私たちは、博士に感謝します。有益なコメントをロバートBonneauとニールクリステンセン。 この作品は、医学資金のペンシルバニア州立大学によってサポートされていました。

1。 BDC2.5マウスの脾臓およびリンパ節由来のT細胞の単離<ol><li>糖尿病誘発性CD4 + T細胞のドナーとして6週齢前糖尿病女性BDC2.5マウスを使用してください。尿糖測定（下記参照）によって決定されたマウスは糖尿病フリーでなければなりません。 </li><li> CO 2窒息を使用して、各マウスを安楽死させると脾臓を除去し、無菌条件下で腋窩と上腕リンパ節。脾臓を削除するには、70％エタノールで毛皮を浸し、その後カットして肌を撤回。脾臓は、マウスの左側にある暗赤色器官として見えるようになります。小さなハサミを使って腹膜に1インチ切開を加え、そっと小さなピンセットで中央に脾臓を把握する。慎重にできるだけ結合組織および添付脂肪をトリミングおよび脾臓を除去。 </li><li>氷上で15 mlコニカルチューブに10ミリリットルダルベッコ修飾イーグル培地（DMEM）中で脾臓とリンパ節を収集します。 </li><li>第を使用して、単一細胞懸濁液を調製しプランジ​​ャが優しく同じ50ミリリットルコニカルチューブに70μmのセルストレーナー（こしにつき1脾臓、ストレーナ当たり4-6リンパ節）を通ってリンパ器官を押して滅菌を10mlの注射器の電子終わり。 </li></ol>プロセス中に、ストレーナからの細胞の回収を最大化するために1ミリリットルDMEMで数回各ストレーナーをすすいでください。 <ol start="5"><li>室温で7分間300-400 XGで15 mlコニカルチューブと遠心分離機に50ミリリットルコニカルチューブから採取した細胞を移す。 </li><li>赤血球を溶解するために、上清を捨て5mlの塩化アンモニウムカリウム（ACK）緩衝液（pH7.2）中で細胞を再懸濁、及び5分間RTでACK細胞懸濁液をインキュベートする。 </li><li> ACK-細胞懸濁液にDMEMを10mlを加え、上記のようにチューブを遠心。一回10ミリリットルDMEMで細胞ペレットを洗浄します。 </li></ol> 2。糖尿病CD4の蛍光活性化細胞選別BDC2.5 +マウスからのT細胞<ol><li> 5ミリリットルFACS s内の再懸濁細胞バッファ寧トリパンブルー色素排除により生存細胞の数（位相差顕微鏡と血球計数器を用いて）数える。 </li><li> FACS緩衝液を用いて、5×10 7細胞/ mlの細胞懸濁液の音量を調節する。 〜1染色コントロールあたり×10 6細胞（染色1いや、3つのシングル汚れ）を取り外します。非活性化されたトランスジェニックCD4 +抗CD4とT細胞（APC）、抗TCRVβ4（FITC）及び15 mlチューブ中の抗CD62L（PE）モノクローナル抗体（モノクローナル）のためのサンプルを染色。暗闇の中で4℃で20〜30分間、製造元が推奨するのmAb濃度°Cを用いた細胞染色を行う。 </li><li> &gt; 3X染色反応音量でサンプルし、FACSバッファーを持つ単一の染色コントロールを洗ってください。 （1-2×10 7細胞/サンプルをソートするためのMLと300μlの単染色コントロール用）と氷の上で店の隣まで300-400 xgでチューブを遠心、上清を除去し、FACSバッファーで細胞を再懸濁ステップ。 </li><li> 35μmのセルを介して細胞サンプルを渡す細胞塊を除去するストレーナーキャップチューブ。訓練されたオペレータとセルソーターでCD4 + TCRVβ4+ CD62L +細胞のためのサンプルをソートします。を15mlチューブに3ミリリットルDMEMに選別された細胞を収集します。 2×10 4合計イベント/秒のソート率で1.5×10 8細胞（3ドナーマウスから得られた細胞の概数）ソートするには、セットアップを含む2.5-3時間を許可します。 〜2.5×10 6非活性ジェニックCD4 +細胞選別後のマウス当たりT細胞を期待しています。 </li><li>ソートの末尾に選別された細胞の絶対数を記録し、300から400×gで7分間のためにそれらを遠心。上清を廃棄し、再懸濁細胞（2×10 6細胞/ ml）中の滅菌リン酸緩衝養子T細胞転送用生理食塩水（PBS）マグネシウム（Mg 2 + / Ca 2 +の自由）。 </li></ol> 3。糖尿病CD4の養子移入+ BDC2.5マウス由来のT細胞<ol><li>再懸濁FACS精製CD4優しく、1ミリリットルの注射器と18-1½ゲージ針を用いて+1ミリリットルのシリンジをT細胞とをロードします。注射の準備のために、27½ゲージ針で18-1½ゲージの針を交換してください。 </li><li>彼らはグルーミング行動を示すまで加熱ランプに6-8週齢の雌NOD.SCIDレシピエントマウスを公開します。 </li><li>拘束のレシピエントマウスを抑制し、注射部位を消毒するために70％（v / v）のエタノールで自分の尾を拭いてください。 </li><li>外側尾静脈のいずれかに1〜2×10 6 FACS選別された細胞/マウスを（最大で500μlのPBS）に注入します。 </li></ol>プランジ​​ャーを無理に押し込まないでください。針が静脈に適切に配​​置されている場合は、注入はほとんど抵抗なく行われる。 4。高血糖のためにレシピエントマウスの監視とT1D <ol><li> T細胞移入後5日から開始して、NOD.SCIDレシピエントマウスは、製造業者の指示に従って、試薬ストリップ（バイエルDiastix）を使用して、上昇した尿糖を毎日監視される。二つの連続urin持つマウス電子グルコース測定値は&gt; 250ミリグラム/ dlと、糖尿病と考えられている。 </li></ol>

<ol>
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すべてのマウスは、ペンシルベニア州立大学の動物実験のガイドラインに準拠し、委員会を設置し、使用中の薬の特定病原体フリー（SPF）施設のペンシルベニア州立大学で飼育した。 著者は、彼らが競合する経済的利益を持っていないことを宣言します。

T1Dは、糖尿病のNODマウスの糖尿病又はT細胞クローンに由来するTCRトランスジェニックマウスからの脾臓細胞全体またはT細胞サブセットの養子移入することにより効率を変化させることで、レシピエントマウスに誘導することができる。我々はここにNOD.SCIDマウスにFACS精製CD62L + BDC2.5ジェニックCD4 + T細胞を転送することにより、100％の発生率で2週間以内に、レシピエントマウスにT1D誘導する...

NODマウスは、自発的に自己免疫性糖尿病を発症し、広く人間T1D 1,2のための動物モデルとして使用されている。 NODマウスにおけるT1Dの病因は、T細胞とB細胞が続き、樹状細胞やマクロファージによるランゲルハンスの膵島の、生後3-4週で始まる、浸潤によって特徴付けられる。非破壊ペリ膵島炎のこの段階では、3歳の4〜6ヶ月まであからさまな糖尿病になり、インスリン産生膵β細胞の遅い、進行性の破壊につながる。 4,5脾細胞、CD4 +糖尿病NODマウスからの6,7またはCD8 + 8,9 T細胞の転送は、膵島反応性T細胞がT1D病因において中心的な役割を果たすことを示す、免疫不全NODマウスにおいて糖尿病を媒介することが示されている。実験条件に応じて、糖尿病は、これらの研究で数週間にわたって、徐々にレシピエントマウスで開発された。同様に、様々なT細胞クローンは時間とコストがかかることによって導出糖尿病誘発T細胞の培養は、数週間レシピエントマウス7,10への移行後に糖尿病を媒介することが報告されている。 CD4-CD8または拘束性糖尿病誘発T細胞クローンに由来するTCRを発現するトランスジェニックマウスの可用性と、いくつかの研究室は、その後、例えば、マウスからの脾臓T細胞は、レシピエント11-13に糖尿病を転送することができたことを示している。具体的には、BDC2.5 NODマウスは、クロモグラニンA、膵β細胞におけるタンパク質14-16に特異的なTCR BDC2.5、トランスジェニックである。あからさまに糖尿病または前糖尿病BDC2.5マウスからin vitroで活性化または非活性化の全体または画し脾臓細胞での転送効率を11,17-19を変えることで、新生児や免疫不全NODマウスに糖尿病を譲渡。 私たちは、精製されたトランスジェニックCD4を利用し簡単な方法+高効率とconsisteでレシピエントマウスにT1D誘導する前糖尿病BDC2.5マウスからT細胞を記述NCY。トランスジェニックTCRVβ4鎖を発現するナイーブ、膵島抗原特異的CD4 + T細胞はCD4ための蛍光活性化細胞選別（FACS）によって、これらのマウスから単離され+ CD62L + T細胞の多数。精製されたトランスジェニックT細胞を、次いで、官能性T細胞およびB細胞を欠き、膵島炎および糖尿病フリー20アールNOD.SCIDマウスに活性化せずに転送される。レシピエントマウスはT細胞移入後2週間以内に急速に発達T1Dを示す尿糖濃度の上昇のために監視されています。 他の方法とは対照的に不均一な特異性を有する糖尿病誘発性T細胞を移すことを、我々のプロトコルは、FACSソートしたCD4 +ほぼ独占的に糖尿病BDC2.5 TCRを発現したT細胞を使用しています。彼らの均質性に起因して、移入されたT細胞（〜1×10 6細胞/マウス）のほんの数は100％の発生率で2週間以内に急速T1D開発のために必要とされる。我々のプロトコルの別の利点は、irradiatioあるレシピエントマウスのnが、それはいくつかの他の方法のためにあるようにする必要はありません。この方法の潜在的な制限は、糖尿病の両方のCD4とCD8 T細胞サブセットまたは特にCD8 T細胞の寄与の調査を許可していないということです。 記述されたプロトコルは、ナイーブ、単一特異的CD4 +標的臓器への膵島抗原特異Th細胞のホーミングに介入するT細胞と同様に、治療戦略により仲介急速T1D開発を研究するために有用であろう。

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			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>BDC 2.5 TCR transgenic NOD mice (NOD.Cg-Tg(Tcr&alpha;BDC 2.5, Tcr&beta;BDC 2.5)</td>
			<td>JAX</td>
			<td>004460</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NOD.SCID mice (NOD.CB17-Prkdcscid/J)</td>
			<td>JAX</td>
			<td>001303</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dulbecco's Modified Eagle's Medium (DMEM)</td>
			<td>Themo Scientific</td>
			<td>SH30022.01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bayer Diastix</td>
			<td>Fisher Scientific</td>
			<td>AM2803</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>15 ml conical tubes</td>
			<td>Falcon</td>
			<td>352095</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>50 ml conical tubes</td>
			<td>Falcon</td>
			<td>352070</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sterile surgical tweezers</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sterile small pair scissors</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sterile large pair scissors</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>70 &mu;m cell strainers</td>
			<td>Fisher Scientific</td>
			<td>22363548</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>35 &mu;m cell strainer cap tubes</td>
			<td>BD Biosciences</td>
			<td>352235</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ammonium-Chloride-Potassium (ACK) buffer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>0.15 M NH4Cl, 1 mM KHCO3, 0.1 mM Na2EDTA, pH 7.2 in dH2O</td>
		</tr>
		<tr>
			<td>BD FACSFlowTM sheath fluid</td>
			<td>BD Biosciences</td>
			<td>342003</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FACS staining buffer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>PBS, 0.2 mM EDTA, 0.5% BSA/FCS, filter sterilized</td>
		</tr>
		<tr>
			<td>Phase contrast microscope</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-CD4 (APC) mAb</td>
			<td>Biolegend</td>
			<td>1005616</td>
			<td>clone RM4-5</td>
		</tr>
		<tr>
			<td>Anti-TCR V&beta;4 (FITC) mAb</td>
			<td>BD Biosciences</td>
			<td>553365</td>
			<td>clone KT4</td>
		</tr>
		<tr>
			<td>Anti-CD62L (PE) mAb</td>
			<td>BD Biosciences</td>
			<td>553151</td>
			<td>clone MEL-14</td>
		</tr>
		<tr>
			<td>Cell sorter</td>
			<td>BD Biosciences</td>
			<td>&nbsp;</td>
			<td>e.g. BD FACSAria III</td>
		</tr>
		<tr>
			<td>Heat lamp</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mouse restrainer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1 ml syringes</td>
			<td>Becton Dickinson</td>
			<td>309602</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>18-1&frac12; gauge needles (sterile)</td>
			<td>Becton Dickinson</td>
			<td>305196</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>27&frac12; gauge needles (sterile)</td>
			<td>Becton Dickinson</td>
			<td>305109</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

accelerated type 1 diabetes induction in mice by adoptive transfer of diabetogenic cd4+ t cells

非肥満糖尿病（NOD）マウスは自発的に生後12週間後に自己免疫性糖尿病を開発し、人間の1型糖尿病（T1D）の中で最も広範囲に研究の動物モデルである。照射レシピエントマウスでセル転送研究はT細胞が、このモデルでT1D発症に極めて重要であることを確立した。我々は、本明細書に急速に精製され、プライマリCD4 +膵島特異的T細胞受容体（TCR）NOD.SCIDレシピエントマウスへのBDC2.5トランスジェニック前糖尿病NODマウス由来のT細胞の養子免疫伝達によってT1D誘導するための簡単​​な方法を述べる。糖尿病誘発T細胞の単離および養子移入は、同じ日以内に完了することができるこの技術の主な利点は、受信者の照射が必要とされないし、T1Dの高い発生率は、T細胞の転送後2週間以内に誘発される。したがって、T1Dにおける病因および治療介入の研究では、不均一なT細胞集団またはクローンに頼る方法よりも速い速度で進むことができます糖尿病NODマウス由来。

我々の結果は、このような膵臓リンパ節などの二次リンパ器官への家へのT細胞のために重要であるCD62Lを発現するトランスジェニックBDC2.5細胞の単離を示す。今回の知見は、さらに急速に転送し、効率的にNOD.SCIDレシピエントマウスにT1Dにするには、この単一特異的T細胞集団の強力な能力を実証する。 糖尿病CD4 + BDC2.5マウス由来のT細胞の単離は、図2に示さ?...

Watch this Scientific Journal Video about Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells at JoVE.com

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

私たちは、膵島抗原特異、プライマリーCD4 + T細胞の養子移入によって2週間以内に、マウスにおける1型糖尿病（T1D）を誘導するための再現可能な方法を提供する。

糖尿病CD4の養子移入+ T細胞によるマウスの加速された1型糖尿病の誘導

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of ...

accelerated-type-1-diabetes-induction-mice-adoptive-transfer

Research

JoVE Journal

Immunology and Infection

16.6K ビュー.  Pennsylvania State University College of Medicine. 私たちは、膵島抗原特異、プライマリーCD4 + T細胞の養子移入によって2週間以内に、マウスにおける1型糖尿病（T1D）を誘導するための再現可能な方法を提供する。

ビデオ: Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

ナチュラルキラー細胞傷害性アッセイにおけるHIV - 1感染プライマリCD4 + T細胞を標的細胞としての準備と使用

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

免疫・感染学

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

HIV-1感染CD4の生得センシングを評価<sup&gt; +</sup&gt;使用して形質細胞様樹状細胞によるT細胞<em&gt;エクスビボ</em&gt;共培養系。

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

エフェクターCD4の養子移入によるマウス腸の炎症の誘導<sup&gt; +</sup&gt; CD45RB<sup&gt;高い</sup&gt;免疫不全マウスへのT細胞

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

The methods described herein for activation of na&#239;ve CD4+ T cells in suspension and their adherence in coverslips for confocal microscopy analysis allow the spatial localization and visualization of gangliosides involved in CD4+ T cell activation, that complement expression profiling experiments such as flow cytometry, western blotting or real-time PCR. The quantification of ganglioside expression through flow cytometry and their cellular localization through microscopy can be obtained by the use of anti-ganglioside antibodies with high affinity and specificity. Nonetheless, an adequate handling of cells in suspension involves the treatment of culture plates to promote the necessary adherence required for fluorescence or confocal microscopy acquisition. In this work, we describe a protocol for determining GD3 and GD2 ganglioside expression and colocalization with the TCR during na&#239;ve CD4+ T cell activation. Also, real-time PCR experiments using &#60;40,000 cells are described for the determination of the GD3 and GM2/GD2 synthase genes, demonstrating that gene analysis experiments can be performed with a low number of cells and without the need of additional low input RNA kits.

Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

We describe a standard antibody staining protocol for use in microscopy to determine the membrane expression and localization of gangliosides in resting and activated human na&#239;ve CD4+ T cells. Also described are real-time PCR experiments using &#60;40,000 cells that do not require additional low input RNA kits.

CD4の調製<sup&gt; +</sup&gt;顕微鏡によるGD3およびGD2ガングリオシドの膜発現の分析のためのT細胞

The methods described herein for activation of na&#239;ve CD4+ T cells in suspension and their adherence in coverslips for confocal microscopy analysis ...

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.

TGF-βを含むプロトコルを使用したナイーブCD4 + T細胞からのヒトCD4 + FOXP3 +誘導された調節性T細胞（iTregs）のインビトロ分化における

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

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human central nervous system (CNS) autoimmune demyelinating disease, creation of an AQP4-targeted model with both clinical and histologic manifestations of CNS autoimmunity has proven challenging. Immunization of wild-type (WT) mice with AQP4 peptides elicited T cell proliferation, although those T cells could not transfer disease to na&#239;ve recipient mice. Recently, two novel AQP4 T cell epitopes, peptide (p) 135-153 and p201-220, were identified when studying immune responses to AQP4 in AQP4-deficient (AQP4-/-) mice, suggesting T cell reactivity to these epitopes is normally controlled by thymic negative selection. AQP4-/- Th17 polarized T cells primed to either p135-153 or p201-220 induced paralysis in recipient WT mice, that was associated with predominantly leptomeningeal inflammation of the spinal cord and optic nerves. Inflammation surrounding optic nerves and involvement of the inner retinal layers (IRL) were manifested by changes in serial optical coherence tomography (OCT). Here, we illustrate the approaches used to create this new in vivo model of AQP4-targeted CNS autoimmunity (ATCA), which can now be employed to study mechanisms that permit development of pathogenic AQP4-specific T cells and how they may cooperate with B cells in NMO pathogenesis.

Here, we present a protocol to induce paralysis and opticospinal inflammation by transfer of aquaporin-4 (AQP4)-specific T cells from AQP4-/- mice into WT mice. In addition, we demonstrate how to use serial optical coherence tomography to monitor visual system dysfunction.

麻痺、視神経脊髄炎自己抗原アクアポリン 4 の T 細胞特定のマウスの視覚系障害の誘導

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...

Isolation and Adoptive Transfer of High Salt Treated Antigen-presenting Dendritic Cells

Excess dietary salt intake contributes to inflammation and plays a vital role in the development of hypertension. We previously found that antigen-presenting dendritic cells (DCs) can sense elevated extracellular sodium leading to the activation of the NADPH oxidase and formation of isolevuglandin (IsoLG)-protein adducts. These IsoLG-protein adducts react with self-proteins and promote an autoimmune-like state and hypertension. We have developed and optimized state-of-the-art methods to study DC function in hypertension. Here, we provide a detailed protocol for isolation, in vitro treatment with elevated sodium, and adoptive transfer of murine splenic CD11c+ cells into recipient mice to study their role in hypertension.

Here, we present a protocol to isolate dendritic cells from murine spleens by magnetic cell sorting and subsequent adoptive transfer into na&#239;ve mice. The model of high-salt activated dendritic cells was chosen to explain the step-by-step procedures of adoptive transfer and flow cytometry.

分離と養子伝高塩処理樹状細胞の抗原提示

Excess dietary salt intake contributes to inflammation and plays a vital role in the development of hypertension. We previously found that ...

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear cells (PBMCs). The development of stable, non-toxic, fluorescent dyes such as carboxyfluorescein succinimidyl ester (CFSE) allows for rare, antigen-specific T cells to be distinguished from bystanders by diminution in fluorescent staining, as detected by flow cytometry. This method has the following advantages over alternative approaches: (i) it is very sensitive to low-frequency T cells, (ii) no knowledge of the antigen or epitope is required, (iii) the phenotype of the responding cells can be analyzed, and (iv) viable, responding cells can be sorted and used for further analysis, such as T cell cloning.

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Presented here is a protocol for measuring proliferating CD4+ T cells in response to antigenic proteins or peptides using dye dilution. This assay is particularly sensitive to rare antigen-specific T cells and can be modified to facilitate cloning of antigen-specific cells.

カルボキシフルオレスチンスクシニミジルエステルを用いたヒト抗原特異的CD4+T細胞の増殖の定量化

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear ...

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase (G6PI)-Induced RA Mice

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T (iNKT) cells. Patients with active RA present fewer iNKT cells, defective cell function, and excessive polarization of Th1. In this study, an RA animal model was established using a mixture of hGPI325-339 and hGPI469-483 peptides. The iNKT cells were obtained by in vivo induction and in vitro purification, followed by infusion into RA mice for adoptive immunotherapy. The in vivo imaging system (IVIS) tracking revealed that iNKT cells were mainly distributed in the spleen and liver. On day 12 after cell therapy, the disease progression slowed down significantly, the clinical symptoms were alleviated, the abundance of iNKT cells in the thymus increased, the proportion of iNKT1 in the thymus decreased, and the levels of TNF-&#945;, IFN-&#947;, and IL-6 in the serum decreased. Adoptive immunotherapy of iNKT cells restored the balance of immune cells and corrected the excessive inflammation of the body.

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase G6PI-Induced RA Mice

This protocol uses G6PI mixed peptides to construct rheumatoid arthritis models that are closer to that of human rheumatoid arthritis in CD4+ T cells and cytokines. High purity invariant natural killer T cells (mainly iNKT2) with specific phenotypes and functions were obtained by in vivo induction and in vitro purification for adoptive immunotherapy.

グルコース-6-リン酸イソメラーゼ(G6PI)誘発RAマウスにおけるiNKT細胞の導入免疫療法

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T ...

A High-Throughput Multiplexed Screening for Type 1 Diabetes, Celiac Diseases, and COVID-19

An ongoing clinical trial, Autoimmunity Screening for Kids (ASK), is the first screening study in the general population for type 1 diabetes (T1D) and celiac disease in the United States. With the coronavirus disease 2019 (COVID-19) pandemic, the epidemiology of COVID-19 in the general population and knowledge about the association between COVID-19 infection and T1D development are urgently needed. The currently standard screening method of the radio-binding assay (RBA) has met two great challenges: low efficiency with a single assay format and low disease specificity with a large proportion of low-affinity antibodies generated in screening. With the platform of the multiplex electrochemiluminescence (ECL) assay we established previously, a novel 6-Plex ECL assay was developed that combines, in a single well, all four islet autoantibodies (IAbs) to insulin, glutamic acid decarboxylase (GAD65), insulinoma antigen 2 (IA-2), and Zinc transporter 8 (ZnT8) for T1D, transglutaminase autoantibodies (TGA) for celiac disease, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor-binding domain (RBD) antibodies for COVID-19. The assay was validated in blind using 880 samples from the ASK study, including 325 positive samples and 555 all antibody-negative samples, and compared with the standard RBAs and a single ECL assay. With the advantages of high efficiency, low cost, and low serum volume, this assay has been accepted as the primary screening tool for the ASK study.

In line with the urgent need for screening for type 1 diabetes, celiac disease, and coronavirus disease 2019, we developed a high throughput 6-Plex electrochemiluminescence assay to simultaneously detect all four islet autoantibodies, tissue transglutaminase autoantibodies, and antibodies to the receptor binding domain of severe acute respiratory syndrome coronavirus 2.

1型糖尿病、セリアック病、およびCOVID-19のハイスループット多重化スクリーニング

An ongoing clinical trial, Autoimmunity Screening for Kids (ASK), is the first screening study in the general population for type 1 diabetes (T1D) and ...