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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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=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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redefining+chronic+viral+infection.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interplay+between+regulatory+T+cells+and+PD-1+in+modulating+T+cell+exhaustion+and+viral+control+during+chronic+LCMV+infection.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ubiquitin+ligase+Peli1+negatively+regulates+T+cell+activation+and+prevents+autoimmunity.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+infection+with+respiratory+syncytial+virus+impairs+regulatory+T+cell+function+and+increases+susceptibility+to+allergic+asthma.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropilin-1+distinguishes+natural+and+inducible+regulatory+T+cells+among+regulatory+T+cell+subsets+in+vivo.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basis+of+CTLA-4+function+in+regulatory+and+conventional+CD4(+)+T+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulatory+T+cells+suppress+in+vitro+proliferation+of+virus-specific+CD8++T+cells+during+persistent+hepatitis+C+virus+infection.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nuclear+orphan+receptor+Nr4a2+induces+Foxp3+and+regulates+differentiation+of+CD4.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+alloantibodies+induce+a+postnatal+immune+response+that+limits+engraftment+following+in+utero+hematopoietic+cell+transplantation+in+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+CD103+identifies+human+regulatory+T-cell+subsets.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LAG-3+expression+defines+a+subset+of+CD4(+)CD25(high)Foxp3(+)+regulatory+T+cells+that+are+expanded+at+tumor+sites.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+GARP+selectively+identifies+activated+human+FOXP3++regulatory+T+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-2-independent+and+TNF-alpha-dependent+expansion+of+Vbeta5++natural+regulatory+T+cells+during+retrovirus+infection.">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 disease development, their suppressive activity needs to be measured.
Here, we describe a protocol to analyze the ex vivo phenotype of Treg cells and measure their suppressive activity using an in vitro co-culture system of CD8+ T cells and Treg cells. Critical steps in the current protocol are analysis and isolation of Treg cells. Live and freshly isolated Treg cells show clear suppressive activity in vitro. We also examined the phenotypes of Tconv and Treg cells during a chronic virus infection. Tconv cells, i.e., Foxp3-CD4+ and CD8+ T cells showed a decrease in cellular activity after the chronic virus infection (Figure 1). The PD-1 expression was upregulated in both the Tconv cell populations. Increase in Treg cell number and up-regulation of PD-1 expression are the hallmarks of immune response during a chronic virus infection. Additionally, anti-bodies for other inhibitory receptors such as TIM-3 and CTLA-4 can be used in combination with PD-1 to examine the T cell phenotype by FACS after choric virus infection.
In order to investigate the suppressive activity, Tresp cells and Treg cells were co-cultured at a ratio of 1 to 1. Co-culture with Treg cells from chronically infected mice significantly reduced CD8+ T cell proliferation and IFN-&#947; secretion when compared to Treg cells from na&#239;ve mice. This implies that the proliferation and cytokine production of CD8+ T cells co-cultured with Treg cells are inversely related to the suppressive function of Treg cells. Although this protocol recommends the use of a magnetic cell separation system for Treg cell isolation, contamination with non-Treg cells cannot be ruled out. The Treg cells obtained from Foxp3-GFP reporter mice28-30 are better candidates to accurately investigate the function of Treg cells than magnetic cell separation system, as CD25 is often overexpressed on activated CD4+ Tconv as well as Treg cells.
The suppressive function of Treg cells has been evaluated by various in vitro suppression assays16,28,31-34. The advantage of the in vitro suppression assay is that it evaluates the direct effect of Treg cells on the inhibition of the CD8+ T cell response, because only Tresp cells are co-cultured with Treg cells. In addition to CD8+ T cells, the CD25-CD4+ T cells can be used instead of Tresp cells25. The effects of Treg or Tconv cells on immune cells such as dendritic cells or natural killer cells can be evaluated by varying the experimental conditions.
Molecules such as the CD10335, LAG-336, glycoprotein A repetitions predominant37, CD4338, CD11a38, or killer cell lectin-like receptor subfamily G member 138 have been used as markers of activated Treg cells in specific diseases. In this protocol, we used PD-1 as an indicator to identify activated Treg cells during chronic virus infection. This protocol can be used for multifaceted analyses (phenotypic and functional) of Treg cells under specific conditions.

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 border="0" cellpadding="0" cellspacing="0" width="976">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">FITC Rat Anti-Mouse CD4</td>
			<td style="width:67px;">RM4-5</td>
			<td style="width:173px;">BD Biosciences</td>
			<td style="width:104px;">553047</td>
			<td style="width:249px;">Please determine appropriate concentration. In this protocol, this reagent was diluted 100X in FACS buffer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytofix/Cytoperm</td>
			<td></td>
			<td>BD Biosciences</td>
			<td>554714</td>
			<td>Use this reagent for cell surface staining.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">U-Bottom Tissue Culture Plates</td>
			<td></td>
			<td>BD Biosciences</td>
			<td>353077</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fixation buffer</td>
			<td></td>
			<td>BD Biosciences</td>
			<td align="right">554655</td>
			<td>Use this reagent for cell surface staining.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FITC Rat Anti-Mouse CD25</td>
			<td>7D4</td>
			<td>BD Biosciences</td>
			<td>553072</td>
			<td>Please determine appropriate concentration. In this protocol, this reagent was diluted 100X in FACS buffer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell strainer, 70mm</td>
			<td></td>
			<td>BD Biosciences</td>
			<td>352350</td>
			<td>Use this strainer for grinding the whole spleen.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell strainer, 40mm</td>
			<td></td>
			<td>BD Biosciences</td>
			<td>352340</td>
			<td>Use this strainer for filtering the cells before column enrichment.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Brilliant Violet 421 Anti-mouse CD279 (PD-1)</td>
			<td>29F.1A12</td>
			<td>BioLegend</td>
			<td>135217</td>
			<td>Please determine appropriate concentration. In this protocol, this reagent was diluted 100X in FACS buffer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Brilliant Violet 605 Anti-Mouse CD4</td>
			<td>RM4-5</td>
			<td>Biolegend</td>
			<td align="right">100547</td>
			<td>Please determine appropriate concentration. In this protocol, this reagent was diluted 100X in FACS buffer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">APC Anti-Mouse/Rat Foxp3&#160;</td>
			<td>FJK-16s</td>
			<td>eBioscience</td>
			<td>17-5773</td>
			<td>Please determine appropriate concentration. In this protocol, this reagent was diluted 100X in FACS buffer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Foxp3 / Transcription Factor Staining Buffer Set</td>
			<td></td>
			<td>eBioscience</td>
			<td>00-5223</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PerCP-Cyanine5.5 Anti-Mouse CD8a</td>
			<td>53-6.7</td>
			<td>eBiosicence</td>
			<td>45-0081</td>
			<td>Please determine appropriate concentration. In this protocol, this reagent was diluted 100X in FACS buffer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse IFN-gamma Platinum ELISA</td>
			<td></td>
			<td>eBiosicence</td>
			<td>BMS606</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640</td>
			<td></td>
			<td>GE Life Sciences</td>
			<td>SH30027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS (1X)</td>
			<td></td>
			<td>GE Life Sciences</td>
			<td>SH30256</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ACK Lysing Buffer</td>
			<td></td>
			<td>Gibco</td>
			<td>A10492-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Glutamine, 200mM solution</td>
			<td></td>
			<td>Gibco&#160;</td>
			<td>25030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin, 10,000U/mL</td>
			<td></td>
			<td>Gibco&#160;</td>
			<td>10378-016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit</td>
			<td></td>
			<td>Life technologies</td>
			<td>L-34975</td>
			<td>Please determine appropriate concentration. In this protocol, this reagent was diluted 500X in FACS buffer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD8a+ T Cell Isolation Kit, mouse</td>
			<td></td>
			<td>Miltenyibiotec</td>
			<td>130-104-075</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD4+CD25+ Regulatory T Cell Isolation Kit, mouse</td>
			<td></td>
			<td>Miltenyibiotec</td>
			<td>130-091-041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MACS Separation Columns, LD columns</td>
			<td></td>
			<td>Miltenyibiotec</td>
			<td>130-042-901</td>
			<td>Use this column for Treg cell isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MACS Separation Columns, LS columns</td>
			<td></td>
			<td>Miltenyibiotec</td>
			<td>130-042-401</td>
			<td>Use this column for CD8+ T cell and Treg cell isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA, 0.5M (pH 8.0)</td>
			<td></td>
			<td>Promega</td>
			<td>V4231</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-Mercaptoethanol</td>
			<td></td>
			<td>Sigma Life Science</td>
			<td>M7522</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td></td>
			<td>Thermo Fisher Scientific</td>
			<td>SH30919.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CellTrace Violet Cell Proliferation Kit</td>
			<td></td>
			<td>Thermo Fisher Scientific</td>
			<td>C34557</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD Canto II flowcytometer</td>
			<td></td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Flow cytometer*</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flowjo</td>
			<td></td>
			<td>TreeStar</td>
			<td></td>
			<td>Flow cytometry software&#8224;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hematocytomer</td>
			<td></td>
			<td>Marienfeld superior</td>
			<td></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, upper panel) and Foxp3-CD8+ Tconv (Figure 1B, upper panel) cells. The frequency of Foxp3+CD4+ Treg cells was two times higher in the LCMV CL13-infected mice than in the na&#239;ve mice (Figure 1A). In particular, most of the Foxp3+CD4+ Treg cells displayed the activated phenotype, expressing high levels of PD-1 at this time point (Figure 1A).
For the in vitro suppression assay, a considerable number of CD8+ Tconv cells and CD4+CD25+ Treg cells were required. To obtain 1 x 107 cell proliferation tracking violet dye-labeled CD8+ T cells, splenocytes from two spleens of na&#239;ve mice were pooled. To obtain 2 x 106 or 3 x 106 of CD4+CD25+ Treg cells, at least three spleens from naive and LCMV CL13-infected mice, respectively, were pooled. CD8+ T cells as Tresp cells were separated successfully without significant contamination with other immune cells (Figure 2A). Treg cells could also be isolated, with a dominant population of CD25+CD4+ T cells with more than 80% purity (Figure 2B).
To compare the suppressive function of na&#239;ve and chronic Treg cells, the isolated Treg and Tresp cells were incubated together under stimulation with anti-CD3/CD28-coated beads at 37 &#176;C for 3 days. % inhibition of CD8+ Tresp cell proliferation was increased in a Treg cell dese-dependent manner (Figure 3A). When CD8+ Tresp cells were co-cultured with chronic Treg cells in a ratio of 1:1, proliferation of CD8+ Tresp cells were significantly inhibited (Figure 3B). IFN-&#947; production by CD8+ Tresp cells were also significantly inhibited when CD8+ Tresp cells were co-cultured with activated Treg cells from chronically infected mice rather than with resting Treg cells from na&#239;ve mice in a Treg cell dose-dependent manner (Figure 3C).
<img alt="Figure 1" src="/files/ftp_upload/54138/54138fig1.jpg" /> 
Figure 1: Phenotypes of CD4+ and CD8+ T cells in the spleen of na&#239;ve mice or LCMV CL13-infected mice at 16 d p.i. (A) Expression of PD-1 or CD25 on Foxp3-CD4+ Tconv and Foxp3+CD4+ Treg cells. (B) Expression of PD-1 or CD25 on Foxp3-CD8+ Tconv cells. Splenic lymphocytes were stained with antibodies against CD4, CD8, CD25, PD-1, and Foxp3. Data are representative of three independent experiments. Panel A has been modified from25 as a reference. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54138/54138fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54138/54138fig2.jpg" /> 
Figure 2: Purities of Tresp cells isolated from na&#239;ve mice and Treg cells isolated from na&#239;ve or LCMV CL13-infected mice. (A) Percentage of CD8+ T cells after isolation. (B) Percentage of CD25-expressing CD4+ Treg cells after isolation. Each quadrant in (B) was determined by the expression levels of CD4 and CD25. Data are representative of three independent experiments. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54138/54138fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54138/54138fig3.jpg" /> 
Figure 3: Effect of activated Treg cells on CD8+ T cell response. (A) % inhibition of CD8+ T cells co-cultured with Treg cells in a Treg cell dose-dependent manner. (B) Proliferation of CD8+ T cells co-cultured with Treg cells in 1:1 ratio. (C) IFN-&#947; production of CD8+ T cells co-cultured with Treg cells in a Treg cell dose-dependent manner. Proliferation of CD8+ T cells was measured by dilution of cell proliferation tracking violet dye in proliferated CD8+ T cells and IFN-&#947; secretion was evaluated by ELISA. Cell proliferation tracking violet dye-labeled CD8+ T cells were stimulated with anti-CD3/CD28-coated beads for 3 days in the absence or presence of Treg cells isolated from either na&#239;ve or chronically infected mice. In the histogram, the numbers without and with parenthesis indicate the proliferation percentage of CD8+ T cells co-cultured with and without Treg cells, respectively. Filled gray peaks in the histogram indicate proliferation of CD8+ T cells co-cultured without Treg cells. Each group was designed in triplicates. The bars represent mean + SEM. This figure has been modified from25 as a reference. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54138/54138fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

9.7K 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

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

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 ...

Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative neurology. This directive has arisen from the extensive evidence that brain repair is achieved after focal or systemic NPC transplantation in several preclinical models of neurological diseases.
These experimental data have identified the cell delivery route as one of the main hurdles of restorative stem cell therapies for brain diseases that requires urgent assessment. Intraparenchymal stem cell grafting represents a logical approach to those pathologies characterized by isolated and accessible brain lesions such as spinal cord injuries and Parkinson's disease. Unfortunately, this principle is poorly applicable to conditions characterized by a multifocal, inflammatory and disseminated (both in time and space) nature, including multiple sclerosis (MS). As such, brain targeting by systemic NPC delivery has become a low invasive and therapeutically efficacious protocol to deliver cells to the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system (CNS).
This alternative method of cell delivery relies on the NPC pathotropism, specifically their innate capacity to (i) sense the environment via functional cell adhesion molecules and inflammatory cytokine and chemokine receptors; (ii) cross the leaking anatomical barriers after intravenous (i.v.) or intracerebroventricular (i.c.v.) injection; (iii) accumulate at the level of multiple perivascular site(s) of inflammatory brain and spinal cord damage; and (i.v.) exert remarkable tissue trophic and immune regulatory effects onto different host target cells in vivo.
Here we describe the methods that we have developed for the i.v. and i.c.v. delivery of syngeneic NPCs in mice with experimental autoimmune encephalomyelitis (EAE), as model of chronic CNS inflammatory demyelination, and envisage the systemic stem cell delivery as a valuable technique for the selective targeting of the inflamed brain in regenerative neurology.

The transplantation of neural stem/progenitor cells (NPCs) holds great promises in regenerative neurology. The systemic delivery of NPCs has turned into effective, low invasive, and therapeutically very efficacious protocol to deliver stem cells in the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system.

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative ...

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 ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Here a protocol is presented to measure bacterial capture by CD4+ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with respect to how viral members of the microbiome affect subsequent microbe interactions with these virus-infected host cells. The co-habitation of virus with bacteria and fungi is principally present on the mucosal surfaces of the oral cavity and genital tract. Mucosal cells, particularly those with persistent chronic or persistent latent viral infections, could have a significant impact on members of the microbiome through virus alteration in number and type of receptors expressed. Modification in host cell membrane architecture would result in altered ability of subsequent members of the normal flora and opportunistic pathogens to initiate the first step in biofilm formation, i.e., adherence. This study describes a method for quantitation and visual examination of HSV&#39;s effect on the initiation of biofilm formation (adherence) of S. aureus and C. albicans.

A method is described herein for the determination of inter-Kingdom association and competition (bacterial and fungal) for adherence to virus-infected HeLa cell monolayers. This protocol can be extended to multiple combinations of prokaryotes, eukaryotes, and viruses.

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with ...

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 ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.