#956;L</td>
			<td colspan="2">1x</td>
			<td>500 &#956;L</td>
		</tr>
		<tr>
			<td colspan="3">Sodium pyruvate solution (100x)</td>
			<td colspan="2">1 mM</td>
			<td>500 &#956;L</td>
			<td colspan="2">1 mM</td>
			<td>500 &#956;L</td>
			<td colspan="2">1 mM</td>
			<td>500 &#956;L</td>
			<td colspan="2">1 mM</td>
			<td>500 &#956;L</td>
		</tr>
		<tr>
			<td colspan="3">Anti-Mouse IFN gamma (1 mg/mL)</td>
			<td colspan="2">5 &#181;g/mL</td>
			<td>250 &#956;L</td>
			<td colspan="2"></td>
			<td></td>
			<td colspan="2">10 &#181;g/mL</td>
			<td>500 &#956;L</td>
			<td colspan="2">10 &#181;g/mL</td>
			<td>500 &#956;L</td>
		</tr>
		<tr>
			<td colspan="3">Anti-Mouse IL-4 (2 mg/mL)</td>
			<td colspan="2">5 &#181;g/mL</td>
			<td>125 &#956;L</td>
			<td colspan="2"></td>
			<td></td>
			<td colspan="2">5 &#181;g/mL</td>
			<td>250 &#956;L</td>
			<td colspan="2">10 &#181;g/mL</td>
			<td>250 &#956;L</td>
		</tr>
		<tr>
			<td colspan="3">Mouse rIL-1 beta (20 &#181;g)</td>
			<td colspan="2">20 ng/mL</td>
			<td>NA</td>
			<td colspan="2"></td>
			<td></td>
			<td colspan="2"></td>
			<td></td>
			<td colspan="2"></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">Mouse rIL-6 (20 &#181;g)</td>
			<td colspan="2">20 ng/mL</td>
			<td>NA</td>
			<td colspan="2"></td>
			<td></td>
			<td colspan="2">50 ng/mL</td>
			<td>NA</td>
			<td colspan="2">50 ng/mL</td>
			<td>NA</td>
		</tr>
		<tr>
			<td colspan="3">Mouse rIL-23 (50 &#181;g)</td>
			<td colspan="2">50 ng/mL</td>
			<td>NA</td>
			<td colspan="2"></td>
			<td></td>
			<td colspan="2"></td>
			<td></td>
			<td colspan="2">10 ng/mL</td>
			<td>NA</td>
		</tr>
		<tr>
			<td colspan="3">Mouse TGF beta (100 &#181;g)</td>
			<td colspan="2">3 ng/mL</td>
			<td>NA</td>
			<td colspan="2"></td>
			<td></td>
			<td colspan="2">1 ng/mL</td>
			<td>NA</td>
			<td colspan="2">1 ng/mL</td>
			<td>NA</td>
		</tr>
		<tr>
			<td colspan="3">&#160;Murine IL-2 (5 &#181;g)</td>
			<td colspan="2">20 ng/mL</td>
			<td>NA</td>
			<td colspan="2"></td>
			<td></td>
			<td colspan="2"></td>
			<td></td>
			<td colspan="2"></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">RPMI 1640</td>
			<td colspan="2">NA</td>
			<td>To 50 mL</td>
			<td colspan="2">NA&#160;</td>
			<td>To 50 mL</td>
			<td colspan="2"></td>
			<td></td>
			<td colspan="2">NA</td>
			<td>To 50 mL</td>
		</tr>
		<tr>
			<td colspan="3">Total</td>
			<td colspan="2">NA</td>
			<td>50 mL</td>
			<td colspan="2">NA</td>
			<td>50 mL</td>
			<td colspan="2"></td>
			<td></td>
			<td colspan="2">NA</td>
			<td>50 mL</td>
		</tr>
	</tbody>
</table>
Table 1: Target pathogenic Th17 cell culture, Th0 cell culture, classical non-pathogenic and pathogenic Th17 cell culture media.

Author Spotlight: Achieving High-Purity In Vitro Differentiation of Th17 Cells Using Cytokine Concentration Modulation

This protocol describes the differentiation of na&#239;ve CD4+ T cells into pathogenic Th17 cells in vitro. Specifically, when combined with a multi-parameter flow cytometry-based approach, 90% purity of pathogenic Th17 cells can be obtained from na&#239;ve CD4+ T cells using this differentiation method.

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

In vitro T cell differentiation techniques are essential for both functional and mechanistic investigations of CD4+ T cells. Pathogenic Th17 cells have ...

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Research

JoVE Journal

Immunology and Infection

2.7K Views.  Southeast University. Ons onderzoeksdoel is om ons te richten op de gerichte differentiatie van Th17-cellen, en we streven ernaar een eenvoudige methode te verkennen voor re-intro-differentiatie van Th17-cellen, die dient als een traditionele voorwaarde voor re-intro-experimenten die verband houden met sommige ziekte-geassocieerde Th17-cellen. Veel studies hebben de methoden voor herintroductie van Th17-cellen genoemd, en het recente onderzoek heeft zich gericht op kleine molecula...

Video: Auteur in de schijnwerpers: het bereiken van zeer zuivere in vitro differentiatie van Th17-cellen met behulp van cytokineconcentratiemodulatie

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 ...

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of human Type 1 diabetes (T1D). Cell transfer studies in irradiated recipient mice have established that T cells are pivotal in T1D pathogenesis in this model. We describe herein a simple method to rapidly induce T1D by adoptive transfer of purified, primary CD4+ T cells from pre-diabetic NOD mice transgenic for the islet-specific T cell receptor (TCR) BDC2.5 into NOD.SCID recipient mice. The major advantages of this technique are that isolation and adoptive transfer of diabetogenic T cells can be completed within the same day, irradiation of the recipients is not required, and a high incidence of T1D is elicited within 2 weeks after T cell transfer. Thus, studies of pathogenesis and therapeutic interventions in T1D can proceed at a faster rate than with methods that rely on heterogenous T cell populations or clones derived from diabetic NOD mice.

We provide a reproducible method to induce type 1 diabetes (T1D) in mice within two weeks by the adoptive transfer of islet antigen-specific, primary CD4+ T cells.

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of ...

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&#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 ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

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 ...

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor (GM-CSF)-producing T Helper (THGM) Cells

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (THGM) cell is a newly identified T helper cell subset that predominantly secretes GM-CSF without producing interferon (IFN)&#947; or interleukin (IL)-17 and is found to play an essential role in the autoimmune neuroinflammation. A method of isolation of naive CD4+ T cells from a single-cell suspension of splenocytes and THGM cell generation from naive CD4+ T cells would be a useful technique in the study of T cell-mediated immunity and autoimmune diseases. Here we describe a method that differentiates mouse naive CD4+ T cells into THGM cells promoted by IL-7. The outcome of the differentiation was assessed by the analysis of the cytokines expression using different techniques, including intracellular cytokine staining combined with flow cytometry, a quantitative real-time polymerase chain reaction (PCR), and enzyme-linked immunosorbent assays (ELISA). Using the THGM differentiation protocol as described here, about 55% of the cells expressed GM-CSF with a minimal expression of IFN&#945; or IL-17. The predominant expression of GM-CSF by THGM cells was further confirmed by the analysis of the expression of GM-CSF, IFN&#945;, and IL-17 at both mRNA and protein levels. Thus, this method can be used to differentiate naive CD4+ T cells to THGM cells in vitro, which will be useful in the study of THGM cell biology.

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor GM-CSF-producing T Helper THGM Cells

Here, we present a protocol to differentiate murine granulocyte-macrophage-colony-stimulating-factor-producing T helper (THGM) cells from naive CD4+ T cells, including isolation of naive CD4+ T cells, differentiation of THGM, and analysis of differentiated THGM cells. This method can be applied to studies of the regulation and function of THGM cells.

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (THGM) cell is a newly identified T helper cell subset that predominantly ...

Retroviral Transduction of Guide RNA for Gene Editing in Th17 Differentiation

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

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

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

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

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

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