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...

<ol>
	<li>Hori, S., Nomura, T., Sakaguchi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+regulatory+T+cell+development+by+the+transcription+factor+Foxp3.">Control of regulatory T cell development by the transcription factor Foxp3.</a> Science. 299 (5609), 1057-1061 (2003).</li><li>Sakaguchi, S., Sakaguchi, N., Asano, M., Itoh, M., Toda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunologic+self-tolerance+maintained+by+activated+T+cells+expressing+IL-2+receptor+alpha-chains+(CD25).+Breakdown+of+a+single+mechanism+of+self-tolerance+causes+various+autoimmune+diseases.">Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases.</a> J Immunol. 155 (3), 1151-1164 (1995).</li><li>Huang, C. 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=Role+of+LAG-3+in+regulatory+T+cells.">Role of LAG-3 in regulatory T cells.</a> Immunity. 21 (4), 503-513 (2004).</li><li>McHugh, R. S., 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=CD4(+)CD25(+)+immunoregulatory+T+cells:+gene+expression+analysis+reveals+a+functional+role+for+the+glucocorticoid-induced+TNF+receptor.">CD4(+)CD25(+) immunoregulatory T cells: gene expression analysis reveals a functional role for the glucocorticoid-induced TNF receptor.</a> Immunity. 16 (2), 311-323 (2002).</li><li>Takahashi, 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=Immunologic+self-tolerance+maintained+by+CD25(+)CD4(+)+regulatory+T+cells+constitutively+expressing+cytotoxic+T+lymphocyte-associated+antigen+4.">Immunologic self-tolerance maintained by CD25(+)CD4(+) regulatory T cells constitutively expressing cytotoxic T lymphocyte-associated antigen 4.</a> J Exp Med. 192 (2), 303-310 (2000).</li><li>Manigold, 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=Foxp3+CD4+CD25++T+cells+control+virus-specific+memory+T+cells+in+chimpanzees+that+recovered+from+hepatitis.">Foxp3+CD4+CD25+ T cells control virus-specific memory T cells in chimpanzees that recovered from hepatitis.</a> C. 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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=Helminth+secretions+induce+de+novo+T+cell+Foxp3+expression+and+regulatory+function+through+the+TGF-beta+pathway.">Helminth secretions induce de novo T cell Foxp3 expression and regulatory function through the TGF-beta pathway.</a> J Exp Med. 207 (11), 2331-2341 (2010).</li><li>Taylor, M. D., van der Werf, N., Maizels, R. 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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=Specific+recruitment+of+regulatory+T+cells+in+ovarian+carcinoma+fosters+immune+privilege+and+predicts+reduced+survival.">Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival.</a> Nat Med. 10 (9), 942-949 (2004).</li><li>Dittmer, U., 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=Functional+impairment+of+CD8(+)+T+cells+by+regulatory+T+cells+during+persistent+retroviral+infection.">Functional impairment of CD8(+) T cells by regulatory T cells during persistent retroviral infection.</a> Immunity. 20 (3), 293-303 (2004).</li><li>Robertson, S. J., Messer, R. J., Carmody, A. B., Hasenkrug, K. 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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4+CD25++T+cells+regulate+virus-specific+primary+and+memory+CD8++T+cell+responses.">CD4+CD25+ T cells regulate virus-specific primary and memory CD8+ T cell responses.</a> J Exp Med. 198 (6), 889-901 (2003).</li><li>Suvas, S., Azkur, A. K., Kim, B. S., Kumaraguru, U., Rouse, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4+CD25++regulatory+T+cells+control+the+severity+of+viral+immunoinflammatory+lesions.">CD4+CD25+ regulatory T cells control the severity of viral immunoinflammatory lesions.</a> J Immunol. 172 (7), 4123-4132 (2004).</li><li>Veiga-Parga, 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=On+the+role+of+regulatory+T+cells+during+viral-induced+inflammatory+lesions.">On the role of regulatory T cells during viral-induced inflammatory lesions.</a> J Immunol. 189 (12), 5924-5933 (2012).</li><li>Wherry, E. 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=Molecular+signature+of+CD8++T+cell+exhaustion+during+chronic+viral+infection.">Molecular signature of CD8+ T cell exhaustion during chronic viral infection.</a> Immunity. 27 (4), 670-684 (2007).</li><li>Jin, H. 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=Cooperation+of+Tim-3+and+PD-1+in+CD8+T-cell+exhaustion+during+chronic+viral+infection.">Cooperation of Tim-3 and PD-1 in CD8 T-cell exhaustion during chronic viral infection.</a> Proc Natl Acad Sci U S A. 107 (33), 14733-14738 (2010).</li><li>Punkosdy, G. 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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 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<...

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 ...

The overall goal of this procedure is to evaluate the in vitro suppressive function of regulatory T cells, isolated from chronic virus infected mice. This method can help answer questions about the phenotype and in vitro suppressive function of activated regulatory T cells during a chronic virus infection. This technique can evaluate the direct effect of regulatory T cells on the individual with a positive response to T cells in vitro.This is a great advantage of this technique. Begin by transferring five times ten to the fifth total splenic lymphocytes in 50 microliters of facs buffer into each well of a new U bottom 96 well plate, followed by 150 microliters of facs buffer. Centrifuge the plate and discard the supernatant.Then agitate the cell pellets and wash the cells two more times in 200 microliters of facs buffer per well. After the second wash, resuspend the pellets in 50 microliters of antibody cocktail per well, for 20 minutes in the dark at four degrees Celsius. Then wash the cells two times in facs buffer, and decant the supernatant.Next, fix the cells for 20 minutes in the dark at four degrees Celsius with 100 microliters of fixation buffer, followed by two washes with permeabilization wash buffer. After the second wash, resuspend the cells in 50 microliters of anti-FOXP3 antibody. To prepare the anti CD3 CD28 coated beads, transfer the appropriate volume of magnetic beads into a 15 milliliter tube, and add an equal volume of PPS.Gently mix the solution by pipetting, and centrifuge the magnetic beads. At the end of the wash, discard the supernatant, and dilute the beads in 50 microliters of complete medium per well. Next, seed 50 microliters of one times ten to the fifth CD4 positive CD25 positive regulatory T cells per well in a 96 well U bottom plate, followed by 50 microliters of one times ten to the fifth CD8 positive responder T cells per well.When all of the cells have been plated, add 50 microliters of the diluted anti CD3 CD28 coated beads to each well, and add up to 50 microliters of medium to bring the total volume of each well up to 200 microliters. Then cover the plate with foil and place it at 37 degrees Celsius in a carbon dioxide incubator for 72 hours. To analyze the CD8 positive T cell cytokine production, after three days of culture, transfer the supernatants into another 96 well plate and analyze the supernatants by ELISA, according to the manufacturer's instructions.To analyze the proliferation of the CD8 positive T cells, wash the plate of cells with facs buffer three times. Then resuspend the pellets in 50 microliters of an appropriate antibody cocktail against proliferated CD8 positive T cells for 20 minutes in the dark at four degrees Celsius. At the end of the incubation, wash the cells two times in facs buffer and fix them for 20 minutes in the dark at four degrees Celsius with 100 microliters of fixation buffer.Finally, wash the cells two more times in facs buffer and resuspend the pellets in 200 microliters of facs buffer. Then measure the percentage of phylate dilabelled CD8 positive T cell proliferation by flow cytometry. In splenic lymphocytes obtained from naive and infected mice, PD-1 is specifically up regulated in both conventional FOXP3 negative CD4 positive and FOXP3 negative CD8 positive cells of LCMV clone 13 infected mice.The frequency of FOXP3 positive CD4 positive regulatory T cells is two times higher in LCMV clone 13 infected mice than in naive mice, with most of the FOXP3 positive CD4 positive cells also displaying an activated PD-1 high phenotype. CD8 positive responder T cells can be separated successfully without significant contamination by other immune cells. After regulatory T cell isolation, a dominant population of CD25 positive CD4 positive T cells with more than 80 percent purity can be obtained.The stimulation of isolated regulatory and responder T cells by anti CD3 CD28 coated beads results in the inhibition of CD8 positive responder T cells in a dose dependent manner. Indeed, when CD8 positive responder T cells are co-cultured with chronic regulatory T cells at a one to one ratio, the CD8 positive responder T cell proliferation is significantly inhibited. Interferon gamma production by CD8 positive responder cells is also significantly inhibited when CD8 positive responder T cells are co-cultured with activated regulatory T cells from chronically infected mice, compared to co-culture with resting regulatory T cells from naive mice.We have found this technique can be completed in 80 hours if it is performed properly. While you are attempting this procedure, it is important to remember to use freshly isolated regulatory T cells immediately after their isolation. After this development, this technique paved the way for researchers in the field of medicine to explore the function of regulatory T cells in a chronic inflammatory disease.After watching this video, you should have a good understanding of how to set up an in vitro regulatory T cell suppression procedure, using a regulatory and responder T cell co-culture system.

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

Research

JoVE Journal

Immunology and Infection

9.7K Views.  Yonsei University. The overall goal of this procedure is to evaluate the in vitro suppressive function of regulatory T cells, isolated from chronic virus infected mice. This method can help answer questions about the phenotype and in vitro suppressive function of activated regulatory T cells during a chronic virus infection. This technique can evaluate the direct effect of regulatory T cells on the individual with a positive response to T cells in vitro.This is a great advantage of this technique. Begin ...

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&amp;#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 ...

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 ...

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.

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

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

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

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

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

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

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be difficult. To overcome this challenge, a methodology combining indexed flow cytometry and high-throughput multiplexed quantitative polymerase chain reaction (qPCR) was developed. The objective was to identify and characterize simian immunodeficiency virus (SIV)-infected cells present within rhesus macaques. Through quantitation of surface protein by fluorescence-activated cell sorting (FACS) and mRNA by qPCR, virus-infected cells are identified by viral gene expression, which is combined with host gene and protein measurements to create a multidimensional profile. We term the approach, targeted Single-Cell Proteo-transcriptional Evaluation, or tSCEPTRE. To perform the method, viable cells are stained with fluorescent antibodies specific for surface markers used for FACS isolation of a cell subset and/or downstream phenotypic analysis. Single cells are sorted followed by immediate lysis, multiplex reverse transcription (RT), PCR pre-amplification, and high throughput qPCR of up to 96 transcripts. FACS measurements are recorded at the time of sorting and subsequently linked to the gene expression data by well position to create a combined protein and transcriptional profile. To study SIV-infected cells directly ex vivo, cells were identified by qPCR detection of multiple viral RNA species. The combination of viral transcripts and the quantity of each provide a framework for classifying cells into distinct stages of the viral life cycle (e.g., productive versus non-productive). Moreover, tSCEPTRE of SIV+ cells were compared to uninfected cells isolated from the same specimen to assess differentially expressed host genes and proteins. The analysis revealed previously unappreciated viral RNA expression heterogeneity among infected cells as well as in vivo SIV-mediated post-transcriptional gene regulation with single-cell resolution. The tSCEPTRE method is relevant for the analysis of any cell population amenable to identification by expression of surface protein marker(s), host or pathogen gene(s), or combinations thereof.

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Described is a methodology to quantitate the expression of 96 genes and 18 surface proteins by single cells ex vivo, allowing for the identification of differentially expressed genes and proteins in virus-infected cells relative to uninfected cells. We apply the approach to study SIV-infected CD4+ T cells isolated from rhesus macaques.

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be ...

In Vivo Augmentation of Gut-Homing Regulatory T Cell Induction

Inflammatory bowel disease (IBD) is an inflammatory chronic disease in the gastrointestinal tract (GUT). In the United States, there are approximately 1.4 million IBD patients. It is generally accepted that a dysregulated immune response to gut bacteria initiates the disease and disrupts the mucosal epithelial barrier. We recently show that gut-homing regulatory T (Treg) cells are a promising therapy for IBD. Accordingly, this article presents a protocol for in vivo augmentation of gut-homing Treg cell induction. In this protocol, dendritic cells are engineered to produce locally high concentrations of two molecules de novo, active vitamin D (1,25-dihydroxyvitamin D or 1,25[OH]2D) and active vitamin A (retinoic acid or RA). We chose 1,25(OH)2D and RA based on previous findings showing that 1,25(OH)2D can induce the expression of regulatory molecules (e.g., forkhead box P3 and interleukin-10) and that RA can stimulate the expression of gut-homing receptors in T cells. To generate such engineered dendritic cells, we use a lentiviral vector to transduce dendritic cells to overexpress two genes. One gene is the cytochrome P450 family 27 subfamily B member 1 that encodes 25-hydroxyvitamin D 1&#945;-hydroxylase, which physiologically catalyzes the synthesis of 1,25(OH)2D. The other gene is the aldehyde dehydrogenase 1 family member A2 that encodes retinaldehyde dehydrogenase 2, which physiologically catalyzes the synthesis of RA. This protocol can be used for future investigation of gut-homing Treg cells in vivo.

Here we present a protocol for in vivo augmentation of gut-homing regulatory T cell induction. In this protocol, dendritic cells are engineered to locally produce high concentrations of the active vitamin D (1,25-dihydroxyvitamin D or 1,25[OH]2D) and the active vitamin A (retinoic acid or RA) de novo.

Inflammatory bowel disease (IBD) is an inflammatory chronic disease in the gastrointestinal tract (GUT). In the United States, there are approximately 1.4 ...