The author would like to thank the NIH Tetramer Core Facility for tetramer production. The author would also like to thank E.D. O&#8217;Connor and K.E. Marquez at the UCSD Human Embryonic Stem Cell Core Facility flow cytometry laboratory for assistance in cell sorting. This work was funded by National Institutes of Health grant K08AI085039 (G.P.M.).

注意：このプロトコルは、ヒトボランティアから末梢血サンプルを使用する必要がある。ヒト被験者を持つすべての研究はヘルシンキ宣言（2013）1996年の医療保険の携行性と責任法の遵守を確実にするために人間学部治験審査委員会によって審査され、承認されるべきである。 全血由来のT細胞の単離<ol><li>開始する前に、室温に密度勾配媒体を温める。アリコート2-4無菌の15ミリリットルの円錐遠心管に密度勾配培地4ml（1チューブは、それぞれ10ミリリットルに希釈した血液の総容積のために使用される）。 </li><li>コー​​ティングされた1-2ヘパリンナトリウムスプレー（緑·トップ）静脈採血管中の血液の10〜20ミリリットル得る。訓練された個人の監督下でヒト検体を収集し、治験審査委員会によるとプロトコルを承認した。 </li><li> 70％エタノールでチューブの外側を拭きます。注意深くFiのの上部を取り外し収集チューブをlledおよび無菌50ml遠心チューブに血液をデカントする。 </li><li>各採血管10 mlの滅菌リン酸緩衝生理食塩水（PBS）を加える。 、PBSを収集デカント全血に追加し、穏やかに混合する。 </li><li> 25μlのヒトT細胞濃縮カクテル/ 2ミリリットルの合計容量を追加します。 20分間室温でインキュベートする。 </li><li>密度勾配媒体の上に静かに1希釈した血液10 mlピペットを用いて、1を10mlまで層。密度勾配媒体の表面を乱さないように注意してください。 </li><li>遠心分離機は、20 Oで20分間、1200×gでの血液及び密度勾配媒体を層状</li><li>密度勾配媒体及び密度勾配培地と希釈血漿の間の界面での白血球の層との界面を乱さないように注意しながら、遠心機からチューブを取り外す。を5mlピペットを用いて、慎重に白血球層を収集し、新たな滅菌50mlコニカル槽に移す電子メール。 </li><li> 50mlに収集されたPBLの音量を持参し、穏やかに混合するために、PBSを追加します。 </li><li> 20 Oで5分間600×gで遠心分離デカントします。 </li><li>緩衝液（1％BSAを含有する滅菌濾過したPBS）をソーティングサイトメトリ10ml中のペレット化した細胞を無菌の流れを再懸濁する。細胞懸濁液のサンプル10μlを、血球計算板を用いて細胞をカウントするために1：10（90μlに追加）パンブルーを希釈（期待利回り1 -全血の4×10 6細胞/チューブ）。 <ol><li> 5ミリリットルチューブに移し、1ミリリットルのアリコートを取り出し、四量体によって標識されていないT細胞の解析のために氷上に保つ。氷上で細胞懸濁液を保管してください。 </li></ol></li></ol>アロ抗原特異的T細胞の2磁気濃縮<ol><li> 1にアロ抗原のpMHC四量体を希釈：無菌ソートバッファ100。 </li><li> 20℃。で5分間600×gで遠心細胞懸濁液（1.11ステップ9 ml）をデカントします。 </li><li>ペレット化に希釈したアロ抗原のpMHC四量50μlのを追加細胞（10 7細胞まで）。穏やかなピペッティングにより混和する。無菌5ミリリットルチューブに移す。室温で30分間インキュベートする。 </li><li> 2ミリリットルソートバッファを追加します。 20 Oで5分間600×gで遠心分離デカントします。 </li><li> 100μlのソートバッファで細胞を懸濁します。 10μlのビオチン選択カクテルを加え、室温で15分間インキュベートする。 </li><li> 5μlの磁性ナノ粒子を添加し、室温で10分間インキュベートする。 </li><li>ソートバッファおよびピペッティングにより穏やかに混合2.5ミリリットルを追加します。 5ミリリットルチューブに移し、100μlのアリコートを外し、前増菌細胞の分析のために氷上に保つ。 </li><li>細胞分離マグネット中の細胞懸濁液を含む5 mlチューブを置き。キャップは、チューブの上に緩くする必要があります。室温で5分間インキュベートする。 </li><li>優しくチューブからキャップを取り外します。一緒にチューブと磁石を保持し、新鮮な5ミリリットルチューブにチューブの内容をデカント。にチューブの内容をタップするか、振らないでください液体の最後の水滴を除去。 </li><li>磁石からチューブを外します。 2ミリリットルの冷たいソートバッファを追加し、ピペッティングにより穏やかに混合。 2血球計数器を用いて細胞をカウントし、トリパンブルーを（10μlに追加）：サンプルの細胞懸濁液10μlを、1が希薄。 </li></ol>単一細胞フローサイトメトリー細胞選別のためのT細胞の調製<ol><li> （、四量体標識されていない四量体で標識された非濃縮、および四量体で標識された濃縮された）は、細胞の全てのチューブに冷たいソートバッファを追加し3ミリリットルにボリュームをもたらすために。 </li><li> 4°Cので5分間600×gで遠心分離し、デカントバッファー、細胞を回収した。 </li><li> 25μlの冷たいソートバッファでペレット化した細胞を再懸濁。 5μlのヒトFcブロックを追加します。 20分間氷上でインキュベートする。 </li><li>フローサイトメトリーソーティングのための抗体を調製する。 15μlのソートバッファ、15μlの抗CD5、15μlの抗CD14、および15μlの抗CD19を混ぜる。 </li><li>細胞の各チューブに抗体混合液20μlを追加します。 20分間氷上でインキュベートする。 </li><li>各チューブに冷たいソートバッファの2ミリリットルを追加します。 </li><li> 4°Cので5分間600×gで遠心分離し、デカントバッファー、細胞を回収した。 </li><li>並べ替えバッファ内の1 -2×10 6細胞/ mlの濃度で細胞を再懸濁する。 </li><li>ヒトT細胞培養培地のアリコートを100μl（2mMのグルタマックスを補充したIscoveのDMEM、10 mMのHEPES、50μg/ mlのゲンタマイシン、50μM2-メルカプトエタノール、10％熱不活性化ヒトAB血清、および2.5 / mlの組換えヒトIL-2）を96ウェル丸底細胞培養プレートの各ウェルに。氷の上に保管してください。 </li></ol>単一細胞フローサイトメトリーソーティングによる四量体で標識されたT細胞の4.絶縁<ol><li>アロ抗原のpMHC四量体標識したT細胞（ 図2.A）を識別するために、フローサイトメトリー·ゲーティング·パラメータを確立します。 、ダブレットを排除するためのゲーティング戦略を確立するために、前増四量体標識された画分を使用してリンパ球にゲート、B細胞、およびCD14を発現するCD19を除外する単球を発現し、そしてCD5発現によりT細胞を同定する。 （四量体によって標識されていないサンプルからの細胞は、このゲート内に入る必要があります- - CD19 CD5 + CD14の0％を含む）四量体結合性T細胞の同定のためのゲーティングを確立するために、蛍光として四量体で標識されていないサンプルから1を引いたコントロールを使用します。 注：十分に厳しくないゲーティング戦略が過度に制限ゲーティング戦略は、単離されたT細胞の数を減少させる一方で、抗原特異的でない非T細胞又はT細胞の単離をもたらす。 </li><li>単セルのソートのためのプレートのパラメータをプログラムする。各ウェルに四量体+細胞- CD19 - 1 CD5 + CD14を配置するソーターを指示します。ウェル（ウェルにソートされない細胞）を1ネガティブコントロールが含まれていないようにセットアッププレートと1ポジティブコントロールを直接（100 CD5 + CD14 - CD19 -四量体-細胞）各行の（ 図2.B </strong&gt;）。 </li><li>確立されたフローサイトメトリーゲーティング戦略とプレートセットアップを用いて、フローサイトメトリー細胞選別機を使用して直接96ウェルプレートに、個々の四量体結合性T細胞を単離する。 </li></ol> 5.文化とアロ抗原特異的T細胞クローンの拡大<ol><li>細胞選別、20℃。で5分間600×gで遠心分離板の完了後6％CO 2インキュベーター中で37°Cの時の場所培養プレート。 </li><li> 30秒間ボルテックス刺激抗CD3 /抗CD28マイクロビーズは、ビーズを再懸濁する。 </li><li>収集されたT細胞の刺激に必要な活性化因子ビーズの量（活性化因子ビーズ/ウェルの0.5μl）を計算します。無菌5ミリリットルチューブに刺激ビーズの計算されたボリュームを転送します。 1ミリリットルのヒトT細胞培養培地およびボルテックスを追加する。 </li><li>磁石でビーズ懸濁液とチューブを置きます。キャップは、チューブの上に緩くする必要があります。室温で2分間インキュベートする。 </li><li>そっと取り外しチューブからキャップ。新鮮な5ミリリットルチューブに、一緒にデカントチューブ内容物をチューブと磁石を保持している。チューブの内容をタップするか、振らないでください。 </li><li>磁石からチューブを外します。ヒトT細胞培養培地を追加します（マイクロビーズ100μlの培地/ 0.5μlを最初に添加）。 </li><li>アリコートを96ウェルプレートの各ウェルにベータービーズ懸濁液100μl。 6％CO 2インキュベーター内で37°Cの時に培養プレートをインキュベートする。 </li><li>顕微鏡検査によって毎日の細胞増殖を監視します。培養7日間（ 図3.A） -増殖細胞の小さなクラスターが5の後、顕微鏡で観察することができる。 注：この時点でのT細胞培養物の可視化は困難である可能性があり、この段階で観察できる細胞クラスターが存在しないことは、成長しているT細胞の欠如を示すことがないかもしれない。 </li><li>細胞単離後14日目に、慎重に文化の一番のオフとメ​​ディアの100μlのを除去することにより、フィード培養は100μと交換; lの新鮮なヒトT細胞培養培地。 6％CO 2インキュベーター内で37°Cの時に培養プレートをインキュベートし続ける。 </li><li>顕微鏡検査と培養液の色を評価することによって毎日の細胞増殖を監視します。 3日後に培地交換 - 大細胞クラスターは2微視的に見えるはずです。巨視的に、細胞ペレットを培地交換後14日の間に目に見えるようになるはずです。 </li><li>細胞単離に続いて28日目に、顕微鏡検査によって成長しているクローンを同定。 200μlのヒトT細胞培養培地を含む48ウェル組織培養プレートの個々のウェルに成長陽性の96ウェルプレート培養の200μlの体積を転送する。 </li><li>刺激抗CD3 /抗CD28マイクロビーズ（ - 5.6のセクション5.2に記載のように調製）12.5μlのを含む100μlを添加する。 6％CO 2インキュベーター内で37°Cの時に培養プレートをインキュベートする。 </li><li>微視的元によって毎日の培養増殖をモニター培地の色のアミノ化および評価。 T細胞培養物が&gt; 2×10 6 / mlに達したとき（通常は3~5日間、再刺激した後、メディアが開始された日を推定することができる淡黄色の回転）を、24ウェル組織培養プレートのウェルに、転送文化そして500μlのヒトT細胞培地を追加します。 </li><li> 10-14日目のpMHC四量体の分析が結合（セクション3および4.1に記載されてラベル付けおよびゲーティング戦略を使用して）フローサイトメトリーによりアロ抗原特異性を評価するために、T細胞培養液200μlを収集し、再刺激に従ってください。 </li></ol> 6.長期再刺激および培養T細胞クローンの<ol><li>継続的に14日ごとにすることができる所望の特異性を有するクローン性T細胞培養物を再刺激し、展開します。最後の抗CD3 / CD28マイクロビーズ刺激後14日目に無菌の5ミリリットルチューブにT細胞培養を収集します。 2.5ミリリットルの容積まで、単一のチューブ内で同一のT細胞クローンの複数のウェルを組み合わせる。 </li&gt; <li> 2.5ミリリットルの最終容量を持参し、ピペッティングにより穏やかに混合するためにメディアを追加します。 </li><li>培養物から古いマイクロビーズを除去して細胞分離磁石にT細胞培養を含む5 mlチューブを置きます。キャップは、チューブの上に緩くする必要があります。室温で5分間インキュベートする。 </li><li>優しくチューブからキャップを取り外します。一緒にチューブと磁石を保持する新鮮の5mlチューブに細胞懸濁液をデカントする。タップまたは液体の最後の滴を除去するためにチューブの内容を振らないでください。 </li><li> 4ミリリットルに最終容量を持って来るためにメディアを追加します。サンプル10μlを血球計数器を用いて細胞をカウントする。 </li><li>室温で5分間、600×gで遠心分離することによってT細胞を回収する。 </li><li>上清を除去。 10 6細胞/ mlのヒトT細胞培養培地で再懸濁し、T細胞。 24ウェル組織培養プレートの各ウェルに移し1mlアリコートを。 </li><li>刺激性抗CD3 /αの12.5μLを含む100μlのメディアを追加することによってT細胞を再刺激するNTI-CD28マイクロビーズ（セクション5.2で説明したように - 5.6）。 6％CO 2インキュベーター内で37°Cのでインキュベートする。 </li></ol>

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+receptor+T+cells+mediate+pathologic+alloreactivity+in+patients+with+acute+graft-versus-host+disease.">Dual receptor T cells mediate pathologic alloreactivity in patients with acute graft-versus-host disease.</a> Sci. Transl. Med. 5 (188), (2013).</li><li>Altman, J. D., Reay, P. A., Davis, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+functional+peptide+complexes+of+class+II+major+histocompatibility+complex+proteins+from+subunits+produced+in+Escherichia+coli.">Formation of functional peptide complexes of class II major histocompatibility complex proteins from subunits produced in Escherichia coli.</a> Proc. Natl. Acad. Sci. USA. 90 (21), 10330-10334 (1993).</li><li>Sabatino, J. J., Huang, J., Zhu, C., Evavold, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+prevalence+of+low+affinity+peptide-MHC+II+tetramer-negative+effectors+during+polyclonal+CD4++T+cell+responses.">High prevalence of low affinity peptide-MHC II tetramer-negative effectors during polyclonal CD4+ T cell responses.</a> J. Exp. Med. 208 (1), 81-90 (2011).</li></ol>

The authors declare no competing financial interests.

T cell alloreactivity is a long-studied and clinically-relevant phenomenon. The robust proliferative and effector responses of T cells to allogeneic stimulation has enabled extensive analyses of human T cell responses in vitro through relatively straightforward mixed lymphocyte reactions of peripheral blood T cells against inactivated allogeneic cells. However, these primary alloreactive T cell responses are oligoclonal, comprised of a large number of individual T cells responding to specific alloantigens. This ...

Tリンパ球は、適応免疫系の重要な構成要素である。 T細胞だけでなく、直接のエフェクター機構の様々を通して病原体に対する防御免疫応答を媒介するだけでなく、積極的な免疫学的自己寛容を維持し、免疫系の他の細胞の応答を指示するための責任がある。これらの機能は、T細胞受容体（TCR）ライゲーション、サイトカインおよびケモカインを含む統合された信号の数、および代謝産物1を介して導かれる。これらの信号のうち、TCRは、適応免疫におけるT細胞の役割を定義する特徴的な特異性を提供するので、特に重要である。 TCRは、T細胞エフェクター機能を開始する信号を提供するために、高度に特異的かつ高感度な方法でMHC（ヒトオルソログHLA）分子（pMHC複合体）によって提示された線状ペプチド抗原と相互作用する。 pMHC複合リガンドとTCRとの相互作用の生化学的パラメータだけでなく、Tに対する特異性を提供する細胞の活性化だけでなく、その後のT細胞機能2に定性的な影響を与える。したがって、T細胞の機能を研究することは、多くの場合に定義された抗原特異性を有するクローン性T細胞の応答を調べることが必要である。 約10 12αβT細胞を含むヒトT細胞区画、推定された10 7個含まれている- 10 8の異なるαβTCRs3-4。この多様なレパートリーは、防御免疫のためのT細胞応答を必要とするであろう潜在的な病原体からのペプチドの広大な配列を認識するための機会を提供する。その抗原5の前に免疫応答の非存在下で10 -7 -それは、自己MHCにより提示される特定の外来抗原に応答するT細胞の頻度は、10 -4のオーダーであると推定される。ナイーブT細胞レパートリーは、ペプチド抗原および制限が反応提示自己MHCを認識する能力を確保するために、胸腺選択することによって成形される自己ペプチド抗原に対するivity、防御免疫2を仲介するための潜在的な有用性を最大化する。しかし、この設計された反応性は、比較的大きな周波数に違反して、10 -3 -免疫学的にナイーブな個体由来のT細胞の10 -4は 、外来MHC分子だけでなく、それらが提示する内因性ペプチドの両方を認識し、同種異系の細胞による刺激に応答する6。同種異系のpMHCリガンドの認識は、自己MHCによって提示された外来抗原の認識と構造的に類似している。 TCRは同種異系MHC分子だけでなく、提示されたペプチド7の両方で重要な生化学的相互作用を行う。同種細胞8の表面上に存在するpMHC複合体の多様性から、同種刺激の結果に対するT細胞の応答の強固な性質。これは、各MHC 9、約2×10 4個の異なる内因性のペプチド抗原を提示すると推定される。このB同種刺激に対する応答のreadthは、T細胞の同種反応性に起因する、そのような宿主病（GVHD）に対する同種移植片拒絶または移植片として臨床的に関連する病理の重要な側面である。 ヒトT細胞のアロ反応性応答の研究は、伝統的に同種異系細胞での刺激の後に、ナイーブT細胞のポリクローナル応答を調べることに頼っている。限界希釈と組み合わされ、同じ同種異系の細胞株での反復刺激は同種異系HLA 10の定義された認識にクローン性T細胞を生成することができる分析。しかしながら、このアプローチは、HLAがT細胞の広範なレパートリーを刺激する特定の同種のために存在する内因性pMHC複合体の大規模で多様なレパートリーとして、個々の同種のpMHCリガンドへの応答を調べるための問題である。このバルク集団の刺激および限界希釈手法が望まをReactiでT細胞を単離するために多数のクローンのスクリーニングを必要とするシングルのpMHCリガンドに対するVITY。さらに、個々の同種のpMHCリガンドに応答するT細胞の頻度は、所与の抗原に応答したヒトT細胞クローンの効率的な生成に対する障壁を提示ナイーブT細胞集団の中でも比較的低い。 ポリクローナル集団からの抗原特異的T細胞の同定および単離は、フルオロフォアで標識されたpMHC複合多量体11の開発によって使用可能になっている。このアプローチは、ストレプトアビジン標識フルオロフォアに結合することによって標識される組換え可溶性ビオチン化MHC分子にロードされ、特定のペプチド抗原を利用する。のpMHCの多量体は、可溶性のpMHCリガンドのTCRの本質的に低い（μm）との親和性を補償、アビディティーを増加させる。標識細胞は、フローサイトメトリーによって同定し、単離することができる。しかしながら、このアプローチは依然として典型的にナイーブT細胞集団の中の抗原特異的T細胞の低頻度によって制限されほとんどのフローサイトメーター上で正確な同定と定量限界未満の桁違い。この制限に対処するために、四量体標識された細胞についてのpMHC四量体標識およびその後の磁気ビーズの濃縮方法は、12を開発してきた。この方法は、信頼できる検出、計数、および低周波の抗原特異的T細胞の単離を実証した。 このプロトコルは、具体的には、個々の同種異系のpMHCリガンドに反応するヒトT細胞クローンを生成するための効果的なプロトコルについて説明します。このプロトコルは、細胞をフローサイトメトリー選別および単ソートされた細胞（からのT細胞クローンの産生を可能にするために、ヒトT細胞のin vitro培養のための堅牢な方法ではアロ抗原特異的ヒトT細胞の単離のためのpMHC（HLA）多量体標識および濃縮を適用する図1の概要）。

<table border="0" cellpadding="0" cellspacing="0" width="907">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Name of Reagent/ Equipment</td>
			<td style="width:223px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:292px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">Sodium heparin venous blood collection tube 16 x 100 mm</td>
			<td style="width:223px;">Becton, Dickenson and Company</td>
			<td style="width:119px;">366480</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Lymphoprep</td>
			<td style="width:223px;">Stemcell Technologies</td>
			<td style="width:119px;">7801</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:273px;">Rosette Sep Human T Cell Enrichment Kit</td>
			<td style="width:223px;">Stemcell Technologies</td>
			<td style="width:119px;">15061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Dulbecco&#39;s PBS, 1x without Ca or Mg</td>
			<td style="width:223px;">Corning</td>
			<td style="width:119px;">21-031-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Bovine serum albumin</td>
			<td style="width:223px;">Sigma-Aldrich</td>
			<td style="width:119px;">A7906</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">EDTA</td>
			<td style="width:223px;">Sigma-Aldrich</td>
			<td style="width:119px;">E6635</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Fluorophore-labeled pMHC tetramer</td>
			<td style="width:223px;">NIH Tetramer Facility</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">EasySep Biotin Selection Kit</td>
			<td style="width:223px;">Stemcell Technologies</td>
			<td style="width:119px;">18553</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">EasySep Selection magnet</td>
			<td style="width:223px;">Stemcell Technologies</td>
			<td style="width:119px;">18000</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:273px;">TruStain FcX Human Fc blocking solution</td>
			<td style="width:223px;">Biolegend</td>
			<td style="width:119px;">422301</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Anti-CD5 PE-Cy7 (clone UCHT2)</td>
			<td style="width:223px;">Biolegend</td>
			<td style="width:119px;">300621</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Anti-CD14 FITC (clone HCD14)</td>
			<td style="width:223px;">Biolegend</td>
			<td style="width:119px;">325603</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Anti-CD19 FITC (clone HIB19)</td>
			<td style="width:223px;">Biolegend</td>
			<td style="width:119px;">302205</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">Iscove&#39;s DMEM, without b-ME or L-glutamine</td>
			<td style="width:223px;">Corning</td>
			<td style="width:119px;">15-016-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">HEPES</td>
			<td style="width:223px;">Corning</td>
			<td style="width:119px;">25-060-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">b-Mercaptoethanol&#160;</td>
			<td style="width:223px;">Life Technologies</td>
			<td style="width:119px;">21985-023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Glutamax</td>
			<td style="width:223px;">Life Technologies</td>
			<td style="width:119px;">35050061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Gentamicin sulfate (50 mg/ml)</td>
			<td style="width:223px;">Omega Scientific</td>
			<td style="width:119px;">GT-50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Human AB serum, male donor</td>
			<td style="width:223px;">Omega Scientific</td>
			<td style="width:119px;">HS-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Recombinant human IL-2</td>
			<td style="width:223px;">Peprotech</td>
			<td style="width:119px;">AF 200-02</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">Dynabeads Human T-Activator CD3/CD28</td>
			<td style="width:223px;">Life Technologies</td>
			<td style="width:119px;">11131D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;"></td>
			<td style="width:223px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Media</td>
			<td style="width:223px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Cell sorting buffer</td>
			<td style="width:223px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">PBS, pH 7.4</td>
			<td style="width:223px;">1 L</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">BSA</td>
			<td style="width:223px;">10g</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">EDTA (0.5 M)</td>
			<td style="width:223px;">2 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;"></td>
			<td style="width:223px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Human T Cell Culture Medium</td>
			<td style="width:223px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Iscove&#39;s DMEM</td>
			<td style="width:223px;">351.6 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Heat-inactivated human AB serum</td>
			<td style="width:223px;">40 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">HEPES (1 M)</td>
			<td style="width:223px;">4 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Glutamax (100 x)</td>
			<td style="width:223px;">4 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Gentamicin (50 mg/ml)</td>
			<td style="width:223px;">0.4 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">b-mercaptoethanol (14.3 M)</td>
			<td style="width:223px;">1.4 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Recombinant human IL-2 (1 mg/ml)</td>
			<td style="width:223px;">1 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

戦略をソーティングサイトメトリー、磁気ビーズ濃縮および単一セルの流れを経由して特異アロ抗原に定義され、このプロトコルは、クローン性ヒトT細胞培養物の生成を説明しています。 図1は 、プロセスの概要を提供します。 <img alt="図1" src="/files/ftp_upload/52257/52257fig1highres.jpg" /> 図1：プロトコルの概要ここで?...

Watch this Scientific Journal Video about Generation of Human Alloantigen-specific T Cells from Peripheral Blood at JoVE.com

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

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 ...

generation-of-human-alloantigen-specific-t-cells-from-peripheral-blood

Research

JoVE Journal

Immunology and Infection

12.9K ビュー.  University of California, San Diego. 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.

ビデオ: Generation of Human Alloantigen-specific T Cells from Peripheral Blood

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

拡大、精製、及びヒト末梢血NK細胞の機能的評価

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

免疫・感染学

Induction of Alloantigen-specific Anergy in Human Peripheral Blood Mononuclear Cells by Alloantigen Stimulation with Co-stimulatory Signal Blockade

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic diseases. Unfortunately, transplantation of alloreactive donor T cells which recognize and damage healthy patient tissues can result in Graft-versus-Host Disease (GvHD)1. One challenge to successful AHSCT is the prevention of GvHD without associated impairment of the beneficial effects of donor T cells, particularly immune reconstitution and prevention of relapse. GvHD can be prevented by non-specific depletion of donor T cells from stem cell grafts or by administration of pharmacological immunosuppression. Unfortunately these approaches increase infection and disease relapse2-4. An alternative strategy is to selectively deplete alloreactive donor T cells after allostimulation by recipient antigen presenting cells (APC) before transplant. Early clinical trials of these allodepletion strategies improved immune reconstitution after HLA-mismatched HSCT without excess GvHD5, 6. However, some allodepletion techniques require specialized recipient APC production6, 7and some approaches may have off-target effects including depletion of donor pathogen-specific T cells8and CD4 T regulatory cells9.One alternative approach is the inactivation of alloreactive donor T cells via induction of alloantigen-specific hyporesponsiveness. This is achieved by stimulating donor cells with recipient APC while providing blockade of CD28-mediated co-stimulation signals10.This "alloanergization" approach reduces alloreactivity by 1-2 logs while preserving pathogen- and tumor-associated antigen T cell responses in vitro11. The strategy has been successfully employed in 2 completed and 1 ongoing clinical pilot studies in which alloanergized donor T cells were infused during or after HLA-mismatched HSCT resulting in rapid immune reconstitution, few infections and less severe acute and chronic GvHD than historical control recipients of unmanipulated HLA-mismatched transplantation12. Here we describe our current protocol for the generation of peripheral blood mononuclear cells (PBMC) which have been alloanergized to HLA-mismatched unrelated stimulator PBMC. Alloanergization is achieved by allostimulation in the presence of monoclonal antibodies to the ligands B7.1 and B7.1 to block CD28-mediated costimulation. This technique does not require the production of specialized stimulator APC and is simple to perform, requiring only a single and relatively brief ex vivo incubation step. As such, the approach can be easily standardized for clinical use to generate donor T cells with reduced alloreactivity but retaining pathogen-specific immunity for adoptive transfer in the setting of AHSCT to improve immune reconstitution without excessive GvHD.

This paper describes a simple technique to induce alloantigen-specific anergy in human peripheral blood mononuclear cells. The technique can be applied clinically to generate non-alloreactive donor cells. Infusion of these cells could improve immune reconstitution and reduce toxicity after allogeneic hematopoietic stem cell transplantation.

共刺激シグナル遮断によるアロ抗原刺激によるヒト末梢血単核球のアロ抗原特異的アネルギーの誘導

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic ...

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

Expansion of Human Peripheral Blood  &amp;#947;&amp;#948; T Cells using Zoledronate

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

ゾレドロネートを使用してヒト末梢血γδT細胞の拡大

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

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

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

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

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

初代ヒトナイーブおよびメモリーT細胞から誘導した調節性T細胞の生成

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

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

の臨床応用<em&gt;眠れる森の美女</em遺伝的に末梢及び臍帯血由来のT細胞を変更する&gt;と人工抗原提示細胞

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

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

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

全血からのヒト単球由来樹状細胞の生成

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

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

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

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

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

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

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

The protocol describes a technique to study the ability of primary polyclonal human T cells to form synaptic interfaces using planar lipid bilayers. We use this technique to show the differential synapse formation capability of human primary T cells derived from lymph nodes and peripheral blood.

末梢血およびリンパ組織から主な人間の T 細胞のシナプスのインタ フェースの評価

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...

Flotation-Based T Cell Isolation, Activation, and Expansion from Human Peripheral Blood Mononuclear Cell Samples Using Microbubbles

The process of isolating T cells from peripheral blood mononuclear&#160;cells (PBMCs) to establish ex vivo cultures is crucial for research, clinical testing, and cell-based therapies. In this study, a simple, novel protocol to isolate, activate, and expand T cells from PBMCs ex vivo is presented. This study utilizes functionalized buoyancy-activated cell sorting (BACS) technology to isolate and activate T cells. Briefly, the protocol involves the positive selection of CD3+ cells from leukopak-derived PBMCs, followed by a 48 h co-stimulation with pre-conjugated anti-CD28-bound streptavidin microbubbles (SAMBs) prior to transduction in 24-well plates. Functionalized microbubbles offer a unique opportunity to buoyantly activate cells, leading to proliferative phenotypes that allow for expansion with minimal exhaustion. This technique offers reduced exhaustion because the co-stimulatory microbubbles remain buoyant and return to the top of the culture medium, thus reducing the amount of time that the expanding cells are in contact with the co-stimulatory factors. The results indicate that the isolated and cultured T cells receive enough stimulation to activate and proliferate but not to an extent that leads to overactivation, which then leads to exhaustion, as demonstrated by the presence of excessive PD-1.

The objective of this study is to demonstrate the feasibility of flotation-based separation to isolate, activate, and expand primary human T cells.

マイクロバブルを用いたヒト末梢血単核細胞サンプルからの浮遊選鉱ベースのT細胞の単離、活性化、および増殖(英語)

The process of isolating T cells from peripheral blood mononuclear&#160;cells (PBMCs) to establish ex vivo cultures is crucial for research, clinical ...

Differentiation of Functional Osteoclasts from Human Peripheral Blood CD14+ Monocytes

Osteoclasts (OCs) are bone-resorbing cells that play a pivotal role in skeletal development and adult bone remodeling. Several bone disorders are caused by increased differentiation and activation of OCs, so the inhibition of this pathobiology is a key therapeutic principle.Two key factors drive the differentiation of OCs from myeloid precursors: macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL). Human circulating CD14+ monocytes have long been known to differentiate into OCs in vitro. However, the exposure time and the concentration of RANKL influence the differentiation efficiency. Indeed, protocols for the generation of human OCs in vitro have been described, but they often result in a poor and lengthy differentiation process. Herein, a robust and standardized protocol for generating functionally active mature human OCs in a timely manner is provided. CD14+ monocytes are enriched from human peripheral blood mononuclear cells (PBMCs) and primed with M-CSF to upregulate RANK. Subsequent exposure to RANKL generates OCs in a dose- and time-dependent manner. OCs are identified and quantified by staining with tartrate acid-resistant phosphatase (TRAP) and light microscopy analysis. Immunofluorescence staining of nuclei and F-actin is used to identify functionally active OCs. In addition, OSCAR+CD14&#8722; mature OCs are further enriched via flow cytometry cell sorting, and OC functionality quantified by mineral (or dentine/bone) resorption assays and actin ring formation. Finally, a known OC inhibitor, rotenone, is used on mature OCs, demonstrating that adenosine triphosphate (ATP) production is essential for actin ring integrity and OC function. In conclusion, a robust assay for differentiating high numbers of OCs is established in this work, which in combination with actin ring staining and an ATP assay&#160;provides a useful in vitro model to evaluate OC function and to screen for novel therapeutic compounds that can modulate the differentiation process.

Differentiation of Functional Osteoclasts from Human Peripheral Blood CD14+ Monocytes

Osteoclasts are key bone-resorbing cells in the body. This protocol describes a reliable method for the in vitro differentiation of osteoclasts from human peripheral blood monocytes. This method can be used as an important tool to further understand osteoclast biology in homeostasis and in diseases.

機能性破骨細胞のヒト末梢血CD14+ 単球との鑑別

Osteoclasts (OCs) are bone-resorbing cells that play a pivotal role in skeletal development and adult bone remodeling. Several bone disorders are caused ...