我々は、有用な議論のためにアーロンSelyaに感謝します。この作品は、JS、ジョンズホプキンスマラリア研究所からのパイロット助成金やMOへの防衛助成PC 040972の部にNIHの助成金AI29575、CA108835、AI077097によってサポートされていました

1。作り、HLA - A2 - IgをベースaAPC <ol><li>滅菌ホウ酸緩衝液を準備する<ol><li> 0.1Mを作るために水にホウ酸を溶かす。 pHを7.0に調整する。 </li><li> 4で0.22μm滅菌フィルターや店舗をフィルタ℃に</li></ol></li><li>滅菌ビーズ洗浄バッファーを準備します。 <ol><li> 956ミリリットルPBS W / Oマグネシウムやカルシウムを取る。 </li><li> 30ミリリットルのヒトAB血清を追加。 </li><li> EDTA 2mMの最終濃度を追加。 </li><li> 0.1gsodiumアジを追加。 </li><li> 4で0.22μmの滅菌フィルターや店舗をフィルタ℃に</li></ol></li><li> 、在庫から約4億ビーズ（ビーズは、血球計数器でカウントすることができます）インビトロジェンM - 450エポキシビーズを1mlを取り、滅菌、スクリュートップガラスバイアルに入れ。 </li><li>ビーズは、バイアルの側面に付着しながら、DYNAL磁石MPC - 1に対するバイアルを入れ、吸引して上清を取り除く。ホウ酸緩衝液を1mlで一回ビーズを洗浄する。 </li><li> 1ミリリットルホウ酸緩衝液およびHLA - A2 - Igの二量体と抗ヒトCD28モノクローナル抗体（クローン9.3）の20μgの20μgの混合物でビーズを再懸濁します。 </li><li>ビーズ共役への蛋白質：ローテーター上のガラスバイアルを入れ、4℃で回転℃で24時間。 </li><li> MPC - 1マグネットにチューブを置き、すべてのホウ酸緩衝液を取り除く。 </li><li> 1ミリリットルビーズ洗浄バッファーで2回ビーズを洗浄してください。 </li><li> 1ミリリットルビーズ洗浄バッファーでビーズをインキュベートし、4℃で回転させる℃で24時間。ビーズ洗浄バッファーは、ヒトAB血清が含まれているので、それは、ビーズ上のすべての残留タンパク質結合部位をブロックします。 </li><li>ガラス瓶から上清を除去する、1mlの新鮮なビーズの洗浄緩衝液で置き換えてください。 </li></ol> 2。 aAPCとペプチド負荷、ストレージの品質管理<ol><li> FACSチューブに洗浄液100μlのFACSへ〜5 × 10 5ビーズを追加し、抗マウスIgG1 - PE（HLA - A2 - IgのFc部分を認識）および抗マウスIgG2a - FITC（認識1μlの1μlのと染色抗CD28モノクローナル抗体のFc部分）。洗浄バッファーFACSで20分間染色した後、再び洗浄し、フローサイトメーター（図4）によってすぐに染色の結果をお読みください。 </li><li>ビーズ上にペプチド負荷：1ミリリットル滅菌PBSでガラスバイアルに二回ビーズを洗浄する。 1ミリリットル滅菌PBSで再懸濁しは、CMVペプチド​​（1mg/ml）の10μlを加える</li><li>血球計算盤によるビーズをカウントし、日付と濃度とのバイアルにラベルを付けます。 </li><li> aAPCは、HLA - A2 -グロブリン二量体の上にペプチド結合の十分な時間を確保するために、4℃で少なくとも3日後にペプチドインキュベーション使用するための準備が整いました。ビーズは4℃で保存され、少なくとも6ヶ月は継続することができます。 </li></ol> 3。人間のCTLの分離<ol><li> 10 BDのヘパリンナトリウムバキュテイナ管に健康的なHLA - A2陽性ドナーからの新鮮な末梢血の〜100ミリリットルを収集する。 21ゲージ針または溶血を防ぐために、大きい方を使用してください。 </li><li>室温で10分間300xgで遠心分離</li><li>慎重に吸引して上部のプラズマレイヤーを削除します。プラズマは、培地のためのサプリメントとして使用することができます。 </li><li>滅菌PBSで収集した血漿を交換し、滅菌T75培養フラスコまたは50ミリリットルコニカルチューブに血液を移す。上下にピペッティングによりよくPBSで血を混ぜる。 </li><li>一度、すべての血液が収集され、4つの追加50ミリリットルコニカルチューブを準備し、をFicoll - Paqueプラスの15ミリリットルを追加します。 </li><li>ゆっくりオーバーレイ各チューブのフィコールの上に血液細胞の30〜35ミリリットル。フィコールと血液細胞との間のインターフェースが異なるしてください。 </li><li>室温で20分間500xgで遠心分離します。ブレーキ&quot;オフ&quot;と層の間に明確なインタフェースを維持するために可能な限り低加速を回します。 </li><li> seriologicalピペットを使用して、慎重にPBMC層を吸引し、新鮮な50ミリリットルコニカルチューブにPBMCを収集する。すべてのPBMCを10分間400xgでPBSとスピンの30ミリリットルを追加収穫されている場合。上清を捨て、すべての残留フィコールを削除するには、30ミリリットルのPBSでもう一度洗浄する。 </li><li>製造業者のプロトコルに従ってミルテニーヒトCD8 + T Cellアイソレーションキットを使用することによりCD8 + T細胞の分離に進みます。細胞-このキットは非常に（通常&gt; 95％）CD8を枯渇させることによりCD8 +細胞のために豊かに。 </li><li> CD8 + T細胞を数える。予想される純度は95％以上でなければなりません。この使用2 × 10 5細胞を確認し、CD4/3/8 FACS解析を実行する。残りのCTLは、どちらの抗原特異的aAPC刺激に対してすぐに使用することも、将来使用するために凍結することができます。 </li></ol> （4） 体外 aAPCベースの培養系で <ol><li>培地を準備<ol><li>完全RPMI培地に加え、プラズマ、8％のT細胞増殖因子自家5％のドナー：TF（ラボ4で作られたT細胞増殖因子、）2X培地用。 </li><li&gt;ドナー自己血漿は、熱不活化ヒトAB血清で置換することができる。 </li></ol></li><li>再懸 ​​濁し、完全RPMI培地中のTF 2X培地を加えた8ミリリットルの8ミリリットルで100万CTLは、1を追加× 10 6 aAPCを、よく混ぜる。 </li><li> 96ウェルU底の組織培養プレート上にプレートの細胞へのマルチチャンネルピペッターを使用してください。 （ウェルあたり160μlを） </li><li> 37培養細胞° C、7日間、5％CO 2 incubater。 80μlの/ウェルTF 2X培地で4日目に細胞を養う。 </li><li>細胞は7日目に収穫する準備が整いました。収穫後は、磁石から細胞を配置し、古いaAPCを取り外します。 </li><li>抗原特異性は、製造業者のプロトコルに従ってテトラマー染色により決定することができる。表現型の染色と細胞内サイトカイン染色は、私たちの以前の研究の3に従って実行されます。 </li><li>収穫された細胞は、同じ条件の下で再びaAPCと色を保ち続けることができます。細胞数や抗原特異性は、反復刺激後に増加すると予想される。 </li></ol> 5。代表的な結果： HLA A2 - Igおよび抗CD28接合後のaAPCの例を図4に示されています。成功したタンパク質コンジュゲーションは、対応する抗体染色の明確なシフトによって明らかである。末梢血中のCMV特異的CTLの頻度がaAPCを介した刺激の単週後に、通常は0.5から1パーセントですが、特異度は55に到達することができます - 93％（図2および3）。抗原特異的CTLの拡張は、さまざまなドナーの間で非常に可変することができますが、結果は同じドナー内で再現可能です。外挿によって、CMV特異的細胞の拡大は、前駆体のレベルを直接ex vivoで （データは示さず）に比べて倍の数千になることができます。細胞内サイトカイン染色（図3）これらの拡張されたCTLは、長期細胞培養と有意な増殖の後、多官能性ではなく、疲れていることを示しています。 <img src="/files/ftp_upload/2801/2801fig1.jpg" alt="図1"/> 図1。同種HSCTにおける養子免疫療法のためのヒトCTLのaAPCベースのex vivoでの展開の代表的なフローチャート<img src="/files/ftp_upload/2801/2801fig2.jpg" alt="図2"/> 文化の一週間後にaAPCによって生成されたCMV特異的CTLの図2。代表テトラマー染色の結果<img src="/files/ftp_upload/2801/2801fig3.jpg" alt="図3"/> 図3。aAPCによって生成されたCMV特異的CTLの代表的な細胞内サイトカイン染色の結果（CMV特異性は61％であった） <img src="/files/ftp_upload/2801/2801fig4.jpg" alt="図4"/> 図4 M - 450エポキシビーズの代表的な染色の結果抗マウスIgG1 - PEと抗マウスIgG2の- FITCで染色したタンパク質コンジュゲーション後の

<ol>
	<li>Walter, E. A. <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, 1038-1038 (1995).</li><li>Rosenberg, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adoptive+cell+transfer:+a+clinical+path+to+effective+cancer+immunotherapy.">Adoptive cell transfer: a clinical path to effective cancer immunotherapy.</a> Nat Rev Cancer. 8, 3669-3669 (2008).</li><li>Oelke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+induction+and+expansion+of+antigen-specific+cytotoxic+T+cells+by+HLA-Ig-coated+artificial+antigen-presenting+cells.">Ex vivo induction and expansion of antigen-specific cytotoxic T cells by HLA-Ig-coated artificial antigen-presenting cells.</a> Nat Med. 9, 619-619 (2003).</li><li>Oelke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+antigen-presenting+cells:+artificial+solution+for+real+diseases.">Artificial antigen-presenting cells: artificial solution for real diseases.</a> Trends Mol Med. 11, 412-412 (2005).</li></ol>

利害の衝突は宣言されません。

我々はここで説明するaAPCシステムは、抗原の多様性に対する人間のCTLのex vivoでの拡大のための効率的なシステムです。特別なケアは、タンパク質共役および96ウェルプレート培養でaAPCとCTLの均一な分布の品質に関しては注意が必要です。我々は百万倍の4に抗原特異的CTLを展開した中に、8週間以上のためにCTLを拡大することができたこのアプローチを使用する。細胞株や他の無細胞プラットフォーム5活用した様々な人工APCシステムが行われているが、公表されたデータによると、すべてのシステムは、異なるアプリケーションをサポート拡張し、特異性に関して持つ独自のプロファイルを持っています。 CTLの質は量と同様に重要であるので重要なのは、、私たちのシステムによって生成されたCMV特異的CTLのpolyfunctionalityは、優れた抗ウイルス効果を付与することが期待されています。

<table border="1"><tbody><tr><th>試薬</th><th>会社</th><th>カタログ番号</th></tr><tr><td>バキュテイナ管（ヘパリンを含む） </td><td>ベクトンディッキンソン</td><td> 367874 </td></tr><tr><td>ヒトCD8 + T Cellアイソレーションキット</td><td>国内でのみ有効</td><td> 130-094-156 </td></tr><tr><td>ダイナビーズM - 450エポキシ</td><td>インビトロジェン</td><td> 140.11 </td></tr><tr><td> DYNAL MPC - 1マグネット</td><td>インビトロジェン</td><td> 120 - 01D </td></tr><tr><td>をFicoll - Paqueプラス</td><td> GEヘルスケア</td><td> 17-1440-03 </td></tr><tr><td> RPMI培地1640 </td><td>ギブコ</td><td> 11875 </td></tr><tr><td> HLA - A2 -グロブリン二量体X </td><td>ベクトンディッキンソン</td><td> 551263 </td></tr><tr><td> iTAgMHC四量体（HLA - A2 - CMV）- PE </td><td>ベックマンコールター</td><td> T20099 </td></tr><tr><td>ファルコン明確な96穴マイクロプレート</td><td>ベクトンディッキンソン</td><td> 353077 </td></tr><tr><td>ラット抗マウスIgG2a - FITC </td><td>ベクトンディッキンソン</td><td> 553390 </td></tr><tr><td>ヤギ抗マウスIgG1 - PE </td><td>インビトロジェン</td><td> P21129 </td></tr><tr><td>ヒト血清型AB </td><td>アトランタ生物学的製剤</td><td> S40110 </td></tr><tr><td>マウス抗ヒトCD8a - FITC </td><td>シグマアルドリッチ</td><td> F0772 </td></tr><tr><td>マウス抗ヒトCD8a - APC </td><td>ベクトンディッキンソン</td><td> 340684 </td></tr><tr><td>マウス抗ヒトIFNγ- FITC </td><td>ベクトンディッキンソン</td><td> 340449 </td></tr><tr><td>マウス抗ヒトTNFα- PE </td><td>ベクトンディッキンソン</td><td> 340512 </td></tr></tbody></table>

hla-ig based artificial antigen presenting cells for efficient ex vivo expansion of human ctl

最適なエフェクター機能を持つCTLは、様々な細胞内感染症や癌に対する保護を媒介に重要な役割を果たしている。しかし、個人が抑制免疫微小環境を示すことがあると、活性化CTLとは対照的に、彼らの自家抗原提示細胞が耐性化する傾向にあることまたは抗原特異的CTLをanergize。結果として、まだ実験段階にもかかわらず、CTLベースの養子免疫療法は、がんやウイルス感染など、様々な疾患に対する有望な治療になるために進化してきました。初期の実験では、ex vivoで拡張されたCMV（サイトメガロウイルス）特異的CTLは、免疫不全の同種骨髄移植患者におけるCMV感染症の治療のために使用されている。それはこれらの患者の生命を脅かすCMVのウイルス血症、拡張されたCTLを受けている患者のどれもがCMVに関連する病気を開発するのが一般的ですが、抗CMV免疫の養子転送CTL 1で確立されている暗示。有望な結果はまた、悪性黒色腫が観察されており、がん2の他のタイプに延長することができる。 人間のCTLを刺激し、拡大し、ex vivoでの多くの方法がありますが、現在のアプローチは、コストと技術的な限界によって制限されます。例えば、現在のゴールドスタンダードは、自家DCの使用に基づいています。これにより、各患者は、白血球のかなりの数を寄付することが必要とも非常に高価で面倒です。また、DC拡張CTLのin vitroでの特性の詳細は、これらが唯一の次善のエフェクター機能3を持っていることを明らかにした。 ここでは、養子免疫療法（図1）のためのヒトCMV特異的CTLのex vivoでの拡大のための高効率aAPCベースのシステムを提示する。 aAPCは、ヒトHLA - A2 - Igの二量体と抗CD28mAb 4セルサイズの磁気ビーズを結合させることによって行われた。 aAPCが行われると、彼らは興味のある様々なペプチドでロードされ、月間は継続することができます。このレポートでは、aAPCはCMV、pp65を（NLVPMVATV）から支配的なペプチドを負荷した。一週間のためのaAPCと健康なドナーからのヒトCD8 + CTLを精製培養した後、CMV特異的CTLは、98％（図2）に特異性で劇的に増加し、10,000人以上の倍に増幅することができる。より多くのCMV特異的CTLが必要な場合は、さらなる拡張が容易にaAPCで反復刺激することによって達成することができます。表現型および機能的特徴は、これらの拡張された細胞は、エフェクター、メモリー表現型を持つと（図3）TNFαとIFNγの両方を大量に作る示しています。

Watch this Scientific Journal Video about HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL at JoVE.com

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

抗原特異的T細胞の誘導と拡大のための新しいDCの独立した方法が記載されている。 HLA A2 - Igはベースの人工的な抗原提示細胞（aAPC）が効率的に多様な抗原特異性のCTLを拡大するHLA - A2制限されたペプチドがロードされます。この技術は、CTLベースの養子免疫療法のための大きな可能性を秘めている。

HLA - Igは効率のためのベースの人工抗原提示細胞生体外で</emヒトCTLの>拡大

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals ...

hla-ig-based-artificial-antigen-presenting-cells-for-efficient-ex

Research

JoVE Journal

Immunology and Infection

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

15.3K ビュー.  Johns Hopkins University. 抗原特異的T細胞の誘導と拡大のための新しいDCの独立した方法が記載されている。 HLA A2 - Igはベースの人工的な抗原提示細胞（aAPC）が効率的に多様な抗原特異性のCTLを拡大するHLA - A2制限されたペプチドがロードされます。この技術は、CTLベースの養子免疫療法のための大きな可能性を秘めている。

ビデオ: HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation. This labeling has classically been used to study proliferation and migration. In the method presented here, we have shortened the timeline after adoptive transfer to look at survival and killing of epitope specific CFSE labeled target cells4-6. The level of specific killing of a CD8 + T cell clone can indicate the quality of the response, as their quantity may be misleading. Specific CD8+ T cells can become functionally exhausted over time with a decline in cytokine production and killing7,8. Also, certain CD8 + T cell clones may not kill as well as others with differing TCR specificities9. For effective Cell Mediated Immunity (CMI), antigens must be identified that produce not only adequate numbers of responding T cells, but also functionally robust responding T cells. Here we assess the percent cell specific killing of two peptide specific T cell clones in BALB/c mice.

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Many infections elicit a strong CTL response, but occasionally, the quantity of responding cells does not correlate to control of the pathogen1. One measure of CTL quality is their ability to kill specifically2. CFSE labeling of target cells can be used to investigate this CTL response quality in vivo3,4.

抗原特異的インビボでの</emCFSE標識した標的細胞を用いた>キリングアッセイ

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation.  ...

免疫・感染学

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide complete protection against the development or recurrence of breast cancer. To overcome this problem by increasing the frequency of tumor-reactive T cells, adoptive immunotherapy has been employed. A variety of protocols have been used for the expansion of tumor-specific T cells. These protocols, however, are restricted to the use of tumor antigens ex vivo for the activation of antigen-specific T cells. Very recently, common gamma chain cytokines such as IL-2, IL-7, IL-15, and IL-21 have been used alone or in combination for the enhancement of anti-tumor immune responses3. However, it is not clear what formulation would work best for the expansion of tumor-reactive T cells. Here we present a protocol for the selective activation and expansion of tumor-reactive T cells from the FVBN202 transgenic mouse model of HER-2/neu positive breast carcinoma for use in adoptive T cell therapy of breast cancer. The protocol includes activation of T cells with bryostatin-1/ionomycin (B/I) and IL-2 in the absence of tumor antigens for 16 hours. B/I activation mimics intracellular signals that result in T cell activation by increasing protein kinase C activity and intracellular calcium, respectively4. This protocol specifically activates tumor-specific T cells while killing irrelevant T cells. The B/I-activated T cells are cultured with IL-7 and IL-15 for 24 hours and then pulsed with IL-2. After 24 hours, T cells are washed, split, and cultured with IL-7 + IL-15 for additional 4 days. Tumor-specificity and anti-tumor efficacy of the ex vivo expanded T cells is determined.

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

An efficient protocol for the ex vivo expansion of tumor-reactive T cells from tumor-draining lymph nodes or other secondary lymphoid tissues of tumor-bearing hosts is described. This protocol selectively expands tumor-specific T cells for use in adoptive immunotherapy of breast cancer.

ブリオスタチンの1/Ionomycinによる腫瘍反応性T細胞のex vivoでの拡張と共通γ鎖のサイトカインの策定

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide ...

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 ...

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

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

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

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

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 ...

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

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

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

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

ナチュラルキラーT細胞の細胞（AAPC）媒介活性化と増殖を提示する人工抗原

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

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 ...

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 ...

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

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

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

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

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

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

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

表現型分析のためのヒト皮膚からリンパ球単離と<em&gt; ex vivoで</em&gt;細胞培養

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/- recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts.
This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

T細胞への抗原提示細胞による抗原の提示を研究するための抗原駆動性大腸炎モデルの開発

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...

HLA - Igは効率のためのベースの人工抗原提示細胞生体外で拡大