この作品は、細胞治療（契約NIH - NHLBI（HB - 10 - 03）HHSN26820100000C）（CMR）、細胞ベースの治療のための専門センターの生産支援によってサポートされている補助金NIH - NHLBI 1 U54 HL081007（CMR）、ASBMT若手研究者賞（UGおよびJV）、臨床研究賞（UG）の白血病とリンパ腫協会特別研究員、およびエイミーStrelzer Manasevit Scholarの賞（AML）。

1。直流nucleofection <ol><li>セルGenix IL4（1000U/ml）を添加したメディア、GMCSF（800IU/ml）、さらに、DCの成熟のサイトカインIL4を（1000Uを使用して24時間熟成を使用して5日間培養プラスチック接着を、使用して濃縮されている収穫単球由来樹状細胞、 / ml）を、GMCSF（800IU/ml）、IL6100ng/ml、TNF -α10ng/ml、IL1 -β10ng/mlとPGE2（1μg/mlの）1、3ミリリットルの転送ピペットで穏やかに再懸濁による。 </li><li>トリパンブルー、ないより少ない6 0.5 × 10以上と3倍の15ミリリットルチューブに転送し、これ以上の6 × 10以上の細胞/チューブを使用して実行可能な樹状細胞を数える。 </li><li> 10分@ 200グラムのための樹状細胞を遠心する。 37℃/ 5％CO 2インキュベーターでプレートを扱う12ウェル組織培養の3つのウエルに2ml/well -この時間の間に事前に暖かいセルGenixメディアは、DCの成熟のサイトカイン（DC成熟のメディア）を補充した。 </li><li>一度細胞が回転し終わったら、上清を吸引し、5μgのDNA /チューブの最終濃度で管のそれぞれに関連するDNAプラスミドを追加します。この場合、チューブ＃3をコードするプラスミドチューブ＃1、ヘキソン-ペントン管に＃2〜IE1 - pp65を、およびEBNA1 - LMP2 - BZLF1を追加します。 </li><li> Amaxaのnucloefection液の100μLと再懸樹状細胞とDNAには、よく混ぜるとnucleofectionのキュベットに移す。 </li><li> 4D nucleofectorの場所のキュベットは、プログラムCB150（Amaxa /ロンザ）を選択し、スタートを押してください。 </li><li> nucleofectionがキュベットに2ミリリットル予め温めておいたセルGenix DCの成熟のメディアの500μLを追加した直後は、上下に2〜3回ピペッティングにより穏やかに混合し、あらかじめ温めておいたの残りの1.5mlを含む調製12ウェルプレートにnucleofected DCを転送するDCの成熟のメディア。 37譲渡℃/ 5％CO 2インキュベーターをさらに12 - 18hrsのために。 </li></ol> 2。 T細胞刺激<ol><li>収穫と30Gyでnucleofected DCをカウントし、照射。 CTLの培地10mlの（45％RPMI、45％のクリック数EHAA、10％FBS、2mMのグルタミン）とCTLメディアのmlあたり再懸@ 3 × 10 5のDCで1回洗浄します。 </li><li>プール各プラスミドを含む樹状細胞の7.5x10 5の最小値（2.5ミリリットル）と15x10 5の最大値（5ml）をして、インキュベータ内に配置されるG -レックスのデバイスにプールされたDCを転送する。 </li><li>応答細胞の調製については以前に​​凍結末梢血またはDCの選択（遵守またはCD14選択）した後に残っている非接着単核細胞のいずれかを使用してください。細胞を解凍、温めておいた培地に移す、CTL媒体で1回洗浄します。 CTLメディアで細胞を再懸濁し、細胞をカウントし、mlあたり2 × 10 6細胞の濃度にそれらをもたらす。 30000U IL4と15x10 6個の細胞または7.5ミリリットルとサプリメントを（。1000U/ml -最終濃度）に乗り、300ng IL7（10ng/ml -最終濃度。）。 </li><li> G -レックスと30ミリリットルの合計体積に対するCTLメディアとバイオリアクターまでトップにPBMCの7.5ミリリットルを（15x10 6個の細胞）に転送します。 </li><li>文化37の6-7日のためのG -レックス℃/ 5％CO 2の加湿インキュベーター。 </li></ol> 3。 T細胞の拡大<ol><li>日6-7、メディアの吸引10ミリリットルで、その後10ミリリットルピペットでメディアの残りの20ミリリットルの細胞を混合し、トリパンブルーを用いて生細胞を数える。ある場合は&lt;50x10 6は新鮮な培地+サイトカインを補給。 &gt; 50x10 6個の細胞が存在する場合、細胞懸濁液10mlのを削除し、新しいG - Rexに転送し、新鮮なCTLメディア+サイトカインとの両方G -レックスズを養う。 </li><li>追加の4-6日の培養。一度十分な細胞は、将来使用するためにCTLと低温保存する過剰の表現型および機能解析を行う、拡張されています。 </li></ol> 4。代表的な結果： 私たちのFDA承認multivirus特異的CTL生成プロセスの概略図を図1に示されています。ウイルス反応性T細胞を刺激するadenovectorsとEBV - LCLを使用する慣習multivirus CTLのプロトコルには対照2では我々はDNAプラスミドを感染性ウイルスの材料を交換したそのウイルス3のそれぞれから派生する複数の抗原をコード化する。 trivirusのCTL​​を刺激するために我々は3つのmulticistronicプラスミドエンコーディングヘキソンとペントンアデノウイルスの、CMVのIE1およびpp65を、およびEBNA1、LMP2、およびEBVのBZLF1を設計した。これらの抗原は、私たち自身の細胞やT細胞は、ADV -ヘキソンとペントン2,4-6に対して向けられることを示す他のグループの臨床結果を奨励し、CMV - 1E1、およびCMV - pp65をに基づいて選択された生体 7 の保護です。 EBVの場合、EBNA1は免疫CD4 +すべてのEBV関連悪性腫瘍で、通常のEBVに感染したB細胞で発現するT細胞の標的抗原である8,9、LMP2は、複数のHLAタイプにわたり、免疫原性であり、ほとんどのEBV悪性腫瘍に発現し、10,11 BZLF1中ほとんどの個体からのCD4 +およびCD8 + T細胞を刺激し、共同のために可能性が重要である免疫、即時初期の溶菌サイクルの抗原をコードしているウィルス12複製細胞のntrol。さらに私たちの製造方法を最適化するために我々は、CTL刺激13,14用minimalized、抗生物質フリー（FDA準拠）プラスミドを生成したネイチャーテクノロジーとのコラボレーション。としてIFNγのELISPOTにより測定された最適化されたDNAプラスミドに応答して高い細胞生存率（データは示していない）3。 図2に示すように、ウイルス特異的T細胞のその周波数を維持しながら、我々は一貫して&gt; 35％のnucleofection効率を達成するため、この戦略を使用して、大きい同じ抗原（N = 8アデノウイルスは、n = 4 CMV、およびn = 2 EBV）を発現する従来のpShuttleベースの発現プラスミドへの応答に比べて。 Rの比（n = 2のドナー）：DCの最適な比率：1:50の比は1：20 Sに比べて準最適な活性化を生成した図3に示すように、PBMCは、強力なT細胞刺激のために重要であった。広範な抗原特異性を有する十分なCTLの数値の生産は、すべての3つのウイルスに対する臨床的有効性の前提条件です。これは、 図4Bに示すように、文化へのIL4とIL7の加算がレパートリーと特異性を増加させる一方、従来の24ウェルプレート（ 図4A）15 日と比較して優れたT細胞の拡張をサポートするG -レックス、でCTL培養することによって達成されるCMV - pp65を由来HLA - A2に対する反応性T細胞の頻度がrestictedどこNLVペプチドは、IL4および/ ​​またはIL7 16,17の存在下または非存在下で生成された文化で評価した。我々はフローサイトメトリー解析、細胞内サイトカイン染色/IFNγELISPOT、および凍結保存/注入のための最終的な製品でCR 51放出アッセイを実行する拡張された細胞の表現型および機能的能力を評価する。一般的に生成された細胞はCD4 +および両方のT細胞区画において検出可能な抗原特異性CD8 + T細胞の混合集団とポリクローナルです。 CTLは、 生体内 （ 図5） に移植片対宿主病（GVHD）を誘導してはならないことを示す、ウイルス部分的にHLA一致の目標負ではないウイルス抗原を発現する標的細胞を殺すができるようになりました。 <img src="/files/ftp_upload/2736/2736fig1.jpg" alt="図1"/> 図1。rctlの生成プロトコル。最初に、樹状細胞は、ウイルス抗原をコードするプラスミドでnucleofectedし、Rにおける自家末梢血単核球と混合される：10または20:1のS。細胞は、その後、収穫、IL4とIL7の存在下で10〜14日間G -レックスに拡大して数え、機能、アイデンティティーと不妊の検査、およびその後の臨床使用のために凍結保存されています。 <img src="/files/ftp_upload/2736/2736fig2.jpg" alt="図2"/> 図2。最適化されたDNAプラスミドは 、in vitro での優れたT細胞の活性化を誘発する。 DCは、最適化、FDAに準拠したプラスミド符号化ヘキソンとペントン（前売）、IE1およびpp65を（CMV）、およびEBNA1、LMP2、そしてBZLF1（EBV）または同じ抗原をコードする従来のpShuttleプラスミドでnucleofectedていた。これらはT細胞を刺激するために使用され、特異性は、IFNγのELISPOT 10日のポスト刺激により分析した。 <img src="/files/ftp_upload/2736/2736fig3.jpg" alt="図3"/> 図3最適なDC：。CTL活性化のためのT細胞の比率。 2ドナーからの樹状細胞は、すべての3つの最適化されたプラスミドをnucleofectedしてから、1：20または1:50 DCで自家末梢血単核球を刺激するために使用されていました：PBMCの比率。再活性化T細胞の頻度は、IFNγのELISPOTにより、10日目に評価した。 <img src="/files/ftp_upload/2736/2736fig4.jpg" alt="図4"/> 図4。高めるサイトカインを用いたG -レックスのT細胞の拡大。パネルは、G -レックスのデバイスと同様に顕微鏡で評価したガス透過性膜、上のCTL外観を示しています。プレートVS G -レックス扱わ大会の組織培養で得セルの出力との間の比較も示されています。パネルBは、無サイトカインの存在下で拡大培養において達成CMV量体正のCTL、単独でIL4、IL7アローンおよびIL4 + IL7の周波数を示しています。 <img src="/files/ftp_upload/2736/2736fig5.jpg" alt="図5"/> 図5。拡張されたCTLの表現型と機能。とCD8 +（42％ -細胞毒性） - パネルには、CD4 +（ヘルパー45％）の混合物とのポリクローナルな拡張multivirus CTLの表現型の代表的な例を示してT大多数（95％）が発現し、そのうちの細胞、メモリマーカーCD45RO + / CD62L +。 パネルBは、これらの細胞はすべての刺激抗原に特異的であり、IFNγの産生と抗原刺激後にTNFαを検出するために細胞内サイトカイン染色によって評価される多官能性であることを示しています。 パネルCは、拡張されたCTLは、測定されたとして機能していることを示していますCR 51アッセイによる。自家単独で、またはヌルベクトルまたはCMV - pp65をを発現するアデノウィルスベクターで形質LCLは、タールとして使用された取得。同種反応性は、ターゲットとして同種のPHA芽球を用いて評価した。

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+restoration+of+immunity+against+Epstein-Barr+virus+infection+by+adoptive+transfer+of+gene-modified+virus-specific+T+lymphocytes.">Long-term restoration of immunity against Epstein-Barr virus infection by adoptive transfer of gene-modified virus-specific T lymphocytes.</a> Nature Medicine. 2, 551-555 (1996).</li><li>Rooney, C. M., Smith, C. A., Ng, C., Loftin, S. K., Li, C., Krance, R. A., Brenner, M. K., Heslop, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+gene-modified+virus-specific+T+lymphocytes+to+control+Epstein-Barr+virus-related+lymphoproliferation.">Use of gene-modified virus-specific T lymphocytes to control Epstein-Barr virus-related lymphoproliferation.</a> Lancet. 345, 9-13 (1995).</li><li>Einsele, H., Roosnek, E., Rufer, N., Sinzger, C., Riegler, S., Loffler, J., Grigoleit, U., Moris, A., Rammensee, H. G., Kanz, L., Kleihauer, A., Frank, F., Jahn, G., Hebart, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infusion+of+cytomegalovirus+(CMV)-specific+T+cells+for+the+treatment+of+CMV+infection+not+responding+to+antiviral+chemotherapy.">Infusion of cytomegalovirus (CMV)-specific T cells for the treatment of CMV infection not responding to antiviral chemotherapy.</a> Blood. 99, 3916-3922 (2002).</li><li>Walter, E. A., Greenberg, P. D., Gilbert, M. J., Finch, R. J., Watanabe, K. S., Thomas, E. D., Riddell, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+cellular+immunity+against+cytomegalovirus+in+recipients+of+allogeneic+bone+marrow+by+transfer+of+T-cell+clones+from+the+donor.">Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T-cell clones from the donor.</a> N. Engl. J. Med. 333, 1038-1044 (1995).</li></ol>

フアンF.ベラは、ウィルソンウルフの製造のためのコンサルタントです。他の著者は、関連する開示はない。

ウイルス感染症は、自分の病気やその治療によって免疫不全患者ではかなりの罹患率と死亡率を占めている。 HSCT後は、例えば、EBVやCMV、だけでなく、呼吸器合胞体ウイルス（RSV）などの呼吸器系ウイルスなどによって永続的なヘルペスウイルスによって引き起こされる感染症は、よく、知られているが、前売、BKウイルスによって引き起こされる感染症の重要性、そして人間のヘルペスウイルス（HHV）-6は、最近では評価されている。薬理学的薬剤はいくつかの感染症の標準治療であるが、彼らは実質的な毒性を持って、耐性変異株を生成し、そして頻繁に無効である。対照的に、幹細胞のドナー由来のウイルス特異的T細胞は、造血幹細胞移植（HSCT）の設定2,5,6,18-21のウイルス感染や病気の予防と治療のために安全かつ効果的であることが証明されている。しかし、T細胞免疫療法の広範な実装は、最終的にCTL産生に必要なコスト、複雑さと時間によって制限されます。 現在の原稿に記載されてmultivirus CTLを生成するために私たちの斬新かつ迅速なアプローチは、実質的に免疫不全宿主の標準治療になるための戦略を可能にする、ウイルス性疾患に対する細胞傷害性T細胞治療の実現可能性を増やす必要があります。 APCのようなプラスミドnucleofected DCの使用は、異なるDC集団はそれぞれのプラスミド3用に利用されているため、単一細胞内で発現している複数のウイルス抗原から実際にウイルスベクターやから競争することなくMHCクラスIおよびIIの両方に抗原提示を可能にします。 IL - 4月7日の使用も拡大しそれに応じて抗原特異的T細胞16,17の応答の頻度とレパートリーを増やすに役立つT細胞の生存や増殖、。最後に、G -レックスの文化は劇的に培養中のT細胞のアポトーシスを減らすことができます。ガス交換（のO 2とCO 2のうちは）より多くの栄養素を供給し、廃棄物を希釈し、細胞を上記培地の深みを許可しながら、低酸素症を防止する、フラスコの底にガス透過性のシリコン膜を横切って発生します。このプラットフォームはまた、防御抗原が同定されている場合など、追加のウイルスに拡張することができます。

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th></tr><tr><td> CellGenix </td><td> CellGenix </td><td> 2005 </td></tr><tr><td> IL4 </td><td> R + Dシステムズ</td><td> 204-IL/CF </td></tr><tr><td> IL7 </td><td>ペプロテック</td><td> 200から15 </td></tr><tr><td> Hyclone RPMI 1640 </td><td>サーモサイエンティフィック</td><td> SH30096.01 </td></tr><tr><td>ヒトAB血清</td><td>バレーバイオメディカル（株） </td><td> HP1022 </td></tr><tr><td> Nucleofector </td><td> Amaxa /ロンザ</td><td> AAF - 1001B＆AAF - 1001X </td></tr><tr><td> Nucleofectionキット</td><td> Amaxa /ロンザ</td><td> V4XP - 3012 </td></tr><tr><td>プラスミド</td><td> NTC </td><td> N / A </td></tr><tr><td> GM - CSF </td><td> R＆D </td><td> 215-GM/CF </td></tr><tr><td> IL1 </td><td> R＆D </td><td> 201 - LB - 025 </td></tr><tr><td> IL6 </td><td> R＆D </td><td> 206 - IL - CF </td></tr><tr><td> TNFα </td><td> R＆D </td><td> 210 - TA - 010 </td></tr><tr><td> PGE2 </td><td>シグマ</td><td> P6532 - 1mgの</td></tr><tr><td> G -レックス</td><td>ウィルソンウルフ製造</td><td> AY11 - 00027 </td></tr></tbody></table>

generation of multivirus-specific t cells to prevent/treat viral infections after allogeneic hematopoietic stem cell transplant

ウイルス感染症は同種造血幹細胞移植（HSCT）受信者の罹患率と死亡率を引き起こす。我々と他の人が正常に生成し、エプスタインバーウイルス（EBV）、サイトメガロウイルス（CMV）と単球とを用いてアデノウイルス（ADV）のためのT -細胞特異的に注入しているEBV形質転換リンパ芽球様細胞を（EBV - LCL）アデノウイルスベクターでの遺伝子的に修飾された抗原提示細胞（APC）。として注入した後、いくつかのログで増殖した2 × 10 5 / kgのtrivirus特異的細胞傷害性Tリンパ球（CTL）などのいくつかと他の利用可能な治療法に耐性をさらに深刻なウイルス性疾患の予防と治療に見えた。準備のための - この励みアプローチの広範な実装は、高い生産コスト、製造の複雑さと長時間（合計10〜14週CTLを製造するためのEBV - LCLの生成、および4-8週間4-6週間）によって制限されます。これらの制限を克服するために我々は新しい、GMP準拠のCTL生産のプロトコルを開発しました。最初に、我々は抗原提示のようなDNAプラスミドエンコーディングLMP2、EBNA1とBZLF1（EBV）、ヘキソンとペントン（前売）、およびpp65をとIE1（CMV）とnucleofected樹状細胞（DC）を使用するT細胞を刺激するadenovectorsの代わりに細胞。これらのAPCは、すべての刺激的な抗原に特異的なT細胞を再アクティブ化します。第二に、文化はIL - 4の存在下でT細胞を活性化（1,000 U / ml）とIL - 7（10ng/ml）が増加し、私たちの行で特定のT細胞のレパートリーと周波数を維持します。第三に、我々はこのようにEBV - LCLSのための要件を削除し、技術者の介入を減らし、単一の刺激の後に大規模な細胞数の拡大と生存を促進する新しい、ガス透過性培養装置を（G -レックス）を使用しています。これらの変更を実装することによって我々は今&gt; 90％削減され10 6細胞あたりのコストで、EBV、CMV、および前売をターゲットと多重CTLを生成し、可能な追加するために拡張できるアプローチを使用してわずか10日ではなく10週間で防御ウイルス抗原。私たちのFDA承認のアプローチは、リスクの高い同種HSCTレシピエントの予防と治療のアプリケーションのための値である必要があります。

Watch this Scientific Journal Video about Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant at JoVE.com

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

の世代のための迅速、簡単かつコスト効率の良いプロトコルドナー由来のCMV、前売またはEBV感染症を発症する危険性がある同種造血幹細胞移植（HSCT）の受信者に注入するためmultivirus特異的CTL（rctlを）。この製造プロセスはGMPに準拠しており、専門センターを超えてT細胞免疫療法の広範な実施を確保すべきである。

同種造血幹細胞移植後のウイルス感染を予防/治療するMultivirus -特異的T細胞の生成

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

generation-multivirus-specific-t-cells-to-preventtreat-viral

Research

JoVE Journal

Immunology and Infection

17.1K ビュー.  Baylor College of Medicine. の世代のための迅速、簡単かつコスト効率の良いプロトコルドナー由来のCMV、前売またはEBV感染症を発症する危険性がある同種造血幹細胞移植（HSCT）の受信者に注入するためmultivirus特異的CTL（rctlを）。この製造プロセスはGMPに準拠しており、専門センターを超えてT細胞免疫療法の広範な実施を確保すべきである。

ビデオ: Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

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 ...

免疫・感染学

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

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&amp;#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

初代ヒトナイーブおよびメモリーT細胞から誘導した調節性T細胞の生成

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

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 ...

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

精製造血幹細胞/前駆細胞のDNAの変異を特定する

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

マウスにおける同種のCD4 T細胞応答を研究する尾皮膚の移植

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

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

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-&#947;). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.

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 ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

発生とのマルチ表現型ハイコンテンツスクリーニング<em&gt; Q熱リケッチア</em&gt;トランスポゾン変異体

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

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.

自己免疫に対する幹細胞由来の抗原特異的制御性T細胞の開発

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal of immunosuppression. The mixed leukocyte reaction (MLR), limiting dilution assays, and trans-vivo delayed-type hypersensitivity (DTH) assay have all been applied to this question, but these methods have limited predictive ability and/or significant practical limitations that reduce their usefulness. Imaging flow cytometry is a technique that combines the multiparametric quantitative powers of flow cytometry with the imaging capabilities of fluorescent microscopy. We recently made use of an imaging flow cytometry approach to define the proportion of recipient T cells capable of forming mature immune synapses with donor antigen-presenting cells (APCs). Using a well-characterized mouse heart transplant model, we have shown that the frequency of in vitro immune synapses among T-APC membrane contact events strongly predicted allograft outcome in rejection, tolerance, and a situation where transplant survival depends on induced regulatory T cells. The frequency of T-APC contacts increased with T cells from mice during acute rejection and decreased with T cells from mice rendered unresponsive to alloantigen. The addition of regulatory T cells to the in vitro system reduced prolonged T-APC contacts. Critically, this effect was also seen with human polyclonally expanded, naturally occurring regulatory T cells, which are known to control the rejection of human tissues in humanized mouse models. Further development of this approach may allow for a deeper characterization of the alloreactive T-cell compartment in transplant recipients. In the future, further development and evaluation of this method using human cells may form the basis for assays used to select patients for immunosuppression minimization, and it can be used to measure the impact of tolerogenic therapies in the clinic.

This paper describes a method for measuring alloreactivity in a mixed population of T cells using imaging flow cytometry.

イメージングフローサイトメトリーを使用したT細胞同種反応性の測定

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal ...

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

ジカ ウイルス特異 Immunoprivileged 臓器感染 Ifnar1-/-マウスの T 細胞応答の評価

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...