This work was supported by grants BFU2011-29450, BFU2008-04342/BMC from the Spanish Ministry of Science and Innovation and PIES201020I046 from Consejo Superior de Investigaciones Cient&#236;ficas (CSIC).

注意：実験手順は、大学自治・デ・マドリッドの研究倫理委員会によって承認され、スペイン語、欧州のガイドラインに従って動物福祉と健康の大学自治・デ・マドリードヘッドの監督の下で行われました。マウスは、特定病原体不在（SPF）は、ハウジングで飼育され、それらは、二酸化炭素（CO 2）吸入法を用いて、訓練を受けた有資格者により安楽死させました。 1.マウスの骨髄由来DC分化および感染注： 図1は、この最初のステップをまとめたものです。すべての手順は、メディア、楽器、ピペットチップ及び培養皿だけ滅菌を使用して、この時点からフード内で行われるべきです。 <ol><li>マウス骨髄由来DCの分化<ol><li> 1 C57 / BL6 マウス 22から脛骨と大腿骨を解剖し、RPMI培地10mlでプラスチックの皿に移します。 </lI&gt; <li>滅菌はさみで、各骨端をカットし、特徴的な赤の鮮やかな色をしている骨髄を、公開しています。 </li><li>滅菌ペトリに無菌注射器を用いて0.5％ウシ血清アルブミン（BSA）および5mMエチレンジアミン四酢酸（EDTA）（PBS / BSA / EDTA緩衝液）を含有するリン酸緩衝生理食塩水（PBS）10mlで骨の内部に吹き込みます皿。 </li><li>冷却遠心機（4℃）で600×gでチューブ中の細胞懸濁液と遠心分離機を収集します。 </li><li>塩化アンモニウム-カリウム（ACK）溶解緩衝液（0.15 M NH 4 Clを 、10mMのKHCO 3および0.1mM EDTA）を1mlで細胞を再懸濁し、赤血球を除去するために室温で30秒間インキュベートします。 </li><li>塊は、濾過により除去されたので、同じ速度（600×gで）で遠心分離する前に、骨片および細胞塊を除去するために、セルストレーナー（70ミクロン）を介して、10％ウシ胎児血清（FBS）を含むRPMI 1640培地10mlを加え、濾過します。 </li><li>細胞をカウントし、AND RPMI 10％FBS（50μMのβ-メルカプトエタノールを含有し、1mMピルビン酸ナトリウム）、5×10 5細胞/ mlに調整し、20 ngの/ mlの組換えマウス顆粒球-マクロファージコロニー刺激因子（RM-GM-CSF）を追加最終濃度として。 </li><li> CO 2インキュベーター中で、滅菌、微生物学的品質、15 cmでペトリ皿、培養にこの懸濁液を加える（37℃、5％CO 2）。 </li><li> 3日ごとに、20 ng / mlでRM-GM-CSFを含有する新鮮な培地で、細胞密度を5×10 5細胞/ mlで、非接着細胞およびPBS中の5mM EDTAで剥離した細胞を再懸濁両方をスピンダウン。 </li></ol></li><li> DCおよび抗原負荷の成熟<ol><li> 9日目に、20 ngの/ mlのRM-GM-CSFを含有する培地中濃度の5×10 5細胞/ mlで細胞を再懸濁。その後、細胞に高MHC-IIの発現を可能にし、24時間非組織培養処理滅菌15センチメートルペトリ皿にインキュベートすることを20ng /リポポリサッカライド（LPS）のミリリットルを追加します。 </li><li> 1の後LPS刺激の日は、フローサイトメトリーによってDCの分化を確認するために、マウスのCD11c、MHCIIおよびGR-1に対する抗体で、いくつかのDC細胞を染色します。 DCのフローサイトメトリー分析の例を図3A及び 3Bに示されています。 <ol><li>サンプルあたり50μlのPBS / BSA / EDTA緩衝液中の最終濃度2.5μgの/ mlの抗マウスCD16 / CD32モノクローナル抗体を用いたDC（5×10 5細胞 /サンプル）をインキュベートします。それぞれPBS / BSA / EDTA緩衝液で500：100：1：200および1その後、1で異なる蛍光色素とコンジュゲートMHCII（IA / IE）、のCD11cおよびGR-1に対する抗体の50μlを添加します。 </li><li>最後に、PBS / BSA / EDTAバッファーでDCを洗浄し、製造業者のプロトコールに従って、フローサイトメトリーによって分析します。 注：細胞が十分に分化している場合は、細胞が抗原負荷の準備ができています。 </li></ol></li><li>ウォッシュ（抗生物質を含まない）RPMI、10％FBSを含む樹状細胞および10μg/ mlのOTII OVAp（OVA 323-339でそれらをインキュベート; ISQA37ºCで最小1時間、5×10 6のDCあたりRMPI 10％FBS培地1ml中のプラスチックチューブにVHAAHAEINEAGR）。陰性対照として、OVApとインキュベートすることなく、DCを残します。 </li></ol></li><li> DCの感染<ol><li> CO 2インキュベーター（37℃、5％CO 2）で1時間10細菌（DCあたり10細菌）の感染多重度（MOI）での樹状細胞に感染します。 </li><li>ウォッシュDC細胞は、細胞外細菌の大部分を洗浄するためにRMPI、10％FBSおよび450×gで遠心分離にPBSおよび1倍で3倍。最後に、20×10 6細胞/ mlで（抗生物質を含まない）RMPI 10％FBS培地中で細胞を再懸濁し。 </li></ol></li></ol> OTIIトランスジェニックマウスからのCD4 + Tリンパ球の2の単離注： 図1は、この第二段階をまとめたものです。リンパ節は、リンパ節におけるCD4 +リンパ球の割合ので、CD4 + T細胞を単離する代わりに脾臓を使用すべきですS（〜50％）、脾臓（〜25％）よりも大きいので、精製はより有効であろう。 <ol><li>リンパ節の単一細胞懸濁液を作製<ol><li> OTIIトランスジェニックマウスから23ノード鼠径部、腋窩、上腕、子宮頸部および腸間膜リンパを削除し、RPMI、10％FBS培地10mlでプラスチックの皿に移します。 </li><li> 2つや消し顕微鏡スライドを使用して無菌フード下のリンパ節を挽きます。 1顕微鏡スライドのつや消し側のリンパ節を置き、臓器がグラウンドされるまで、第2のスライドのつや消し側でこすります。 </li><li> PBS / BSA / EDTA緩衝液中の単一細胞懸濁液を洗​​ってください。 </li><li>セルストレーナー（70μm）は、結合組織を除去し、PBS / BSA / EDTA緩衝液で洗浄するためにかかわらず、細胞をフィルタリングします。 </li><li> ACK溶解緩衝液1ml中に細胞を再懸濁し、赤血球を除去するために室温で1分間インキュベートします。 </li></ol></li><li> CD4 + T細胞の単離<ol><li>ステップ2.1.3カウントのように再び洗浄し細胞を、PBS / BSA / EDTA緩衝液中の100×10 6細胞/ mlで再懸濁します。 CD4 + T細胞の後の負の選択のために氷上で30分間、250：CD8、IgM抗体、B220、CD19、MHCクラスII（I-AB）、のCD11b、CD11cの1でDX5に対するビオチン化抗体を追加します。 </li><li> PBS / BSA / EDTA緩衝液中で細胞を洗浄し、氷上で15分間、製造業者のプロトコールで推奨濃度でストレプトアビジンマイクロビーズを用いた細胞（100×10 6細胞/ ml）をインキュベートします。 </li><li> PBS / BSA / EDTA緩衝液中で細胞を洗浄し、CD4 + T細胞の単離の前に細胞ストレーナー（30ミクロン）かかわらず、それらをフィルタリングします。 </li><li>製造業者のプロトコルに従って、磁気細胞分離機を用いて陰性選択によってCD4 + T細胞を単離します。 </li><li>細胞をカウントし、（抗生物質を含まない）RPMI、10％FBS培地に4×10 6細胞/ mlに調整します。 </li><li>流れにより純度を確認して修正し、氷単離したCD4 + T細胞 （3×10 5細胞 ）を続けますサイトメーター24を説明します。 CD4 + T細胞の精製の ​​一例は、図3Cに示されています。 </li></ol></li></ol>ゲンタマイシン保護アッセイ3. T細胞Transinfection測定注： 図2は、プロトコルのこの第三の工程をまとめたものです。 <ol><li> T細胞Transinfection <ol><li> CO 2インキュベーター（37℃、5％における免疫シナプス形成を可能にするために30分間（DCを1時間感染させ、自由な細菌を除去するために洗浄した）感染したDC細胞（1）を有する単離されたCD4 + T細胞をインキュベートCO 2）。 </li><li>追加単離されたCD4 + T細胞（2×10 6細胞）を、24ウェル培養プレートに感染したDC（2×10 6細胞 ）を0.1mlの0.5 mlです。陰性対照として、30分間、10細菌のMOIで直接0.5 mlのCD4 + T細胞に感染します。 </li><li>使用したT細胞から感染したDCを分離する追加のコントロールを含めますDC-Tの物理的接触を妨げる（孔径3ミクロンを有する）ポリカーボネートトランスウェル障壁。ウェル（24ウェルプレート）の下の区画に0.1トランスウェル内部のDC mlのCD4 + T細胞の0.5ミリリットルを追加します。 </li><li>最後に、RMPI培地で完全に全てのサンプルにおいて同等のボリュームを作成する各ウェルの0.6ミリリットルがあるまで。 </li><li>免疫シナプス形成の30分後、ゲンタマイシン100μg/ mlを添加し、CO 2インキュベーター中で細胞を収集する前に1時間インキュベート（37℃、5％CO 2）。 </li></ol></li><li>再分離CD4 + T細胞の<ol><li>非接着細胞を収集し、PBS / BSA / EDTA緩衝液で再懸濁します。 DCの大部分は、プラスチック培養皿に付着したままです。ボルテックスチューブを静かに細胞分解を確実にします。ゲンタマイシンがうまく働いていることを示すためのコントロールとして細胞上清500μlのを保管してください。 </li><li>ステップ2.2と一緒にDCおよびT細胞を含む試料からCD4 + T細胞を再単離します、が、DCのほとんどがプレートに付着している必要があります。氷の上にコントロールサンプルを保管してください。アカウントに2％未満の汚染との唯一の実験を行ってください。 transinfection後のCD4 + T細胞の精製の ​​一例が図3Dに示されています。 </li></ol></li><li>溶解性T細胞および細菌中寒天プレート上でそれらをシード。 <ol><li> PBSで2回洗浄CD4 + T細胞。 </li><li>細胞をカウントし、PBS中で2×10 6細胞/ mlで再懸濁。 </li><li> T細胞からの細胞内細菌を放出する、1×10 6細胞/ mlで、それらを溶解するT細胞を500μlの0.1％トリトンX-100の500μLを加えます。 </li><li>溶解した細胞の3連続小数の希釈を行い、細菌培地寒天プレート上で各希釈液50μlをシード。 4の部分に板を分割し、各部分に溶解した細胞の異なる希釈をシード。代表的な結果を図4に示します 。 <ol><li>対照として、またプレートゲンタマイシンがうまく働いていることを確認するために、寒天のコンジュゲートの細胞上清を保管していました。また、追加の対照として、感染したDCをプレート。ケースでは、低DCの汚染は（&lt;2％）、低直流汚染に対応するコロニー形成単位（CFUの）を引く起こります。 </li></ol></li></ol></li></ol>フローサイトメトリーによるT細胞Transinfection 4.定量注： 図2は、この手順をまとめたものです。 <ol><li> T細胞Transinfection <ol><li>ステップ1.3のようにのBMDCに感染するが、この場合には、GFP発現する細菌を使用しています。 </li><li>製造業者のプロトコルに従って、37℃で30分間、細胞トラッカークロロメチルアミノクマリン（CMAC）と（ステップ2.2でリンパ節から単離された）ステインCD4 + T細胞ºC。 </li><li>二回（抗生物質を含まない）RMPI 10％のFBSで標識されたCD4 + T細胞を洗浄します。 </li><li>感染した樹状細胞をインキュベートし、標識CD4 +と（細菌と1時間のインキュベーション後）30分間のT細胞は、ステップ3.1.1で説明するすべてのコントロールを含む37ºCで免疫シナプスの形成を可能にします。 </li></ol></li><li>感染CD4 + T細胞の染色<ol><li> DC-T免疫シナプス形成の30分後、細胞を回収し、PBS / BSA / EDTAでそれらを洗浄。 </li><li> PBS / BSA / EDTA緩衝液、静かにチューブをボルテックスすることにより独立した凝集細胞で2回洗浄した後。 </li><li>室温で15分間、3％PFAを含むPBSを0.2ml /チューブでサンプルを修正しました。その後、PBSでそれらを洗ってください。 </li><li>樹状細胞のFc受容体をブロックするために、氷上で15分間、PBS / BSA / EDTAを50μl/サンプル中の抗マウスCD16 / CD32モノクローナル抗体（2.5μgの/ ml）でインキュベートします。 </li><li>細胞外細菌を染色し、氷上で30分間50μL/ PBS / BSA / EDTAのサンプル中の抗細菌抗体（最終濃度として10 / mlの）ウサギを追加します。 </li><li> PBS / BSAは/ EDTAで細胞を洗浄し、フィコエリトリン（PE）とコンジュゲートしたマウスのCD11cに対する抗体およびSEでインキュベート氷上で30分間、PBS / BSA / EDTAを100μl/サンプル中の200：1でアレクサフルーア647のような他の蛍光色素と結合したウサギに対するcondary抗体。 </li><li>細胞を洗浄し、フローサイトメトリーによってサンプルを分析。ただ一つのサンプルを補償し、設定25フローサイトメトリーを調整するために実験で使用した各蛍光色素で標識の非蛍光細菌などネガティブコントロールとサンプルを使用してtransinfected T細胞のサンプルを含めることを忘れないでください。代表的な分析は、 図5に示されています。 注：細菌は、GFPを発現しない細胞内および細胞外細菌を区別するために、細胞外細菌をラベル、修正し、5分間PBS中の0.5％トリトンX-100を含む試料を透過した場合。その後、異なる蛍光色素で総細菌（細胞内+外）を染色します。 </li></ol></li></ol>

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J., Allison, J., Heath, W. R., Carbone, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+TCR+expression+in+transgenic+mice+constructed+using+cDNA-based+alpha-+and+beta-chain+genes+under+the+control+of+heterologous+regulatory+elements.">Defective TCR expression in transgenic mice constructed using cDNA-based alpha- and beta-chain genes under the control of heterologous regulatory elements.</a> Immunology and Cell Biology. 76 (1), 34-40 (1998).</li><li>Matheu, M. P., Sen, D., Cahalan, M. D., Parker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+bone+marrow+derived+murine+dendritic+cells+for+use+in+2-photon+imaging.">Generation of bone marrow derived murine dendritic cells for use in 2-photon imaging.</a> Journal of Visualized Experiments: JoVE. (17), (2008).</li><li>Inaba, K., Inaba, M., 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=Generation+of+large+numbers+of+dendritic+cells+from+mouse+bone+marrow+cultures+supplemented+with+granulocyte/macrophage+colony-stimulating+factor.">Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor.</a> Journal of Experimental Medicine. 176 (6), 1693-1702 (1992).</li><li>Th&#246;ne, F., Schwanh&#228;usser, B., Becker, D., Ballmaier, M., Bumann, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FACS-isolation+of+Salmonella-infected+cells+with+defined+bacterial+load+from+mouse+spleen.">FACS-isolation of Salmonella-infected cells with defined bacterial load from mouse spleen.</a> Journal of Microbiological Methods. 71 (3), 220-224 (2007).</li><li>Vaudaux, P., Waldvogel, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gentamicin+antibacterial+activity+in+the+presence+of+human+polymorphonuclear+leukocytes.">Gentamicin antibacterial activity in the presence of human polymorphonuclear leukocytes.</a> Antimicrobial Agents and Chemotherapy. 16 (6), 743-749 (1979).</li><li>Zhang, X., Goncalves, R., Mosser, D. M., Coligan, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+14,+The+isolation+and+characterization+of+murine+macrophages.">Chapter 14, The isolation and characterization of murine macrophages.</a> Current protocols in immunology. , Unit 14.1 (2008).</li><li>Bedoya, S. K., Wilson, T. D., Collins, E. 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The authors have nothing to disclose.

T細胞またはTリンパ球は細胞性免疫において中心的な役割を果たし、適応免疫応答26に属している白血球の一種です。 T細胞は 、インビトロで感染されるのに不応性であるが、いくつかの報告は、それらがインビボ14,15 に感染させることができることを示しています。免疫シナプス中のAPCとT細胞の緊密な連絡先は、HIV 11のようないくつかの?...

病原体は、ホストに感染すると、細菌排除のために必要な自然免疫と適応免疫応答の活性化は、通常はあります。自然免疫は、ほとんどの感染を防ぐ防御の最前線です。自然免疫は、微生物の広範なグループの間で保存されている正確な方法要素（病原体関連分子パターン、PAMPS）1に区別します。先天性免疫機構は、食細胞（マクロファージ、好中球、および樹状細胞）、マスト細胞、好酸球、好塩基球、およびナチュラルキラー細胞を含む皮膚、化学障壁（抗菌ペプチド、リゾチーム）と先天的白血球、などの物理的障壁を含みます2。これらの細胞は、コンタクトを介して、または病原体巻き込むと殺害を含んで食作用を介してそれらを攻撃することによってのいずれか、特定して病原体を排除します。このシステムは、pに対する免疫記憶を与える適応免疫とは対照的に、生涯の防衛を許可していませんathogens。適応免疫系は、防衛の2行目で、認識し、複数の微生物および非微生物物質3の特定の抗原に反応することができます。適応免疫系の主要な構成要素は、BおよびT細胞を含むリンパ球です。 B細胞は、病原体または外来性タンパク質に対する抗体を分泌する、体液性応答に関与しています。しかし、T細胞は、サイトカイン分泌または殺す病原体に感染した細胞を4との免疫応答を調節する、細胞性免疫を表します。 主要組織適合複合体（MHC）5-7によって細胞表面に提示される抗原に貪食病原体及びプロセス細菌成分を認識することができ、樹状細胞またはマクロファージ、先天性免疫系の構成成分を含む、抗原提示細胞（APC）。 APCが病原体を貪食した後、彼らは通常、彼らはTと対話流入領域リンパ節への移行します細胞。 Tリンパ球は、そのT細胞レセプターによって特異的ペプチド-MHC複合体を認識することができます。免疫シナプス（IS）は、抗原提示8,9の間に抗原負荷APCとリンパ球との界面に発生します。いくつかの細菌は、食作用を生き残るためには、APCの内部に体系的に普及させることができます。このビューでは、感染したAPCは、細菌の貯水池や細菌の拡散10を容易に 「トロイの木馬」としての役割を果たす。 IS形成の過程で起こりAPCおよびリンパ球の間の密接な接触はまた、膜の一部の交換、遺伝物質およびエキソソームのためのプラットフォームとして機能し、T細胞を感染させるためにいくつかのウイルスのハイジャックすることができます。このプロセスはtransinfection 11-13と呼ばれています。 いくつかの病原性細菌（ リステリア菌 、 サルモネラ菌や赤痢菌）は 、in vivoでのTリンパ球に侵入し、その挙動14-16を変更することができます。我々は持っています最近、Tリンパ球は、抗原提示16の過程で以前に感染した樹状細胞（DC）からtransinfectionによって細菌を捕捉することができることを説明しました。 T細胞の直接感染より（1,000-4,000x）極めてより効果transinfectionによる細菌捕獲。 T細胞はtransinfectionはT細胞によって駆動されるプロセスよりも示す病原体及び非病原体細菌を捕捉します。驚くべきことに、細胞が急速に捕捉された細菌を殺し、プロの食細胞16よりもので、より効率的にやったT（東工大）transinfected。免疫学の定説を破るこれらの結果は、適応免疫の細胞は、先天性免疫のおそらく排他的であった機能を実行することができることを示しています。加えて、我々は、TIT細胞が前炎症性サイトカインを大量に分泌し、 インビボでの細菌感染から保護することが示されました。 ここでは、細菌transinfectionプロセスを研究するために使用される様々なプロトコルを提示マウスモデル。このモデルは、細菌に感染した骨と特異的に相互作用するI-AB 17の文脈においてOVA（OVAp）ペプチド323-339に特異的なTCRを負担OTIIトランスジェニックマウスからのCD4 + T細胞の使用に基づきますmarrow-安定した免疫シナプスを形成OVApでロード由来DC（BMDCを）18,19、。 T細胞transinfectionを可視化し、蛍光顕微鏡を使用して追跡することができます。さらに、フローサイトメトリー、緑色蛍光タンパク質（GFP）16,20を発現する細菌によって放出される蛍光を利用して、感染した細胞を検出するために使用することができます。さらに、T細胞transinfectionより敏感なアプローチ、多数のイベントの測定を可能にゲンタマイシン生存アッセイにより定量することができます。ゲンタマイシンは、真核細胞に浸透することができない抗生物質です。したがって、この抗生物質を使用して、抗生物質添加FRを生き残った細胞内細菌の分化を可能にします21を殺されたオム外のもの。

<table border="0" cellpadding="0" cellspacing="0" width="743">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">RPMI</td>
			<td style="width:131px;">Fisher Scientific</td>
			<td style="width:104px;">SH3025501</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">r-GMCSF</td>
			<td style="width:131px;">Peprotech</td>
			<td style="width:104px;">315-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">LPS</td>
			<td style="width:131px;">SIGMA</td>
			<td style="width:104px;">L2630-10mg</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Na Pyruvate</td>
			<td style="width:131px;">Thermo Scientific</td>
			<td style="width:104px;">SH3023901</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">2-ME</td>
			<td style="width:131px;">Gibco</td>
			<td style="width:104px;">31350-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">OVAp OTII (323&#8211;339)</td>
			<td style="width:131px;">GenScript</td>
			<td style="width:104px;">---</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Cell Strainer 70uM</td>
			<td style="width:131px;">BD</td>
			<td align="right" style="width:104px;">352350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;30uM Syringe Filcons Sterile</td>
			<td>BD</td>
			<td align="right">340598</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">AutoMacs Classic</td>
			<td style="width:131px;">Miltenyi Biotec</td>
			<td style="width:104px;">130-088-887</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Gentamicin</td>
			<td style="width:131px;">Normon</td>
			<td style="width:104px;">624601.6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Transwell</td>
			<td style="width:131px;">Costar</td>
			<td align="right" style="width:104px;">3415</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">LB</td>
			<td style="width:131px;">Pronadisa</td>
			<td align="right">1231</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Agar</td>
			<td style="width:131px;">Pronadisa</td>
			<td align="right">1800</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:341px;">Paraformaldehyde 16%</td>
			<td style="width:131px;">Electron Microscopy Sciences</td>
			<td align="right" style="width:104px;">15710</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Triton X-100</td>
			<td style="width:131px;"></td>
			<td style="width:104px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">CD8 biot</td>
			<td style="width:131px;">BD Biosciences</td>
			<td align="right" style="width:104px;">553029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">IgM Biot</td>
			<td style="width:131px;">ImmunoStep</td>
			<td style="width:104px;">Clone RMM-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">B220 Biot</td>
			<td style="width:131px;">BD Biosciences</td>
			<td align="right" style="width:104px;">553086</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">CD19 biot</td>
			<td style="width:131px;">BD Biosciences</td>
			<td align="right">553784</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">MHC-II Biot (I-A/I-E)</td>
			<td style="width:131px;">BD Biosciences</td>
			<td align="right">553622</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">CD11b biot</td>
			<td style="width:131px;">Immunostep</td>
			<td style="width:104px;">11BB-01mg</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">CD11c biot</td>
			<td style="width:131px;">Immunostep</td>
			<td>11CB3-01mg</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">DX5 biot</td>
			<td style="width:131px;">BD Biosciences</td>
			<td align="right" style="width:104px;">553856</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Gr-1 biot</td>
			<td style="width:131px;">BD Biosciences</td>
			<td align="right" style="width:104px;">553125</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">CD16/CD32</td>
			<td style="width:131px;">ImmunoStep</td>
			<td>M16PU-05MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">anti Salmonella</td>
			<td style="width:131px;">ABD Serotec</td>
			<td style="width:104px;">8209-4006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">CD11cPE</td>
			<td style="width:131px;">BD Biosciences</td>
			<td align="right" style="width:104px;">553802</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">CD4-APC</td>
			<td style="width:131px;">Tonbo Biosciences</td>
			<td style="width:104px;">20-0041-U100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gr-1 APC</td>
			<td style="width:131px;">BD Biosciences</td>
			<td align="right">553129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MHC-II (I-A/I-E) FITC</td>
			<td style="width:131px;">BD Biosciences</td>
			<td align="right">553623</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa-Fluor 647 Goat Anti-Rabbit IgG (H+L) Antibody, highly cross-adsorbed</td>
			<td>Invitrogen</td>
			<td>A-21245</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">CMAC (7-amino-4-chloromethylcoumarin)</td>
			<td style="width:131px;">Life technologies</td>
			<td>C2110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">BSA</td>
			<td style="width:131px;">SIGMA</td>
			<td style="width:104px;">A7030-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Streptavidin MicroBeads</td>
			<td style="width:131px;">Miltenyi Biotec</td>
			<td style="width:104px;">130-048-101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">BD FACSCanto II</td>
			<td style="width:131px;">BD Biosciences</td>
			<td style="width:104px;">---</td>
			<td></td>
		</tr>
	</tbody>
</table>

t cells capture bacteria by transinfection from dendritic cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

ここで、我々は派生-DCを感染した骨髄からマウスT細胞細菌transinfectionを実行する方法を説明し、どのように2つの異なるアプローチを介した細菌transinfectionを測定する：フローサイトメトリーおよびゲンタマイシン生存アッセイ図1は、細胞を得るための手順をまとめたものです。 DCは、9日間のGM-CSFとの骨髄細胞をインキュベートすることによって生成され...

Watch this Scientific Journal Video about T Cells Capture Bacteria by Transinfection from Dendritic Cells at JoVE.com

T Cells Capture Bacteria by Transinfection from Dendritic Cells

ここで、プロトコルは、事前に感染した樹状細胞（DC）からtransinfection介して抗原提示の間に発生するCD4 + T細胞による細菌の取り込みを測定するために提示されています。初代細胞、DC、DC / T細胞複合体の形成の感染、および細菌のT細胞トランスフェクションの測定の分離：私たちは、必要なステップを実行する方法を示しています。

T細胞は、樹状細胞からTransinfectionによって細菌をキャプチャ

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

t-cells-capture-bacteria-by-transinfection-from-dendritic-cells

Research

JoVE Journal

Immunology and Infection

11.8K ビュー.  Spanish National Biotechnology Centre (CNB-CSIC). ここで、プロトコルは、事前に感染した樹状細胞（DC）からtransinfection介して抗原提示の間に発生するCD4 + T細胞による細菌の取り込みを測定するために提示されています。初代細胞、DC、DC / T細胞複合体の形成の感染、および細菌のT細胞トランスフェクションの測定の分離：私たちは、必要なステップを実行する方法を示しています。 

ビデオ: T Cells Capture Bacteria by Transinfection from Dendritic Cells

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

細胞運命を以下に<em&gt; E大腸菌の</emラムダファージによる感染後&gt;

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

免疫・感染学

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 ...

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

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

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

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

初代ヒトナイーブおよびメモリーT細胞から誘導した調節性T細胞の生成

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

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

マウスの腸からの樹状細胞およびマクロファージの単離とキャラクタリゼーション

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches

Peyer's patches (PPs) are integral components of the gut-associated lymphoid tissues (GALT) and play a central role in intestinal immunosurveillance and homeostasis. Particulate antigens and microbes in the intestinal lumen are continuously sampled by PP M cells in the follicle-associated epithelium (FAE) and transported to an underlying network of dendritic cells (DCs), macrophages, and lymphocytes. In this article, we describe protocols in which murine PPs are (i) dissociated into single cell suspensions and subjected to flow cytometry and (ii) prepared for cryosectioning and immunostaining. For flow cytometry, PPs are mechanically dissociated and then filtered through 70 &mu;m membranes to generate single cell suspensions free of epithelial cells and large debris. Starting with 20-25 PPs (from four mice), this quick and reproducible method yields a population of &gt;2.5 x 106 cells with &gt;90% cell viability. For cryosectioning, freshly isolated PPs are immersed in Optimal Cutting Temperature (OCT) medium, snap-frozen in liquid nitrogen, and then sectioned using a cryomicrotome. Tissue sections (5-12 &mu;m) are air-dried, fixed with acetone or methanol, and then subjected to immunolabeling.

There is an increasing interest in understanding the immunological functions of specific subpopulations of cells in Peyer's patches (PPs), the primary inductive sites of gut-associated lymphoid tissues. Here we outline parallel protocols for preparing PP single cell preparations for flow cytometric analysis and PP cryosections for immunostaining.

マウスのパイエル板のリンパ球や樹状細胞を単離し、免疫染色

Peyer's  patches (PPs) are integral components of the gut-associated lymphoid tissues  (GALT) and play a central role in intestinal immunosurveillance and ...

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 ...

Isolation of Double Negative &#945;&#946; T Cells from the Kidney

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in various immune responses. DN T cells are a unique immune cell type that has been implicated in regulating immune and autoimmune responses and tolerance to allotransplants1-6. DN T cells are, however, rare in peripheral blood and secondary lymphoid organs (spleen and lymph nodes), but are major residents of the normal kidney. Very little is known about their pathophysiologic function7 due to their paucity in the periphery. We recently described a comprehensive phenotypic and functional analysis of this population in the kidney8 in steady state and during ischemia reperfusion injury. Analysis of DN T cell function will be greatly enhanced by developing a protocol for their isolation from the kidney.
Here, we describe a novel protocol that allows isolation of highly pure ab CD4+ CD8+ T cells and DN T cells from the murine kidney. Briefly, we digest kidney tissue using collagenase and isolate kidney mononuclear cells (KMNC) by density gradient. This is followed by two steps to enrich hematopoietic T cells from 3% to 70% from KMNC. The first step consists of a positive selection of hematopoietic cells using a CD45+ isolation kit. In the second step, DN T cells are negatively isolated by removal of non-desired cells using CD4, CD8, and MHC class II monoclonal antibodies and CD1d &#945;-galcer tetramer. This strategy leads to a population of more than 90% pure DN T cells. Surface staining with the above mentioned antibodies followed by FACs analysis is used to confirm purity.

DNabT cells are rare among peripheral T cells; however, they are abundant in certain non-lymphoid tissues. Difficulty of isolating DN T cells from non-lymphoid tissue hinders their functional analysis despite increasing recognized pathophysiologic significance. We describe a novel protocol for isolation of highly purified DN T cells from murine kidney.

腎臓からの二重否定αβT細胞の分離

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in ...

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 ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

T細胞の導出<em&gt;体外</em&gt;マウス胚性幹細胞からの

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

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

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

末梢血からのヒトアロ抗原特異的T細胞の生成

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

T細胞は、樹状細胞からTransinfectionによって細菌をキャプチャ

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