私たちは、博士に感謝しています。試薬を提供するためのマルクス・マンツとアービングワイズマン。我々はまた、それぞれ、フローサイトメトリー分析によって提供されるサービスとファーストと医学の国立台湾大学で第二のコア研究所とNTU病院の細胞選別基盤施設を認めます。この作品は、科学技術省、台湾（MOST 102から2320-B-002から030-MY3）と国民健康研究所、台湾（NHRI-EX102-10256SIとNHRI-EX103-10256SI）によってサポートされていました。

C57BL / 6野生型マウスは、国立実験動物センター（NLAC）、台湾から購入しました。すべてのマウスは、特定病原体を含まない条件下で飼育し、維持しました。プロトコルおよび動物使用手順を見直し、医学の国立台湾大学（許可番号：20120075）の施設内動物管理使用委員会によって承認されました。実験を行っている間に加えて、研究者は、動物のすべての痛みの可能性を減らすため、苦痛、または苦痛を行いました。手袋を着用して説明されているすべての手順は室温で行いました。 AC-6フィーダー細胞の調製注：（I.ワイズマン、スタンフォード大学によって提供された）AC-6.21（AC-6要するに）間質細胞ライン18は、RPMI中で維持されるべきである15％の熱不活性化ウシ胎児血清（FBS）を補充しました。フィーダー細胞として機能するには、AC-6細胞は、γ線を照射している3000ラド（30Gy）1日との共培養の前に、その増殖を防ぐために。注目メンテナンス中に過密放置すれば、AC-6の細胞は、その分化支持能を失う傾向がある、またはその通過数は20を超えている場合。 <ol><li> 1ミリリットルダルベッコのリン酸緩衝生理食塩水（DPBS）と吸引によってDPBSを削除すると1回洗浄AC-6細胞。 37 O℃で3~5分間、0.8 mlのトリプシン溶液（0.05％トリプシンおよびDPBSで0.5mMのEDTA）とAC-6細胞を、5％CO 2を処理した後、培養液の10容量にして、それを希釈することによって反応を停止させます単一細胞懸濁液を作ります。 </li><li> 5.9x10のAC-6細胞/ウェルを12ウェルプレートに4細胞をシードし、37°C ​​の 、5％CO 2、O ​​/ Nでインキュベートします。 注意：AC-6細胞の密度はのCDC生成を有利に働く低密度として非常に重要です。 4細胞/ウェル5.9x10の密度で播種は、AC-6のCLPからのpDCの導出に最適である次の日、によって密集度に到達することができます。 </li><li> γ-照射を使用して3000ラド（30グレイ）のAC-6細胞を照射します。 注意：AC-6を照射するために使用される用量は、増殖から細胞を防ぎ、しかものCLPからのDCの分化に必要なサイトカインおよび細胞 - 細胞接触を提供するのに十分に長い生存を保つために最適化されます。 </li><li>培地を吸引し、1mlの完全RPMI（10％熱不活性化FBS、50μg/ mlのゲンタマイシン、βメルカプトエタノール、50μMを含むRPMI）と交換してください。 </li></ol>マウスから2アイソBM細胞<ol><li> CO 2窒息と頸椎脱臼によりマウスを生け贄に捧げます。解剖トレイにマウスを置き、70％エタノールで滅菌します。半ば腹部に切開を行い、下肢をカバーする皮膚を含むマウスの先端部から皮膚を除去。 </li><li>半無菌フード内でマウスを解剖。大腿骨と脛骨を解除するには、膝関節以下上記の大腿骨と脛骨をクリップ。はさみを使用して、骨から筋肉を外し、完全RPMI 5ミリリットルを含む6センチペトリ皿に骨を置きます。 </li><li>無菌培養フードに移動します。完全RPMIで27G針に接続された3ミリリットル注射器を埋めます。はさみを使用して、骨の両端を切断し、静かにそれぞれの端から一度完全RPMIを注入することによって、骨の外に骨髄をフラッシュするために27Gの針を挿入します。 </li><li>細胞を上下に3-5回、針なし注射器を用いて培養皿で穏やかにピペッティングにより単一細胞懸濁液を準備します。 </li><li> 500×gで5分間、細胞を遠心分離し、上清をデカント。 </li><li> 1ミリリットルACKバッファー（150mMのNH 4 Clを 、10 mMのKHCO 3、0.1mMのEDTA）で1分間インキュベートし、完全RPMIの10容量で希釈して反応を停止で溶解赤血球。細胞が重力によって死んだ細胞塊および組織破片をダウンペレット化するために、5分間放置します。 注意：これは原因で生細胞の沈降に細胞の損失になりますように5分以上を立たないでください。 </li><li>ゆっくりと破片を残して新しいチューブに細胞含有上清をデカント元のチューブ内の背後にあります。 </li><li>その後、上清を除去し、廃棄し、細胞をペレットに500×gで5分間遠心します。 </li><li> 100μlのFACS緩衝液（1×PBS + 2％FBS + 1mMのEDTA）中（通常は4-6×10 7のBM細胞は、1つのマウスから得られる）総BM細胞を再懸濁します。 </li></ol> 3のCLPのFACS分析およびソート<ol><li> Fc受容体をブロックするために、1〜2分間抗CD16 / 32（ハイブリドーマ上清クローン2.4G2、50μL/反応または1-2μgの/反応）、FACS緩衝液中の細胞への（ステップ2.9）を追加します。 注：抗CD16 / 32はまた、顆粒球、単球およびBリンパ球などのFc受容体を発現する細胞に対する抗体の非特異的結合を防止するために添加されたFcブロックと呼ば​​れます。 </li><li>同時に周囲光を回避し、氷上で15分間（4×10 7細胞）以下の蛍光色素標識抗体で細胞を染色します。抗体：抗CD3を含むPE結合系統マーカー（0.2μgの各）（17A2）、抗CD8（53-6.7）、抗B220（RA3-6B2）、抗CD19（eBio1D3）、抗CD11b（M1 / 70）、抗GR-1（RB6-8C5）、抗Thy1.1（HIS51）、抗NK1.1（PK136）、抗TER119（TER-119）、および抗MHC-II（NIMR-4）、0.2μgの抗c-Kitの-PerCPを-のCy5.5 （2B8）、1μgの抗SCA-1-FITC（D7）、0.4μgの抗M-CSFR-APC（AFS98）、および0.2μgの抗IL-7Rα-PE-Cy7の（A7R34）。 </li><li> 3ミリリットルFACS緩衝液で細胞を洗浄し、500×gで5分間遠心します。 </li><li> 40μMのセルストレーナーを通して300μlのFACS緩衝液およびフィルタ細胞で細胞を​​再懸濁します。 </li><li>残りの細胞を回収し、同じセルストレーナーを使用してチューブをソートする無菌FACSにフィルタリングするために、追加の100μlのFACS緩衝液でチューブを洗浄します。 </li><li>信号検出のための適切なフィルタおよび電圧を使用して染色した後すぐにフローサイトメトリー分析を行います。 PE-およびPE-Cy7の-labelded抗体および633nmのラスを検出するために561 nmレーザー、FITC-、PerCPを-のCy5.5標識抗体を検出するために488nmのレーザーを使用してERは、APC標識抗体を検出します。 </li><li>セルソーターを用いて、IL-7Rα+（ステップ3.2で染色されたように） -のc-kit は int SCA-1 INT ​​M-CSFR -以下のマーカーLINに従ってソートアウトのCLP。クッションとして完全RPMI 8 mlを含む15mlチューブで選別された細胞を収集します。 </li><li>実行終了時にセルソーターによって示されるように選別された細胞の絶対数を記録します。一般的に、5×10 4のCLPは1マウスのBMから取得することができます。 </li></ol> 4.共培養のCLPとAC-6 <ol><li> 、500×gで10分間、ステップ3.7からの選別された細胞を遠心分離し、上清を除去し、5×10 4細胞/ mlの周りに細胞密度を得るために十分な完全RPMIで細胞ペレットを再懸濁します。シード500細胞/ウェルのステップ1.4で調製したフィーダー細胞を含む12ウェルプレートに。 </li><li> 100 ngの追加/ mlのFL 19（HU-のFlt3L-IgはM.マンツ、Universiによって提供される発現系を使用して、社内で発生しましたTY病院チューリッヒ、スイス）と37°Cの 、顕微鏡下でのDC開発の定期的な視覚的な監視と5％CO 2でインキュベートします。 注：市販の組換えヒトまたはマウスFL FLは、DCの発達をサポートするHU-のFlt3L-Igのための代替物として使用することができます。 </li><li> 12日目に上清中の細胞を収集し、得​​られた上清を組み合わせた0.5ミリリットル完全RPMI培地で1回、ウェルを洗浄します。 0.5ミリリットルに新鮮な培地を追加し、セルスクレーパーで接着細胞をスクラップ。 </li><li> 500×gで5分間の部品や遠心機の両方からの細胞含有培地を組み合わせます。 </li><li> 50μlのFACS緩衝液中で上清をデカントし再懸濁した細胞。 50μlの抗CD16 / 32ハイブリドーマ上清を加え、Fc受容体をブロックするために1~2分間インキュベートします。 </li><li>列挙し、次抗体（0.05μgの各）抗CD11cの-APC（N418）、抗CD11b-FITCおよび抗B220-PEで、すべての細胞を染色。 </li><li>洗って説明するように、細胞を遠心分離ステップ3.3でD。 </li><li> 、100μlのFACS緩衝液中で細胞を再懸濁したCD11c +の上のゲートとのCDC（のCD11c +のCD11b + B220 - ）のために分析したpDC（CD11cの+のCD11b - B220 +）の17。 </li></ol>

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Authors have nothing to disclose.

ここでは、のCLPの少数から、特にDCの堅牢な世代のためのin vitro培養系を説明し、pDCに。この培養系の独自性は、フィーダーとしてのAC-6細胞、ストローマ細胞株の使用によるものです。このアプローチは、IL-7、SCF、M-CSFおよびFL 20などのサイトカインが、また、DCの発達をサポートするのに必要な細胞-細胞接触21だけでなく、提供することが示されています。 AC-6細胞は?...

樹状細胞（DC）は、1免疫応答の制御に重要な役割を果たしているプロフェッショナル抗原提示細胞です。 DCは不均一であるが、それらは、大きく2つの集団に分類することができ、従来のDC（のCDC）および形質のDC（pDCに）2,3。リンパ器官に加えて、のCDCとのpDCは、肺、腸および皮膚2を含む組織中に見出されます。 CDCとのpDCの形態はのCDCは、デンドライト状の突起と以上のプラズマ細胞様であることのpDCの形状を呈すると、異なります。また、一般的なマウスDCマーカーCD11cのは、より高度のpDC上でのCDCより上で発現されます。 pDCに2が行うよりも、MHCクラスIIの高いレベルと共刺激分子を発現どちらのCD24 +のCDC、 -のCDCとのCD11b -また、のCDCは、さらにのCD11b + CD24に分けることができます。成熟のpDCは、一方で、選択シグレックHおよびBST2 4を発現することが示されています。 Functionally、のCDCは、pDCにはよりも優れた抗原提示細胞です。しかし、pDCには、私は、ウイルス感染または炎症性刺激5時に型インターフェロンの膨大な量を生成することができます。 両方のCDCとのpDCは短命であるため、常に骨髄（BM）6,7内の前駆細胞から補給する必要があります。致死的に照射したマウスに共通の骨髄前駆細胞（のCMP）とリンパ球系共通前駆細胞（のCLP）の養子移入はのCDCとのpDCは、両方の系統8-10から生成することができることを実証しています。しかし、RAG1 / 2を発現し、IgH遺伝子座11,12に再配置DJセグメントを所有するpDCのサブセットがあります。これらの細胞はまた、B220マーカーの発現は、核酸感知受容体（TLR7 / TLR9）、および転写因子（SPIBとBcl11a）13を含む Bリンパ球との分子の類似性を共有すると、すべてがリンパ系列の署名であると考えられています。したがって、CLPsがあるため、系統の類似のpDCのインビトロ生成のための良い選択かもしれません。 ヒトおよびマウスの両方でのCDCとのpDCの頻度が非常に低いが6、DCは、特にたCDCは、（例えば、GM-CSF 11,14またはFlt3リガンドなどのサイトカインの存在下で、BMまたは前駆細胞からインビトロで生成することができるFL ）フィーダーフリーシステム11,15,16を使用 。しかし、残念ながら、それは、FL 11,15,16 を用いてインビトロでのpDCの多くを生成することは不可能です。以前に我々は、pDCには、効率的にAC-6フィーダシステム17を使用してのCLP からインビトロで生成することができることを実証しました。培養系においてAC-6間質細胞株を使用することの利点は、それが細胞 - 細胞接触とのCLPからのpDCの多数の生成をサポートするサイトカインの分泌を提供することです。このシステムの生産は非常に堅牢ですが、下記の手順を慎重にレプリケーションが私を必要とされますn個の順序のpDCの効率的な生成を確実にします。

<table><tbody><tr><td>Anti-mouse Ly6g/Ly6c (PE), clone RB6-8C5</td><td>Biolegend</td><td>108408</td><td>linage marker</td></tr><tr><td>Anti-mouse NK1.1 (PE), clone PK136</td><td>Biolegend</td><td>108708</td><td>linage marker</td></tr><tr><td>Anti-mouse&amp;nbsp; CD11b (PE), cloneM1/70</td><td>Biolegend</td><td>101208</td><td>linage marker</td></tr><tr><td>Anti-mouse CD19 (PE), clone eBio1D3</td><td>Biolegend</td><td>115508</td><td>linage marker</td></tr><tr><td>Anti-mouse B220 (PE), clone RA3-6B2</td><td>Biolegend</td><td>103208</td><td>linage marker/FACS</td></tr><tr><td>Anti-mouse CD3 (PE), clone 17A2</td><td>Biolegend</td><td>100308</td><td>linage marker</td></tr><tr><td>Anti-mouse CD8a (PE), clone 53-6.7</td><td>Biolegend</td><td>100707</td><td>linage marker</td></tr><tr><td>Anti-mouse MHC-II (PE), clone NIMR-4</td><td>Biolegend</td><td>107608</td><td>linage marker</td></tr><tr><td>Anti-mouse Ter119 (PE), clone TER-119</td><td>Biolegend</td><td>116208</td><td>linage marker</td></tr><tr><td>Anti-mouse Thy1.1 (PE), clone HIS51</td><td>eBioscience</td><td>12-0900-83</td><td>linage marker</td></tr><tr><td>Anti-mouse M-CSFR (APC), clone AFS98</td><td>Biolegend</td><td>135510</td><td>FACS</td></tr><tr><td>Anti-mouse c-Kit (PerCP/Cy5.5), clone 2B8</td><td>Biolegend</td><td>105824</td><td>FACS</td></tr><tr><td>Anti-mouse Sca-1 (FITC), clone D7</td><td>Biolegend</td><td>108106</td><td>FACS</td></tr><tr><td>Anti-mouse IL-7Ra (PE/Cy7), clone A7R34</td><td>Biolegend</td><td>135014</td><td>FACS</td></tr><tr><td>Anti-mouse CD11c (PerCP/Cy5.5), clone N418</td><td>Biolegend</td><td>117328</td><td>FACS</td></tr><tr><td>Anti-mouse CD11b (FITC), clone M1/70</td><td>Biolegend</td><td>101206</td><td>FACS</td></tr><tr><td>FACSAria III</td><td>BD Biosciences</td><td></td><td>Cell sorter</td></tr><tr><td>FACS sorting tube&amp;nbsp;</td><td>BD Biosciences</td><td>352054</td><td></td></tr><tr><td>FlowJo</td><td>FlowJo LLC</td><td></td><td>Flow analysis sofrware</td></tr></tbody></table>

in vitro generation of murine plasmacytoid dendritic cells from common lymphoid progenitors using the ac-6 feeder system

形質細胞様樹状細胞（pDC）感染に応答して、または炎症応答の間に活性化された細胞を産生Iインターフェロン強力型（IFN-I）です。残念ながら、pDCの機能の研究は、リンパ器官で、その低周波によって妨げられ、in vitroでのDC生成のための既存の方法は、主にpDCを超えるのCDCの生産を好みます。ここでは、効率的にin vitroでのリンパ球系共通前駆細胞（のCLP）からのpDCを生成するためのユニークなアプローチを提示します。具体的には、プロトコルは、骨髄からのCLPを浄化し、Flt3リガンドの存在下でγ線を照射し、AC-6フィーダー細胞との共培養により、pDCにを生成する方法の詳細を説明しました。この培養系のユニークな特徴は、のCLPは、AC-6細胞の下に移動して、石畳の領域形成細胞になることのpDCを拡大するための重要なステップです。形態学的に異なるDCは、すなわちたpDCとのCDCは、7の組成物で約2週間後に生成されました最適な条件の下で0から90パーセントのpDCに。通常、この方法によって生成されたpDCの数はおおよそのCLPの数の100倍がシードされます。したがって、これは、確実に、これらの細胞の発達と機能にさらなる研究を容易にするために必要なのpDCを大量に生成すると、新規システムです。

4-6x10 7 BM細胞の総数は、典型的には、大腿骨一6~8週齢の野生型C57BL / 6マウスの脛骨から単離されます。 CLPを整理するには、総BM細胞は、系統マーカー（CD3、CD8、B220、CD19、CD11bの、GR-1、Thy1.1、NK1.1、TER119、およびMHC-II）に対するPE結合抗体で染色され、抗-c-Kitの-PerCPを/ Cy5.5を、抗SCA-1-FITC、抗M-CSFR-APCおよび抗IL-7Rα-PE / Cy7の、分析し、セルソーターでソート。 CLPのための選別戦略は<stron...

Watch this Scientific Journal Video about In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System at JoVE.com

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

In this study we present an in vitro culture system that can efficiently generate pDCs by co-culturing common lymphoid progenitors with AC-6 feeder cells in the presence of Flt3 ligand.

AC-6フィーダシステムを使用してリンパ球系共通前駆細胞からマウス形質細胞様樹状細胞のインビトロ生成に

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during ...

in-vitro-generation-murine-plasmacytoid-dendritic-cells-from-common

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

8.4K ビュー.  National Taiwan University College of Medicine. In this study we present an in vitro culture system that can efficiently generate pDCs by co-culturing common lymphoid progenitors with AC-6 feeder cells in the presence of Flt3 ligand. 

ビデオ: In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

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

Culture of myeloid dendritic cells from bone marrow precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors. 

This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.

骨髄前駆細胞から骨髄樹状細胞の培養

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to ...

生物学

Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% CD11c high dendritic cells in culture that home to the draining lymph node after subcutaneous injection and present antigen to antigen specific T cells (see video). Additionally, we use Essen Instruments Incucyte to track dendritic cell maturation, where, at day 10, the morphology of the cultured cells is typical of a mature dendritic cell and &lt;85% of cells are CD11chigh. The study of antigen presentation in peripheral lymph nodes by 2-photon imaging revealed that there are three distinct phases of dendritic cell and T cell interaction1, 2. Phase I consists of brief serial contacts between highly motile antigen specific T cells and antigen carrying dendritic cells1, 2. Phase two is marked by prolonged contacts between antigen-specific T cell and antigen bearing dendritic cells1, 2. Finally, phase III is characterized by T cells detaching from dendritic cells, regaining motility and beginning to divide1, 2. This is one example of the type of antigen-specific interactions that can be analyzed by two-photon imaging of antigen-loaded cell tracker dye-labeled dendritic cells.

Antigen presentation in secondary lymphoid organs by dendritic cells is crucial for the initiation of the T cell mediated adaptive immune response. Here we demonstrate the culture of bone marrow derived murine dendritic cells, activation, and labeling for 2-photon imaging.

2光子イメージングで使用するための骨髄由来マウス樹状細胞の世代

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% ...

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer&#39;s patch (PP). Common dendritic cell progenitors (CDPs, lin- c-kitlo CD115+ Flt3+) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1+ and recipients being CD45.2+. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.

This protocol describes experimental procedures to assess the differentiation of plasmacytoid dendritic cells in Peyer&#8217;s patch from common dendritic cell progenitors, using techniques involving FACS-mediated cell isolation, hydrodynamic gene transfer, and flow analysis of immune subsets in Peyer&#8217;s patch.

造血前駆細胞の養子移入を使用して、パイエル板におけるマウス形質細胞様樹状細胞の開発を評価

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

マウスの樹状細胞サブセットの単離のための効率的かつ高収率方法

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

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

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

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

代謝表現型が異なる未熟、熟女と寛容原性樹状細胞の生成

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

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 Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have recently been recognized to be more heterogeneous than previously appreciated. These cultures routinely contain moDC as well monocyte-derived macrophages (moMac), and even some less developed cells such as monocytes. The goal of this protocol is to provide a consistent method for identification and separation of the many cell types present in these cultures as they develop, so that their specific functions may be further investigated. The sorting strategy presented here separates cells first into four populations based on expression of Ly6C and CD115, both of which are expressed transiently by cells as they develop in GM-CSF-driven culture. These four populations include Common myeloid progenitors or CMP (Ly6C-, CD115-), granulocyte/macrophage progenitors or GMP (Ly6C+, CD115-), monocytes (Ly6C+, CD115+), and monocyte-derived macrophages or moMac (Ly6C-, CD115+). CD11c is also added to the sorting strategy to distinguish two populations within the Ly6C-, CD115- population: CMP (CD11c-) and moDC (CD11c+). Finally, two populations may be further distinguished within the Ly6C-, CD115+ population based on the level of MHC class II expression. MoMacs express lower levels of MHC class II, while a monocyte-derived DC precursor (moDP) expresses higher MHC class II. This method allows for the reliable isolation of several developmentally distinct populations in numbers sufficient for a variety of functional and developmental analyses. We highlight one such functional readout, the differential responses of these cell types to stimulation with Pathogen-Associated Molecular Patterns (PAMPs).

Here we provide a method for identifying and isolating large numbers of GM-CSF driven myeloid cells using high speed cell sorting. Five distinct populations (Common myeloid progenitors, granulocyte/macrophage progenitors, monocytes, monocyte-derived macrophages, and monocyte-derived DCs) can be identified based on Ly6C and CD115 expression.

大量の骨髄前駆細胞と新規セルソーティング戦略を使用してマウスの骨髄から樹状細胞前駆体の生成

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have ...

Development and Functional Characterization of Murine Tolerogenic Dendritic Cells

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state against self-antigens as well as antigens derived from commensal organisms. The disruption of this immune homeostasis can lead to chronic inflammation and to the development of autoimmunity. Dendritic cells (DCs) are the professional antigen-presenting cells of the innate immune system involved in activating na&#239;ve T cells to initiate immune responses against foreign antigens. However, DCs can also be differentiated into TolDCs that act to maintain and promote T cell tolerance and to suppress effector cells contributing to the development of either autoimmune or chronic inflammation conditions. The recent advancement in our understanding of TolDCs suggests that DC tolerance can be achieved by modulating their differentiation conditions. This phenomenon has led to tremendous growth in developing TolDC therapies for numerous immune disorders caused due to break in immune tolerance. Successful studies in preclinical autoimmunity murine models have further validated the immunotherapeutic utility of TolDCs in the treatment of autoimmune disorders. Today, TolDCs have become a promising immunotherapeutic tool in the clinic for reinstating immune tolerance in various immune disorders by targeting pathogenic autoimmune responses while leaving protective immunity intact. Although an array of strategies has been proposed by multiple labs to induce TolDCs, there is no consistency in characterizing the cellular and functional phenotype of these cells. This protocol provides a step-by-step guide for the development of bone marrow-derived DCs in large numbers, a unique method used to differentiate them into TolDCs with a synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid-difluoro-propyl-amide (CDDO-DFPA), and the techniques used to confirm their phenotype, including analyses of essential molecular signatures of TolDCs. Finally, we show a method to assess TolDC function by testing their immunosuppressive response in vitro and in vivo in a preclinical model of multiple sclerosis.

Here, we present a protocol to develop and characterize tolerogenic dendritic cells (TolDCs) and evaluate their immunotherapeutic utility.

開発とマウス免疫寛容樹状細胞の機能解析

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state ...

医学

In Vitro Generation of Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors

Source: Chang, S., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/53211">In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System.</a> J. Vis. Exp. (2015)
This video demonstrates an in vitro assay to generate plasmacytoid dendritic cells (pDCs) from common lymphoid progenitor cells (CLPs). Upon co-culturing purified CLPs with &#947;-irradiated feeder cells in the presence of Fms-like tyrosine kinase-3 ligand (FL), the CLPs differentiate into non-adherent pDCs, the presence of which is confirmed using flow cytometry.

一般的なリンパ系前駆細胞からの形質細胞様樹状細胞のin vitro作製

Source: Chang, S., et al. In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System. J. ...