1。 PBMCの分離<ol><li>ヘパリンナトリウムでBDバキュテイナCPTの細胞調製チューブへの血液（7.5〜8.0ミリリットル）を描く。チューブはヘパリンナトリウム抗凝固剤とフィコール- Hypaque密度流体、および2つの液体を分離するポリエステルゲル障壁を、含まれています。 1800 × gで20分間水平ローター（スイングアウトの頭）で、室温（18℃〜25℃）でチューブ/血液サンプルを遠心分離します。ブレーキをオフ遠心切り替えます。 </li><li>遠心後、レイヤーの順序は、（上から下まで見て）次のように行われます。）プラズマ - b）の末梢血単核細胞（PBMC）および血小板 - c）の密度のソリューション - D）ポリエステルゲル - E）、顆粒球 - F）赤血球（ 図1）。 </li><li>細胞層を乱すことなく、相間上記プラズマの5〜10 mmを残して、プラズマ層の​​割合を収集する。プラズマは、文化（セクション2.5）に使用することができます。 </li><li>ピペットと15 mlのコニカルチューブに移すとの相間で濃縮分画（PBMC）を回収する。 </li><li> 5回チューブを反転させ、生理食塩水（PBS）リン酸緩衝し、400 × gで5分間遠心操作して、10mlでPBMCを洗ってください。 </li><li>二回の洗浄ステップを繰り返し、次にPBS 5 mlに細胞ペレットを再懸濁します。細胞数を決定します。通常1.3 × 10 6細胞を全血1 mlのから回収されています。 </li><li>フローサイトメトリー（セクション3）（ 図2）でPBMCにおけるγδT細胞の頻度と表現型を決定します。 </li></ol> 2。 γδT細胞の拡大<ol><li>室温で400 × gで5分間、15 mlコニカルチューブに細胞懸濁液を遠心し、上清を捨てる。 </li><li>それぞれ、1000 IU / mlおよび5μMの最終濃度にヒトIL - 2（IL - 2）とゾレドロネート（ゾメタ）を追加することにより、培養液（CM）を準備する。 ALyS203（細胞科学技術研究所）またはOpTmizer（Invitrogen社）のメディアは、γδT細胞（参照6〜9の詳しい情報）の良好な拡張をサポートしています。ゾメタは、液体形態（4 mg/5-mlバイアル）で提供されています。 5μM溶液を調製し、培養液30mlにゾメタの50μlを加える。 </li><li>再懸 ​​濁し、培養液中の細胞ペレットと1 × 10 6細胞/ mlに調整します。 </li><li> 24ウェルプレートの各ウェルに1 × 10 6細胞を含むCMのピペットで1ミリリットル。大規模な文化のために、細胞はプレートのウェル、皿、またはフラスコの表面領域に応じて2 0.5 × 10 6細胞/ cmで播種することができます。 </li><li>それは文化のボリューム（24ウェルプレートの各ウェルに100μL）の約10％になるように自己血漿（セクション1.3）、プールしたヒトAB血清、またはFCSを追加。加湿37℃、5％CO 2 24〜48時間のためのインキュベーターでプレートを置きます。 </li><li> 0.5〜2 × 10 6細胞/ mlの細胞密度で培養を維持する。ヒトIL - 2のみ（1000 IU / ml）を（ゾメタなし）ごとに2〜3日を含む新鮮な培地を追加し、必要に応じて新しい井戸やフラスコに培養細胞を譲渡する、細胞増殖の程度（ 図3）によると。供給血漿または血清中濃度が1％以上を維持できるように、培地に血清。 </li><li>収穫の日12月14日で細胞や頻度、表現型、およびフローサイトメトリーにより、γδT細胞の機能を決定します（下記参照）。 </li></ol> 3。フローサイトメトリーによる表現型解析<ol><li>蛍光活性化細胞選別（FACS）のチューブに2 × 10 5細胞を含む200μlのサンプルを移す。 </li><li>冷PBS 2 mlを加え、400 x gで5分間遠心します。その後、50μlのFACS緩衝液（PBS + 1％FCS + 0.1％アジ化ナトリウム）でペレットを懸濁します。サンプル（モノクローナル抗体は、表1にリストされている）に、各抗体の5μlを添加する。 </li><li> 20分間暗所で氷上でインキュベートする。 </li><li>各サンプルに2 mlのFACS緩衝液を追加してから、渦。 4℃で400 × gで5分℃でサンプルを遠心上清を注意深くデカントする。 </li><li> 300μlのFACS緩衝液と渦で、細胞を再懸濁します。フローサイトメーター（ 図2）上のサンプルを分析する。 </li></ol> 4。 IFN -γ産生アッセイ10 <ol><li>アッセイの前日、RPMI 1640培地+10％FCS（RPMI - 10）ゾメタ（5μM）（以下、Z - Daudiを指定）と一晩の培養を3から5 × 10 5 Daudi細胞/ mlで刺激細胞を準備する。 </li><li> 2 × 10 6細胞/ mlでRPMI - 10 Z -ダウディ再懸濁しますを収集する。丸底96ウェルプレートの各ウェルにZ -ダウディ（2 × 10 5）100μlを追加します。 </li><li>で20μg/ mlでブレフェルジンを含むRPMI - 10に2 × 10 6細胞/ mlでγδT細胞を準備します。各ウェルを含むZ - Daudi細胞にγδT細胞懸濁液（2 × 10 5）100μlを転送したり、井戸（のみRPMI - 10、またはホルボール12 -ミリステート13 -アセテートの20 ng / mlのとRPMI - 10の100μlを制御する[PMA]イオノマイシンのプラス2μg/ mlの）。 </li><li>数回上下にピペッティングして混ぜる。 37℃、5％CO 2インキュベーターで4時間インキュベートする。 </li><li> 4℃で400 × gで5分間プレートを遠心し、冷PBS 200μlでペレットを懸濁します。 </li><li> FACSチューブへサンプルを移す。冷PBS 4 mlを加え、4℃で400 × gで5分間チューブを遠心℃に</li><li> FITC結合抗TCRVγ9（5μl）をとPE/Cy5-conjugated抗CD3 mAb（2.5μl）を持つFACS緩衝液50μlにペレットを再懸濁します。室温で15分間遮光してインキュベートする。 </li><li> IntraPrepの試薬1の100μlを加え、室温で15分間インキュベートする。室温で400 × gで5分間、各チューブと遠心分離機にPBS 4 mlを加え。 </li><li>吸引により上清を除き、IntraPrep試薬2の100μlを加える。振とうせずに室温で5分間インキュベートする。 </li><li>試験管に、PE結合抗- IFN -γモノクローナル抗体の5μlを添加する。室温で15分間遮光してインキュベートする。 </li><li>室温で400 × gで5分間、各チューブと遠心分離機にPBS 4 mlを加え。吸引により上清を除去し、FACS緩衝液0.5 mlに細胞ペレットを再懸濁します。 </li><li>フローサイトメーターで細胞を分析する。 CD3 +TCRVγ9+細胞のゲートとIFN -γ（ 図4）の発現を調べる。 </li></ol> 5。代表的な結果： 培養開始時にPBMCにおけるγδT細胞の割合を決定することが重要です。 図に示す。 2、PBMC中のCD3の割合+TCRVγ9+γδT細胞は、0日目1.6％であった。中央メモリー表現型-支配的な集団は、CD27 + CD45RA +ナイーブまたはCD27 + CD45RAでした。 γδT細胞が効率的に刺激されたとき、彼らは日3-5（ 図3、B）上にクラスターを形成。クラスターの形成が遅延したときは、次のような他の細胞型、の成長CD4 +またはCD8 +αβT細胞やNK細胞がγδT細胞（ 図3 CとD）の成長を支配することができます。文化の14日後、γδT細胞の頻度は、成功したγδT細胞の培養における培養細胞以上の93.8パーセント（ 図2 E）に増加した。培養γδT細胞アップレギュレートNKG2DとCD69の発現（ 図2 G及びH）。彼らは、CD27を表示- CD45RA -エフェクターメモリー表現型（ 図2 F）。 γδT細胞の機能は、サイトカイン産生および細胞毒性に関して評価した。細胞内IFN -γ染色γδT細胞は、PMA /イオノマイシンの治療またはZ - Daudi細胞に反応してIFN -γを産生することが明らかにそのゾレドロネートの治療後に蓄積されたIPP（ 図4）。これらの結果は、ゾレドロネートが効率的に機能するγδT細胞を刺激し、展開することができますを示している。 <table border="1"><tbody><tr><td> チューブ </td><td> FITC </td><td> PE </td><td> ECD </td><td> PE/Cy5 </td></tr><tr><td> 1 </td><td> CD3 </td><td> CD19 </td><td> CD45 </td><td> CD14 </td></tr><tr><td> 2 </td><td> CD3 </td><td> TCRαβ </td><td> CD4 </td><td> CD8 </td></tr><tr><td> 3 </td><td> CD3 </td><td></td><td></td><td> CD56 </td></tr><tr><td> 4 </td><td> TCRVγ9 </td><td> TCRαβ </td><td> CD45 </td><td> CD3 </td></tr><tr><td> 5 </td><td> TCRVγ9 </td><td> NKG2D </td><td></td><td></td></tr><tr><td> 6 </td><td> TCRVγ9 </td><td> CD69 </td><td></td><td></td></tr><tr><td> 7 </td><td> TCRVγ9 </td><td>マウスIgG1 </td><td></td><td></td></tr><tr><td> 8 </td><td> TCRVγ9 </td><td></td><td> CD45RA </td><td> CD27 </td></tr><tr><td> 9 </td><td> TCRVγ9 </td><td></td><td>マウスIgG1 </td><td>マウスIgG1 </td></tr></tbody></table> 表1。γδT細胞のマルチカラー染色に使用されるモノクローナル抗体。当研究室で行わγδT細胞の表現型解析の例を図に示します。 2。 <img src="/files/ftp_upload/3182/3182fig1.jpg" alt="図1"/> 図1。PBMCの分離。血液（7.5〜8.0ミリリットル）をヘパリンナトリウムでBDバキュテイナCPTの細胞調製チューブに引き込まれて、直接1800 × gで20分間遠心分離する。遠心分離後、得られた層は、boの上から見たttom：）プラズマ - b）のPBMCおよび血小板 - c）の密度のソリューション - D）ポリエステルゲル - E）顆粒球 - f）の赤血球。 <img src="/files/ftp_upload/3182/3182fig2.jpg" alt="図2"/> 図2。γδT細胞の典型的な表面表現型。 PBMCを14日間ゾレドロネートとIL - 2で刺激した。細胞はで染色したFITC標識抗TCRVγ9とγδT細胞（およびE）の拡大を監視するために抗CD3 PE/Cy5-labeled。 γδT細胞はTCRVγ9の発現により同定した、とCD27とCD45RA（BとF）、NKG2D（CとG）、またはCD69（DおよびH）のそれらの発現を調べた。 <img src="/files/ftp_upload/3182/3182fig3.jpg" alt="図3"/> 図3。代表γδT細胞の培養。 PBMCをIL - 2（1000 IU / ml）とゾレドロネート（5μM）で刺激した。代表的なフィールドは、（IX71倒立顕微鏡[オリンパス] × 200）表示されます。 γδT細胞のクラスターと集計は、γδT細胞が正常に展開された3日目（A）と5日目（B）、で観察することができます。 γδT細胞増殖は、（CとD）十分でない場合には対照的に、クラスターまたは凝集物が観察されなかった。 <img src="/files/ftp_upload/3182/3182fig4.jpg" alt="図4"/> 図4。IFN -γ産生。 γδT細胞は、RPMI - 10（A）またはPMA /イオノマイシン（B）または4時間Z -ダウディ（C）とともにインキュベートした。最初に、TCRVγ9の表面発現は、染色した後、IFN -γ産生は、細胞内IFN -γ染色により検討した。 <img src="/files/ftp_upload/3182/3182fig5.jpg" alt="図5"/> 図5。γδT細胞培養の速度論。培養細胞の（A）の絶対数、（B）γδT細胞、および指定された時点で、γδT細胞の（C）絶対数の割合。

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利害の衝突は宣言されません。

ここで紹介する方法は、PBMCからのγδT細胞の効率的な展開を可能にします。ゾレドロネートで活性化と拡大γδT細胞とIL - 2は、サイトカイン産生と細胞毒性によって反射された完全なエフェクター機能を、開発する。それが合成phosphoantigensのブロモヒドリンピロリン酸（BrHPP）と2 - メチル-3 - ブテニル-1 - ピロリン酸（2M3B1PP）ことが報告されてもγδT細胞を展開していますが、それらは市販さ?...

<table border="1"><tbody><tr><th>試薬名</th><th>会社</th><th>カタログ番号</th><th>コメント（省略可能） </th></tr><tr><td>ゾメタ</td><td>ノバルティスファーマK. K </td><td></td><td>ゾレドロネート</td></tr><tr><td> PROLEUKIN </td><td>ノバルティスファーマ</td><td></td><td>ヒト組換えIL - 2 </td></tr><tr><td> BDバキュテイナCPTナトリウムヘパリンによる細胞調製チューブ</td><td> BD </td><td> 362753 </td><td></td></tr><tr><td> RPMI1640 </td><td>インビトロジェン</td><td> 21870-076 </td><td></td></tr><tr><td> ALyS203 -培地</td><td>細胞科学技術研究所</td><td> 0301から7 </td><td></td></tr><tr><td> OpTmizer </td><td>インビトロジェン</td><td> 0080022SA </td><td></td></tr><tr><td>ブレフェルジン</td><td>シグマ</td><td> B5936 - 200UL </td><td></td></tr><tr><td>ホルボール12 - ミリステート13 - アセテート（PMA） </td><td>シグマ</td><td> P1585 - 1mgの</td><td></td></tr><tr><td>イオノマイシン</td><td>シグマ</td><td> 13909 - 1mLの</td><td></td></tr><tr><td> IntraPrep </td><td> BECKMAN COULTER </td><td> A07803 </td><td></td></tr><tr><td>抗ヒトCD3 - FITCまたはPE/Cy5 </td><td> BECKMAN COULTER </td><td> A07746 FITC A07749 PE/Cy5 </td><td></td></tr><tr><td>抗ヒトCD4 - ECD </td><td> BECKMAN COULTER </td><td> 6604727 </td><td></td></tr><tr><td>抗ヒトCD8-PE/Cy5 </td><td> BECKMAN COULTER </td><td> 6607011 </td><td></td></tr><tr><td>抗ヒトCD14-PE/Cy5 </td><td> BECKMAN COULTER </td><td> A07765 </td><td></td></tr><tr><td>抗ヒトCD19 - PE </td><td> BECKMAN COULTER </td><td> A07769 </td><td></td></tr><tr><td>抗ヒトCD45 - ECD </td><td> BECKMAN COULTER </td><td> A07784 </td><td></td></tr><tr><td>抗ヒトCD56-PE/Cy5 </td><td> BECKMAN COULTER </td><td> A07789 </td><td></td></tr><tr><td>抗ヒトTCRαβ- PE </td><td> BECKMAN COULTER </td><td> A39499 </td><td></td></tr><tr><td>抗ヒトTCRVγ9- FITC </td><td> BECKMAN COULTER </td><td> IM1463 </td><td></td></tr><tr><td>抗ヒトCD27-PE/Cy5 </td><td> BECKMAN COULTER </td><td> 6607107 </td><td></td></tr><tr><td>抗ヒトCD45RA - ECD </td><td> BECKMAN COULTER </td><td> IM2711 </td><td></td></tr><tr><td>抗ヒトCD69 - PE </td><td> BD </td><td> 555531 </td><td></td></tr><tr><td>抗ヒトNKG2D - PE </td><td> BECKMAN COULTER </td><td> A08934 </td><td></td></tr><tr><td>抗humalIFNγ- PE </td><td> BECKMAN COULTER </td><td> IM2717U </td><td></td></tr><tr><td>マウスIgG1アイソタイプコントロール- PE </td><td> BECKMAN COULTER </td><td> A07796 </td><td></td></tr><tr><td>マウスIgG1アイソタイプコントロール、ECDまたはPE/Cy5 </td><td> BECKMAN COULTER </td><td> A07797 A07798 </td><td></td></tr></tbody></table>

expansion of human peripheral blood  &gamma;&delta; t cells using zoledronate

人間のγδT細胞が認識し、ストレス誘発抗原のさまざまに反応する、それによって生来の広範な抗腫瘍および抗感染活動を展開することができます。末梢血中のγδT細胞の1大多数がVγ9Vδ2T細胞受容体を持っている。これらの細胞は主要組織適合性複合体に依存しない方法で抗原を認識し、強力な細胞傷害性とTh1細胞のようなエフェクター機能を開発する。1したがって、γδT細胞は、癌免疫療法のための魅力的な候補エフェクター細胞である。 Vγ9Vδ2T細胞は、次のようなphosphoantigensに対応する（E）-4 -ヒドロキシ-3 -メチル-ブト-2 -エニルピロリン酸（HMBPP）、イソプレノイド生合成を介して細菌で合成され、生成される2、イソペンテニルピロリン酸（IPP）、メバロン ​​酸経路を介して真核細胞で。生理的な状態で3、非形質転換細胞におけるIPPの世代は、γδT細胞の活性化のために十分ではありません。腫瘍細胞でのメバロン ​​酸経路の調節不全は、IPPの蓄積とγδT細胞の活性化につながる。3 aminobisphosphonatesが（そのようなパミドロネートまたはゾレドロネートなど）ファルネシルピロリン酸合成酵素（FPPS）、メバロン ​​酸経路でIPPの下流で作用する酵素の細胞内レベルを抑制するので、 IPPとγδT細胞の認識にsensitibityは、治療上aminobisphosphonates増加させることができる。 IPPの蓄積は私たちにaminobisphosphonatesとγδT細胞を活性化することによって、癌の免疫療法を可能にすること、aminobisphosphonatesの薬理学的に関連する濃度で腫瘍細胞よりもnontransfomred細胞に効率的ではありません。おもしろい4 PBMCがaminobisphosphonatesで処理されるとき、IPPのために効率的で、単球に蓄積これらの細胞による薬物取り込み。IPPとなる抗原提示細胞を蓄積し、末梢血でVγ9Vδ2T細胞を刺激する5単球6これらのメカニズムに基づいて、我々は、ゾレドロネートおよびインターロイキンを使用して、γδT細胞培養物の大規模展開のための技術を開発-2（IL - 2）。γδT細胞の拡大のための7他の方法は、合成phosphoantigensのブロモヒドリンピロリン酸（BrHPP）8または2 -メチル-3 -ブテニル-1 -ピロリン酸（2M3B1PP）を利用する。これらのメソッドの9すべてがexを許可養子免疫療法における使用のためのγδT細胞の多数の結果in vivoでの展開、。ただし、ゾレドロネートがFDA承認された市販の試薬です。ゾレドロネート-拡張されたγδT細胞はCD27を表示- CD45RA -エフェクターメモリー表現型とそれらの機能は、IFN -γ産生アッセイによって評価することができる7。

Watch this Scientific Journal Video about Expansion of Human Peripheral Blood  Î³Î´ T Cells using Zoledronate at JoVE.com

Expansion of Human Peripheral Blood  &#947;&#948; T Cells using Zoledronate

末梢血単核細胞（PBMC）からのγδT細胞を拡大する方法が記載されている。 PBMC由来のγδT細胞を刺激し、ゾレドロネートとインターロイキン-2（IL - 2）を使用して展開されます。 γδT細胞の大規模の拡大は、癌の自家細胞免疫療法に適用することができます。

ゾレドロネートを使用してヒト末梢血γδT細胞の拡大

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

expansion-of-human-peripheral-blood-t-cells-using-zoledronate

Research

JoVE Journal

Immunology and Infection

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

26.5K ビュー.  University of Tokyo Hospital. 末梢血単核細胞（PBMC）からのγδT細胞を拡大する方法が記載されている。 PBMC由来のγδT細胞を刺激し、ゾレドロネートとインターロイキン-2（IL - 2）を使用して展開されます。 γδT細胞の大規模の拡大は、癌の自家細胞免疫療法に適用することができます。

ビデオ: Expansion of Human Peripheral Blood  γδ T Cells using Zoledronate

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

拡大、精製、及びヒト末梢血NK細胞の機能的評価

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

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&#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 ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

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 ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

末梢血からのヒトアロ抗原特異的T細胞の生成

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease and treatment. It has the potential to predict therapeutic efficacy and identify novel therapeutic targets. Whole blood staining utilizes unmanipulated blood, which minimizes artifacts that can occur during sample preparation. However, whole blood staining must also be done on freshly collected blood to ensure the integrity of the sample. Additionally, it is best to prepare antibody cocktails on the same day to avoid potential instability of tandem-dyes and prevent reagent interaction between brilliant violet dyes. Therefore, whole blood staining requires careful standardization to control for intra and inter-experimental variability.
Here, we report deployment of an automated liquid handler equipped with a two-dimensional (2D) barcode reader into a standard process of making antibody cocktails for flow cytometry. Antibodies were transferred into 2D barcoded tubes arranged in a 96 well format and their contents compiled in a database. The liquid handler could then locate the source antibody vials by referencing antibody names within the database. Our method eliminated tedious coordination for positioning of source antibody tubes. It provided versatility allowing the user to easily change any number of details in the antibody dispensing process such as specific antibody to use, volume, and destination by modifying the database without rewriting the scripting in the software method for each assay.
A proof of concept experiment achieved outstanding inter and intra- assay precision, demonstrated by replicate preparation of an 11-color, 17-antibody flow cytometry assay. These methodologies increased overall throughput for flow cytometry assays and facilitated daily preparation of the complex antibody cocktails required for the detailed phenotypic characterization of freshly collected anticoagulated peripheral blood.

Whole blood Immunophenotyping is indispensable for monitoring the human immune system. However, the number of reagents required and the instability of specific fluorochromes in premixed cocktails necessitates daily reagent preparation. Here we show that semi-automated preparation of staining antibody cocktail helps establish reliable immunophenotyping by minimizing variability in reagent dispensing.

ヒト末梢血の免疫表現型分析のための抗体のカクテルを準備する半自動のアプローチ

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

ヒト子宮頸管ガンマ・デルタT細胞の単離およびフローサイトメトリー分析

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

デキストランは、マウスとヒト NK 細胞のレンチ ウイルスの伝達効率を向上します。

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and multiplexing ability. Using fluorescent nano-diamond fiducial markers, we describe the drift correction and alignment procedures required to obtain high precision in single molecule localization microscopy. In addition, a new multiplexing strategy, madSTORM, is described in which multiple molecules are targeted in the same cell using sequential binding and elution of fluorescent antibodies. madSTORM is demonstrated on an activated T cell to visualize the locations of different components within a membrane-bound, multi-protein structure called the T cell receptor microcluster. In addition, application of madSTORM as a general tool for visualization of multi-protein structures is discussed.

We demonstrate a method to image multiple molecules within heterogeneous nano-structures at single molecule accuracy using sequential binding and elution of fluorescently labeled antibodies.

ゾレドロネートを使用してヒト末梢血γδT細胞の拡大

要約

さらに動画を探す

この動画の章

拡大、精製、及びヒト末梢血NK細胞の機能的評価

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

の臨床応用

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

HIV-1感染CD4の生得センシングを評価

末梢血からのヒトアロ抗原特異的T細胞の生成

ヒト末梢血の免疫表現型分析のための抗体のカクテルを準備する半自動のアプローチ

ヒト子宮頸管ガンマ・デルタT細胞の単離およびフローサイトメトリー分析

デキストランは、マウスとヒト NK 細胞のレンチウイルスの伝達効率を向上します。

madSTORMを用いたT細胞の高度に多重化された超解像イメージング

ゾレドロネートを使用してヒト末梢血γδT細胞の拡大

要約

さらに動画を探す

この動画の章

拡大、精製、及びヒト末梢血NK細胞の機能的評価

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

の臨床応用

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

HIV-1感染CD4の生得センシングを評価

末梢血からのヒトアロ抗原特異的T細胞の生成

ヒト末梢血の免疫表現型分析のための抗体のカクテルを準備する半自動のアプローチ

ヒト子宮頸管ガンマ・デルタT細胞の単離およびフローサイトメトリー分析

デキストランは、マウスとヒト NK 細胞のレンチ ウイルスの伝達効率を向上します。

madSTORMを用いたT細胞の高度に多重化された超解像イメージング

デキストランは、マウスとヒト NK 細胞のレンチウイルスの伝達効率を向上します。