この作品は、NIH（N01 HHSN272201100019C、U19 AI 089992、およびNCRR / GCRCプログラムM01-RR00125）によって部分的にサポートされていました。著者らは競合する経済的利益を宣言しないと博士マークShlomchik、エールセルソーターの中核施設のディレクターに感謝します。

1。免疫細胞の単離と刺激<ol><li>地元の施設内倫理委員会のガイドラインの下で書かれたインフォームドコンセントの後に健康なボランティアから10〜50ミリリットルヘパリン添加血液を取得します。老化や病気の病因の研究については、各ボランティアや患者のための排除と選択基準は明示的に決定する必要があります。 </li><li>ヒト末梢血単核細胞（PBMC）CPT管またはをFicoll-Paque Plusを使用し、製造元の指示に従って（GEヘルスケア、NJ）分離し、分離7日（試薬、 表1）の実験を実施しています。 </li><li>インキュベートしたPBMC（3×10 6 /チューブ）を単独で0.6mlの培地に1.5 mlチューブまたはTLRリガンドまたは他の活性剤、例えばで刺激TLR7 / 8リガンドR848（0.6 mlの培地を加えた10mMのTLR7 / 8リガンドの0.6μL 10〜60分（InvivoGen、カリフォルニア州）9の37℃でR848、最終濃度10μM）を。 </li></ol> 2。研究室セルのeling <ol><li>ラベルの細胞系譜と蛍光標識抗体を用いて生細胞懸濁液の他の表面マーカー。ラベル3×10 4で20分6 PBMCを°単球またはDC系統（ 表2）比表面積抗体のカクテルと1.5mlのエッペンドルフチューブにPBS / 2％FBSを100μlのC。最適な抗体の希釈は、ロット間のばらつきを最小限に抑えるためにバッチで処理する必要があり、予備実験やサンプルで決定することができます。ベンダーの抗体を0.1 mg / mlの株は、抗体5μlの（1:20希釈）を使用します。楽器の蛍光チャネルを設定するだけで一つのカラーコンジュゲートで標識した細胞の別々のチューブを使用しています。 </li><li>表面標識した後、室温で10分間、4％PFA / PBSで細胞を固定します。ストレージの分析の時まで、検定の日まで-80℃で凍結バッファで500×gで再懸濁した細胞（90％FBSはDMSOの10％を含む）°Cで遠心し細胞。 </li><li>アセスメント、プロセスバッチoのfのばらつきを最小限に抑えるために一緒にドナーのグループからの未処理と刺激した細胞。分析の日に、融解細胞をすばやく37℃の水浴、凍結バッファを削除する500分の1 gで遠心する。 </li><li> 100μlのBDパーマ/ Wash Bufferで（BD Biosciences社、NJ）で、細胞内のサイトカインや他のマーカーの検出のために再懸濁し、細胞懸濁液を、細胞を透過します。例の細胞内シグナル伝達コンポーネントのラベルを、RTで20分間ウサギ抗NF-κB（p65の）抗体（最終濃度10μg/ mlで、サンタクルーズバイオテクノロジー、CA）の1:20希釈、500 xgで遠心し、上清を吸引し、 100μlのBDパーマに再懸濁した細胞は、/ヤギ​​抗ウサギIgG-Alexa647（Invitrogen社、カリフォルニア州）の1:250希釈を含むバッファを洗浄し、室温で20分間インキュベートします。 </li><li>イメージングの直前に、DAPI（0.2μg/ mlで、Invitrogen社、CA）またはヨウ化プロピジウム（、20 ng / mlで、Invitrogen社、CA PI）で核を対比染色。 </li></ol> 3。のImageStream分析<ol><li>人間のサンプル間のばらつきを低減するためにバッチ処理されたサンプルでのImageStreamでイメージングを行っています。 488 nmの、658 nmと405 nmでの250 mWで10 mWの200 mWを次のようにレーザーの電源の機器の設定であった。 </li><li>データファイル（ 図1）を分析するには、通常のPI強度と高いPIのアスペクト比（幅：高さ比）のイベントの最初のゲートは、単一の破片の細胞（R1）（低PI強度）または多細胞イベント（区別するために高いPI強度と低アスペクト比）。単球はCD14陽性細胞のゲート内にあります。 MDCはLIN-1マーカー（R3）のCD11cは、低CD4（R2）のために、また、低い高である細胞のゲート内にあります。 </li><li>各細胞の活性化の程度の効果的な定量的尺度を提供するためにIDEASソフトウェア（Amnis、WA）を使用します。核への移行を示すために、核の色素PI（R4）と細胞質の転写因子NF-κBの類似性（あるいは共局在）を決定します。 NF-κB/PIlocaliの高い相関zationは高い類似度スコアに反映され、アクティベーション10の程度を示しています。 </li><li>細胞の相対的な機能の効率性を示すために、異なる治療群または患者集団間の類似性の比を比較する。絶対値または倍の差の統計的比較によりサンプル間でデータを定量化する。人口ヒストグラムのキーセグメント（ 図2）からの細胞の画像を表示するためのアイデアソフトウェアを使用します。 </li></ol> 4。代表的な結果我々は、TLR7 / 8リガンド、R848（ 図1）PBMCを刺激することにより、モデルウイルスのリガンドに応答してシグナル伝達経路を定量化しています。我々は、サンプルのグループ（ 図2）細胞内局在の画像や人口統計の両方を収集しました。のImageStreamは、末梢血単核球とイメージ細胞応答ゲートからではなく、ソートされていない細胞集団から直接ゲート細胞サブセットに私たちをことができます。ここでは、TLRの効果を定量-LIN1でNF-κBの核局在化（P65）にignaling CD11cは、薄暗いCD4 +骨髄系樹状細胞（MDC）が。ベースライン時、我々は細胞質内転写因子NF-κB（p65の）を持つ非刺激細胞の割合が高いを観察した。刺激した後、処理した細胞は、NF-κB（p65の（）の中央値の類似度スコアは、それぞれ1.52と3.01であり、 図刺激後に核内へ移行することを示し、核染色PIとNF-Bの有意に高い類似性スコア（P65）を持っています。 2）。同様の結果がTLR5刺激単球9に記載されています。 <img alt="図1" src="/files/ftp_upload/3741/3741fig1.jpg" /> 図1。染色と人間の樹状に刺激の効果を定量化するためのゲーティング戦略 。 NF-κB転写因子は、数多くの免疫系遺伝子の発現を調節する。本研究では、TLR7 / 8リーガ後の代表的な一主題から樹状にお​​けるNF-κB（p65の）の核局在化を測定する<html> ND刺激。合焦単一細胞は、高い核のアスペクト比（R1）とPI陽性のイベントにゲーティングによって同定した。 MDCは（LIN1-、CD4dimは、CD11c +）R3のゲートであった。 NF-κB（p65の）の核局在化はR4にプロットされます。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/3741/3741fig1large.jpg" target="_blank">拡大図を表示するには、ここをクリックしてください</a> 。 <img alt="図2" src="/files/ftp_upload/3741/3741fig2.jpg" /> 図2。 。刺激後の樹状におけるNF-κBの移行 NF-κB（p65の）の転座核への刺激後（R4）が未処理と刺激樹状の集団に描かれている中央値の類似性のスコアは、未処理サンプルでは1.52および3.01です。刺激されたサンプル（A）。未処理またはR848刺激の細胞集団を同時に収集したデジタル画像は代表的な細胞や細胞核（B）に転NF-Bの強度を示しています。_upload/3741/3741fig2large.jpg &quot;ターゲット=&quot; _blank &quot;&gt;拡大図を表示するには、ここをクリックしてください。

<ol>
	<li>Linton, P. J., Dorshkind, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+changes+in+lymphocyte+development+and+function.">Age-related changes in lymphocyte development and function.</a> Nat. Immunol. 5, 133-139 (2004).</li><li>Shaw, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysregulation+of+Human+Toll-like+Receptor+Function+in+Aging.">Dysregulation of Human Toll-like Receptor Function in Aging.</a> Ageing Research Reviews. 10, 346-353 (2011).</li><li>Medzhitov, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptors+and+innate+immunity.">Toll-like receptors and innate immunity.</a> Nature Rev. Immunol. 1, 135-145 (2001).</li><li>van Duin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-associated+Defect+in+Human+TLR1+Function+and+Expression.">Age-associated Defect in Human TLR1 Function and Expression.</a> J. Immunol. 178, 970-975 (2007).</li><li>Panda, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-associated+decrease+in+TLR+function+in+primary+human+dendritic+cells+predicts+influenza+vaccine+response.">Age-associated decrease in TLR function in primary human dendritic cells predicts influenza vaccine response.</a> J. Immunol. 184, 2518-2527 (2010).</li><li>Kong, K. -. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysregulation+of+TLR3+impairs+the+innate+immune+response+to+West+Nile+virus+in+the+elderly.">Dysregulation of TLR3 impairs the innate immune response to West Nile virus in the elderly.</a> J. Virol. 82, 7613-7623 (2008).</li><li>Qian, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+interferon+signaling+in+dendritic+cells+from+older+donors+infected+in+vitro+with+West+Nile+virus.">Impaired interferon signaling in dendritic cells from older donors infected in vitro with West Nile virus.</a> J. Infect. Dis. 203, 1415-1424 (2011).</li><li>Hayden, M. S., Ghosh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NF-kappaB+in+immunobiology.">NF-kappaB in immunobiology.</a> Cell research. 21, 223-244 (2011).</li><li>Qian, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-associated+elevation+in+TLR5+leads+to+increased+inflammatory+responses+in+the+elderly.">Age-associated elevation in TLR5 leads to increased inflammatory responses in the elderly.</a> Aging Cell. 11, 104-110 (2011).</li><li>George, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+measurement+of+nuclear+translocation+events+using+similarity+analysis+of+multispectral+cellular+images+obtained+in+flow.">Quantitative measurement of nuclear translocation events using similarity analysis of multispectral cellular images obtained in flow.</a> Journal of immunological. 311, 117-129 (2006).</li></ol>

利害の衝突が宣言されません。

トランスレーショナル研究のためのImageStreamの使用における重要なステップは、関連する比較グループと抗体特異性の最適化と検証の選択です。標識した標的の対象群間の差は、ヒストグラムとセル画像を介して容易に明らかになるであろう。関連する受容体とシグナル伝達経路の変化の徹底的な分析と組み合わせることで、これらのデータは、専門的なコホートで免疫不全の根底にあるメカニズムに貴重な洞察を提供します。技術は、関連するターゲットのための十分な親和性の特異的な抗体の可用性によって制限されます。さらに、測定器は、マルチユーザー機能に最も適していると解析ソフトウェアは、マスターにいくつかの注意が必要です。 この技術は、ヒトの疾患のトランスレーショナル研究のための強力な手法です。ゲノム配列と配列技術の進歩は、mに関与する変異体の同定を可能にした任意の疾患、まれにノミネートされた遺伝子の作用メカニズムを識別しません。我々の研究は、刺激後のNF-κBの細胞質比、核などの個々の被験者のトランスレーショナル研究のための青写真を提供しています。血の少量のサンプルから、我々は、対象自身の細胞の系統固有の方法で処理したり、差動信号を識別することができます。この方法を使用して、我々は高齢化9細胞シグナル伝達の若者と高齢者と明らかに変化の大規模コホートの単球での細胞応答を定量化しています。のImageStreamは、そのような治療応答と非応答者として多くの研究で、治療前と後の個々の被験者の、またはフレアと寛解時に細胞内の主要なメカニズムを強調するために、より広く適用されるかもしれません。対象研究の下にグループ、セルのタイプ、および調査するための細胞メカニズムの選択可能な豊富な修正を加えて、のImageStreamの将来のアプリケーションが多く、shoulです。dは、多くの翻訳の分野で大きな進歩を可能にします。

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td>をFicoll-Paqueプラス</td><td> GEヘルスケア</td><td> 17-1440-03 </td></tr><tr><td> R848 </td><td> Invivogen </td><td> tlrl-r848 </td></tr><tr><td>パラホルムアルデヒド（16％） </td><td>電子顕微鏡学</td><td> 15710 </td></tr><tr><td> BDはペルミ/洗浄</td><td> BDバイオサイエンス</td><td> 554723 </td></tr><tr><td> CD14-PE </td><td>ベイバイオサイエンス</td><td> 12-0149-41 </td></tr><tr><td> CD4-PacBlue </td><td> BD Pharmingen社</td><td> 68436 </td></tr><tr><td>リネージュカクテル1（LIN 1）-FITC </td><td> BDバイオサイエンス</td><td> 340546 </td></tr><tr><td> CD11cは-PE </td><td> BDバイオサイエンス</td><td> 347637 </td></tr><tr><td> CD123-PE </td><td> BDバイオサイエンス</td><td> 340545 </td></tr><tr><td>ウサギ抗NF-κB（p65の） </td><td>サンタクルス</td><td> SC-372 </td></tr><tr><td> Alexa647のF（ab &#39;）2ヤギ抗ウサギIgG </td><td>インビトロジェン</td><td> A21246 </td></tr><tr><td> DAPI </td><td>インビトロジェン</td><td> D21490 </td></tr><tr><td>のImageStream X </td><td> Amnis </td><td></td></tr></tbody></table> 表1特異的試薬および装置。 <table border="1"><tbody><tr><td> 細胞型 </td><td> FITC </td><td> PE </td><td> アレクサ647 </td><td> PacBlue </td></tr><tr><td>単球</td><td></td><td> CD14 </td><td> NF-κB（p65の） </td><td></td></tr><tr><td> MDC </td><td> LIN1 </td><td> CD11cは</td><td> NF-κB（p65の） </td><td> CD4 </td></tr><tr><td> pDCで</td><td> LIN1</td><td> CD123 </td><td> NF-κB（p65の） </td><td> CD4 </td></tr></tbody></table>シグナル伝達経路の細胞系譜染色のための表2抗体パネル単球：CD14 + MDC：LIN1-、CD4dimは、CD11c + PDC：LIN1-、CD4dim、CD123 + LIN1（系列）マーカーはCD3、CD14、CD19、CD20、単球、NK細胞、T＆B細胞のCD56マーカーが含まれています。

quantitative imaging of lineage-specific toll-like receptor-mediated signaling in monocytes and dendritic cells from small samples of human blood

免疫状態の個々の変動が感染症への対応を決定し、疾患の重症度および予後に貢献しています。エージングは、ウイルスおよび細菌感染症に対する感受性の上昇と関連付けられ、体液によく文書化減少と同様に細胞性免疫応答の1,2のワクチンに反応性を減少させる。我々は最近、Toll様受容体（TLR）は、微生物感染およびトリガ抗菌宿主の防御応答3を検出する自然免疫系の主要なコンポーネントでエージングの効果を評価しています。健康な人間のドナーの大規模コホートでは、我々は、高齢者の末梢血単球は、抗原提示において重要であるセルを特定のTLRは4と同様の削減TLRレベルと樹状細胞のシグナル伝達応答（DCS）の発現と機能が低下している示した自然免疫と獲得免疫の5との間のリンケージ。我々は、マクロファージではTLR3の調節不全を示しているウエストナイルウイルス6,7の感染に応答して、高齢者ドナーからの樹状細胞によるIFNのd低い生産。 免疫老化の我々の理解および治療的介入へのパラマウントは、特定の細胞応答の種類とシグナル伝達の機構（s）の詳細な理解です。 FACSまたはイムノブロットによって初代培養細胞や測量細胞マーカーのイメージングを介して免疫応答の従来の研究では、しかしながら、これらの研究は一般の患者から入手可能な少量のサンプル量と複数の複雑な実験技術を実施することができないことに技術的に制限され、大幅に我々の理解を進めてきたヒトサンプル。のImageStreamは、同時高解像度のデジタル画像の定量的なフローサイトメトリーを組み合わせたため、患者の感受性の効率的な捕獲のための同時期に複数の細胞集団の調査を容易にします。ここでは、TLR7 / 8を評価するために、樹状細胞でのImageStreamの使用方法を示していますリン酸化と免疫応答8の重要な多数の遺伝子の転写を開始する重要な転写因子、NF-κBの核移行の活性化を介する増加します。この技術を用いて、我々はまた、最近では9高齢化で変更された免疫応答性に貢献するかもしれない古いドナーから単球におけるTLR5シグナリングとNF-κB経路の以前に認識されていない変化を示している。

Watch this Scientific Journal Video about Quantitative Imaging of Lineage-specific Toll-like Receptor-mediated Signaling in Monocytes and Dendritic Cells from Small Samples of Human Blood at JoVE.com

Quantitative Imaging of Lineage-specific Toll-like Receptor-mediated Signaling in Monocytes and Dendritic Cells from Small Samples of Human Blood

我々は（のImageStream技術の使用を記述する<a href="http://www.amnis.com/" target="_blank"&gt; www.amnis.com</a&gt;）、これは明確に定義された患者のコホートからの主な免疫細胞の細胞メカニズムを定量化するために、同時高解像度のデジタル画像で定量的なフローサイトメトリーを組み合わせたものです。我々の研究は、対象のコホートから少量のサンプルで系統特異的な細胞応答を定量化するためのトランスレーショナル研究のための青写真を提供しています。

ヒト血液の小さなサンプルから単球および樹状細胞のlineage特異的Toll様受容体を介するシグナル伝達の定量的イメージング

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome.  Aging is associated with an ...

quantitative-imaging-lineage-specific-toll-like-receptor-mediated

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

35.0K ビュー.  Yale University School of Medicine. 我々は（のImageStream技術の使用を記述する www.amnis.com）、これは明確に定義された患者のコホートからの主な免疫細胞の細胞メカニズムを定量化するために、同時高解像度のデジタル画像で定量的なフローサイトメトリーを組み合わせたものです。我々の研究は、対象のコホートから少量のサンプルで系統特異的...

ビデオ: Quantitative Imaging of Lineage-specific Toll-like Receptor-mediated Signaling in Monocytes and Dendritic Cells from Small Samples of Human Blood

Detection of  Neu1 Sialidase Activity in Regulating TOLL-like Receptor Activation

Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of microbial (e.g., viruses, bacteria and parasite) infections, they also play an important role in the pathophysiology of infectious diseases, inflammatory diseases, and possibly in autoimmune diseases. Thus, the intensity and duration of TLR responses against infectious diseases must be tightly controlled. It follows that understanding the structural integrity of sensor receptors, their ligand interactions and signaling components is essential for subsequent immunological protection. It would also provide important opportunities for disease modification through sensor manipulation. Although the signaling pathways of TLR sensors are well characterized, the parameters controlling interactions between the sensors and their ligands still remain poorly defined. We have recently identified a novel mechanism of TLR activation by its natural ligand, which has not been previously observed 1,2. It suggests that ligand-induced TLR activation is tightly controlled by Neu1 sialidase activation. We have also reported that Neu1 tightly regulates neurotrophin receptors like TrkA and TrkB 3, which involve Neu1 and matrix metalloproteinase-9 (MMP-9) cross-talk in complex with the receptors 4. The sialidase assay has been initially use to find a novel ligand, thymoquinone, in the activation of Neu4 sialidase on the cell surface of macrophages, dendritic cells and fibroblast cells via GPCR G&alpha;i proteins and MMP-9 5. For TLR receptors, our data indicate that Neu1 sialidase is already in complex with TLR-2, -3 and -4 receptors, and is induced upon ligand binding to either receptor. Activated Neu1 sialidase hydrolyzes sialyl &alpha;-2,3-linked &beta;-galactosyl residues distant from ligand binding to remove steric hinderance to TLR-4 dimerization, MyD88/TLR4 complex recruitment, NFkB activation and pro-inflammatory cell responses. In a collaborative report, Neu1 sialidase has been shown to regulate phagocytosis in macrophage cells 6. Taken together, the sialidase assay has provided us with powerful insights to the molecular mechanisms of ligand-induced receptor activation. Although the precise relationship between Neu1 sialidase and the activation of TLR, Trk receptors has yet to be fully elucidated, it would represent a new or pioneering approach to cell regulation pathways.

The sialidase assay is a simple technical approach that will elucidate novel molecular mechanism(s) of TLR sensors of microbial infections and involvement in inflammatory diseases at the receptor level on the cell surface of live macrophages.

Toll様受容体の活性化を調節するのNeu1シアリダーゼ活性の検出

Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of ...

免疫・感染学

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 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 ...

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. Macrophages express surface Toll-like receptors (TLRs), which recognize molecular patterns conserved through evolution in a wide range of microorganisms. TLRs play a central role in macrophage activation which is usually associated with gene expression alteration. Macrophages are critical in many diseases and have emerged as attractive targets for therapy. In the following protocol, we describe a procedure to isolate murine peritoneal macrophages using Brewer&#8217;s thioglycollate medium. The latter will boost monocyte migration into the peritoneum, accordingly this will raise macrophage yield by 10-fold. Several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. However, peritoneal macrophages were shown to be more mature upon isolation and are more stable in their functionality and phenotype. Thus, macrophages isolated from murine peritoneal cavity present an important cell population that can serve in different immunological and metabolic studies. Once isolated, macrophages were stimulated with different TLR ligands and consequently&#160;gene expression was evaluated.

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

Toll様受容体刺激時の遺伝子発現解析を行うためにマウス腹腔マクロファージの単離

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

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 ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

Fcγ受容体媒介食作用を評価するために、単球単層アッセイの使用

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

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

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

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

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

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

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labeled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.
It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Here, we describe a protocol to assess antifungal activity of primary human immune cells in real-time using fluorescent Aspergillus reporter conidia in conjunction with live-cell video microscopy and flow cytometry. Generated data provide insight into host cell-Aspergillus interactions such as fungicidal activity, phagocytosis, cell migration and inhibition of fungal growth.

に応答したヒト一次好中球及び単球による抗カビ活動のライブイメージング<em&gt; A。フミガーツス</em

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

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 ...

Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low numbers of DCs present per tissue. Yet, as the phenotype and function of DCs is modified by the tissue environment, it is necessary to analyze tissue resident DC populations, since blood derived DCs incompletely reflect the complexities of DCs in tissues. Here we present a protocol to isolate DCs from the human female reproductive tract (FRT) using hysterectomy specimens that allows both phenotypical and functional analyses. The protocol consists of tissue digestion to generate a single cell mixed cell suspension, followed by positive magnetic bead selection. Our tissue digestion protocol does not cleave surface markers, which allows phenotypical and functional analysis of DCs in the steady state, without overnight incubation or cell activation. This protocol can be adapted for the isolation of other immune cell types or isolation of DCs from other tissues.

We describe here a method to isolate and purify dendritic cells from different anatomical compartments in the human female reproductive tract for the evaluation of their phenotypical and functional characteristics. This method can be adapted to isolate other immune cells or dendritic cells from other mucosal tissues.

人間の女性の生殖器官から樹状細胞の表現型および機能研究の分離

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low ...