Name: phenotypic and functional analysis of activated regulatory t cells isolated from chronic lymphocytic choriomeningitis virus-infected mice
Uploaded: 2016-06-22T15:00:00
Description: 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

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).

本研究では、マウスは、延世大学の延世大学実験動物研究センターの特定病原体フリーの施設で維持しました。全ての動物実験は、延世大学で延世大学実験動物研究センターの国際動物実験委員会によって承認されたプロトコルを使用して、韓国の食品医薬品局（FDA）のガイドラインに従って行いました。 ソリューションの調製<ol><li> 2％RPMI培地は、RPMIで1％まで2％ペ?...

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T reg細胞の少数が、マウスおよびヒトに存在するが、免疫応答を調節し、免疫寛容の維持において重要な役割を果たしているように、それらの機能を理解することが重要です。 T reg細胞の数と抑制機能は、慢性ウイルス感染15-20だけでなく、癌の進行13,14の間に増加します。これはおそらく、継続的な抗原刺激によるものです。抗原の永続性と病気の開発中のT...

調節性T（TのREG）細胞は、それらの発生と機能1用の転写因子としてフォークヘッドボックスP3（Foxp3の）を発現します。加えて、T reg細胞は、CD25 2、リンパ球活性化遺伝子-3（LAG-3）3、グルココルチコイド誘導腫瘍壊死因子受容体4、および細胞傷害性Tリンパ球関連タンパク質4（CTLA-4）5などの様々な他の分子を発現しますそれらの表面または細胞内領域に。このような強化された抑制機能を表示するウイルス6,7、細菌8,9、および寄生虫10-12、または癌発生13,14の過程で、T reg細胞が活性化された細胞に分化になる、などの病原体の様々な種類の慢性感染の間エフェクターCD4 +及びCD8 + T細胞を標的とします。論文の数は拡大し、活性化したT reg細胞が損なわCD8 + T細胞RESPONSに寄与することが示唆されています友人レトロウイルス（FV）感染15-17の間に電子。 FV誘発性T reg細胞は、IFN-γまたはグランザイムBの発現およびCD8 + T細胞の細胞傷害反応性15-17を阻害します 。また、単純ヘルペスウイルス感染モデルでは、ウイルス特異的CD8 + T細胞および免疫病原性CD4 + T細胞の18-20の浸潤によって深刻な組織損傷の拡大が生じたCD4 + CD25 + T reg細胞の枯渇が報告されました。 リンパ球性脈絡髄膜炎ウイルス（LCMV CL13）のクローン13株を用いて慢性的に感染したマウス21〜24は、広く、慢性ウイルス感染の間にエフェクターT細胞（T effの）およびT reg細胞の表現型および機能を特徴づけるために使用されています。永続LCMV感染の間に、ウイルス特異的T effの細胞が次第に彼らのエフェクター機能を失い、疲れT（TのEXH）細胞になります。一方、Treg細胞は、ウイルス特異的T細胞応答25を抑制する能力を強化します。 T effは細胞の機能容量の低下は、T effは細胞上の阻害性受容体のアップレギュレーションは、抗原提示細胞の変化した機能、免疫調節性サイトカインの産生、および頻度の増加、またはT REGの強化機能など、いくつかの要因によって説明することができます細胞26。 T細胞の抑制に関与する因子の中でも、プログラムされた細胞死は、タンパク質1（PD-1）発現のT EXH細胞およびT reg細胞が広く、抗原の持続性および抑制性環境の特徴として考えられてきました。最近では、T reg細胞のPD-1経路及びアブレーションの遮断が強化されたT細胞の機能をもたらし、LCMV慢性感染27の間のウイルス負荷を減少させたことが報告されました。また、T reg細胞は、LCMV 23,25によるマウスの慢性感染の間に活性化されています</sアップ&gt;とその抑制機能は25強化されます。 PD-1は、高T reg細胞ならびにT EXH細胞上に発現し、T reg細胞によって発現されるPD-1のレベルは、T細胞増殖25を阻害するそれらの抑制機能の強さと相関します。 ここでは、LCMV CL13とナイーブマウスから単離された休止T reg細胞に感染したマウスから単離した活性化T reg細胞の特性を比較する方法を記載しています。さらに、我々は活性化T reg細胞を分離し、それらのex vivoでの表現型を調べるだけでなく、in vitroでのそれらの抑制活性を測定するための一連の処理を説明します。

<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

転写因子としてのFoxp3を発現する調節性T（T REG）細胞、CD4 + T細胞のサブセットです。 T reg細胞は、免疫応答を調節することによって免疫寛容と恒常性の維持に重要な役割を果たしています。 T reg細胞の主な役割は、エフェクターT（T effの）細胞の増殖ならびにIFN-γ、TNF-α、およびIL-2などのサイトカインの産生を抑制することです。 T effは 、細胞の機能を阻害するT reg細胞の能力は、永続的な病原体感染および癌の発生の間に増強されることが実証されています。休止または炎症条件下でT reg細胞の機能を明確にするために、マウスまたはヒトT reg細胞を使用して、種々のインビトロ抑制アッセイが考案されています。本研究の主な目的は、休止および活性化T regの間の表現型および抑制機能の違いを比較するための方法を開発することです細胞。活性化T reg細胞を単離するために、マウスを、リンパ球性脈絡髄膜炎ウイルス（LCMV）クローン13（CL13）、LCMVの慢性株に感染させました。 LCMV CL13感染マウスの脾臓から単離されたT reg細胞は、ナイーブマウスから単離されたT reg細胞を休止期と比較して活性化された表現型および強化された抑制活性の両方を示しました。ここでは、T reg細胞を休止期から活性化したT reg細胞を区別するためのex vivoでの表現型分析のための基本的なプロトコルを記述します。さらに、我々は、完全に活性化したT reg細胞の抑制活性の測定のためのプロトコルを説明します。

私たちは、静脈内にLCMV CL13の2×10 6 PFUでそれらを注入することにより、持続的なウイルス感染マウスを作製しました。 T reg細胞および慢性ウイルス感染の間にT CONV細胞における表現型の変化を調べるために、ナイーブ、感染マウスから得られた脾臓リンパ球を、種々の抗体で染色し、フローサイトメトリーにより分析しました。 CD4 + T?...

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.

表現型および慢性リンパ球性脈絡髄膜炎ウイルスに感染したマウスから単離された活性化制御性T細胞の機能解析

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 ...

Research

JoVE Journal

Immunology and Infection

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

Generation of Induced Regulatory T Cells from Primary Human NaÃƒÆ’Ã‚Â¯ve and Memory T Cells

初代ヒトナイーブおよびメモリーT細胞から誘導した調節性T細胞の生成

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

HIV-1に感染した個体からの制御性T細胞を増殖するための新しいツール

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

慢性EAEを有するマウスにおける神経幹/前駆細胞の全身注射

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

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

T細胞の導出<em&gt;体外</em&gt;マウス胚性幹細胞からの

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

末梢血からのヒトアロ抗原特異的T細胞の生成

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

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

表現型分析のためのヒト皮膚からリンパ球単離と<em&gt; ex vivoで</em&gt;細胞培養

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

T細胞は、樹状細胞からTransinfectionによって細菌をキャプチャ

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

細菌や真菌によってウイルス感染細胞上のバイオフィルムの開始の決意

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