著者は、この原稿のデータを貢献するためのマイケル·チョウ、ビンビン大、そして梁暁を確認したいと思います。また、本研究で使用した試薬の寛大な贈り物のためにドクタージョングレイを認める。 PBは、国立がんセンターからポスドクでサポートされています。この研究は、国立衛生研究所（R01AI68978、P01CA132681とRCA170820A）、ビル＆メリンダ·ゲイツ財団、トランスレーショナル医学とカリフォルニアHIV / AIDSからの助成金のための共同センターから並進加速助成金からの助成金からの補助金によって支えられて研究プログラム。

DC-LV安定産生細胞は、必要にレンチウイルスコンポーネントgagpol、REVおよびTETオフシステムを含むGPRパッケージングセルライン11に基づいて構築されています。まず、レトロウイルス形質導入は、テトラ依存SVGmu糖タンパク質をコードするGPRSパッケージング細胞株を生成するために使用される。その後、コンカテマーアレイトランスフェクションは、GFPのようなレンチウイルスベクターでトランスジーンGPRS細胞系をトランスフェクトするために使用される。 LV-MGFPとして指定され、この安定した産生細胞系は、GFPに対するDC-LVワクチンを産生する能力についてin vitroおよびin vivoで試験することができる。 1。テト依存SVGmu細胞株を生成するプラスミドPRX-SVGmuは、DC特有の糖タンパク質SVGmuがレトロウイルスプラスミドpRetroX-テトオフ1（ 図1C）のTTA-高度なプロモーターの下流にクローニングされている構造です。 D10での培養293T細胞（ダルベッコ改変イーグル培地で10％ウシ胎児血清、2mMのL-グルタミン）。ドキシサイクリン（1 ngの/ ml）およびピューロマイシン（2μgの/ mL）でD10で文化GPRのパッケージング細胞株。すべての細胞は5％CO 2で加湿37℃のインキュベーターで培養する。 <ol><li> 16-18時間前に一過性トランスフェクション、プレート2×10細胞は、トランスフェクション時に90％の密集度に近づくように、6 cmの組織培養皿でD10 4mlの6 293T細胞。 </li><li> レトロウイルスプラスミドのトランスフェクション ：ミックス100μlの1.25 MのCaCl 2溶液、滅菌ミリQ水と以下のプラスミド：5μgのPRX-SVGmu、2.5μgのPGAGポル、2.5μgのpVSV-G。最終容量は500μLです。 5ミリリットルポリスチレン丸底チューブに、500μlの2X HBSを液（50mM HEPES、10mMのKClを、12 mMのブドウ糖、280のNaCl、1.5mMののNa 2 HPO 4·7H 2 O、pHは7.05）を追加します。ガラスパスツールピペットを用いて精力的にバッファをバブリングしながら、2X HBSバッファーにプラスミド混合物を滴下を追加します。マイルを追加した後xtureは、別の30秒間バブリング続ける。その後、6 cmの培養皿で293T細胞上に混合物全体を追加し、37℃でインキュベート℃、 </li><li> 4時間トランスフェクション後、慎重に4ミリリットル予熱したD10と培養皿に培地を交換してください。 </li><li> 感染スピン ：トランスフェクション後48時間を、収穫SVGmuエンコード0.45μmのフィルターを通して上澄みを渡すことによって、上清中のレトロウイルス粒子を。 2×10 4細胞/ウェルで24ウェル皿にプレートGPRパッケージング細胞を。 1,050×gで90分間、25℃で皿にGPRのパッケージング細胞（2ミリリットル/ウェル）、遠心上清を細胞にフィルタ追加（2ミリリットル/ウェル）スピン感染後ドキシサイクリン（1 ngの/ ml）で新鮮なD10に培地を変更します。 </li><li> 72時間後、トランスフェクションは、ドキシサイクリン（1 ngの/ ml）およびピューロ（2 ngの/ ml）でD10で形質導入パッケージング細胞の培養を展開します。 </li><li> 48時間ドキシサイクリンずにSVGmu、文化細胞の発現を確認する。測定表面EX抗シンドビス血清を用いたフローサイトメトリーとSVGmuの下り坂。これらの細胞は、GPRSのパッケージング細胞株として指定されている。 </li></ol> 2。コンカテマーアレイトランスフェクションによってDC-LVの産生細胞を構築レンチウイルストランスファープラスミドTL20-GFPは自己不活性化レンチウイルストランスファーベクタープラスミドのDox-調節ウイルスRNAゲノム発現系と7 TET子（ 図1C）11とサイトメガロウイルス（CMV）エンハンサーの交換にpCL20c-MSCV-GFPに基づく、12,15。プラスミドPGK-BLEは弱いPGKプロモーター2によって駆動ブレオマイシン耐性（BLE）カセットです。 <ol><li> 50で制限酵素SfiIでとダイジェスト20μgのプラスミドTL20-GFP℃まで37℃PflMIとダイジェスト20μgのプラスミドPGK-BLE℃まで</li><li>アガロースゲル電気泳動によりDNA断片（PGK-BLEカセットが1011 bpであるとベクトルTL20-GFPは6861 bpである）浄化。 </li><li> TL20-GFPベクターおよびPGK-を連結25:1のモル比（NEB T4 DNAリガーゼ）におけるBLEカセット。室温で一晩インキュベートする。 </li><li>一晩ライゲーション後、ライゲーション混合物（キアゲンDNeasy血液および組織キット）からDNAを精製する。精製されたDNAの量を確保するには、5μgの程度である。 </li><li>トランスフェクションの前に16〜18時間、密集度は、トランスフェクションの時点で約90％となるように6 cmの組織培養皿にプレートGPRSパッケージング細胞。 </li><li> 6 cmの組織培養皿の中のGPRSパッケージング細胞に精製コンカテマーDNA5μgのをトランスフェクトするために、ステップ1.2で説明したリン酸カルシウムトランスフェクション法を使用します。 4時間トランスフェクション後、慎重に培地を除去し、ドキシサイクリン（1 ngの/ mL）で4ミリリットル予熱されたD10と交換してください。 </li><li>トランスフェクション後48時間では、ドキシサイクリン（1 ngの/ ml）およびピューロマイシン（2μgの/ ml）をD10にメディアを変更してください。培養培地中でゼオシン（50μgの/ ml）を添加することによりトランスフェクトされたクローンを選択する。セルコローニまで約2週間培養細胞エスは、皿の底部に見ることができる。 </li><li>皿の下部にある細胞コロニーにラベルを付けます。 、24ウェル組織培養皿を取る数井戸やmlのD10はゼオシン（50μgの/ ml）を、ドキシサイクリン（1 ngの/ ml）を、およびピューロマイシン（2μgの/ ml）を含む各ウェル2に追加。形質細胞の培地を吸引し、1分未満のためにコロニーのそれぞれにトリプシンの一滴を追加します。その後、同じコロニーにD10の一つ以上の滴を追加します。ピペットでつずつのコロニーつをピックアップし、24ウェル組織培養プレートの別々のウェルに移す。 </li><li>文化やウイルスの産生能の評価のためにゼオシン（50μgの/ ml）を、ドキシサイクリン（1 ngの/ ml）およびピューロマイシン（2μgの/ ml）を含むD10内のすべての細胞クローンを展開します。 </li></ol> 3。各細胞クローンのウイルス産生を評価<ol><li>コンフルエントそのようドキシサイクリンずにD10で6 cmの組織培養皿で産生細胞とプレートの周りに4×10 6個の細胞をトリプシン処理90％を超える。毎日新鮮な予備加熱D10で培地を交換し、DOXを削除した後に力価アッセイ72時間のための培地を収集します。 </li><li> 0.45μmのフィルターを用いて培地をフィルタリングすることで、ウイルス上清を収穫。 </li><li>プレート標的細胞は×10 4細胞/ウェル1、96ウェル培養皿に陰性対照としてDC-SIGN受容体（ 例えば、派生293T.hDC-SIGNまたはマウス骨髄樹状細胞16）および293Tを発現する。ウェルにウイルス上清（100μL/ウェル）の2倍連続希釈を追加します。通常、希釈液6-8は、GFP陽性細胞を添加したウイルスの量に線形の関係にある形質導入された範囲に到達するのに十分である。 </li><li>細胞を遠心分離し、ステップ1.4で説明したように培地を交換してください。たBMDCを、10％FBSおよびGM-CSF（1時20 J558L馴化培地）を含むRPMI培地で培養されるべきである。 </li><li>フローサイトメトリー4-6日後に形質導入​​により形質導入細胞におけるGFPの発現を測定します。レンチウイルスvectoGFP陽性細胞とベクトル量の割合は線形関係にあるときにrの力価は、ベクターの希釈範囲で算出される。その後のアプリケーションのための最高のウイルス産生と細胞クローンを選択してください。 </li></ol> 4。レンチウイルスベクターを生産し、集中<ol><li> 15cmの組織培養皿で培養プロデューサー細胞株（LV-MGFP参照）。 </li><li>ドキシサイクリンなし新鮮D10において密集度90％以上で15cmの組織培養皿に産生細胞とプレート細胞をトリプシン処理。毎日新鮮な、予備加熱D10と培地を変更します。 </li><li>ピークウイルス産生（ユーザによって決定される）の時点で、ウイルス上清を採取し、0.45μmのフィルターを使用して、それをフィルタリングします。 90分間50,000 xgでパラフィルムと遠心、4℃で密閉し、厚い壁32.5ミリリットルの遠心チューブにフィルター上清をロードします。徹底的に50μlのか、アプリケーションに応じて、PBSまたはHBSSの適切な音量でペレットを再懸濁します。&lt;/李&gt; </ol> 5。 インビボでマウスを免疫化し、抗原特異的免疫応答の分析<ol><li>セクション4で説明されているように高力価のウイルスを生成します。 </li><li>ベクター懸濁液（蹠あたり25μL）と蹠経路を介してBALB / cマウス（メス、古い6-8週）を注入する。 </li><li> 2週間の免疫後、脾臓や収穫脾細胞を分離する。 GFPエピトープペプチドで培養脾細胞とGFP特異的CD8 +の存在下予め17（ 図3B）に記載のように、細胞内サイトカイン染色法を用いてT細胞を分析する。 </li></ol>

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<p class="jove_content"&gt;著者は、彼らが競合する金融利害関係を持たないことを宣言します。</p

ここでは、安定して規制システムオフテトラ下にあるすべてのコンポーネントレンチウイルスで形質導入された293T細胞を用いたレンチウイルスベクターを大量に製造する方法を概説している。現在までに、レンチウイルスベクターを製造するためのほとんどのプロトコル（たとえば、18を参照）は、標準リン酸カルシウム一過性トランスフェクションに依存している。このアプロー?...

多くのベクター系は、遺伝子送達のために開発されている。レンチウイルスに基づくベクターは、最も一般的に研究ウイルスシステムの間でされています。それらは効率的に分割し、非分裂細胞1の両方を形質導入宿主ゲノムへの組込みに起因する長期発現を達成するため、2ほとんどのヒト集団における低天然の抗ベクトル耐性を示し、かつに対して低電位を有することができるので、これらのベクターは、有利で ​​ある挿入突然変異3,4から遺伝毒性。 レンチウイルスの産生は、常に安全性の懸念に彩られています。レンチウイルスベクターは、一般に、HIV-1、AIDSの病原体から誘導される。 lentivectorの個々の成分（転写、エンベロープ、およびパッケージングプラスミド）の一過性トランスフェクション実験室の設定で遺伝物質を送達する一般的で柔軟な手段である。しかし、臨床応用のためのスケールアップ一過性トランスフェクションは、面倒でmAですyは複製能レンチウイルス5,6の開発につながる。これらのハードルを克服するために、いくつかの安定したパッケージングおよびプロデューサー細胞株は6-11開発されている。これらのいずれかの行、GPR包装ライン11は 、テトラサイクリンにより調節される魅力的な利点を有する。本稿では、特に樹状細胞（DC-のLV）12に向けて対象としている自己不活性化レンチウイルスベクターを製造するためにこのシステムを適応させる方法を示しています。 樹状細胞（DC）は、免疫システムの中で最も堅牢な抗原提示細胞である。それらは、直接、プログラムを開始し、腫瘍特異的免疫応答を調節する13ため、それらは、癌ワクチン開発に大きな関心の対象となっている。 DCは、ペプチド又はDNAワクチンよりも強い抗腫瘍免疫応答を誘発する可能性を秘めている含むようにワクチン接種プロトコルを組み込む。最近、我々はレンチウイルスベクターを開発したことspecificallyは修正されたシンドビスウイルス糖タンパク質、SVGmu 14を通じて樹状細胞をターゲットとしています。これらのベクターは、それらが樹状細胞に高い特異性を示し、非特異的、VSVG偽型ベクターより強い免疫応答を生成するという点でユニークです。 ここでは、これらのDC-標的化レンチウイルスベクターを大量に製造する方法を報告している。我々は、これらのDC-DCを論理ボリュームが感染し、強力なCD8 + T細胞免疫応答を生成することができることを実証している。動物に関わるすべての手順は、USC Intitutional動物のケアと使用委員会の承認の下に、人道的に行われた。 インビボおよび臨床実験で行うためには、高力価でウイルスを生産する細胞株を作成することが重要である。ここで説明しているとおりに伝達し、トランスフェクションの手順を実行すると、このようなクローンを生成する可能性を最大化します。

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>DMEM</td>
			<td>Mediatech Inc.</td>
			<td>10-013-CV</td>
			<td></td>
		 </tr>
		 <tr>
			<td>FBS</td>
			<td>Sigma-Aldrich</td>
			<td>F2442</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Glutamine</td>
			<td>Mediatech Inc.</td>
			<td>25-005-Cl</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Doxycycline</td>
			<td>Clontech Laboratories, Inc.</td>
			<td>631311</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Zeocin</td>
			<td>Invitrogen</td>
			<td>R250-01</td>
			<td>Toxic</td>
		 </tr>
		 <tr>
			<td>Puromycin</td>
			<td>Sigma-Aldrich</td>
			<td>P8833</td>
			<td></td>
		 </tr>
		 <tr>
			<td>T4 DNA Ligase</td>
			<td>NEB</td>
			<td>M0202S</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DNeasy Blood &amp; Tissue Kit</td>
			<td>Qiagen</td>
			<td>69506</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

a tetracycline-regulated cell line produces high-titer lentiviral vectors that specifically target dendritic cells

レンチウイルスベクター（論理ボリューム）は、多くの種類の細胞への遺伝物質を送達する強力な手段である。 LVが大量に生成し、これらのHIV-1由来のベクターに関連する安全上の懸念のためには、困難である。本稿では、自己不活性化のLVの高い力価を製造​​する方法を報告している。私たちは、レトロウイルス樹状細胞特異的糖タンパク質、SVGmu含むすべてのウイルス成分を、表現できる細胞株、GPRSを生成するために安定したプロデューサー細胞株GPR TET-OFF形質。その後、我々はLV転送をコードするプラスミドの選択マーカーと組み合わせてレポーター遺伝子GFPをトランスフェクトするコンカテマーDNAトランスフェクションを使用します。得られたクローンのいくつかは、我々は一過性のトランスフェクションでは達成ものより10倍高い力価でLVを生成することができます。さらに、これらのウイルスは、in vitroで効率的樹状細胞を形質導入し、我々のレポーター抗原に対する強力なT細胞免疫応答を生成する。この方法では、良いオプションのFOかもしれないrは、がんや感染症の臨床研究のための強力なLVに基づくワクチンを生産する。

この方法で説明した安定な細胞系は、特に樹状細胞を標的とするレンチウイルスベクターを大量に製造することができる。 図1Bに示すように、個々のクローンの単離質12が変化する安定な細胞株を得た。テストした26クローンのうち、8は、一過性トランスフェクションによって生成されたSVG偽型レンチウイルスベクターのための典型的なベンチマークであるミリリットル?...

Watch this Scientific Journal Video about A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells at JoVE.com

A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells

ここでは、レトロウイルス形質導入およびテトラサイクリンの不存在下でレンチウイルスベクター（LV）の成分を発現できる細胞株を作成するためのコンカテマートランスフェクションを使用する。このLVは、GFPをコードし、樹状細胞上の受容体に特異的な糖タンパク質、SVGmuと偽されています。

テトラサイクリン調節細胞株は、具体的に樹状細胞を標的とする高力価レンチウイルスベクターを生成

Lentiviral  vectors (LVs) are a powerful means of delivering genetic material to many types  of cells. Because of safety concerns associated with these ...

a-tetracycline-regulated-cell-line-produces-high-titer-lentiviral

Research

JoVE Journal

Immunology and Infection

14.4K ビュー.  University of Southern California, Los Angeles. ここでは、レトロウイルス形質導入およびテトラサイクリンの不存在下でレンチウイルスベクター（LV）の成分を発現できる細胞株を作成するためのコンカテマートランスフェクションを使用する。このLVは、GFPをコードし、樹状細胞上の受容体に特異的な糖タンパク質、SVGmuと偽されています。

ビデオ: A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection of pathogenic antigen by na&iuml;ve DCs is through pattern recognition receptors (PRRs) which are able to recognize specific conserved structures referred to as pathogen-associated molecular patterns (PAMPS). Detection of PAMPs by DCs triggers an intracellular signaling cascade resulting in their activation and transformation to mature DCs. This process is typically characterized by production of type 1 interferon along with other proinflammatory cytokines, upregulation of cell surface markers such as MHCII and CD86 and migration of the mature DC to draining lymph nodes, where interaction with T cells initiates the adaptive immune response2,3. Thus, DCs link the innate and adaptive immune systems. 
The ability to dissect the molecular networks underlying DC response to various pathogens is crucial to a better understanding of the regulation of these signaling pathways and their induced genes. It should also help facilitate the development of DC-based vaccines against infectious diseases and tumors. However, this line of research has been severely impeded by the difficulty of transfecting primary DCs4.
Virus transduction methods, such as the lentiviral system, are typically used, but carry many limitations such as complexity and bio-hazardous risk (with the associated costs)5,6,7,8. Additionally, the delivery of viral gene products increases the immunogenicity of those transduced DCs9,10,11,12. Electroporation has been used with mixed results13,14,15, but we are the first to report the use of a high-throughput transfection protocol and conclusively demonstrate its utility.
In this report we summarize an optimized commercial protocol for high-throughput transfection of human primary DCs, with limited cell toxicity and an absence of DC maturation16. Transfection efficiency (of GFP plasmid) and cell viability were more than 50% and 70% respectively. FACS analysis established the absence of increase in expression of the maturation markers CD86 and MHCII in transfected cells, while qRT-PCR demonstrated no upregulation of IFN&beta;. Using this electroporation protocol, we provide evidence for successful transfection of DCs with siRNA and effective knock down of targeted gene RIG-I, a key viral recognition receptor16,17, at both the mRNA and protein levels.

We present our optimized high-throughput nucleofection protocol as an efficient way of transfecting primary human monocyte-derived dendritic cells with either plasmid DNA or siRNA without causing cell maturation. We further provide evidence for successful siRNA silencing of targeted gene RIG-I at both the mRNA and protein levels.

細胞の成熟せずに樹状細胞の効率的なトランスフェクションに最適化されたプロトコル

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection ...

免疫・感染学

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

細菌付着の研究ではせん断応力を導入する

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

マウス肺樹状細胞の分離

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

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

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

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

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

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

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

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

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

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

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

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

Generation of a Novel Dendritic-cell Vaccine Using Melanoma and Squamous Cancer Stem Cells

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H mice using ALDEFLUOR/ALDH as a marker, and tested their immunogenicity using the cell lysate as a source of antigens to pulse dendritic cells (DCs). DCs pulsed with ALDHhigh CSC lysates induced significantly higher protective antitumor immunity than DCs pulsed with the lysates of unsorted whole tumor cell lysates in both models and in a lung metastasis setting and a s.c.&#160;tumor growth setting, respectively. This phenomenon was due to CSC vaccine-induced humoral as well as cellular anti-CSC responses. In particular, splenocytes isolated from the host subjected to CSC-DC vaccine produced significantly higher amount of IFN&#947; and GM-CSF than splenocytes isolated from the host subjected to unsorted tumor cell lysate pulsed-DC vaccine. These results support the efforts to develop an autologous CSC-based therapeutic vaccine for clinical use in an adjuvant setting.

Evaluated in syngeneic immunocompetent hosts, cancer stem cell (CSC) based dendritic cell (DC) vaccine demonstrated significantly higher antitumor immunity than traditional DC vaccines pulsed with heterogeneous bulk tumor cells.

黒色腫と扁平上癌幹細胞を用いた新規樹状細胞ワクチンの生成

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

ヒト胸腺からの骨髄樹状細胞と上皮細胞の単離

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. The immune system is a constant balance in maintaining tolerance to self-molecules and reacting rapidly to pathogens. Dendritic cells (DCs) are powerful professional antigen presenting cells that link the innate immune system to the adaptive immune system and balance the adaptive response between self and non-self. Depending on the maturation signals, immature dendritic cells can be selectively stimulated to differentiate into immunogenic or tolerogenic DCs. Immunogenic dendritic cells provide proliferation signals to antigen-specific T cells for clonal expansion; while tolerogenic dendritic cells regulate tolerance by antigen-specific T-cell deletion or clonal expansion of regulatory T-cells. Due to this unique property, dendritic cells are highly sought after as therapeutic agents for cancer and autoimmune diseases. Dendritic cells can be loaded with specific antigens in vitro and injected into the human body to mount a specific immune response both immunogenic and tolerogenic. This work presents a means to generate in vitro from monocytes, immature monocyte derived dendritic cells (moDCs), tolerogenic and mature moDCs that differ in surface marker expression, function and metabolic phenotypes.

Immature dendritic cells can be selectively differentiated into tolerogenic or mature dendritic cells to regulate the balance between immunity and tolerance. This work presents a means to generate from immature monocyte derived dendritic cells (moDCs), in vitro tolerogenic and mature moDCs that differ in metabolic phenotypes.

代謝表現型が異なる未熟、熟女と寛容原性樹状細胞の生成

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. ...

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. Among them, reversible lysine acetylation is one of the most widely distributed in all domains of life. Although numerous mass spectrometry-based acetylome studies have been performed, further characterization of these putative acetylation targets has been limited. One possible reason is that it is difficult to generate purely acetylated proteins at desired positions by most classic biochemical approaches. To overcome this challenge, the genetic code expansion technique has been applied to use the pair of an engineered pyrrolysyl-tRNA synthetase variant, and its cognate tRNA from Methanosarcinaceae species, to direct the cotranslational incorporation of acetyllysine at the specific site in the protein of interest. After first application in the study of histone acetylation, this approach has facilitated acetylation studies on a variety of proteins. In this work, we demonstrated a facile protocol to produce site-specifically acetylated proteins by using the model bacterium Escherichia coli as the host. Malate dehydrogenase was used as a demonstration example in this work.

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Genetic code expansion serves as a powerful tool to study a wide range of biological processes, including protein acetylation. Here we demonstrate a facile protocol to exploit this technique for generating homogeneously acetylated proteins at specific sites in Escherichia coli cells.

大腸菌Site-Specifically アセチル化蛋白質を生成する安易なプロトコル

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&amp;#945;

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

超高感度単一分子の開発と検証配列人間のインターフェロン-α のデジタルの酵素免疫測定法

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...