著者らは、ポリICLCの贈り物のためにアンドレスサラザール（Oncovir株）に感謝したいと思います。

1。 PBMCを4の単離と凍結保存<ol><li>無菌スパイクプラズマ転送セットを使用して、白血球搬出袋のアクセスポートのいずれ。 60ミリリットル注射器を用いて、無菌の500mlボトルに患者から得られた白血球搬出を転送する。 </li><li>室温RPMIを用いて元のボリュームを2倍に白血球搬出の音量を調整します。徹底的に混ぜる。 </li><li>優しくフィコール - Paque PLUSのボトルを混ぜて。滅菌50ミリリットルコニカルチューブに12ミリリットルフィコール - Paque PLUSを追加します。 </li><li>フィコールを含む各滅菌50ミリリットルコニカルチューブに希釈白血球搬出製品の優しく層30ミリリットル。フィコールと細胞懸濁液との間のインターフェースを邪魔しないように注意してください。 </li><li>白血球搬出のすべてが積層されるまで手順1.3と1.4を繰り返します。 </li><li>ブレーキなしで室温で20分間、1,000×gで50ミリリットルコニカルチューブを遠心。 </li><li>慎重に各チューブとtransfe由来のPBMCの濁った層を収穫新しい無菌の50mlチューブにR。 </li><li> 50ミリリットルの最終体積に各チューブにRPMIを追加します。反転により穏やかに混合。 </li><li> 4時10分°Cフルブレーキ付、500×gで、細胞を遠心分離します。 </li><li>各チューブから上清を取り除きます。各チューブからの細胞ペレットを再懸濁し、50 mlの最終容積にRPMIを追加します。反転により穏やかに混合。 </li><li> 500×4で6分間遠心℃で細胞を遠心</li><li>各チューブから上清を取り除きます。再懸濁し、1 50ミリリットルコニカルチューブに一緒にプールに細胞懸濁液を。 </li><li>もっとRPMIで50 mlにプールされた末梢血単核球の体積を持ち出す。反転により穏やかに混合。 </li><li> 300×4で6分間遠心℃で細胞を遠心</li><li>上清を取り除きます。 50ミリリットルRPMIで細胞ペレットを再懸濁します。均一な細胞懸濁液を確実にするために穏やかに混合する。 </li><li>セルの数をカウントし、細胞生存率を決定します。生存細胞の総数を計算します。 </li><li> 300で細胞を遠心×4で6分間遠心℃、凍結メディアを準備します。ヒトAB血清（バレーバイオメディカル）において、凍結培地は、10％ジメチルスルホキシド（DMSOミルテニーバイオテク社）で構成されています。 </li><li>上清を取り除きます。寒い凍結メディアで2×10 8細胞/ mlの最終濃度で細胞を再懸濁します。 </li><li> 1.8ミリリットルのクライオバイアルに1ミリリットルのアリコートを作る。 </li><li>制御された速度冷凍庫にクライオバイアルに移し、凍結実行、プログラム1を開始します。実行の最後に、直ちに液体窒素冷凍庫の気相に凍結クリオバイアルを移す。 </li></ol> 2。 0日目：単球から樹状細胞の分化<ol><li>滅菌50ミリリットルコニカルチューブにRPMI / 1％自己血漿30mlのを追加します。 </li><li> 37℃の水浴中で穏やかに撹拌によって凍結したPBMCの融解アリコート。完全に解凍すると、無菌の50ミリリットルコニカルチューブにバイアルの内容を転送します。徹底的に混ぜる。 </li><li> 6分間、細胞を遠心分離室温で500×gで。上清を除去し、RPMI / 1％自己血漿少量のペレット化した細胞を再懸濁します。次いで50 mlの最終容積にもっとRPMI / 1％自己血漿を追加します。徹底的に混ぜる。 </li><li> 500で6分×gで室温では再び細胞を遠心分離。上清を吸引し、50ミリリットルRPMI / 1％自己血漿でペレットを再懸濁します。徹底的に混ぜる。 </li><li>セルの数をカウントし、細胞生存率を決定します。生存細胞の総数を計算します。 </li><li>プレート1.40 40 RPMIのミリリットル/ 225センチメートル2 EasyFlaskへ1％自家血漿中×10 8細胞。すべての細胞がメッキされているまで繰り返します。 </li><li> 1のためにその側にフラットEasyFlasksをインキュベート- 37で2時間°組織培養インキュベーターでCは単球がフラスコに付着できるように、5％CO 2を含む。 </li><li>インキュベーションが完了したら、インキュベーターからEasyFlaskを除去し、非接着細胞USIを除去するために二回洗っNG温めRPMI 30mlの。 </li><li>二回目の洗浄後、400〜1,000 IU / mlのIL-4および100〜1,000 IU / mlのGM-CSF 5-8でRPMI / 1％自己血漿40 mlを加える。このプロトコルのために、我々は400 IU / mlのIL-4、100 IU / mlのGM-CSFを使用しています。 </li><li> 37℃で2日間EasyFlasksをインキュベート°組織培養インキュベーター中でCが5％CO 2を含む。 </li></ol> 3。 2日目：IL-4およびGM-CSFを有する樹状細胞の給餌<ol><li> 400〜1,000 IU / mlのIL-4および100〜1,000 IU / mlのGM-CSFで各EasyFlaskにRPMI / 1％自己血漿4 mlを加え。渦巻くとその側に揺動混ぜる。 </li><li> 37℃の組織培養インキュベーター中、5％CO 2を含むでより多くの3日間EasyFlasksを（5日まで）インキュベートする。 </li></ol> 4。 5日目：未成熟樹状細胞の収穫<ol><li>精力的にフラスコを渦巻くことで、非粘着性と緩く接着細胞を再懸濁するために上下にピペッティングにより文化を収穫。転送新しい50 mlコニカルチューブに回収した細胞。 </li><li>室温で6分間、500×gでチューブを遠心する。上清を取り出し、50ミリリットルRPMI / 1％自己血漿で細胞ペレットを再懸濁します。徹底的に混ぜる。 </li><li>セルの数をカウントし、細胞生存率を決定します。生存細胞の総数を計算します。 </li><li>室温で6分間、500×gでチューブを遠心する。 3ミリリットルRPMI / 6ウェル組織培養プレートで400 IU / mlのIL-4および100 IU / mlのGM-CSFと1％の自己血漿中のウェル当たりプレート1-2×10 6細胞。 </li><li> 37℃でプレートをインキュベート℃で組織培養インキュベーターでは2晩、5％のCOを含む。 </li></ol> 5。 6日目：樹状細胞の成熟と抗原読み込み<ol><li> 10μgの/ mlのKLHに6ウェル培養プレート中のウェルの1/3を入れる。渦巻プレートが混合する。 KLHにのみ第1のDCワクチンに使用されるためKLHは、ウェルの1/3に添加する。その後のDCワクチンの詐欺唯一の腫瘍抗原ペプチドをTainの。 </li><li> DCは異なる刺激を使用して成熟し刺激することができる。臨床試験で用いられている最も一般的な成熟刺激サイトカインのカクテル（IL-1β、TNFαおよびIL-6）及びプロスタグランジンE2（PGE 2）9,10されている。より最近では、Toll様受容体（TLR）アゴニストの使用は人気11,12を集めている。このプロトコルでは、ウェルに2 mg / mlのポリイノシン-ポリシチジル酸-ポリ-L-リジンカルボキシメチルセルロース（ポリICLC）を追加し、よく混ぜる。ポリICLCは、ポリ-L-*-リジンおよびカルボキシメチルセルロース（Oncovir社製）で安定化されているポリ-ICの臨床グレードの製剤である。 </li><li>各ウェルクロス競争13を回避する唯一の単一のペプチドを受け、それらの指定されたウェルには100μg/ mlの長いペプチド抗原（NY-ESO-1および/ ​​またはMelan-A/MART-1）を追加します。 </li><li> 37℃のプレートをインキュベートし、組織培養におけるCインキュベーターがCO 5％を含む&lt;サブ&gt; 2 O / N </li></ol> 6。 7日目：樹状細胞の凍結保存<ol><li>自己血漿および10％DMSOを使用してDC凍結溶液を調製。任意の粒子状物質を除去するために4℃で20分間2,000のDC凍結ソリューション×gで遠心します。新しいラベル15ミリリットルコニカルチューブに上清を移します。 4℃で保存し、使用するまでC。 </li><li>一晩のインキュベーションが終了し、収穫、プール50ミリリットルコニカルチューブにペプチドをパルスした樹状細胞を含むすべての井戸。 </li><li> 6分間500×gでチューブを遠心する。上清を取り除きます。 RPMI / 1％自己血漿5mlの細胞ペレットを再懸濁します。 RPMI / 1％自己血漿で50 mlに総量を持ち出す。徹底的に混ぜる。 </li><li>セルの数をカウントし、細胞生存率を決定します。生存細胞の総数を計算します。 </li><li>室温で6分間、500×gでチューブを遠心する。上清を取り出し、5ミリリットル殺菌の各ペレットを再懸濁しル0.9％のNaCl、USPおよび新しい15ミリリットルコニカルチューブに移す。より滅菌0.9％のNaCl、USPは14 mlに各細胞懸濁液を持参してください。反転によってキャップとミックス。 </li><li>二回前の手順を繰り返します。最後の遠心分離後、上清を除去し、ペレットを乱すことなく、細胞ペレットにできるだけ近くになって。 </li><li> 4のDC凍結溶液中の細胞ペレットを再懸濁し- 20×10 6細胞/ mlとアリコートそれぞれ1.8ミリリットルのクライオチューブバイアルに1ミリリットル。 </li><li> DCワクチンのアリコートを下に凍結し、長期保存のために液体窒素冷凍庫の気相中にすぐに転送するために、制御された速度冷凍庫、プログラム1を使用します。 </li></ol> 7。ロットリリーステスト<ol><li>それは典型的には5〜6週間ワクチン調製後、研究では患者への注射のためにリリースされる前に、DCワクチンの各バッチが評価され、特定のロットリリース基準（ 表1）を満たしている必要があります。 </li><li> 生存：自動細胞計数器を用いたDCワクチンの有効性を評価する。最小許容生存仕様は&gt; 70％である。 </li><li> アイデンティティ：フローサイトメトリーによる成熟DC（CD11cは+ CD14-CD83 +）の身元を評価する。のCD11c +細胞の割合は&gt; 50％であるべきである。 CD11c陽性の割合は+およびCD14 +細胞が&lt;30％のCD11c +、の割合がCD14-、およびCD83 +細胞がなければならない&gt;ことが50％。べき</li><li> 不妊：直接文化やグラムの汚れで嫌気と好気性細菌や真菌の存在のためのテストのDCワクチン。インジケータセルラインと直接培養系及びDNA蛍光色素染色アッセイを用いてマイコプラズマの存在を評価します。これらのテストでは、最小の仕様セットは成長がすべての培養物から観察されないということである。 </li><li> エンドトキシン：キネティック-QCLカイネティック発色LALアッセイを用いてエンドトキシンの内容を評価し、それが最低基準を満たすために&lt;50 EU / mlのでなければなりません。 </li><lI&gt; 機能：同種T細胞とのインキュベーションによって混合リンパ球反応においてDC機能を評価します。刺激されていない樹状細胞と比較して増殖の存在が（ 図3）が報告されている。 </li></ol>

<ol>
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V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+generation+of+broad+T-cell+immunity+in+humans+after+a+single+injection+of+mature+dendritic+cells.">Rapid generation of broad T-cell immunity in humans after a single injection of mature dendritic cells.</a> J. Clin. Invest. 104, 173-180 (1999).</li><li>Schuler-Thurner, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+induction+of+tumor-specific+type+1+T+helper+cells+in+metastatic+melanoma+patients+by+vaccination+with+mature,+cryopreserved,+peptide-loaded+monocyte-derived+dendritic+cells.">Rapid induction of tumor-specific type 1 T helper cells in metastatic melanoma patients by vaccination with mature, cryopreserved, peptide-loaded monocyte-derived dendritic cells.</a> J. Exp. Med. 195, 1279-1288 (2002).</li></ol>

NIH（AI061684、AI071078、AI044628、およびK08 AI084578（ミラー））、ビル＆メリンダ·ゲイツ財団、ドリス·デューク慈善財団、がん研究所、ループス研究のための同盟、そしてエメラルド：著者は、次の中から財政支援を受けている財団。ニーナBhardwajは、免疫力を操作するために、樹状細胞の調製および使用に関連した特許に関する共同発明者である。著者らは、他の関連する提携または任意の組織または団体の金融関心を持つか、または離れて開示されているものから原稿で議論主題または材料と金融紛争を有する金融関わりを持っていません。

フェーズIおよびIIは、単球由来樹状細胞の臨床試験は、患者しかしながら、臨床的成功は1に限定されているにおける免疫応答を誘導することが示されている。これは、腫瘍免疫療法用に最適DCを生成する方法に関するコンセンサスが不足している一因であってもよい。臨床グレードのDCを生成する多数の方法があるが、これらの方法は、単球、成熟を誘導するために使用される刺激、...

<table border="1">
<tbody>
 <tr>
 <td fo:width="2in">Reagent/Supplies/Equipment</td>
 <td>Manufacturer</td>
 <td>Catalog No.</td>
 <td></td>
 </tr>
 <tr>
 <td>RPMI-1640 medium with L-glutamine</td>
 <td>BioWhittaker</td>
 <td>12-702F</td>
 <td></td>
 </tr>
 <tr>
 <td>1M HEPES buffered saline</td>
 <td>BioWhittaker</td>
 <td>17-737E</td>
 <td></td>
 </tr>
 <tr>
 <td>Phosphate buffered saline (PBS)</td>
 <td>BioWhittaker</td>
 <td>17-516F</td>
 <td></td>
 </tr>
 <tr>
 <td>Human albumin, 25% solution USP</td>
 <td>Aventis Behring</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Ficoll-Hypaque PREMIUM</td>
 <td>GE Healthcare</td>
 <td>17-5442-03</td>
 <td></td>
 </tr>
 <tr>
 <td>Human AB serum</td>
 <td>Valley Biomedical</td>
 <td>HP1022</td>
 <td></td>
 </tr>
 <tr>
 <td>Sterile saline USP</td>
 <td>Hospira</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>CryoMACS DMSO </td>
 <td>Miltenyi Biotec</td>
 <td>170-076-303</td>
 <td></td>
 </tr>
 <tr>
 <td>Leukine GM-CSF, 0.5 mg/ml</td>
 <td>Berlex</td>
 <td>A02266</td>
 <td></td>
 </tr>
 <tr>
 <td>MACS GMP IL-4</td>
 <td>Miltenyi Biotec</td>
 <td>170-076-101</td>
 <td></td>
 </tr>
 <tr>
 <td>Hiltonol, Poly-ICLC, 2 mg/ml</td>
 <td>Oncovir</td>
 <td>NA</td>
 <td></td>
 </tr>
 <tr>
 <td>VACMUNE KLH</td>
 <td>Biosyn</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>225 sq cm EasyFlasks</td>
 <td>Nalgene Nunc</td>
 <td>159934</td>
 <td></td>
 </tr>
 <tr>
 <td>Falcon 6-well tissue culture plates</td>
 <td>Becton Dickinson</td>
 <td>353046</td>
 <td></td>
 </tr>
 <tr>
 <td>1.8 ml CryoTube vials</td>
 <td>Nalgene Nunc</td>
 <td>377267</td>
 <td></td>
 </tr>
 <tr>
 <td>Controlled Rate Freezer</td>
 <td>Thermo</td>
 <td>CryoMed</td>
 <td></td>
 </tr>
 </tbody>
</table>

preparation of tumor antigen-loaded mature dendritic cells for immunotherapy

臨床研究は、抗原·ロードDCワクチンは腫瘍1のための安全かつ有望な治療法であり、それらの臨床的有効性が確立されて残っていることが確立しているが。方法は、良好な製造技術（GMP）のガイドラインに準拠して作成、後述するように、臨床研究のためのDC 2を大量に生成するための最も一般的なex vivoでの調製方法の最適化である。 提案手法では、樹状細胞を刺激するための合成TLR 3アゴニストポリイノシン - ポリシチジル酸 - ポリ-L-リジンカルボキシメチルセルロース（ポリICLC）を利用する。 CD83およびCD86のアップレギュレーションにより評価されるポリICLCはヒトDCのための最も強力な個々の成熟刺激であることが確立我々の以前の研究では、インターロイキン12の誘導（IL-12）、腫瘍壊死因子（TNF）、インターフェロンγ誘起タンパク質10（IP-10）、interleukmin 1（IL-1）、I型インターフェロン（IFN）、および最小のインターロイキン10（IL-10）産生。 </p&gt; 樹状細胞は、白血球搬出により得られた凍結末梢血単核細胞（PBMC）から区別される。 PBMCを、フィコール勾配遠心分離によって単離し、アリコートで凍結する。 1日目では、PBMCを解凍され、℃の中の組織培養インキュベーターで、37〜2時間インキュベートした後、プラスチック表面に付着した単球を選択するために組織培養フラスコに播種。インキュベーション後、リンパ球が洗い落とされ、接着単球を未成熟DCに分化するインターロイキン-4（IL-4）および顆粒球マクロファージコロニー刺激因子（GM-CSF）の存在下で5日間培養する。 6日目に、未成熟DCは、ワクチンの品質の制御として機能し、ワクチン3の免疫原性を高めることがキーホールリンペットヘモシアニン（KLH）タンパク質でパルスされています。 DCは、成熟に刺激ペプチド抗原を搭載し、一晩インキュベートする。 7日目に、細胞を洗浄し、含有する、1mlのアリコートで凍結4 -制御された速度冷凍庫を使って20×10 6細胞。 DCのバッチのロットリリーステストが行​​われ、それらが患者に注入される前に、最低限の仕様を満たしている必要があります。

10との間 - 出発したPBMCの20％は培養期間の終了時にDCへと分化する。成熟DCはあるのCD11c +、CD14-、CD83 +、CD40 +、およびCCR7 +（ 図1）。それらは、MHCクラスIおよびII分子および共刺激分子CD80およびCD86の高いレベルを発現する。他のTLRアゴニスト14と比較して、ポリICLCまた、PDL-1の低レベルを誘導した。さらに、これらのポリIC-成熟DCを分泌する大IL-12の量（ 図2及...

Watch this Scientific Journal Video about Preparation of Tumor Antigen-loaded Mature Dendritic Cells for Immunotherapy at JoVE.com

Preparation of Tumor Antigen-loaded Mature Dendritic Cells for Immunotherapy

腫瘍免疫療法に使用するための自​​己樹状細胞（DC）を大量に生成するための最も一般的に使用される方法が記載されている。この方法は、単球から樹状細胞を区別するためにIL-4およびGM-CSFを使用する。彼らは患者に再び注入される前に、未成熟DCは、成熟した後、抗原でロードに刺激される。

免疫療法のための腫瘍抗原にロードされた成熟樹状細胞の調製

While clinical studies have established that antigen-loaded DC vaccines are safe and promising therapy for tumors 1, their clinical efficacy remains to be ...

preparation-tumor-antigen-loaded-mature-dendritic-cells-for

Research

JoVE Journal

Immunology and Infection

18.1K ビュー.  NYU Langone Medical Center. 腫瘍免疫療法に使用するための自​​己樹状細胞（DC）を大量に生成するための最も一般的に使用される方法が記載されている。この方法は、単球から樹状細胞を区別するためにIL-4およびGM-CSFを使用する。彼らは患者に再び注入される前に、未成熟DCは、成熟した後、抗原でロードに刺激される。

ビデオ: Preparation of Tumor Antigen-loaded Mature Dendritic Cells for Immunotherapy

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

B細胞のCD40 -活性化のモデルマウス

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

免疫・感染学

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 ...

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 CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo to express chimeric antigen receptors specific for glioma antigens (CAR T cells). The expansion and function of adoptively transferred CAR T cells can be potentiated by the lymphodepletive and tumoricidal effects of standard of care chemotherapy and radiotherapy. We describe a method for generating CAR T cells targeting EGFRvIII, a glioma-specific antigen, and evaluating their efficacy when combined with a murine model of glioblastoma standard of care. T cells are engineered by transduction with a retroviral vector containing the anti-EGFRvIII CAR gene. Tumor-bearing animals are subjected to host conditioning by a course of temozolomide and whole brain irradiation at dose regimens designed to model clinical standard of care. CAR T cells are then delivered intravenously to primed hosts. This method can be used to evaluate the antitumor efficacy of CAR T cells in the context of standard of care.

The lymphodepletive and immunomodulatory effects of chemotherapy and radiation standard of care can be leveraged to enhance the antitumor efficacy of T cell immunotherapy. We outline a method for generating EGFRvIII-specific chimeric antigen receptor (CAR) T cells and administering them in the context of glioblastoma standard of care.

ケアの膠芽腫の標準のコンテキストにおける養子療法のためのCAR T細胞の生成

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo ...

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 CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph node (DLN). The mobilization of DCs to the DLN is complex and remains to be fully elucidated during infection. Herein described is the use of an innovative, simple assay that relies on the fluorochrome 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) to track the migration of DCs during footpad infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin (BCG) in C57BL/6 mice. This assay enables the characterization of skin DC sub-populations that actively relocate to the draining, popliteal LN in response to BCG. This protocol originates from a BCG model where migratory skin DCs were identified by flow cytometry. The assay is amiable to the study and identification of DCs or other cells that home to the popliteal LN after inoculation of microbes, their metabolites or other inflammatory stimuli in the footpad, and consequently to study factors that regulate the migration of these cells.

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with &lt;em&gt;Mycobacterium bovis&lt;/em&gt; Bacille Calmette-Gu&amp;#233;rin

An assay in mice to track cell migration from skin to draining lymph node is described which enables the characterization of skin Dendritic cells mobilized to the lymph node after footpad infection with Bacille Calmette-Gu&#233;rin.

での感染の間に流入領域リンパ節へのマウスの皮膚樹状細胞の移動を研究するためにCFSEベースアッセイ<em&gt;マイコバクテリウム・ボビス</em&gt;カルメット - ゲラン

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

全血からのヒト単球由来樹状細胞の生成

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

まれな抗原特異的 B 細胞血液の循環から弁別に対するモノクローナル抗体の生成

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

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 ...

Novel Protocol for Generating Physiologic Immunogenic Dendritic Cells

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university centers worldwide. While ECP&#8217;s clinical efficacy and exemplary safety profile have driven its widespread use, elucidation of the underlying mechanisms has remained a challenge, partly owing to lack of a laboratory ECP model. To overcome this obstacle and create a simple, user-friendly platform for ECP research, we developed a scaled-down version of the clinical ECP leukocyte-processing device, suitable for work with both mouse models, and small human blood samples. This device is termed the Transimmunization (TI) chamber, or plate. In a series of landmark experiments, the miniaturized device was used to produce a cellular vaccine that regularly initiated therapeutic anti-cancer immunity in several syngeneic mouse tumor models. By removing individual factors from the experimental system and ascertaining their contribution to the in vivo anti-tumor response, we then elucidated key mechanistic drivers of ECP immunizing potential. Collectively, our results revealed that anti-tumor effects of ECP are initiated by dendritic cells (DC), physiologically generated through blood monocyte interaction with platelets in the TI plate, and loaded with antigens from tumor cells whose apoptotic cell death is finely titrated by exposure to the photoactivatable DNA cross-linking agent 8-methoxypsoralen and UVA light (8-MOPA). When returned to the mouse, this cellular vaccine leads to specific and transferable anti-tumor T cell immunity. We verified that the TI chamber is also suitable for human blood processing, producing human DCs fully comparable in activation state and profile to those derived from the clinical ECP chamber. The protocols presented here are intended for ECP studies in mouse and man, controlled generation of apoptotic tumor cells with 8-MOPA, and rapid production of physiologic human and mouse monocyte-derived DCs for a variety of applications.

The transimmunization (TI) device or plate and related protocols have been developed to replicate the key features of extracorporeal photochemotherapy (ECP), in an experimental setting, allowing for production of physiologically activated, tunable dendritic cells (DCs) for cancer immunotherapy.

生理学的免疫原性樹状細胞を生成するための新規プロトコル

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university ...