Christian Welker is funded by a grant provided by the J&#252;rgen-Manchot-Stiftung.

倫理文：すべての手順は、ヘルシンキ宣言に従って実施し、テュービンゲン大学で臨床倫理委員会によって承認された（38 / 2009B02と470 / 2013B02プロジェクト）。 末梢血単核細胞（PBMC）の単離1。 <ol><li>千IUのヘパリン硫酸を含む50 mlシリンジを使用して静脈穿刺を介し健常者50〜100ミリリットルを取り、血液1希釈：PBS（pH値= 7.2）と2。 </li><li> 15ミリリットルの血液分離ソリューションのPBS溶液などBiocollとして（D = 1.077グラム/ ml）を50ml中の円錐管：慎重に血液35mlのレイヤ。室温で800×gで15分間遠心します。 注：ブレーキを使用することはできません。 </li><li>ピペットでリンパ球層の細胞を収集し、少なくとも50 mlのPBSで細胞を2回洗浄（; 400×gで12分間遠心分離）。ピペットで遠心分離後、上清を取り除きます。 </li><li>溶解残り懸濁させることによって赤血球およびincub2-4分間低張緩衝液1-5 mlのリンパ球画分をレーティング。 注：溶解緩衝液の曝露とボリュームの持続時間は、使用される溶解バッファーにし、リンパ球画分に残った赤血球の量に依存します。 </li><li> 12分間2​​50×gでPBS（1:10）、遠心分離で細胞を希釈し、その後12分間300×gで一回10mlのPBSで細胞ペレットを再懸濁し、洗います。 </li><li> PBS中のリンパ球を再懸濁し、トリパンブルー溶液を使用して、ノイバウアーカウントチャンバー内の細胞をカウントします。 注：通常、&gt; 1×10 8のリンパ球を新たに末梢血を集めて100ミリリットルから受信されます。 </li></ol> Vδ1T細胞の単離2。 <ol><li> &lt;Vδ1CD4 T細胞の頻度を決定する最初のFACSのための1×10 6単離したリンパ球を取ります。 注：このT細胞の実体の人口規模は、個人とその免疫学的状態に応じ間で大きく異なる可能性があります。 Typic味方、T細胞の約1％がVδ1+であり、Vδ1+細胞の約1-5％がCD4 +です。 <ol><li> PBS、2％FCSおよび5mM EDTAを含んで構成され、1mLのFACS緩衝液で細胞を希釈します。 2分間660×gで遠心分離し、上清をデカント。約100μlのFACS緩衝液の還流量は、その後の染色のために十分です。簡単に言うと渦とFACS染色の特異性を増大させるために室温で5分間、5μlのFcをブロッキング試薬で細胞を培養します。 </li><li> Fcブロッキング試薬を除去することなく、細胞に直接ヒトモノクローナル抗体を追加します。 （1：200）、CD8（1：200）とTCRαβ（1：25）Vδ1（1時50分）、CD3（1時50分）、CD4に対する抗体で細胞を染色することを確認します。対応する免疫グロブリンアイソタイプとアイソタイプコントロールを準備します。暗所で4℃で20分間、抗体と細胞をインキュベートします。 </li><li> （2分間遠心分離; 660×gにて）を1mlのFACS緩衝液中で細胞を洗浄し、supernをデカントatant、残りのバッファ量にFACS取得に進みます。 </li></ol></li><li> 12分間の遠心分離（FACS分析に使用されていない）細胞をペレット化します。 300 gで。 8℃。上清を除去し、1×10 7個の細胞あたり60μlのMACSバッファーpelletin細胞を再懸濁します。 1×10 7個の細胞あたり20μlのFcをブロッキング試薬を加え、4℃で5分間インキュベートします。 </li><li>細胞に直接1×10 7個の細胞あたり10μlのFITC結合抗ヒトVδ1抗体を追加します。暗闇の中で4℃で12分間インキュベートします。 </li><li> 14ミリリットルMACSバッファー（; 300グラムで、8℃12分間遠心分離器）を用いて細胞を洗浄します。完全に上澄みを捨て、1×10 7個の細胞あたり80μlのMACS緩衝液で細胞を再懸濁します。 </li><li> 1-2×10 5個の細胞を取り、FACS分析により標識の検証のために、100μlのFACS緩衝液でそれらを希釈します。さらなる添加剤は、このステップのために必要とされていません。 suffiがあることを確認してくださいcientのVδ1のための明るさの違い-とVδ1+細胞 。そうでない場合は、繰り返し）は2.3）と2.4を繰り返します。 </li><li>残りのセルに1×10 7細胞あたり20μlの抗FITCマイクロビーズを加え、よく混ぜ、4℃で暗所で15分間インキュベートします。その後、（300×gで、12分間の遠心分離; 8°C）を少なくとも10ミリリットルのMACS緩衝液を用いて細胞を洗浄します。 </li><li>遠心分離の間に、500μlのMACS緩衝液で磁石に配置された予備冷却磁気カラムを平衡化。 </li><li> （1×10 8よりも高い細胞数のために、それに応じてこのボリュームをスケールアップ）500μlのMACS緩衝液中で細胞を再懸濁し、カラムに慎重に細胞を適用します。細胞- Vδ1を含むフロースルーを収集します。 </li><li> 500μlのMACS緩衝液でカラムを3回洗浄し、同じチューブに再びフロースルーを収集します。リザーバーはカラム上にバッファを適用する前に、空でなければならないことに注意してください。 </li><li>磁気デバイスから列を削除し、15ミリリットルコニカルチューブにそれを置きます。すばやくしっかりと列にプランジャーを押して1ミリリットルMACS緩衝液でカラムを洗浄します。チューブに溶出液を収集します。 注：&gt; 98％の純度を得るためには、第二列を含むステップ2.7から2.9）を繰り返すことが通常必要です。 </li><li>前と同じ抗体パネルとのFACS分析18によって細胞の純度を確認します（2.1.2を参照）、細胞をカウントします。 </li></ol> Vδ1CD4+ T細胞の単離3 <ol><li> &gt; 30秒間ボルテックスでCD4ビーズ25μlの再懸濁します。 1分間磁石にチューブを配置することにより、1.5 mlの反応チューブに1 mlのMACS緩衝液でビーズ（製造業者によって示されるように最小量）を洗浄します。小規模ピペット（200μL）で慎重に上清を廃棄し、MACSバッファーの元のボリュームにビーズを再懸濁します。 </li><li> 1Vδ1+細胞 （遠心分離をペレット化2分; 300×gで）し、上清を吸引し、500μlのMACSバッファー内のすべてのVδ1+細胞を再懸濁し、ビーズを含むチューブに追加します。勢いよく混合し、（回転子に、例えば ）一定の傾斜で4℃で20分間インキュベートします。 </li><li> 2分間磁石に細胞を含有するチューブを置きます。蓋には名残がないことを確認してください。 注：CD4 +細胞は、磁気ビーズを結合させ、磁石が直面しているチューブの側面に取り付けられています。 </li><li>慎重に、磁気装置内チューブを保持しながら、蓋を開け、CD4含ま上清を収集-小規模のピペットを用いてVδ1+細胞。別のチューブにVδ1+細胞および集団から、おそらく残りのCD4細胞またはビーズを避けるために、再び磁石にこのチューブを置く- CD4を置きます。 <ol><li> CD4を洗う- 5ミリリットルMACS緩衝液中で細胞（12分間遠心; 300×gで）AND100μlの培地あたり1×10 5細胞の濃度で再懸濁します。 CD4 -細胞が容易に（4.2）の下に与えられた条件下で培養することができます。 </li></ol></li><li> 500μlのMACS緩衝液中で細胞を再懸濁し、磁気デバイスに戻し入れ、磁場の外CD4 + 細胞標的にチューブを置きます。繰り返しは、より高い純度を得るために二度3.3から3.5を繰り返します。ピペットで上清を除去</li><li> 100μlの培養培地中のCD4 +細胞（RPMI 1640、10％FCS、1％L-グルタミン、1％ペニシリン/ストレプトマイシン）に再懸濁し、ビーズ取り外し溶液10μlを加えます。一定の傾斜（例えば、回転体で）と室温で45分間インキュベートします。 </li><li> 1分間磁石に細胞を配置します。注意深く小規模ピペットを用いてビーズを含まないVδ1+ CD4 +細胞を含む上清を収集します。 </li><li>磁石の外では、100μlの培地を繰り返してビーズを再懸濁CD4 +細胞の高い細胞数を得るために、3.6倍及び3.7ステップ。 </li><li>細胞をペレット化する（12分間、300×gで遠心分離）し、ピペッティングにより完全に上清を捨てます。新鮮な、予め温めておいたメディアで細胞を再懸濁し、それらを数えます。ステップ2.1.1-2.1.3に示されたFACS分析によってVδ1+ CD4 +細胞の純度を調べます。 </li></ol>限界希釈4.単一細胞クローニング<ol><li> 1.1〜1.6に示すように、同種異系のドナーからの末梢血単核細胞（PBMC）を単離します。 </li><li> γ線照射を用いて、25 mlの培養培地中で80グレイで2.5×10 7の同種異系のPBMCを照射します。 </li><li>照射されたフィーダー細胞をIL-2（200 U / ml）を、IL-7（20ng / ml）およびPHA（2μg/ ml）を添加し、96ウェルU型プレートに配布し、5×10 4フィーダウェルあたり50μl中の細胞。 </li><li> 50μlの0.3細胞の濃度にVδ1+ CD4 +細胞を希釈します。ピペット各ウェル照射された細胞およびサイトカインを保有する細胞溶液の50μlのTE。 注：このように、サイトカインは、100 U / mlのIL-2を10ng / mlのIL-7とを1μg/ mLのPHAの最終濃度に希釈します。 37℃、5％CO 2の加湿雰囲気でインキュベーター中で96ウェルプレートをインキュベートします。 <ol><li>必要に応じて、クローンの同じ培養条件下でバルク培養物としてVδ1+ CD4 +細胞を精製したまま養います。 </li></ol></li><li>サイトカインおよび新鮮な培地で3〜4日ごとに細胞を供給してください。このため、ピペッティングにより50μlの新鮮な培地でウェル中の培地の交換半分。すべての補充にサイトカインの2倍の濃度を追加します。給餌のも、他のすべてのラウンドあたり5×10 4照射フィーダー細胞を追加します。 </li><li>彼らは代謝、したがって、培地およびフォームコロニーの色を変更すると、顕微鏡を用いて、観察最初のクローンは、3〜4週間後に目に見えるようになります。 注：毛皮のためにTHER栽培は、よくクローンを再懸濁し、96ウェルプレートを分離するためにそれらを転送します。 2.1.1-2.1.3に示すように、FACSを介して細胞を分析します。これらの条件の下に保持したクローンは、最終的にαβTCRにVδ1から自分のTCRを変更することがあります。この分化転換のための時点は、クローンからクローンに変化します。 </li></ol>

<ol>
	<li>Bhandoola, A., von, B. H., Petrie, H. T., Zuniga-Pflucker, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Commitment+and+developmental+potential+of+extrathymic+and+intrathymic+T+cell+precursors:+plenty+to+choose+from.">Commitment and developmental potential of extrathymic and intrathymic T cell precursors: plenty to choose from.</a> Immunity. 26 (6), 678-689 (2007).</li><li>Von, B. H., Melchers, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Checkpoints+in+lymphocyte+development+and+autoimmune+disease.">Checkpoints in lymphocyte development and autoimmune disease.</a> Nat. Immunol. 11 (1), 14-20 (2010).</li><li>Prinz, I., 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=Visualization+of+the+earliest+steps+of+gammadelta+T+cell+development+in+the+adult+thymus.">Visualization of the earliest steps of gammadelta T cell development in the adult thymus.</a> Nat. Immunol. 7 (9), 995-1003 (2006).</li><li>Ferrero, I., 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=TCRgamma+silencing+during+alphabeta+T+cell+development+depends+upon+pre-TCR-induced+proliferation.">TCRgamma silencing during alphabeta T cell development depends upon pre-TCR-induced proliferation.</a> J. Immunol. 177 (9), 6038-6043 (2006).</li><li>Krangel, M. S., Carabana, J., Abbarategui, I., Schlimgen, R., Hawwari, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enforcing+order+within+a+complex+locus:+current+perspectives+on+the+control+of+V(D)J+recombination+at+the+murine+T-cell+receptor+alpha/delta+locus.">Enforcing order within a complex locus: current perspectives on the control of V(D)J recombination at the murine T-cell receptor alpha/delta locus.</a> Immunol. Rev. 200, 224-232 (2004).</li><li>Hawwari, A., Krangel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+for+rearranged+variable+gene+segments+in+directing+secondary+T+cell+receptor+alpha+recombination.">Role for rearranged variable gene segments in directing secondary T cell receptor alpha recombination.</a> Proc. Natl. Acad. Sci. U.S.A. 104 (3), 903-907 (2007).</li><li>Hawwari, A., Krangel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+TCR+delta+and+alpha+repertoires+by+local+and+long-distance+control+of+variable+gene+segment+chromatin+structure.">Regulation of TCR delta and alpha repertoires by local and long-distance control of variable gene segment chromatin structure.</a> J. Exp. Med. 202 (4), 467-472 (2005).</li><li>Hawwari, A., Bock, C., Krangel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+T+cell+receptor+alpha+gene+assembly+by+a+complex+hierarchy+of+germline+Jalpha+promoters.">Regulation of T cell receptor alpha gene assembly by a complex hierarchy of germline Jalpha promoters.</a> Nat. Immunol. 6 (5), 481-489 (2005).</li><li>Hager, E., Hawwari, A., Matsuda, J. L., Krangel, M. S., Gapin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+constraints+at+the+level+of+TCRalpha+rearrangement+impact+Valpha14i+NKT+cell+development.">Multiple constraints at the level of TCRalpha rearrangement impact Valpha14i NKT cell development.</a> J. Immunol. 179 (4), 2228-2234 (2007).</li><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 (2), 133-139 (2004).</li><li>Sprent, J., Tough, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphocyte+life-span+and+memory.">Lymphocyte life-span and memory.</a> Science. 265 (5177), 1395-1400 (1994).</li><li>Guy-Grand, D., 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=Extrathymic+T+cell+lymphopoiesis:+ontogeny+and+contribution+to+gut+intraepithelial+lymphocytes+in+athymic+and+euthymic+mice.">Extrathymic T cell lymphopoiesis: ontogeny and contribution to gut intraepithelial lymphocytes in athymic and euthymic mice.</a> J. Exp. Med. 197 (3), 333-341 (2003).</li><li>McClory, S., 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=Evidence+for+a+stepwise+program+of+extrathymic+T+cell+development+within+the+human+tonsil.">Evidence for a stepwise program of extrathymic T cell development within the human tonsil.</a> J. Clin. Invest. 122 (4), 1403-1415 (2012).</li><li>Jbakhsh-Jones, S., Jerabek, L., Weissman, I. L., Strober, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extrathymic+maturation+of+alpha+beta+T+cells+from+hemopoietic+stem+cells.">Extrathymic maturation of alpha beta T cells from hemopoietic stem cells.</a> J. Immunol. 155 (7), 3338-3344 (1995).</li><li>Garcia-Ojeda, M. E., 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=Stepwise+development+of+committed+progenitors+in+the+bone+marrow+that+generate+functional+T+cells+in+the+absence+of+the+thymus.">Stepwise development of committed progenitors in the bone marrow that generate functional T cells in the absence of the thymus.</a> J. Immunol. 175 (7), 4363-4373 (2005).</li><li>Arcangeli, M. L., 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=Extrathymic+hemopoietic+progenitors+committed+to+T+cell+differentiation+in+the+adult+mouse.">Extrathymic hemopoietic progenitors committed to T cell differentiation in the adult mouse.</a> J. Immunol. 174 (4), 1980-1988 (2005).</li><li>Maillard, I., 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=Notch-dependent+T-lineage+commitment+occurs+at+extrathymic+sites+following+bone+marrow+transplantation.">Notch-dependent T-lineage commitment occurs at extrathymic sites following bone marrow transplantation.</a> Blood. 107 (9), 3511-3519 (2006).</li><li>Ziegler, H., 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=Human+Peripheral+CD4(+)+Vdelta1(+)+gammadeltaT+Cells+Can+Develop+into+alphabetaT+Cells.">Human Peripheral CD4(+) Vdelta1(+) gammadeltaT Cells Can Develop into alphabetaT Cells.</a> Front Immunol. 5, 645 (2014).</li><li>Mollet, M., Godoy-Silva, R., Berdugo, C., Chalmers, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computer+simulations+of+the+energy+dissipation+rate+in+a+fluorescence-activated+cell+sorter:+Implications+to+cells.">Computer simulations of the energy dissipation rate in a fluorescence-activated cell sorter: Implications to cells.</a> Biotechnol Bioeng. 100 (2), 260-272 (2008).</li></ol>

The authors have nothing to disclose.

その正磁気細胞分離のためVδ1およびCD4：希少（T-）細胞エンティティ、すなわちVδ1+ CD4 + T細胞の表現型、生物学と機能を研究するために、我々は二つのマーカーを使用していました。 CD4は、Tヘルパー細胞上に発現されるのに対し、Vδ1は、単球および樹状細胞上に低レベルで、および造血前駆細胞上で非常に低レベルで、オーファン受容体です。 高純度の細胞?...

脊椎動物では、細胞性および体液性免疫の部分で構成されている適応免疫は、病原体に対する防御において重要な役割を果たしています。抗原の広範囲の認識は、T細胞に関して胸腺1を中心に製造されているものとするhyperpolymorphic T-およびB細胞受容体（TCR / BCR）によって媒介されます。これには、骨髄由来の造血幹細胞（HSC）は、胸腺に播種し、最終的にすべてのT細胞系統を生じる明確に定義されたステージに沿って分化します。およびCD8 - -胸腺播種前駆細胞は、CD4であり、したがって、未熟、ダブルネガティブ（DN）胸腺細胞の割合を構成しています。胸腺由来の信号は、その系統のコミットメントとαβまたはγδT細胞のいずれかへの分化を誘導します。 DN2 / 3胸腺細胞で機能的に再編成されたTCR-γおよびTCR-δ鎖遺伝子の発現は、細胞増殖のANを駆動δTCR複合体を、γにつながりますDγδT細胞2,3への分化を促進します。これとは対照的に、preTCRのpTを構築するためにpreTαとペアにすることができ、機能TCR-β鎖の再配列は、転写DN3胸腺細胞におけるTCR-γ鎖のサイレンシングおよび CD4 + CD8 +ダブルポジティブ胸腺細胞4への移行を誘導。この段階では、TCR-α鎖の再結合は、このようにこれらの細胞取消不能5-9で生産γδTCRを抑止、TCR-α遺伝子座内にたたずむTCR-δ遺伝子座を削除、発生します。再配置αβTCRsはその後自己免疫（ネガティブ選択）を避けるために、特定のしきい値を超えてはならない弱（ポジティブ選択）、自己MHCに結合するそれらの能力のために選択されます。結合MHCクラスIまたはIIの能力に応じて、選択されたαβT細胞は、単一陽性CD4 +またはCD8 + T細胞にその出口胸腺としてナイーブT細胞を開発します。 しかし、胸腺の退縮はほとんど消灯後思春期10でナイーブT細胞の指数関数的に減少する出力をリードする人生の早い段階で開始します。それにもかかわらず、T細胞プールのサイズは、ポスト胸腺恒常性T細胞の増殖および長寿命の免疫メモリ11の増殖によってのみ部分的に説明できる寿命を通して一定のままです。したがって、胸腺外T細胞の開発が行われなければなりません。最近の研究では、胸腺外で機能αβT細胞12-17を生じサイト-たαβT細胞前駆体を、特徴であるかなりの魅力を得ています。しかし、胸腺から独立したが、αβT細胞に分化胸腺外αβT細胞前駆体についての詳細な知識は、我々は、彼らがそれによって取るルート上に持っている背景のように断片的です。 我々は最近、Vδ1+の小さなT細胞エンティティを特定し</su健常人ドナーの末梢血から分離された胸腺外αβT細胞prognitor 18、などのp&gt; CD4 +γδT細胞は軽度の炎症環境でαβT細胞に分化転換することができます。興味深いことに、潜在的に新規の抗原を認識することができるように、このように、レパートリーの多様性を拡大後の胸腺T細胞の恒常性増殖、Vδ1CD4 +細胞の分化転換新たなT細胞受容体を生成する逆保護よい新たに取得された病原体に対する宿主。これは、T細胞の可塑性に追加され、胸腺外T細胞の開発のために、これまで正しく評価されていない新しい経路を追加します。 目的は、このαβT細胞が胸腺外発達をprecursor`sトリガこれらのマーカーおよび分子を同定するために、リンパ球の供給源からの定量的な分離は、単一細胞クローンの生成及びその効率的な拡張が必須です。

<table border="0" cellpadding="0" cellspacing="0" width="789">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Biocoll Solution</td>
			<td style="width:93px;">Biochrom</td>
			<td style="width:119px;">L 6113</td>
			<td style="width:361px;">lymphocyte separating solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Lysing Buffer</td>
			<td style="width:93px;">BD BioSciences</td>
			<td>555899</td>
			<td>lysis of erythrocytes&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Phosphate-buffered Saline</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td style="width:119px;">D8537&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MACS buffer</td>
			<td style="width:93px;">Miltenyi Biotec</td>
			<td>130-091-222</td>
			<td>supplement with BSA and pre-cool before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">BSA</td>
			<td style="width:93px;">Miltenyi Biotec</td>
			<td>130-091-376</td>
			<td>not mandatorily from this supplier</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">anti-human Vd1 FITC (clone:&#160; TS8.2)</td>
			<td style="width:93px;">Thermo Scientific</td>
			<td>TCR2730</td>
			<td>not mandatorily from this supplier</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">anti-human CD3 PerCP (clone: SK7)</td>
			<td style="width:93px;">BD BioSciences</td>
			<td>345766</td>
			<td>not mandatorily from this supplier or this flurochrome</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">anti-human TCRab PE (clone: T10B9.1A-31)</td>
			<td style="width:93px;">BD BioSciences</td>
			<td>555548</td>
			<td>not mandatorily from this supplier or this flurochrome</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">anti-human CD4 VioBlue (clone: M-T466)&#160;</td>
			<td style="width:93px;">Miltenyi Biotec</td>
			<td>130-097-333</td>
			<td>not mandatorily from this supplier or this flurochrome</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">anti-human CD8 APC-H7 (clone: SK1)</td>
			<td style="width:93px;">BD BioSciences</td>
			<td>641400</td>
			<td>not mandatorily from this supplier or this flurochrome</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Anti-FITC MultiSort Kit</td>
			<td style="width:93px;">Miltenyi Biotec</td>
			<td>130-058-701</td>
			<td>yields better results than anti-FITC MicroBeads</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MS columns</td>
			<td style="width:93px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-042-201</td>
			<td>pre-cool before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">MiniMACS Separator</td>
			<td style="width:93px;">Miltenyi Biotec</td>
			<td>130-042-102</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CD4 Positive Isolation Kit</td>
			<td style="width:93px;">life technologies</td>
			<td>11331D</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and ex vivo culture of v&#948;1+cd4+&#947;&#948; t cells, an extrathymic &#945;&#946;t-cell progenitor

胸腺、αβT細胞および脊椎動物における適応免疫系の主鎖を生成するための主要な器官は、長いαβT細胞の唯一の供給源として考えられてきました。しかし、胸腺退縮は、周囲へのナイーブαβT細胞の劇的に減少出力につながる人生の早い段階で開始します。それにもかかわらず、あっても百歳は、新たに取得した病原体に対する免疫を構築することができます。最近の研究では、機能の胸腺損失を補償することができる経路のが我々の理解はまだルディメンタルあり、胸腺外αβT細胞の発生を示唆しています。 γδT細胞は、組織における主要なT細胞のサブセットを構成する生来のリンパ球です。我々は最近、CD4 +Vδ1+の γδの希少エンティティT細胞が炎症状態にαβT細胞に分化転換できることを示すことによって、γδT細胞サブセットに、これまで正しく評価されていない優れた機能を帰する。ここでは、P末梢血から、この前駆細胞の単離およびその後の培養のためのプロトコルをrovide。 Vδ1細胞を積極我々は、磁気標識技術とCD4 +細胞の乏しい部分を対象と第2のステップが続く、磁気ビーズを使用して、健康なヒトのドナーのPBMCから濃縮されます。第二の標識の磁力が第一の磁性ラベルの1を超え、従って、関心の人口の、効率的な定量的・具体的な正の単離を可能にします。それから、細胞をクローニングし、効率的に拡大するためのFACS解析によって生成されたクローンの同定に必要な技術や文化状況を紹介します。そこで、精製、文化および CD4 +Vδ1+γδT細胞のex vivoでの拡大のための詳細なプロトコルを提供します。この知識は、このαβT細胞progenitor`s生物学に関連する研究のため、IDEに目指す人のための前提条件でありますその転換に関与している分子トリガーをntify。

図1は、種々の段階及び末梢血からVδ1T細胞の単離の結果を示している。 図1Aは、CD3 +リンパ球におけるVδ1+細胞の代表的な分布、ならびにVδ1+集団の共受容体の発現を示します。このドナーでは、Vδ1+細胞 （赤）の周波数は、総リンパ球数の2.3％であり、Vδ1+リンパ球のCD4発現（緑）が2.6％です。要するに、分離のための標的?...

Watch this Scientific Journal Video about Isolation and Ex Vivo Culture of VÎ´1+CD4+Î³Î´ T Cells, an Extrathymic Î±Î²T-cell Progenitor at JoVE.com

Isolation and Ex Vivo Culture of V&amp;#948;1+CD4+&amp;#947;&amp;#948; T Cells, an Extrathymic &amp;#945;&amp;#946;T-cell Progenitor

Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral V&#948;1+CD4+ T cells that is, as we showed recently, an extrathymic &#945;&#946; T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.

単離と<em&gt;エクスビボ</emVδ1の&gt;文化<sup&gt; +</sup&gt; CD4<sup&gt; +</sup&gt;γδT細胞、胸腺外αβT細胞前駆

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been ...

isolation-ex-vivo-culture-v1-cd4-t-cells-an-extrathymic-t-cell

Research

JoVE Journal

Immunology and Infection

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

9.2K ビュー.  Children's University Hospital Tübingen. Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral Vδ1+CD4+ T cells that is, as we showed recently, an extrathymic αβ T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.

ビデオ: Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

移行のヒトTリンパ球の単離、培養および解析インビトロ</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

免疫・感染学

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide complete protection against the development or recurrence of breast cancer. To overcome this problem by increasing the frequency of tumor-reactive T cells, adoptive immunotherapy has been employed. A variety of protocols have been used for the expansion of tumor-specific T cells. These protocols, however, are restricted to the use of tumor antigens ex vivo for the activation of antigen-specific T cells. Very recently, common gamma chain cytokines such as IL-2, IL-7, IL-15, and IL-21 have been used alone or in combination for the enhancement of anti-tumor immune responses3. However, it is not clear what formulation would work best for the expansion of tumor-reactive T cells. Here we present a protocol for the selective activation and expansion of tumor-reactive T cells from the FVBN202 transgenic mouse model of HER-2/neu positive breast carcinoma for use in adoptive T cell therapy of breast cancer. The protocol includes activation of T cells with bryostatin-1/ionomycin (B/I) and IL-2 in the absence of tumor antigens for 16 hours. B/I activation mimics intracellular signals that result in T cell activation by increasing protein kinase C activity and intracellular calcium, respectively4. This protocol specifically activates tumor-specific T cells while killing irrelevant T cells. The B/I-activated T cells are cultured with IL-7 and IL-15 for 24 hours and then pulsed with IL-2. After 24 hours, T cells are washed, split, and cultured with IL-7 + IL-15 for additional 4 days. Tumor-specificity and anti-tumor efficacy of the ex vivo expanded T cells is determined.

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

An efficient protocol for the ex vivo expansion of tumor-reactive T cells from tumor-draining lymph nodes or other secondary lymphoid tissues of tumor-bearing hosts is described. This protocol selectively expands tumor-specific T cells for use in adoptive immunotherapy of breast cancer.

ブリオスタチンの1/Ionomycinによる腫瘍反応性T細胞のex vivoでの拡張と共通γ鎖のサイトカインの策定

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide ...

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration of T cells into lymph nodes is a complex process which involves both cellular and chemical factors including chemokines. Recently, the use of two-photon microscopy has permitted to track T cells in intact lymph nodes and to derive some quantitative information on their behavior and their interactions with other cells. While there are obvious advantages to an in vivo system, this approach requires a complex and expensive instrumentation and provides limited access to the tissue. To analyze the behavior of T cells within murine lymph nodes, we have developed a slice assay 
1, originally set up by neurobiologists and transposed recently to murine thymus 2. In this technique, fluorescently labeled T cells are plated on top of an acutely prepared lymph node slice. In this video-article, the localization and migration of T cells into the tissue are analyzed in real-time with a widefield and a confocal microscope. The technique which complements in vivo two-photon microscopy offers an effective approach to image T cells in their natural environment and to elucidate mechanisms underlying T cell migration.

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes.

広視野と共焦点顕微鏡によるマウスリンパ節スライスにおけるT細胞のex vivoでのイメージング

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration ...

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.

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

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 ...

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

アン<em&gt;エクスビボ</em急性脳スライスにおけるオリゴデンドロサイト指向T細胞攻撃の&gt;モデル

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

表現型分析のためのヒト皮膚からリンパ球単離と<em&gt; ex vivoで</em&gt;細胞培養

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the complexities of the organoid growth characteristics carry significant caveats for the investigator. Specifically, the growth patterns of each individual organoid are highly variable and create a heterogeneous population of epithelial cells in culture. With such caveats, common tissue culture practices cannot be simply applied to the organoid system due to the complexity of the cellular structure. Counting and plating based solely on cell number, which is common for individually separated cells, such as cell lines, is not a reliable method for organoids unless some normalization technique is applied. Normalizing to total protein content is made complex due to the resident protein matrix. These characteristics in terms of cell number, shape and cell type should be taken into consideration when evaluating secreted contents from the organoid mass. This protocol has been generated to outline a simple procedure to culture and treat small intestinal organoids with microbial pathogens and pathogen associated molecular patterns (PAMPs). It also emphasizes the normalization techniques that should be applied when protein analysis are conducted after such a challenge.

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Here, a protocol to harvest, maintain, and treat mouse small intestinal organoids with pathogen associated molecular patterns (PAMPs) and Listeria monocytogenes is described, as well as emphasis on gene expression and proper normalization techniques for protein.

ザ<em&gt; ex vivoで</emマウス腸オルガノイドの&gt;文化とパターン認識受容体刺激

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the ...

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCR&#945; and TCR&#946; chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR.

We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice.

骨髄前駆細胞のレトロウイルス形質導入は、T細胞受容体Retrogenicマウスを生成するために、

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Here we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with infectious bursal disease virus, and the quantification of viral replication.

伝染性ファブリキウス嚢病ウイルスの病原性を研究するためのEx Vivo鶏ファブリキウス嚢細胞培養モデル

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...