This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2015R1A6A3A01020610 to HJP) and a grant from the Korean Health Technology R&amp;D Project, Ministry for Health, Welfare and Family Affairs, Republic of Korea (HI15C0493 to SJH).

In this study, mice were maintained in a specific pathogen-free facility of the Yonsei Laboratory Animal Research Center of Yonsei University. All animal experiments were conducted in accordance with the Korean Food and Drug Administration guidelines using protocols approved by the International Animal Care and Use Committee of the Yonsei Laboratory Animal Research Center at Yonsei University.
1. Preparation of Solutions
<ol>
	<li>Prepare 2% RPMI media by diluting fetal bovine serum (FBS) to 2% and penicillin-streptomycin to 1% in RPMI</li>
	<li>Prepare complete RPMI media. To RPMI media add 10% of FBS, 1% of penicillin-streptomycin, 1% of L-glutamine, and 50 &#956;M 2-mercaptoethanol.</li>
	<li>Prepare fluorescence activated cell sorting (FACS) buffer. To do so, supplement phosphate-buffered saline (PBS) with 2% of FBS.</li>
	<li>Prepare T cell isolation buffer. To do so, supplement PBS with 2% of FBS and 2 mM ethylenediaminetetraacetic acid.</li>
</ol>
2. Isolation of Splenic Lymphocytes
<ol>
	<li>Remove the spleens from na&#239;ve or LCMV CL13-infected mice as described previously19 and place them in 60 mm x 15 mm Petri dishes containing 10 ml of 2% RPMI media. 
	NOTE: In this study experiments, 5 to 6 week-old C57B1L/6J female mice received 2 x 106 plaque-forming units (p.f.u.) of LCMV CL13 through intravenous injection via the tail vein. Sacrifice the mice at day 16 post-infection (16 d p.i.)25. Analyze ge-matched naive mice were on the same day.</li>
	<li>Place a cell strainer into a 50 ml tube and rinse it with 2 ml of 2% RPMI media. Place the spleen on the 70 &#956;m cell strainer and grind using the plunger of a syringe. Rinse the cell strainer with 2 ml of 2% RPMI media and remove it from the 50 ml tube.</li>
	<li>Fill up the tube with 2% RPMI media and centrifuge at 300 x g for 5 min at 4 &#176;C. Discard the supernatant and resuspend the cell pellet in 1 ml of ACK lysing buffer. Incubate the samples at room temperature (RT) for 5 min. 
	NOTE: The volume of ACK lysing buffer indicated above is for one spleen. Scale up the volume accordingly for pooled spleen samples.</li>
	<li>Fill up the tube with 2% RPMI media and centrifuge at 300 x g for 10 min at 4 &#176;C. Discard the supernatant and resuspend the cells at a density of 1 x 107 cells/ml in complete RPMI media. 
	NOTE: For live cell counting, stain cells with trypan blue and count only live cells that are not stained using hemocytometer.</li>
</ol>
3. Phenotyping of Splenic Conventional T (Tconv) Cells and Treg Cells
NOTE: Before Treg cell isolation, examine the phenotype of splenic lymphocytes isolated from na&#239;ve or infected mice by staining the cells with various antibodies and analyzing them by flow cytometry.
<ol>
	<li>Transfer 50 &#956;l of the cells (5 x 105 cells) among total splenic lymphocytes into each well of a new u-bottom 96-well plate and add 150 &#956;l of FACS buffer per well. Centrifuge at 300&#160;x g for 2 min at 4 &#176;C.</li>
	<li>Discard the supernatant. Agitate the cell pellet and add 200 &#956;l of FACS buffer per well. Centrifuge at 300 x g for 2 min at 4 &#176;C (2 times).</li>
	<li>Prepare the antibody cocktail for staining cell surface markers in 50 &#956;l of FACS buffer per well with the following antibodies and reagents. Anti-CD4 violet dye, Anti-CD25 green dye, Anti-PD-1 violet dye, Anti-CD8 PerCP-Cy5.5 dye and cell viability detection reagent near-infrared (IR) fluorescent reactive dye. 
	NOTE: To further analyze the phenotype of activated Treg cells, anti-CD10323,25 can be added for cell surface-marker staining.</li>
	<li>Resuspend the cell pellet with 50 &#956;l of antibody cocktail per well. Incubate for 20 min in the dark at 4 &#176;C. Wash the cells twice with FACS buffer by centrifugation at 300 x g for 2 min at 4 &#176;C.</li>
	<li>After the final washing step, decant the supernatant and fix the cells for 20 min in the dark at 4 &#176;C with 100 &#956;l of fixation buffer prepared according to the manufacturer&#39;s protocol.</li>
	<li>Wash the cells twice with the permeabilization wash buffer (prepared according to manufacturer&#39;s protocol) by centrifugation at 300 x g for 2 min at 4 &#176;C.</li>
	<li>Prepare the antibody solution for intracellular Foxp3 staining, and resuspend the cell pellet with 50 &#956;l of Anti-Foxp3 antibody solution. 
	NOTE: To further analyze the phenotypes of Tconv and Treg cells, anti-CTLA can be added at this step.</li>
	<li>Incubate for 20 min in the dark at 4 &#176;C and repeat Step 3.6. After the final washing step, resuspend the cell pellet in 200 &#956;l of FACS buffer and examine the phenotype of the cells by flow cytometer25.</li>
	<li>Gate the live cell population. To analyze the phenotypes of Tconv cells during LCMV CL13 infection, examine the frequency of Foxp3-PD-1+ cells among CD4+ or CD8+ T cells. 
	NOTE: Based on experimental results, the percentage of Foxp3-PD-1+ is more than 50% in both CD4+ and CD8+ T cell populations at 16 d p.i. with LCMV CL13.
	<ol>
		<li>To analyze the frequency and phenotypes of Treg cells, examine the frequencies of Foxp3+ or Foxp3+PD-1+ cells among CD4+ T cells. 
		NOTE: Based on experimental results, the percentage of Foxp3+ or Foxp3+PD-1+ cells is more than 20% in CD4+ T cell population, respectively.</li>
	</ol>
	</li>
</ol>
4. Isolation of CD4+CD25+ Treg Cells
NOTE: The volumes of all reagents indicated below are for a starting cell number of 1 x 107 total splenocytes.
<ol>
	<li>Prepare the cells as described in section 2 using naive and LCMV chronically infected mice. Wash the cells by adding 10 ml of T cell isolation buffer. Centrifuge at 300&#160;x g for 10 min at 4 &#176;C. Discard the supernatant completely and resuspend the cell pellet in 40 &#956;l of buffer.</li>
	<li>For CD4+ T cell enrichment using the magnetic cell separation system, add 10 &#956;l of biotin-antibody cocktail and mix well. Incubate for 10 min at 4 &#176;C.</li>
	<li>Add 30 &#956;l of buffer, 20 &#956;l of anti-biotin micro-beads for labeling of non-CD4+ T cells and 10 &#956;l of CD25-PE antibody for fluorescent labeling of CD25+ cells. Mix well and incubate for 15 min in the dark.</li>
	<li>Add 2 ml of buffer and pass the cells through a 40 &#956;m cell strainer into a new 50 ml tube to remove cell debris. Wash the cells by centrifugation at 300 x g for 10 min at 4 &#176;C. Discard the supernatant completely and resuspend the cell pellet in 500 &#956;l buffer at a density of up to 1.25&#160;x 108 cells.</li>
	<li>Apply the cells onto the column and collect the unlabeled cells that pass through the column. Wash the column by adding 2 ml of buffer and centrifuge at 300 x g for 10 min at 4 &#176;C. Discard the supernatant completely and resuspend the isolated CD4+ T cells in 90 &#956;l of buffer.</li>
	<li>For magnetic labeling of CD25+ cells, add 10 &#956;l of anti-PE microbeads, mix well, and incubate for 15 min in the dark at 4 &#176;C.</li>
	<li>Add 2 ml of buffer and wash the cells by centrifugation at 300 x g for 10 min at 4 &#176;C. Discard the supernatant completely and resuspend the cell pellet in 500 &#956;l of buffer at a density of up to 1 x 108 cells.</li>
	<li>To enrich the CD4+CD25+ Treg cells, apply the cells onto the column for positive selection, and wash the column by adding 2 ml of buffer. Repeat the wash three times.</li>
	<li>When the column is empty after the final washing step, add 1 ml of buffer onto the column, and flush out the magnetically labeled CD4+CD25+ cells using the plunger.</li>
	<li>Repeat steps 4.8-4.9 to enhance the purity of the isolated CD4+CD25+ Treg cells. Wash the cells with FACS buffer by centrifugation at 300 x g for 10 min at 4 &#176;C. Discard the supernatant and resuspend the isolated CD4+CD25+ Treg cells at a concentration of 2 x 106 cells/ml in complete RPMI media.</li>
	<li>To check the purity of the isolated-CD4+CD25+ Treg cells, use 2 x 105 cells from the total isolated-CD4+CD25+ Treg cells. Stain the cells with 50 &#956;l of FACS buffer containing anti-CD4 FITC and cell viability detecting reagent near-IR fluorescent reactive dye. 
	NOTE: CD25 was already labeled with PE during isolation.</li>
	<li>Incubate for 20 min in the dark at 4 &#176;C. Wash the cells with FACS buffer by centrifugation at 300 x g for 2 min at 4 &#176;C. After washing, resuspend the cell pellet in 100 &#956;l of FACS buffer and check the purity by flow cytometry.</li>
	<li>Gate the live-cell population. To analyze the purity of Treg cells, confirm the percentage of CD4+CD25+ cells. 
	NOTE: Based on experimental results, the percentage of CD4+CD25+ cells among purified cells is over about 80%.</li>
</ol>
5. Isolation of CD8+ T Cells and Labeling of CD8+ T Cells
NOTE: The volumes of all reagents indicated below are for a starting cell number of 1 x 107 total splenocytes.
<ol>
	<li>Prepare cells as described in section 2 using na&#239;ve mice. Wash the cells by adding 10 ml of T cell isolation buffer. Centrifuge at 300 x g for 10 min at 4 &#176;C. Discard the supernatant completely, and resuspend the cell pellet in 40 &#956;l of buffer.</li>
	<li>For CD8+ T cell isolation using the magnetic cell separation system, add 10 &#956;l of biotin-antibody cocktail for non-CD8+ T cell labeling and mix well. Incubate for 5 min at 4 &#176;C.</li>
	<li>Add 30 &#956;l of buffer and 20 &#956;l of anti-biotin microbeads. Mix well and incubate for 10 min at 4 &#176;C.</li>
	<li>Apply the cells onto the column and collect the unlabeled cells that pass through the column. Wash the column by adding 2 ml of buffer three times.</li>
	<li>Centrifuge at 300&#160;x g for 10 min at 4 &#176;C. Discard the supernatant completely and resuspend the isolated CD8+ T cells in 2 ml of buffer.</li>
	<li>To check the purity of the isolated cells, prepare 50 &#956;l of antibody solution in FACS buffer. Resuspend the 2&#160;x 105 cells among isolated CD8+ T cells in 50 &#956;l of antibody solution. 
	NOTE: To prepare antibody solution, add anti-CD8 FITC, and cell viability detection reagent (near-IR fluorescent reactive dye) into FACS buffer.</li>
	<li>Incubate for 20 min in the dark at 4 &#176;C. Wash the cells with FACS buffer by centrifugation at 300 x g for 2 min at 4 &#176;C. After washing, resuspend the cell pellet in 100 &#956;l of FACS buffer, and check the purity of the cells by flow cytometry.</li>
	<li>Gate the live-cell population. To check the purity of CD8+ T cells, confirm the percentage of CD8+ T cells. 
	NOTE: Based on experimental results, the percentage of CD8+ cells among purified cells is over about 90%.</li>
	<li>Add PBS to the isolated CD8+ T cells. Centrifuge at 300 x g for 10 min at 4 &#176;C. Discard the supernatant. Resuspend the isolated CD8+ T cells at a concentration of 1 x 107 cells/ml in PBS.</li>
	<li>To label the CD8+ T cells for the in vitro suppression assay, dilute cell proliferation tracking violet dye in PBS to obtain a concentration of 5 &#956;M at RT. 
	NOTE: The approximate excitation and emission peaks of the cell proliferation tracking violet dye used in the study are 405 and 450 nm, respectively.</li>
	<li>Mix well equal volumes of cell proliferation tracking violet dye (5 &#956;M) and cell suspension (1 x 107 cells/ml of CD8+ T cells) in a 15 ml tube, and incubate at 20 min at 37 &#176;C. Vortex the tube every 10 min.</li>
	<li>Fill up the tube with cold complete RPMI media, and leave the tube for 10 min at RT. Centrifuge at 300 x g for 10 min at RT. Discard the supernatant completely, and resuspend the cells at a concentration of 2 x 106 cells/ml with pre-warmed complete RPMI media. Incubate the cells for 15 min at RT.</li>
</ol>
6. Setting Up the In Vitro Suppression Assay Using CD4+CD25+ Treg and CD8+ T Cells
<ol>
	<li>To prepare anti-CD3/CD28-coated beads, transfer the appropriate volume of magnetic beads to a 15 ml of tube (2.5 &#956;l/1 x 105 cells). Add an equal volume of PBS and mix. Wash by centrifugation at 300&#160;x g for 2 min at 4 &#176;C and discard the supernatant. Dilute the magnetic beads in complete media (50 &#956;l/well).</li>
	<li>Aliquot 50 &#956;l of CD4+CD25+ Treg cells per well of u-bottom 96-well plate (1 x 105 cells/well). Add 50 &#956;l of CD8+ T cells as responder T (Tresp) cells per well (1 x 105 cells/well). Add 50 &#956;l of diluted anti-CD3/CD28-coated beads into per well. 
	NOTE: In this step, label and set up control wells as follows: &#34;unstimulated CD8+ T cell only&#34; with no anti-CD3/CD28-coated beads; &#34;CD8+ T cell only&#34; with anti-CD3/CD28-coated beads; &#34;CD8+ T cell only&#34; with anti-CD3/CD28-coated beads; &#34;Treg cell only&#34; with anti-CD3/CD28-coated beads. Treg cells can be diluted by complete media and co-cultured with Tresp cells in a different ratio of Tresp cells:Treg cells (1:0.25-1:1).</li>
	<li>Add 50 &#956;l or appropriate volume of media into all wells to total volume of 200 &#956;l. Cover the plate with foil and incubate in a CO2 incubator at 37 &#176;C for 72 hr.</li>
</ol>
7. Analysis of CD8+ T Cell Proliferation &#38; Cytokine Production from CD8+ T Cells
<ol>
	<li>For the cytokine production analysis, after 3 days of culture, separate the supernatant of each well into another plate and perform enzyme-linked immunosorbent assay (ELISA). 
	NOTE: The supernatant may be aliquoted and stored at -70 &#176;C. In this experiment, anti-mouse IFN-&#947; antibody-coated plate was used to detect IFN-&#947; production according to the manufacturer s protocol. To determine IFN-&#947; production of proliferating CD8+ T cells on a single cell level, intracellular cytokine staining can be performed.</li>
	<li>After separating the supernatant from each well, wash the plate containing the cells with FACS buffer and centrifuge at 300 x g for 2 min at 4 &#176;C (3 times).</li>
	<li>After washing, discard the supernatant. Resuspend the cell pellet with 50 &#956;l of antibody cocktail for staining of proliferated CD8+ T cells. Incubate for 20 min in the dark at 4 &#176;C. 
	NOTE: To prepare antibody cocktail, add anti-CD4 FITC, anti-CD8 PerCP-Cy5.5, and cell viability detection reagent (near-IR fluorescent reactive dye) into FACS buffer. 
	NOTE: Antibodies against various markers such as CD44 or CD69 can be combined with other antibodies to confirm activation of CD8+ T cells. Remember that CD8+ T cells have already been labeled with cell proliferation tracking violet dye at Step 5.10.</li>
	<li>Wash twice by centrifugation at 300 x g for 2 min at 4 &#176;C. After the final washing step, discard the supernatant, and fix the cells for 20 min in the dark at 4 &#176;C with 100 &#956;l of fixation buffer.</li>
	<li>Wash twice by centrifugation at 300 x g for 2 min at 4 &#176;C. Resuspend the cells with 200 &#956;l of FACS buffer and measure the proliferation of cell proliferation tracking violet dye-labeled CD8+ T cells by flow cytometry.
	<ol>
		<li>Gate the CD8+ T cell population among the live cells. Measure the percentages of divided and undivided cells according to the dilution of the cell proliferation tracking violet dye and CD8+ T cell dilution according to the following equation. % Inhibition = [(% of proliferated CD8+ T cells in the absence of Treg cells - % of proliferated CD8+ T cells in the presence of Treg cells)/(% of proliferated CD8+ T cells in the absence of Treg cells)] x 100. Further analyze the data by using the flow cytometry software25.</li>
	</ol>
	</li>
</ol>

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<li>Punkosdy, G. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulatory+T-cell+expansion+during+chronic+viral+infection+is+dependent+on+endogenous+retroviral+superantigens%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Regulatory T-cell expansion during chronic viral infection is dependent on endogenous retroviral superantigens.</a> Proc Natl Acad Sci U S A. 108 (9), 3677-3682 (2011).</li>
<li>Blackburn, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Coregulation+of+CD8%2B+T+cell+exhaustion+by+multiple+inhibitory+receptors+during+chronic+viral+infection%5BTitle%5D)+AND+%22Nat+Immunol%22%5BJournal%5D)">Coregulation of CD8+ T cell exhaustion by multiple inhibitory receptors during chronic viral infection.</a> Nat Immunol. 10 (1), 29-37 (2009).</li>
<li>Park, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PD-1+upregulated+on+regulatory+T+cells+during+chronic+virus+infection+enhances+the+suppression+of+CD8%2B+T+cell+immune+response+via+the+interaction+with+PD-L1+expressed+on+CD8%2B+T+cells%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">PD-1 upregulated on regulatory T cells during chronic virus infection enhances the suppression of CD8+ T cell immune response via the interaction with PD-L1 expressed on CD8+ T cells.</a> J Immunol. 194 (12), 5801-5811 (2015).</li>
<li>Virgin, H. W., Wherry, E. J., Ahmed, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Redefining+chronic+viral+infection%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Redefining chronic viral infection.</a> Cell. 138 (1), 30-50 (2009).</li>
<li>Penaloza-MacMaster, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interplay+between+regulatory+T+cells+and+PD-1+in+modulating+T+cell+exhaustion+and+viral+control+during+chronic+LCMV+infection%5BTitle%5D)+AND+%22J+Exp+Med%22%5BJournal%5D)">Interplay between regulatory T cells and PD-1 in modulating T cell exhaustion and viral control during chronic LCMV infection.</a> J Exp Med. 211 (9), 1905-1918 (2014).</li>
<li>Chang, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+ubiquitin+ligase+Peli1+negatively+regulates+T+cell+activation+and+prevents+autoimmunity%5BTitle%5D)+AND+%22Nat+Immunol%22%5BJournal%5D)">The ubiquitin ligase Peli1 negatively regulates T cell activation and prevents autoimmunity.</a> Nat Immunol. 12 (10), 1002-1009 (2011).</li>
<li>Krishnamoorthy, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Early+infection+with+respiratory+syncytial+virus+impairs+regulatory+T+cell+function+and+increases+susceptibility+to+allergic+asthma%5BTitle%5D)+AND+%22Nat+Med%22%5BJournal%5D)">Early infection with respiratory syncytial virus impairs regulatory T cell function and increases susceptibility to allergic asthma.</a> Nat Med. 18 (10), 1525-1530 (2012).</li>
<li>Yadav, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neuropilin-1+distinguishes+natural+and+inducible+regulatory+T+cells+among+regulatory+T+cell+subsets+in+vivo%5BTitle%5D)+AND+%22J+Exp+Med%22%5BJournal%5D)">Neuropilin-1 distinguishes natural and inducible regulatory T cells among regulatory T cell subsets in vivo.</a> J Exp Med. 209 (10), 1713-1722 (2012).</li>
<li>Tai, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Basis+of+CTLA-4+function+in+regulatory+and+conventional+CD4%28%2B%29+T+cells%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Basis of CTLA-4 function in regulatory and conventional CD4(+) T cells.</a> Blood. 119 (22), 5155-5163 (2012).</li>
<li>Rushbrook, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulatory+T+cells+suppress+in+vitro+proliferation+of+virus-specific+CD8%2B+T+cells+during+persistent+hepatitis+C+virus+infection%5BTitle%5D)+AND+%22J+Virol%22%5BJournal%5D)">Regulatory T cells suppress in vitro proliferation of virus-specific CD8+ T cells during persistent hepatitis C virus infection.</a> J Virol. 79 (12), 7852-7859 (2005).</li>
<li>Sekiya, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+nuclear+orphan+receptor+Nr4a2+induces+Foxp3+and+regulates+differentiation+of+CD4%5BTitle%5D)+AND+%22T+cells.+Nat+Commun%22%5BJournal%5D)">The nuclear orphan receptor Nr4a2 induces Foxp3 and regulates differentiation of CD4.</a> T cells. Nat Commun. 2 (269), (2011).</li>
<li>Merianos, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Maternal+alloantibodies+induce+a+postnatal+immune+response+that+limits+engraftment+following+in+utero+hematopoietic+cell+transplantation+in+mice%5BTitle%5D)+AND+%22J+Clin+Invest%22%5BJournal%5D)">Maternal alloantibodies induce a postnatal immune response that limits engraftment following in utero hematopoietic cell transplantation in mice.</a> J Clin Invest. 119 (9), 2590-2600 (2009).</li>
<li>Allakhverdi, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expression+of+CD103+identifies+human+regulatory+T-cell+subsets%5BTitle%5D)+AND+%22J+Allergy+Clin+Immunol%22%5BJournal%5D)">Expression of CD103 identifies human regulatory T-cell subsets.</a> J Allergy Clin Immunol. 118 (6), 1342-1349 (2006).</li>
<li>Camisaschi, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((LAG-3+expression+defines+a+subset+of+CD4%28%2B%29CD25%28high%29Foxp3%28%2B%29+regulatory+T+cells+that+are+expanded+at+tumor+sites%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">LAG-3 expression defines a subset of CD4(+)CD25(high)Foxp3(+) regulatory T cells that are expanded at tumor sites.</a> J Immunol. 184 (11), 6545-6551 (2010).</li>
<li>Wang, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expression+of+GARP+selectively+identifies+activated+human+FOXP3%2B+regulatory+T+cells%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Expression of GARP selectively identifies activated human FOXP3+ regulatory T cells.</a> Proc Natl Acad Sci U S A. 106 (32), 13439-13444 (2009).</li>
<li>Myers, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((IL-2-independent+and+TNF-alpha-dependent+expansion+of+Vbeta5%2B+natural+regulatory+T+cells+during+retrovirus+infection%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">IL-2-independent and TNF-alpha-dependent expansion of Vbeta5+ natural regulatory T cells during retrovirus infection.</a> J Immunol. 190 (11), 5485-5495 (2013).</li>
</ol>

S.-J.H. has a patent and receives patent royalties related to the PD-1 pathway. The other authors have no financial conflicts of interest.

Although only a small number of Treg cells exist in mice and humans, it is important to understand their function as they play a crucial role in regulating the immune response and maintaining immune tolerance. The number and suppressive functions of Treg cells increases during a chronic virus infection15-20 as well as cancer progression13,14. This is probably due to continued antigen stimulation. To evaluate the Treg cells function under antigen persistence and disea...

Regulatory T (Treg) cells express forkhead box P3 (Foxp3) as a transcription factor for their development and function1. Additionally, Treg cells express various other molecules such as CD252, lymphocyte-activation gene 3 (LAG-3)3, glucocorticoid-induced tumor necrosis factor receptor4, and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4)5 on their surface or intracellular region. During chronic infection with various kinds of pathogens such as viruses6,7, bacteria8,9, and parasites10-12, or in the course of cancer development13,14, Treg cells become differentiated into activated cells, displaying enhanced suppressive function targeting effector CD4+ and CD8+ T cells. A number of papers have suggested that expanded and activated Treg cells contribute to the impaired CD8+ T cell response during friend retrovirus (FV) infection15-17. FV-induced Treg cells inhibit IFN-&#947; or granzyme B expression and cytotoxic reactivity of CD8+ T cells15-17. Moreover, in a herpes simplex virus infection model, it was reported that depletion of CD4+CD25+ Treg cells resulted in expansion of virus-specific CD8+ T cells and severe tissue damage by infiltration of immunopathogenic CD4+ T cells18-20.
Mice infected chronically with the clone 13 strain of lymphocytic choriomeningitis virus (LCMV CL13)21-24 have been widely used to characterize the phenotype and function of effector T cells (Teff) and Treg cells during chronic virus infection. During persistent LCMV infection, virus-specific Teff cells progressively lose their effector function and become exhausted T (Texh) cells. On the other hand, Treg cells reinforce their ability to suppress virus-specific T cell response25. The decrease in the functioning capacity of the Teff cells can be explained by several factors such as upregulation of inhibitory receptors on Teff cells, altered function of antigen-presenting cells, production of immunoregulatory cytokines, and increased frequency or enhanced function of Treg cells26. Among the factors involved in T cell suppression, programmed cell death protein-1 (PD-1)-expressing Texh cells and Treg cells have been widely considered as the hallmarks of antigen persistence and suppressive environment. Recently, it was reported that blockade of the PD-1 pathway and ablation of Treg cells lead to enhanced T cell function and decreased viral load during LCMV chronic infection27. Furthermore, Treg cells are activated during chronic infection of mice with LCMV23,25 and their suppressive function is strengthened25. PD-1 is highly expressed on Treg cells as well as Texh cells, and the level of PD-1 expressed by Treg cells correlates with the strength of their suppressive function to inhibit T cell proliferation25.
Here, we describe a method to compare the characteristics of activated Treg cells isolated from mice infected with LCMV CL13 and resting Treg cells isolated from na&#239;ve mice. Furthermore, we explain a series of processes to separate activated Treg cells and examine their ex vivo phenotype, as well as measure their suppressive activity in vitro.

<table><tbody><tr><td>FITC Rat Anti-Mouse CD4</td><td>RM4-5</td><td>BD Biosciences</td><td>553047</td></tr><tr><td>Cytofix/Cytoperm</td><td></td><td>BD Biosciences</td><td>554714</td></tr><tr><td>U-Bottom Tissue Culture Plates</td><td></td><td>BD Biosciences</td><td>353077</td></tr><tr><td>Fixation buffer</td><td></td><td>BD Biosciences</td><td>554655</td></tr><tr><td>FITC Rat Anti-Mouse CD25</td><td>7D4</td><td>BD Biosciences</td><td>553072</td></tr><tr><td>Cell strainer, 70 mm</td><td></td><td>BD Biosciences</td><td>352350</td></tr><tr><td>Cell strainer, 40 mm</td><td></td><td>BD Biosciences</td><td>352340</td></tr><tr><td>Brilliant Violet 421 Anti-mouse CD279 (PD-1)</td><td>29F.1A12</td><td>BioLegend</td><td>135217</td></tr><tr><td>Brilliant Violet 605 Anti-Mouse CD4</td><td>RM4-5</td><td>Biolegend</td><td>100547</td></tr><tr><td>APC Anti-Mouse/Rat Foxp3&amp;nbsp;</td><td>FJK-16s</td><td>eBioscience</td><td>17-5773</td></tr><tr><td>Foxp3/Transcription Factor Staining Buffer Set</td><td></td><td>eBioscience</td><td>00-5223</td></tr><tr><td>PerCP-Cyanine5.5 Anti-Mouse CD8a</td><td>53-6.7</td><td>eBiosicence</td><td>45-0081</td></tr><tr><td>Mouse IFN-gamma Platinum ELISA</td><td></td><td>eBiosicence</td><td>BMS606</td></tr><tr><td>RPMI 1640</td><td></td><td>GE Life Sciences</td><td>SH30027</td></tr><tr><td>PBS (1x)</td><td></td><td>GE Life Sciences</td><td>SH30256</td></tr><tr><td>ACK Lysing Buffer</td><td></td><td>Gibco</td><td>A10492-01</td></tr><tr><td>L-Glutamine, 200 mM solution</td><td></td><td>Gibco&amp;nbsp;</td><td>25030</td></tr><tr><td>Penicillin-Streptomycin, 10,000 U/ml</td><td></td><td>Gibco&amp;nbsp;</td><td>10378-016</td></tr><tr><td>LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit</td><td></td><td>Life technologies</td><td>L-34975</td></tr><tr><td>CD8a&lt;sup&gt;+&lt;/sup&gt; T Cell Isolation Kit, mouse</td><td></td><td>Miltenyibiotec</td><td>130-104-075</td></tr><tr><td>CD4&lt;sup&gt;+&lt;/sup&gt; CD25&lt;sup&gt;+&lt;/sup&gt; Regulatory T Cell Isolation Kit, mouse</td><td></td><td>Miltenyibiotec</td><td>130-091-041</td></tr><tr><td>MACS Separation Columns, LD columns</td><td></td><td>Miltenyibiotec</td><td>130-042-901</td></tr><tr><td>MACS Separation Columns, LS columns</td><td></td><td>Miltenyibiotec</td><td>130-042-401</td></tr><tr><td>EDTA, 0.5 M (pH 8.0)</td><td></td><td>Promega</td><td>V4231</td></tr><tr><td>2-Mercaptoethanol</td><td></td><td>Sigma Life Science</td><td>M7522</td></tr><tr><td>Fetal Bovine Serum</td><td></td><td>Thermo Fisher Scientific</td><td>SH30919.03</td></tr><tr><td>CellTrace Violet Cell Proliferation Kit</td><td></td><td>Thermo Fisher Scientific</td><td>C34557</td></tr><tr><td>BD Canto II flowcytometer</td><td></td><td>BD Biosciences</td><td></td></tr><tr><td>Flowjo</td><td></td><td>TreeStar</td><td></td></tr><tr><td>Hematocytomer</td><td></td><td>Marienfeld superior</td><td></td></tr></tbody></table>

phenotypic and functional analysis of activated regulatory t cells isolated from chronic lymphocytic choriomeningitis virus-infected mice

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance and homeostasis maintenance by regulating the immune response. The primary role of Treg cells is to suppress the proliferation of effector T (Teff) cells and the production of cytokines such as IFN-&#947;, TNF-&#945;, and IL-2. It has been demonstrated that Treg cells&#39; ability to inhibit the function of Teff cells is enhanced during persistent pathogen infection and cancer development. To clarify the function of Treg cells under resting or inflamed conditions, a variety of in vitro suppression assays using mouse or human Treg cells have been devised. The main aim of this study is to develop a method to compare the differences in phenotype and suppressive function between resting and activated Treg cells. To isolate activated Treg cells, mice were infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13), a chronic strain of LCMV. Treg cells isolated from the spleen of LCMV CL13-infected mice exhibited both the activated phenotype and enhanced suppressive activity compared with resting Treg cells isolated from na&#239;ve mice. Here, we describe the basic protocol for ex vivo phenotype analysis to distinguish activated Treg cells from resting Treg cells. Furthermore, we describe a protocol for the measurement of the suppressive activity of fully activated Treg cells.

We generated mice with persistent virus infection by injecting them with 2 x 106 p.f.u. of LCMV CL13 intravenously. To investigate the phenotypic changes in Treg cells and Tconv cells during chronic virus infection, splenic lymphocytes obtained from na&#239;ve and infected mice were stained with various antibodies and analyzed by flow cytometry. At 16 d p.i., PD-1 was upregulated in both Foxp3-CD4+ Tconv (Figure 1A<...

Watch this Scientific Journal Video about Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice at JoVE.com

Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

Here, we describe a protocol to analyze the phenotype of regulatory T (Treg) cells isolated from na&#239;ve and chronic lymphocytic choriomeningitis virus-infected mice. In addition, we provide a process to evaluate the suppressive activity of the Treg cells.

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance ...

phenotypic-functional-analysis-activated-regulatory-t-cells-isolated

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Immunology and Infection

9.8K Views.  Yonsei University. Here, we describe a protocol to analyze the phenotype of regulatory T (Treg) cells isolated from naïve and chronic lymphocytic choriomeningitis virus-infected mice. In addition, we provide a process to evaluate the suppressive activity of the Treg cells.

Video: Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to measurable increases in antigen-specific T cell responses in vaccine settings for cancer and infectious pathogens. However, their role in HIV-1 immuno-pathogenesis remains controversial, as they could either serve to suppress deleterious HIV-1-associated immune activation and thus slow HIV-1 disease progression or alternatively suppress HIV-1-specific immunity and thereby promote virus spread. Understanding and modulating Treg function in the context of HIV-1 could lead to potential new strategies for immunotherapy or HIV vaccines. However, important open questions remain on their role in the context of HIV-1 infection, which needs to be carefully studied.
Representing roughly 5% of human CD4+ T cells in the peripheral blood, studying the Treg population has proven to be difficult, especially in HIV-1 infected individuals where HIV-1-associated CD4 T cell and with that Treg depletion occurs. The characterization of regulatory T cells in individuals with advanced HIV-1 disease or tissue samples, for which only very small biological samples can be obtained, is therefore extremely challenging. We propose a technical solution to overcome these limitations using isolation and expansion of Tregs from HIV-1-positive individuals.
Here we describe an easy and robust method to successfully expand Tregs isolated from HIV-1-infected individuals in vitro. Flow-sorted CD3+CD4+CD25+CD127low Tregs were stimulated with anti-CD3/anti-CD28 coated beads and cultured in the presence of IL-2. The expanded Tregs expressed high levels of FOXP3, CTLA4 and HELIOS compared to conventional T cells and were shown to be highly suppressive. Easier access to large numbers of Tregs will allow researchers to address important questions concerning their role in HIV-1 immunopathogenesis. We believe answering these questions may provide useful insight for the development of an effective HIV-1 vaccine.

CD4+ Regulatory T cells are potent immune-modulators and serve important functions in immune homeostasis. The paucity of these cells in peripheral blood makes functional studies challenging, specifically in the context of HIV-1-infection. We here describe a method to isolate and expand functional CD4+ Tregs from peripheral blood from HIV-1-infected individuals.

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

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 ...

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

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

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

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

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

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Functional Characterization of Regulatory Macrophages That Inhibit Graft-reactive Immunity

Macrophage accumulation in transplanted organs has long been recognized as a feature of allograft rejection1. Immunogenic monocytes infiltrate the allograft early after transplantation, mount a graft reactive response against the transplanted organ, and initiate organ rejection2. Recent data suggest that suppressive macrophages facilitate successful long-term transplantation3 and are required for the induction of transplantation tolerance4. This suggests a multidimensional concept of macrophage ontogeny, activation, and function, which demands a new roadmap for the isolation and analysis of macrophage function5. Due to the plasticity of macrophages, it is necessary to provide a methodology to isolate and characterize macrophages, depending on the tissue environment, and to define their functions according to different scenarios. Here, we describe a protocol for immune characterization of graft-infiltrating macrophages and the methods we used to functionally evaluate their capacity to inhibit CD8+ T proliferation and to promote CD4+Foxp3+ Treg expansion in vitro.

Macrophages are plastic cells of the hematopoietic system that have a crucial role in protective immunity and homeostasis. In this report, we describe optimized in vitro techniques to phenotypically and functionally characterize graft-infiltrating regulatory macrophages that accumulate in the transplanted organ under tolerogenic conditions.

Macrophage accumulation in transplanted organs has long been recognized as a feature of allograft rejection1. Immunogenic monocytes infiltrate the ...

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

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity. Many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper T cell development and function. However, studies using mouse models have proven to be highly informative due to the availability of genetic, cellular, and biochemical systems. One genetic approach in mice used by many labs involves retroviral transduction of primary helper T cells. This is a powerful approach due to its relative ease, making it accessible to almost any laboratory with basic skills in molecular biology and immunology. Therefore, multiple genes in wild type or mutant forms can readily be tested for function in helper T cells to understand their importance and mechanisms of action. We have optimized this approach and describe here the protocols for production of high titer retroviruses, isolation of primary murine helper T cells, and their transduction by retroviruses and differentiation toward the different helper subsets. Finally, the use of this approach is described in uncovering mechanisms utilized by microRNAs (miRNAs) to regulate pathways controlling helper T cell development and function.

Many experimental systems have been utilized to understand the mechanisms regulating T cell development and function in an immune response. Here a genetic approach using retroviral transduction is described, which is economic, time efficient, and most importantly, highly informative in identifying regulatory pathways.

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...

Evaluation of T Follicular Helper Cells and Germinal Center Response During Influenza A Virus Infection in Mice

T Follicular Helper (Tfh) cells are an independent CD4+&#160;T cell subset specialized in providing help for germinal center (GC) development and generation of high-affinity antibodies. In influenza virus infection, robust Tfh and GC B cell responses are induced to facilitate effective virus eradication, which confers a qualified mouse model for Tfh-associated study. In this paper, we described protocols in detection of basic Tfh-associated immune response during influenza virus infection in mice. These protocols include: intranasal inoculation of influenza virus; flow cytometry staining and analysis of polyclonal and antigen-specific Tfh cells, GC B cells and plasma cells; immunofluorescence detection of GCs; enzyme-linked immunosorbent assay (ELISA) of influenza virus-specific antibody in serum. These assays basically quantify the differentiation and function of Tfh cells in influenza virus infection, thus providing help for studies in elucidating differentiation mechanism and manipulation strategy.

This paper describes protocols of evaluating Tfh and GC B response in mouse model of influenza virus infection.

T Follicular Helper (Tfh) cells are an independent CD4+&#160;T cell subset specialized in providing help for germinal center (GC) development and ...

Isolating Central Nervous System Tissues and Associated Meninges for the Downstream Analysis of Immune cells

The central nervous system (CNS) is comprised of the brain and spinal cord and is enveloped by the meninges, membranous layers serving as a barrier between the periphery and the CNS. The CNS is an immunologically specialized site, and in steady state conditions, immune privilege is most evident in the CNS parenchyma. In contrast, the meninges harbor a diverse array of resident cells, including innate and adaptive immune cells. During inflammatory conditions triggered by CNS injury, autoimmunity, infection, or even neurodegeneration, peripherally derived immune cells may enter the parenchyma and take up residence within the meninges. These cells are thought to perform both beneficial and detrimental actions during CNS disease pathogenesis. Despite this knowledge, the meninges are often overlooked when analyzing the CNS compartment, because conventional CNS tissue extraction methods omit the meningeal layers. This protocol presents two distinct methods for the rapid isolation of murine CNS tissues (i.e., brain, spinal cord, and meninges) that are suitable for downstream analysis via single-cell techniques, immunohistochemistry, and in situ hybridization methods. The described methods provide a comprehensive analysis of CNS tissues, ideal for assessing the phenotype, function, and localization of cells occupying the CNS compartment under homeostatic conditions and during disease pathogenesis.

This paper presents two optimized protocols for examining resident and peripherally derived immune cells within the central nervous system, including the brain, spinal cord, and meninges. Each of these protocols helps to ascertain the function and composition of the cells occupying these compartments under steady state and inflammatory conditions.

The central nervous system (CNS) is comprised of the brain and spinal cord and is enveloped by the meninges, membranous layers serving as a barrier ...

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

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

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

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

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

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

Retroviral Transduction of Guide RNA for Gene Editing in Th17 Differentiation

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

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

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

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

Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

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