We thank Miltenyi Biotec, Germany for providing reagents and CliniMACS Prodigy equipment for evaluation studies. We thank George T. McNamara (Pediatric department, MD Anderson Cancer Center) for proof reading the manuscript. Grant support: Cancer Center Core Grant (CA16672); RO1 (CA124782, CA120956, CA141303; CA141303); R33 (CA116127); P01 (CA148600); Burroughs Wellcome Fund; Cancer Prevention and Research Institute of Texas; CLL Global Research Foundation; Estate of Noelan L. Bibler; Gillson Longenbaugh Foundation; Harry T. Mangurian, Jr., Fund for Leukemia Immunotherapy; Institute of Personalized Cancer Therapy; Leukemia and Lymphoma Society; Lymphoma Research Foundation; MDACC&#8217;s Sister Institution Network Fund; Miller Foundation; Mr. Herb Simons; Mr. and Mrs. Joe H. Scales; Mr. Thomas Scott; National Foundation for Cancer Research; Pediatric Cancer Research Foundation; William Lawrence and Blanche Hughes Children's Foundation.

無菌条件下での材料の作製（材料および装置表を参照してください） <ol><li> （w / v）の最終濃度0.5％までのヒト血清アルブミン（HSA）を補充したPBS / EDTA緩衝液の3 Lを準備します。 </li><li>臨床グレードの0.9％塩化ナトリウム（NaCl）溶液の1リットルバッグ及びGMPグレードの細胞培養培地の2リットルを調製します。 </li><li>滅菌水8mlでのCMVのpp65の1バイアルを再構成することによりCMV特異的ペプチド抗原カクテルの60ナノモルを準備します。 </li><li>ルアー/スパイクインターコネクタを使用して、50ミリリットルの容量冷凍袋にのCMVのpp65ペプチドカクテルを移し、チューブセットにカクテルのその後の配布を回避するために鉗子をロックで固定します。無菌条件下でのオープンセル濃縮管セット（TS 500）。 </li><li>滅菌管溶接機を使用して、この時点では、ペプチドカクテルバッグクランプを開けないでくださいチューブセットのバルブ2 TS 500用のチューブ接続にペプチドカクテル凍結バッグを接続します。 </li><li> 1×10 9 Tを削除しますNC細胞産物を開始してから、50mlの総体積2.5％HSAを含有するPBS / EDTA緩衝液に懸濁します。 150ミリリットルのトランスファーバッグに携帯製品を注入します。 </li></ol> 2.自動細胞分離システムの製造および使用（材料および装置表を参照してください） <ol><li>細胞濃縮システム（ 図1B）に切り替えて、プログラム「CCS_IFN-γ充実」を選択してください。手順を介してオペレータを案内する指示や写真で画面を示すユーザインタフェースを確認します。 </li><li>パラメータの 「オペレーター」と「 チューブセットP / Nなし」と入力します 。次に、対話型のモニタ画面に表示される指示に従って自動化された細胞濃縮装置にチューブセット500をインストールしてください。 </li><li>デバイスにメディアやバッファを接続するために画面に表示されるステップバイステップのインストラクションに従ってください。レコードカタログ番号とCONNECTIN前試薬のロット番号楽器にグラム。 </li><li>チューブセットの最終チェック後、ペプチドカクテルバッグのクランプを開きます。ミディアムバッグを開き、チューブセットの自動プライミングを開始します。 </li><li>プライミング工程が完了した後、滅菌管溶接機の助けを借りて、貯蔵バッグ（200 mL）中のNaCl緩衝液中にHSA（2.5％）を補足します。滅菌管溶接機を使用して、「アプリケーション・バッグ」に始まる細胞産物を転送します。 </li><li>アダプタを介して各チューブにCCS（IFNγ）の試薬を接続します。濃縮プロセスの前に細胞物質の画分を回収するために好ましい時間を入力します。入力されたすべてのデータ/パラメータの精度を確認し、確認してください。プロセスを開始します。 </li><li>自動細胞濃縮プロセスを開始する前に、品質管理バッグ（QCBは、元の画分（ORI）は、PBS / EDTA緩衝液で希釈した100ミリリットル室コンテンツのうち、約1.3ミリリットルが含まれています）を取り外します。 QCBを密封、重さ、そして4℃で保存します。 </li><li> enrichmeを開始NTプロセス。プロセスの終わりには、標的細胞は、貯蔵バッグから溶出緩衝液の概算体積で溶出されます。 </li><li>非ターゲットセルバッグ（NTCB、陰性分画= NEG）と標的細胞バッグ（TCB、陽性画分= POS）を密封し、各バッグの重量を量ります。重みは、後に細胞数の計算に使用されます。 </li><li>すぐに濃縮手順後にフローサイトメトリー分析のために端数ごとに2つのアリコートを収集し、4℃でのサンプルの残りの部分を格納します。細胞数の決意と濃縮パフォーマンス分析（ 表1）のために他の試料アリコートのための1つの試料アリコートを使用してください。 </li><li>細胞濃縮器からチューブセットを削除します。将来の使用のためにUSBドライブにログファイルを転送します。 注：すべての試薬は、滅菌条件下で調製されるべきです。バイオセーフティII型フードの使用を強くお勧めします。健康から分離された定常状態のアフェレーシス細胞産物（非動員が）を使用しCMV血清陽性ドナーがCMV抗原特異的T細胞を豊かにします。唯一のFDAは、HSAを使用する必要がありますライセンス。細胞調製のためのバッファが低減純度および標的細胞の収率低下をもたらす低いまたは高い周囲温度として25℃まで19℃に維持されるべきです。 </li></ol> 3.セル数の決意<ol><li> 表1に示すように、細胞数のためのQCB、NTCB及びTCBのアリコートを取る。各アリコート（力価1時11分）にCD45-VoBlueを追加し、4℃で10分間、暗所でインキュベートします。 </li><li>元の画分および陰性画分を、陽性画分を450μlの新たに調製した赤血球細胞溶解液に1.5 mlの新たに調製した赤血球溶解液（1×）を加え、そして室温で15分間、全ての画分をインキュベートします。 </li><li>直前の分析、を1μg/ mlの最終濃度になるようにヨウ化プロピジウムを添加（1：100μg/ mlの100希釈）。細胞数およびVIを決定するために、自動細胞カウンターを使用してください能力。フローサイトメトリー分析のために細胞計数デバイス推奨ソフトウェアを使用してください。 、元の負と正の分画用白血球の絶対数を決定します。 注：細胞計数分析のために採取した試料1mlあたり生存白血球の細胞数は、細胞分析装置推奨されるソフトウェアを用いて決定されます。 </li><li> 図2（領域5、生存可能な白血球）に示すように地域を設定します。細胞数を決定するには、次のゲーティング戦略を使用してください。元の画分中の生存白血球は、図2に示されています。 </li><li>次のように示した領域（ 図2、1-6）は、階層的に以下のとおりです。 1：タイムゲート→2：→3シングル細胞：CD45 +細胞 →4：白血球（残骸を除く）→5：実行可能な白血球→6：生存リンパ球 </li><li>ネガティブとポジティブ画分について細胞数を決定するために、同じ手順を繰り返します。全画分の細胞数を計算しますサンプル画分（ 表2）の総体積の希釈係数を考慮することによって。 </li></ol>分離性能の検討4。 <ol><li>予備冷却し、PBS / EDTAバッファー/ 0.5％AB血清でQCB、NTCBおよびTCB分数細胞のアリコートを洗います。 4℃で5分間、300×gで細胞を遠心分離し、上清を吸引します。 </li><li>含む100μlの抗体 - 蛍光色素染色混合物中に再懸濁細胞：CD3-FITC、CD4-APC、CD8-APC-Vio770、CD14-PerCPを、CD20-PerCPを、CD45-VioBlueにおよび抗IFNγ-PE（力価1:11）と4℃で10分間、暗所でインキュベートします。 </li><li> 1 mlの新たに調製した赤血球溶解液（1×）を加え、室温で15分間インキュベートします。 4℃で5分間、300×gで遠心分離し、上清を吸引除去します。 PBS / EDTAバッファ/ 0.5％AB血清の適切な量で細胞を再懸濁します。 </li><li> 100 diluti：1 / mlの直前分析（1の最終濃度にヨウ化プロピジウムを追加）100μg/ mlのの上。試料の純度を評価するために、フローサイトメトリー分析を実行します。 </li><li> CCS濃縮処理後の陽性画分に示されているCD3 + T細胞を決定するための実行可能な白血球ゲーティング戦略のCD3 + T細胞を計算するには、次のゲーティング戦略を使用してください。次のように示した領域（ 図3Aおよび3B、1-6）は、階層的にリンクされます。 1：タイムゲート→2：→3シングル細胞：CD45 +細胞 →4：細胞（残骸を除く）→5：実行可能な白血球→6：生存CD3 +細胞集団 </li><li> CD4 +、CD8 +、CD4 + IFN-γ+およびCD8 + IFN-γ+ CCS濃縮処理後のT細胞（ 表2）の頻度を決定します。 </li><li> Fの下に示すCD4 +、CD8 +、CD4 + IFN-γ+およびCD8 + IFN-γ+ T細胞の頻度を決定するためのゲーティング戦略を使用してまたはCCS処理後の元と濃縮された（取り込まれ）陽性画分。示された領域は、階層的にリンクされ、次のように名前が付けられています。 1：タイムゲート→2：→3シングル細胞：CD45 +細胞 →4：細胞（残骸を除く）→5：実行可能な白血球→6：生存CD3 +細胞→7：CD4 +細胞→7A：CD4 + IFN-γ+細胞（ボックス）→8：CD8 +細胞→8A：CD8 + IFN-γ+細胞 （ボックス） </li></ol>注：階層リンクの最初の6示した領域は、（1-6）を、図3と同様であり、最後の2つの領域は 、図4（6-8a）に示されています。

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MDアンダーソンがんセンターと博士はクーパーの両方がZIOPHARMオンコロジー社、およびIntrexon社の金融関心を持っています。 2015年5月7日に、博士はクーパーはZIOPHARM腫瘍学で最高経営責任者（CEO）に任命されました。博士はクーパーは現在、MDアンダーソンでの客員研究員です。博士はクーパーは彼が人工ヌクレアーゼでサンガモバイオサイエンスと特許を持つInCellerate社を設立し、所有しています。彼はTargazyme株式会社（旧アメリカン幹細胞、株式会社）、GEヘルスケア、フェリング医薬品、運命のTherapeutics、ヤンセンファーマ、ブリストル・マイヤーズスクイブ社と協議。彼はセレクティスの科学諮問委員会です。彼は、ミルテニーバイオテク社から謝礼金を受け取ります。

養子T細胞療法は、B細胞悪性腫瘍4を処置するための実行可能な選択肢として浮上しています。その治療可能性は、複製の老化2を欠く標的抗原特異的T細胞の所望の数の注入に依存します。これは、現在の適正製造基準に準拠して増殖したT細胞の抗原特異的T細胞の純粋な集団を選別することによって達成することができます。二つのソート手順が広く、すなわち、蛍光活性化?...

造血幹細胞移植（HSCT）1は、移植片対腫瘍効果を改善し、日和見感染2に対する免疫を提供するために、養子T細胞療法と組み合わせることができます。注入のための抗原特異的なドナー由来T細胞の生成は、歴史的に熟練者とGMP準拠している専門の施設の使用を必要としてきました。このようなT細胞の送達は、日和見感染症3の解像度と同様に、基礎となる悪性腫瘍4の治療をもたらしました。最近、研究者は実証されているそのわずか数千のウイルス特異的T細胞の養子移入（〜1×10 4から2.5×10 5細胞 / kgを、レシピエントの体重）が正常に同種HSCT 5-9後の日和見のCMV感染症を治療することができます。関連した製造業者の要件にGMP施設の限られた数および細胞の生産に関連する高いコストは、しかしながら、RESTRを有しT細胞療法10を約束に対する患者のアクセスをicted。抗原特異的T細胞を単離するための一つのアプローチは、CD45およびIFN-γを認識する二重特異性試薬を用いてCCSに基づいています。示されているように、この方法は、自動化された細胞濃縮CCS装置（ 図1B）を用いた臨床グレードのCMV特異的T細胞を生成するために使用することができます。 CMV特異的T細胞は、CMV血清反応陽性ドナーからの白血球搬出総核細胞（TNC）でのCMVのpp65抗原のオーバーラップペプチドをインキュベートすることにより生成されます。ヒト白血球抗原（HLA）との関連で表示されるこれらのペプチドは、IFN-γを分泌するTNC内のCMVのpp65特異的T細胞を活性化します。これらのT細胞は、その後、「捕捉される」ことと、磁気的に分離することができます。第一世代の細胞濃縮装置の動作（ 図1A）は、複数のを行うことGMP条件下で細胞培養での人材を必要とし、スタッフのコーディネート「捕捉された」製品を生成するために必要なTEPS。 手順は、通常、連続運転の10〜12時間を必要とするため、担当者はおそらく、GMP施設で2シフト上で動作する必要があります。これらの制約は、現在（ 図1Bに示される）第二世代のデバイスの実装によって回避されます。このデバイスは、最初の生成装置と同等の磁気濃縮を、引き受けるが、unbreachedアプローチで、CCSの他の側面を自動化します。ステップのほとんどは、スタッフが無人達成することができるので、これはかなりのGMPチームの負担を軽減します。デバイスは、閉鎖系として動作するので、さらに、抗原特異的T細胞が捕捉され、機器を起動する前に、白血球搬出単離および材料の製造に含まれる工程を除いて、ベンチトップ上で処理することができます。この第二世代の細胞濃縮装置の完全な計測および機能の詳細については、パブされています11 lished。 ここでは、自動化された細胞濃縮CCSシステムを使用して、定常状態のアフェレーシス産物からのCMVのpp65特異的T細胞を濃縮するための手順について説明します。一旦単離されると、これらのCMV特異的T細胞は、直ちに患者に注入することができます。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>CliniMACS PBS/EDTA Buffer 3 L bag</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>700-29</td>
			<td></td>
		</tr>
		<tr>
			<td>CliniMACS Prodigy Tubing Set TS 500</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-097-182</td>
			<td></td>
		</tr>
		<tr>
			<td>5 L waste bag</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>110-004-067</td>
			<td></td>
		</tr>
		<tr>
			<td>CliniMACS Cytokine Capture System (IFN-gamma)</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>279-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin (Human) 25%&#160;</td>
			<td>Grifols</td>
			<td>58516-5216-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer/Spike Interconnector</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-018-701</td>
			<td></td>
		</tr>
		<tr>
			<td>0.9 % NaCl Solution (1 L)</td>
			<td>Miltenyi Biotec GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MACS GMP PepTivator HCMV pp65</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>170-076-109</td>
			<td></td>
		</tr>
		<tr>
			<td>Water for injections</td>
			<td>Hospira, inc, Lake Forest, IL</td>
			<td>NDC-0409-4887-10</td>
			<td></td>
		</tr>
		<tr>
			<td>MILLEX GV Filter Unit 0.22 &#956;m&#160;</td>
			<td>Millipore</td>
			<td>SLGV033RB</td>
			<td></td>
		</tr>
		<tr>
			<td>TexMACS GMP Medium 2 L bag</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>170-076-306</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer Bag, 150 mL (for cellular starting material)</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-018-301</td>
			<td></td>
		</tr>
		<tr>
			<td>CryoMACS Freezing Bag 50</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>200-074-400</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mL Syringes, sterile</td>
			<td>BD, Laagstraat, Temse, Belgium</td>
			<td>309653</td>
			<td></td>
		</tr>
		<tr>
			<td>CMV sero positive apheresis product</td>
			<td>Key Biologics, LLC, Memphis</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Cytometry Materials</td>
			<td>Manufacturer</td>
			<td>Catalog number</td>
			<td></td>
		</tr>
		<tr>
			<td>AB Serum, GemCell</td>
			<td>Gemini Bio-Products, West Sacramento, USA</td>
			<td>100-512</td>
			<td></td>
		</tr>
		<tr>
			<td>CD3-FITC</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-080-401</td>
			<td></td>
		</tr>
		<tr>
			<td>CD4-APC</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-098-033</td>
			<td></td>
		</tr>
		<tr>
			<td>CD8-APC-Vio770</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-098-065</td>
			<td></td>
		</tr>
		<tr>
			<td>CD14-PerCP</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-098-072</td>
			<td></td>
		</tr>
		<tr>
			<td>CD20-PerCP</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-098-077</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45-VioBlue</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-098-136</td>
			<td></td>
		</tr>
		<tr>
			<td>aIFN-&#947;-PE, human</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-097-940</td>
			<td></td>
		</tr>
		<tr>
			<td>CD3-PE</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-091-374</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium Iodide Solution (100 &#181;g/mL)</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-093-233</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td>Manufacturer</td>
			<td>Catalog Number</td>
			<td></td>
		</tr>
		<tr>
			<td>CliniMACS Prodigy Device&#160;</td>
			<td rowspan="2">Miltenyi Biotec GmbH</td>
			<td rowspan="2">200-075-301</td>
			<td></td>
		</tr>
		<tr>
			<td>Software V1.0.0.RC</td>
			<td></td>
		</tr>
		<tr>
			<td>MACSQuant Analyzer 10</td>
			<td rowspan="2">Miltenyi Biotec GmbH</td>
			<td rowspan="2">130-096-343</td>
			<td></td>
		</tr>
		<tr>
			<td>Software 2.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5415R&#160;</td>
			<td>Eppendorf AG</td>
			<td>22331</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellometer K2</td>
			<td>Nexelom Bioscience, Lawrence, MA</td>
			<td>LB-001-0016</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile tubing welder SCDIIB</td>
			<td>Terumo Medical Corp., Elkton, MA</td>
			<td>7811</td>
			<td></td>
		</tr>
	</tbody>
</table>

automated cell enrichment of cytomegalovirus-specific t cells for clinical applications using the cytokine-capture system

病原体特異的T細胞の養子移入は、同種造血幹細胞移植後に発生するサイトメガロウイルス（CMV）感染症などの日和見感染症を予防および治療するために使用することができます。第三者ドナーを含む同種異系のドナーからのウイルス特異的T細胞は、選択的に、所望のT細胞を増殖するための抗原駆動型の刺激の繰り返しのラウンドを用いて、現在の適正製造基準（cGMPの）に準拠してエクスビボで増殖させることができます。抗原特異的T細胞の同定および単離はまた、γ-インターフェロン（IFN-γ）を分泌するように活性化されたT細胞のサイトカイン捕捉システムに基づいて行うことができます。製造プロセスは、時間がかかり、熟練したオペレータを必要とするが、免疫を回復するのに役立つサイトカイン捕獲システム（CCS）の広範なヒトの適用が制限されています。現在、CliniMACSプロディジーなどの第二世代の細胞濃縮装置の開発自動化され、より少ない労働集約システムを使用してウイルス特異的T細胞を生成するための研究を可能にします。このデバイスは、磁気的、臨床グレードの製品を生成するために、磁気活性化細胞選別技術を用いて、非標識細胞から細胞を分離する標識された、閉じたシステムとして設計され、ベンチトップ上でアクセスして操作することができます。我々は、CMV血清反応陽性ドナーから得られた定常状態のアフェレーシス産物から得られたCMVのpp65特異的T細胞を作製するために、この新しい自動化された細胞濃縮装置の動作を示します。これらの単離されたT細胞は、直接機関と連邦規制監督下で患者に注入することができます。赤血球、T細胞の刺激、抗原特異的T細胞の分離、精製、及び洗浄の除去を含むすべての生物処理工程を完全に自動化されています。このようなデバイスは、ヒト適用のためのT細胞は、専用の適正製造基準（GMPの外で製造することができるという可能性を提起）代わりに、設備やスタッフは、複数の製品を生産するために自動化されたプロトコルを監視することができ、血液銀行施設で製造すること。

本研究では、自動化された細胞の濃縮CCSシステムは、CMVのpp65特異的T細胞の自動化生産のために使用しました。 CMV特異的T細胞は、3つの細胞アフェレーシス産物から濃縮しました。定常状態のアフェレーシス産物は、CMV血清陽性ドナーから2時間かけて回収し、10 10合計核細胞（TNC）を生成しました。 10 9 TNCは、その後4時間のCMVのpp65由来ペプチド（60ナノモル）で活性化し、IFN...

Watch this Scientific Journal Video about Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System at JoVE.com

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

The goal of this protocol is to manufacture pathogen-specific clinical-grade T cells using a bench-top, automated, second generation cell enrichment device that incorporates a closed cytokine capture system and does not require dedicated staff or use of a GMP facility. The cytomegalovirus pp65-specific-T cells generated can be directly administered to patients.

サイトカインキャプチャシステムを用いた臨床応用のためのサイトメガロウイルス特異的T細胞の自動化された細胞濃縮

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection ...

automated-cell-enrichment-cytomegalovirus-specific-t-cells-for

Research

JoVE Journal

Immunology and Infection

12.3K ビュー.  U.T. MD Anderson Cancer Center. The goal of this protocol is to manufacture pathogen-specific clinical-grade T cells using a bench-top, automated, second generation cell enrichment device that incorporates a closed cytokine capture system and does not require dedicated staff or use of a GMP facility. The cytomegalovirus pp65-specific-T cells generated can be directly administered to patients.

ビデオ: Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

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

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

細胞運命を以下に<em&gt; E大腸菌の</emラムダファージによる感染後&gt;

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

プライマリーヒトT細胞のpiggyBacトランスポゾンシステムの変更

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

A basic necessity for researchers studying adaptive immunity with in vivo experimental models is an ability to identify T cells based on their T cell antigen receptor (TCR) specificity. Many indirect methods are available in which a bulk population of T cells is stimulated in vitro with a specific antigen and epitope-specific T cells are identified through the measurement of a functional response such as proliferation, cytokine production, or expression of activation markers1. However, these methods only identify epitope-specific T cells exhibiting one of many possible functions, and they are not sensitive enough to detect epitope-specific T cells at naive precursor frequencies. A popular alternative is the TCR transgenic adoptive transfer model, in which monoclonal T cells from a TCR transgenic mouse are seeded into histocompatible hosts to create a large precursor population of epitope-specific T cells that can be easily tracked with the use of a congenic marker antibody2,3. While powerful, this method suffers from experimental artifacts associated with the unphysiological frequency of T cells with specificity for a single epitope4,5. Moreover, this system cannot be used to investigate the functional heterogeneity of epitope-specific T cell clones within a polyclonal population.
	The ideal way to study adaptive immunity should involve the direct detection of epitope-specific T cells from the endogenous T cell repertoire using a method that distinguishes TCR specificity solely by its binding to cognate peptide:MHC (pMHC) complexes. The use of pMHC tetramers and flow cytometry accomplishes this6, but is limited to the detection of high frequency populations of epitope-specific T cells only found following antigen-induced clonal expansion. In this protocol, we describe a method that coordinates the use of pMHC tetramers and magnetic cell enrichment technology to enable detection of extremely low frequency epitope-specific T cells from mouse lymphoid tissues3,7. With this technique, one can comprehensively track entire epitope-specific populations of endogenous T cells in mice at all stages of the immune response.

This protocol describes the use of peptide:MHC tetramers and magnetic microbeads to isolate low frequency populations of epitope-specific T cells and analyze them by flow cytometry. This method enables the direct study of endogenous T cell populations of interest from in vivo experimental systems.

ペプチドエピトープ特異的T細胞のMHCテトラマーベースの濃縮

A  basic necessity for researchers studying adaptive immunity with in vivo experimental models is an  ability to identify T cells based on their T cell ...

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

T細胞の定量的なハイスループットシングルセル細胞毒性アッセイ

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

の臨床応用<em&gt;眠れる森の美女</em遺伝的に末梢及び臍帯血由来のT細胞を変更する&gt;と人工抗原提示細胞

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

使用して樹状細胞の移行の監視<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H磁気共鳴イメージング

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

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 ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&amp;#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

マウスナイーブCD4<sup&gt; +</sup&gt; T細胞の単離と<em&gt;インビトロ</em&gt; T細胞サブセットへの分化

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.