D.E. and M.B.D. are members of a multidisciplinary research platform (MRP) of Ghent University, and the Ghent Researchers on Unfolded Proteins in Inflammatory Disease (GROUP-ID) consortium.

本研究では、成体（6-8週）の雌C57BL / 6マウスを使用しました。マウスは、ゲント大学のビバリウムのガイドラインに従って飼育し、飼育しました。すべての動物の手順は、動物実験倫理委員会によって承認されました。 マウスの脾臓から単核細胞（MNCを）の調製<ol><li>頸椎脱臼によりマウスを生け贄に捧げます。 </li><li>バックダウン解剖ボード上にマウスを置き、ピンと後肢と前足を修正。 </li><li> 70％で皮膚表面を殺菌（体積/体積）エタノール/ H 2 O </li><li>胸部に腹部正中線に沿って皮膚をカットし、無菌手術器具を使用して脾臓を露出させます。 </li><li>周囲の脂肪組織をトリミングした後、室温で1ミリリットル1×リン酸緩衝生理食塩水（PBS）を含む24ウェルプレートに脾臓を配置します。 </li><li>滅菌メスを用いて脾臓を解剖し、50mlの浴槽内に配置された70ミクロンのナイロンフィルターに作品を転送しますE。 </li><li> 2ミリリットルの注射器のプランジャーを使用して、静かにセルストレーナーを通して脾臓片をマッシュアップし、5mlの1×PBSで洗浄します。 </li><li> 15mlチューブにフィコール - パックの3ミリリットルを追加し、5ミリリットルの細胞懸濁液と密度勾配媒体をオーバーレイ。 </li><li>ブレーキなしの室温で20分間、400×gで遠心分離します。 </li><li> 、新鮮な15mlチューブに相間を移し、4℃で10分間300×gで10ミリリットルの冷1×PBSおよび遠心分離機を追加します。 </li><li> 0.5％ウシ血清アルブミン（BSA）および1mMエチレンジアミン四酢酸（EDTA）（FACS緩衝液）を含むPBS 1×2mlの細胞を再懸濁。小さなチューブに移し、細胞懸濁液10μlを、0.4％の10μLと混合</li></ol> 2.濃縮、検出とマウスのiNKT細胞の精製<ol><li>マウス脾臓CD5positive（CD5 +）リンパ球の濃縮。 <ol><li> （オプション）染色で10 5細胞のFITC結合抗CD5（4μlの1：30希釈の/ 10 6細胞 ）暗所で4℃で30分間、200μlのFACS緩衝液および500で再懸濁し、FACS緩衝液μlの洗浄。 1×PBS中の4 &#39;、6-ジアミジノ-2-フェニルインドール（DAPI）の20μMの作業溶液を調製し、FACS緩衝液中に10μlのその5個の細胞を1追加し、室温で5分間インキュベートします。フローサイトメーターで10 4前濃縮された細胞を取得します。 <ol><li> （ 図1A）、細胞ダブレットと凝集体を排除するためにFSC-WのLO細胞にFCS-HとFCS-Wを使用して、電子的にゲート。そして、電子的にゲートアウトDAPI + FSC-W LO集団内（死）細胞（ 図1B）、および電子的に大きさ（ 図1C）によってリンパ球集団を選択するために、SSC-AとFCS-Aを使用しています。 </li><li>リンパ球ゲート内の細胞を用いて、CD5対SSC-Aをプロットし、 図1Dに示すように、電子的にゲートCD5 +リンパ球。フローサイトメーターのプレ濃縮されたサンプルの残りを取得します。 </lI&gt; </ol></li><li> FACS緩衝液に90μlのあたり10 7細胞に細胞数を調整し、10μlの抗CD5マイクロビーズを追加します。 </li><li> 15分間4ºCでインキュベート、FACSは、500μlの冷たいFACS緩衝液中に再懸濁緩衝液および4ミリリットルで1回洗浄。製造業者の推奨に従って磁気細胞分離（MACS）カラムを使用して、CD5 +リンパ球を濃縮します。 </li><li> FITC結合抗CD5とオプション）染色は、10 5細胞 （2.1.1のように1:30希釈/ 10 6細胞 ）およびDAPI4μlの。フローサイトメーター上の10 4濃縮リンパ球を取得し、2.1.1で作成した電子ゲートことを確認します。濃縮された細胞画分に、CD5 +リンパ球を選択します。フローサイトメーター上の10 5個の細胞を取得し、 図1（e）に示すように富む画分に存在する割合CD5 +リンパ球を分析- H。 </li></ol></li><li>検出およびマウスのiNKT CEの単離FACSによるLLS。 <ol><li>抗CD16 / CD32を含むFACS緩衝液中に約10 6個の細胞 20当たりμLおよびPE結合αGalCerの（1:50希釈/ 10 6細胞の4μL）の濃度で濃縮CD5 +リンパ球を再懸濁/のCD1d四量体（1μgの/ 10 6細胞 ）。 </li><li>暗所でRTで30分間インキュベートします。 </li><li>のマウス抗体（mAb）抗CD3εV500（午前1時20希釈の4μL/ 10 6細胞 ）、抗CD19 E450（1:50希釈の4μL/ 10 6細胞）および抗CD8αのE450（4μLを希釈FACS緩衝液で1:50希釈/ 10 6細胞 ）、CD5 +リンパ球に加え、暗所で4℃で30分間インキュベートします。 </li><li> 10 7細胞/ mlの最終濃度で4mlにFACS緩衝液を4℃で10分間、300×gで遠心分離し、再懸濁することによって1mLのFACS緩衝液で細胞を洗浄します。 </li><li> 30μmのセルストレーナーを通して細胞を濾過し、細胞を氷上に置きます。 </li><li>メーカーの推奨14,15に従って細胞選別のためのフローサイトメーターを調整します。 注：細胞選別のためのフローサイトメーターのキャリブレーションは、トレーニングを必要とし、経験豊富なオペレータ、または訓練を受けた技術者により行うことができます。 </li><li> 10 6個の細胞あたりDAPI20μMのワーキング溶液10μlを加え、室温で5分間インキュベートします。フローサイトメーターで約10 4濃縮CD5 +リンパ球を取得します。 FSC-WのLO細胞の電子的にゲートが、細胞ダブレットと集合体（ 図2A）を除外します。その後、FSC-W LO集団内のゲートアウトDAPI + CD8 + CD19 +細胞 （ダンプチャンネル）（ 図2B）、電子的、および2.1.1.1に記載されているように電子的にリンパ球（ 図2C）を選択し 、SSC-AとFCS-Aを使用します。 <ol><li> CD3εと電子的にゲートαGalCerの/ CD1dのことでプロットαGalCerの/のCD1d四量体図2Dに示すように、四量体+CD3ε+ iNKT細胞。割合αGalCerの/のCD1d四量体+濃縮された細胞画分に存在CD3ε+ iNKT細胞を決定するために、2×10 5 CD5 +濃縮リンパ球よりも多くを獲得しません。 </li></ol></li><li> 「1.5」の流速で70μmのノズルを用いて、70 psiで、フローサイトメーターで2.2.7で作成された電子ゲートを使用して、FACSソートαGalCerの/のCD1d四量体+CD3ε+ iNKT細胞。 1ミリリットルの100％FCSを含むFACSチューブにソートされたiNKT細胞を収集します。続いて、4℃で10分間、300×gで細胞を10 mlのRMPI 1640培地および遠心分離機を用いてFACSチューブの側面から選別iNKT細胞をすすぎます。 </li><li>再懸濁し、1mlのRPMI 1640培地にiNKT細胞を選別し、ソートされたiNKT細胞の純度を評価するために、フローサイトメーター上でμLその40を獲得。リンパ球- FSC-W LO DAPIを選択2.2.7で説明したように、S。 （ 図2E - G）と電子的にゲートαGalCerの/のCD1d四量体+CD3ε+ iNKT細胞図2Hに示すように。 </li></ol></li></ol> 3.文化とマウスのiNKT細胞の増殖 0日目<ol><li>コー​​ト室温で1時間、ウェルあたり精製した抗CD3ε（3μgの/ ml）で平底の96ウェルプレート。 200μlの1×PBSおよび一度200μlで完全RPMI 1640培地（10％容量/容量のFCS、100U / mlのペニシリン - ストレプトマイシン、5.5μMのβメルカプトエタノール）で2回洗浄します。 </li><li> 1.11のように0.4％血球計数器を使用して実行可能なソートiNKT細胞（ステップ2.2.8）の数をカウントし、完全RPMI 1640培地中で5×10 5細胞/ mlで4℃で10分間、300×gで遠心分離及び再懸濁組換えマウスIL-2（10ng / ml）を、組換えマウスIL-12（1 ngの/ ml）および可溶性抗マウスCD28（5μg/ ml）を含有します。プレートの抗CD3εコートしたプレートにウェル10 5細胞 /でiNKT細胞をorted、2日間のCO 2インキュベーター中、37℃でインキュベートします。 </li></ol> 2日目<ol start="3"><li> 2日間の新鮮なコーティングされていない平底96ウェルプレートと文化のCO 2インキュベーター内で37℃での完全RPMI 1640培地中で静止条件下での細胞に細胞を移します。 </li></ol> 4日目<ol start="4"><li>穏やかにピペッティングすることによってプレートから細胞を除去し、新鮮な15mlチューブに細胞を移します。 4°C、組換えマウスIL-7（5 ngの/ ml）を含む完全RPMI 1640培地中で5×10 5細胞/ mlで再懸濁し、で10分間、300×gで遠心分離し、フラット10 5細胞/ウェルをプレートCO 2インキュベーター中、37℃で4日間、96ウェルプレートの底。 </li></ol> 8日目<ol start="5"><li> 300 xにおける新たなチューブに細胞を回収し、遠心分離可溶性抗CD28（5μg/ ml）を含む完全RPMI 1640培地中で5×10 5細胞/ mlで4℃で10分間再懸濁のために、G。 </li><li>コー​​トウェル当たり精製した抗CD3ε（3μgの/ ml）で平底の96ウェルプレートとは3.1のように洗います。プレート10 5細胞/ウェル中の抗CD3εコーティングされたプレートとは、11日目までのCO 2インキュベーター中、37℃でインキュベートします。 </li></ol>日11から18 <ol start="7"><li>繰り返しは3.6から3.4のステップ。 </li></ol>日19-20 <ol start="8"><li>完全RPMI 1640培地中に5×10 5細胞/ mlで300×gで再懸濁で新鮮な15mlチューブ、遠心分離機に細胞を移します。 </li><li>平底96ウェルプレート中にウェル10 5細胞 /プレートで細胞と細胞を実験に使用する前にCO 2インキュベーター中、37℃で2日間休息することを可能にします。 </li></ol> 4.サイトカイン産生のCD1dぷらっとを用いて、マウスのiNKT細胞による電子結合アッセイ<ol><li>コー​​ト平底96ウェルあたり組換えマウスCD1dの100μlの（1×PBS中10μg/ ml）でプレートし、37℃で1時間インキュベートします。 </li><li> 200μlの1×PBSで4回洗浄し、ウェル当たり1×PBS中の2％FCS（ブロッキング溶液）200μlを添加し、37℃で2時間インキュベートします。 </li><li>ブロッキング溶液を除去し、ウェルあたりαGalCerの200μlの（5 ngの/μl）を追加し、16時間、37℃でプレートをインキュベートします。 [αGalCerの溶液の調製のために、車両が96 mg / mlのスクロース、10 mg / mlのデオキシコール酸ナトリウム及び5 mg / mlのTween 20を含むに溶解αGalCerの55μgのに1,085μ1×PBSを追加]。 </li><li>インキュベーション後、αGalCerの液を捨て、200μlの完全RPMI 1640培地に続いて、PBS 1×200μlでも二回洗浄します。 </li><li>完全なRで遠心細胞300×gで10分間4℃で再懸濁し、21日目にin vitroで拡大iNKT細胞を収集0.5×10 6細胞/ mlの濃度でPMI 1640培地。 </li><li>ウェル当たりのiNKT細胞の再懸濁液の200μlを添加し、CO 2インキュベーター内で37℃で72時間インキュベートします。 </li><li>製造業者のプロトコルに従って、酵素結合免疫吸着検定法（ELISA）によって関心の上澄み液と測定サイトカインを収集します。 </li></ol>

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The authors have no competing interests to disclose.

現在のプロトコルにおける重要なステップは、分離およびCD5 +リンパ球 （第1＆2）、（3節）とiNKT細胞の初期めっき（第4節）をFACSソーティングのその後の濃縮を含みます。第1節で実行されるステップの、慎重に異なる細胞間期を遠心分離後に生成されるような密度勾配媒体を介して脾臓細胞懸濁液をレイヤーすることを忘れないでください。磁気細胞分離（第2節）により、CD5 +?...

ネズミ不変ナチュラルキラーT（iNKT細胞）細胞は、CD1dの発現皮質胸腺細胞1,2-によって胸腺内で選択された生来のTリンパ球の異なる集団です。 iNKT細胞は、Vβ8、Vβ7またはのCD1dの文脈における内因性ならびに外来脂質抗原を認識することができるVβ2TCRは3のいずれかと対に不変Vα14-Jα18TCR鎖からなるT細胞受容体（TCR）を発現します。例えば、マウスのiNKT細胞を認識し、内因性isoglobotrihexosylceramide（のiGb3）4と呼ばれる脂質抗原と同様に、αガラクトシルセラミド（αGalCerの）5,6、海綿から単離された糖脂質によって活性化されます。 iNKT細胞のTCR依存性活性化は、適応免疫応答のプライミングを促進し、その結果、iNKT細胞は、リウマチ性疾患7及び癌を含む病理の範囲の改善や開発に機能的関与することが示されています&lt;SUP&gt; 8。現在、合成のiNKT細胞リガンドは、免疫病理学的条件の数を調節することが可能である有望な新しいワクチンアジュバントを構成しています。 これは、以前にiNKT細胞しかしマウス組織から単離した後、in vitroで生成することができることが実証されています。これらの研究の多くは、Vα14TCRトランスジェニック（Tg）は、マウス10、またはのiNKT細胞由来のハイブリドーマ11,12を生成するための胸腺腫、主要な抗原提示細胞（APC）、及び/又は細胞株の使用9を用います 。さらに、マウスの多くは、αGalCerのロードされたCD1dダイマー、および長い培養時間などの試薬 ​​の高いボリュームがいくつか公開されたプロトコル少ない倫理的および経済的に9,13をアピールしてください。 この報告では、単離のために、マウスの脾臓からiNKT細胞の体外拡大して適応する方法を説明します。より具体的には、プロトコルは、方法が記載されていますFACS細胞選別のために必要なマウス、試薬および時間を減少させ、in vitroで 、ソート脾臓iNKT細胞を拡大するために最適化されたアプローチを提案しているマウス脾臓からiNKT細胞を濃縮します。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant murine IL-2</td>
			<td>eBiosciences</td>
			<td>14-8021</td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant murine IL-12</td>
			<td>eBiosciences</td>
			<td>39-8122-65</td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant murine IL-7</td>
			<td>eBiosciences</td>
			<td>14-8071</td>
			<td></td>
		</tr>
		<tr>
			<td>purified anti-mouse CD3e (145-2C11)</td>
			<td>eBiosciences</td>
			<td>16-0032-86</td>
			<td></td>
		</tr>
		<tr>
			<td>purified anti-mouse CD28 (37.51)</td>
			<td>eBiosciences</td>
			<td>16-0281-85</td>
			<td></td>
		</tr>
		<tr>
			<td>e450-conjugated anti-mouse CD19 (eBio1D3)</td>
			<td>eBiosciences</td>
			<td>48-0193-82</td>
			<td></td>
		</tr>
		<tr>
			<td>e450-conjugated anti-mouse CD8&#945; (53-6.7)</td>
			<td>eBiosciences</td>
			<td>48-0081-82</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC-conjugated anti-mouse CD5 (53-7.3)</td>
			<td>eBiosciences</td>
			<td>11-0051-81</td>
			<td></td>
		</tr>
		<tr>
			<td>V500-conjugated anti-mouse CD3&#949; (500A2)</td>
			<td>BD biosciences</td>
			<td>560771</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse CD5 (Ly-1) microbeads</td>
			<td>Macs Miltenyi Biotec</td>
			<td>130-049-301</td>
			<td></td>
		</tr>
		<tr>
			<td>purified anti-mouse CD16/32 (2.4G2)</td>
			<td>Macs Miltenyi Biotec</td>
			<td>130-092-575</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS MS Columns</td>
			<td>Macs Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue, 0.4% (wt/vol)</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 medium</td>
			<td>Gibco</td>
			<td>12633-020</td>
			<td></td>
		</tr>
		<tr>
			<td>1x PBS</td>
			<td>Gibco</td>
			<td>10010-056</td>
			<td>[Ca2+/Mg2+ - free]</td>
		</tr>
		<tr>
			<td>Fetal calf serum</td>
			<td>Gibco</td>
			<td>10270</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Gibco</td>
			<td>25030-123</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P4458-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A7906-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>EDS-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll-Paque plus</td>
			<td>GE Healthcare</td>
			<td>71-7167-00 AF</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate F-bottom</td>
			<td>Greiner-bio one</td>
			<td>657-160</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>Greiner-bio one</td>
			<td>665-180</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Greiner-bio one</td>
			<td>655-180</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml falcon tube</td>
			<td>Greiner-bio one</td>
			<td>188-271</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml falcon tube</td>
			<td>Greiner-bio one</td>
			<td>227-261</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#181;m filter</td>
			<td>Greiner-bio one</td>
			<td>542-070</td>
			<td></td>
		</tr>
		<tr>
			<td>30 &#181;m filter</td>
			<td>Millipore</td>
			<td>SVGP01050</td>
			<td></td>
		</tr>
		<tr>
			<td>MiniMACS separator</td>
			<td>Macs Miltenyi Biotec</td>
			<td>130-042-102</td>
			<td></td>
		</tr>
		<tr>
			<td>MS Columns</td>
			<td>Macs Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
		<tr>
			<td>Water-jacketed CO2 incubator</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection Kit</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSAria III</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

an optimized method for isolating and expanding invariant natural killer t cells from mouse spleen

急速に刺激によりサイトカインを分泌する能力は、インバリアントナチュラルキラーT（iNKT細胞）細胞系統の機能的な特徴です。 iNKT細胞は、したがって、免疫応答を活性化し、ステアリング適応可能な生来のT細胞集団として特徴づけられます。マウスiNKT細胞の培養および増殖のための改良された技術の開発は 、in vitro および in vivo モデル系でのiNKT細胞生物学の研究を容易にします。ここでは、マウスの脾臓iNKT細胞の単離および拡張のための最適化された手順を説明します。 C57BL / 6マウスから脾臓を取り出し、解剖し、緊張し、得られた細胞懸濁液を、密度勾配媒体を介して積層されています。遠心分離後、脾臓単核細胞（MNC）収集され、CD5陽性（CD5 +）リンパ球は、磁気ビーズを使用するために濃縮されます。 CD5 +画分内のiNKT細胞は、その後^で染色します5; GalCerを負荷されたCD1d四量体および蛍光活性化細胞選別（FACS）によって精製しました。 FACSは、組換えマウスサイトカインの組み合わせを用いてインビトロで、その後、最初に培養し、プレートに結合したT細胞受容体（TCR）の刺激を、マウス組換えIL-7の存在下で拡張される前に、iNKT細胞を選別しました。この技術を用いて、約10 8 iNKT細胞は、それらが、インビトロでの機能アッセイのために、または、マウスにおけるインビボ移入実験のために使用することができ、その後、培養の18-20日以内に発生することができます。

密度勾配を使用して、脾臓単核細胞の単離は、約1時間を要し、赤血球（RBC）を溶解するのに必要な試薬の使用を排除します。生存細胞の高い収率が除去される器官の緊張時に発生するこの方法及び破片を用いて得られます。典型的には、脾臓リンパ球のプール内のiNKT細胞の頻度は、しかし、これは使用される動物の年齢、性別、健康状態に応じて変えることができる総Tリンパ球の1と5％の?...

Watch this Scientific Journal Video about An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen at JoVE.com

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

マウスの脾臓から不変ナチュラルキラーT細胞を単離および拡張するための最適化方法

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

an-optimized-method-for-isolating-expanding-invariant-natural-killer

Research

JoVE Journal

Immunology and Infection

11.9K ビュー.  Ghent University Hospital. Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents. 

ビデオ: An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

マウスT細胞におけるT細胞受容体のレトロウイルス導入

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

免疫・感染学

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 ...

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where the CB does not contain appreciable numbers of virus-experienced T cells which can protect the recipient from infection.1-4 We and others have shown that virus-specific CTL generated from seropositive donors and infused to the recipient are safe and protective.5-8 However, until recently, virus-specific T cells could not be generated from cord blood, likely due to the absence of virus-specific memory T cells. 
In an effort to better mimic the in vivo priming conditions of na&iuml;ve T cells, we established a method that used CB-derived dendritic cells (DC) transduced with an adenoviral vector (Ad5f35pp65) containing the immunodominant CMV antigen pp65, hence driving T cell specificity towards CMV and adenovirus.9 At initiation, we use these matured DCs as well as CB-derived T cells in the presence of the cytokines IL-7, IL-12, and IL-15.10 At the second stimulation we used EBV-transformed B cells, or EBV-LCL, which express both latent and lytic EBV antigens. Ad5f35pp65-transduced EBV-LCL are used to stimulate the T cells in the presence of IL-15 at the second stimulation. Subsequent stimulations use Ad5f35pp65-transduced EBV-LCL and IL-2. 
From 50x106 CB mononuclear cells we are able to generate upwards of 150 x 106 virus-specific T cells that lyse antigen-pulsed targets and release cytokines in response to antigenic stimulation.11 These cells were manufactured in a GMP-compliant manner using only the 20% fraction of a fractionated cord blood unit and have been translated for clinical use.

Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly na&#238;ve T cells.

臍帯血から細胞傷害性Tリンパ球の拡大をそのターゲットサイトメガロウイルス、エプスタイン - バーウイルス、およびアデノウイルス

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules2. NKT cells express an invariant TCR&alpha; chain rearrangement: V&alpha;14J&alpha;18 in mice and V&alpha;24J&alpha;18 in humans, which is associated with V&beta; chains of limited diversity3-6, and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d 7. The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with &alpha;-Galactosylceramide (&alpha;-GalCer) and a variety of cytokines8. Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines9,10. In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients11. However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells12, 13. Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is a fundamental requirement of NKT cell activation, antigen: CD1d-Ig complexes provide a reliable method to isolate, activate, and expand effector NKT cell populations.

Artificial Antigen Presenting Cell aAPC Mediated Activation and Expansion of Natural Killer T Cells

Here we describe a method for activating and expanding human NKT cells from bulk T cell populations using artificial antigen presenting cells (aAPC). The use of CD1d-based aAPC provides a standardized method for generating high numbers of functional NKT cells.

ナチュラルキラーT細胞の細胞（AAPC）媒介活性化と増殖を提示する人工抗原

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

効率的なためキラー人工抗原提示細胞（KaAPC）<em&gt;体外</em&gt;ヒト抗原特異的T細胞の枯渇

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

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 ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

ヒト全血中のナチュラルキラー細胞溶解活性のフローサイトメトリー分析

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

生体外でマウスのナチュラル キラー細胞の大量生産のため新規無料の送り装置システム

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

マウス小腸管免疫系からリンパ球を分離すること

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular components responsible for supplying cell energy; however, excess mitochondria also produce reactive oxygen species (ROS) that could cause cell death. Therefore, the number of mitochondria must constantly be adjusted to fit the needs of the cells. This dynamic regulation is achieved in part through the function of lysosomes that remove surplus/damaged organelles and macromolecules. Hence, cellular mitochondrial and lysosomal contents are key indicators to evaluate the metabolic adjustment of cells. With the development of probes for organelles, well-characterized lysosome or mitochondria-specific dyes have become available in various formats to label cellular lysosomes and mitochondria. Multicolor flow cytometry is a common tool to profile cell phenotypes, and has the capability to be integrated with other assays. Here, we present a detailed protocol of how to combine organelle-specific dyes with surface markers staining to measure the amount of lysosomes and mitochondria in different T cell populations on a flow cytometer.

This article illustrates a powerful method to quantify mitochondria or lysosomes in living cells. The combination of lysosome- or mitochondria-specific dyes with fluorescently conjugated antibodies against surface markers allows the quantification of these organelles in mixed cell populations, like primary cells harvested from tissue samples, by using multicolor flow cytometry.

ミトコンドリア、リソソーム T 細胞のマルチカラー フロー フローサイトメトリーを用いた定量化

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular ...