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

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

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

<table 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 vistas.  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 ...