This work was supported by the Swedish Cancer Society (11 0253, 13 0469), the Swedish Research Council (K2012-99X-21959-01-3), Marcus Borgstr&#246;ms Foundation and the Swedish Research Council through a grant to the Uppsala RNA Research Centre (2006-5038-36531-16). We are indebted to the BioVis core facility at Uppsala University for much help with the FACS analysis.

プロトコルは、ヒト患者材料からのリンパ球様細胞の単離を記載し、したがって、倫理的な承認を必要とします。本研究で行われた作業は、ウプサラ倫理審査委員会（DNR。387分の2013）によって付与されました。 ヒトパラティーノ扁桃腺から単核細胞（MNCを）の1の単離注意：血液、組織または体液のようなヒト由来の全てのシールドの材料は、感染した可能性材料とみなされるべきです。したがって、ヒト組織を処理するための推奨されるバイオセーフティ慣行に従うべきです。 注：汚染を避けるために、全ての溶液及び細胞培養装置は、無菌でなければなりません。全ての緩衝液および溶液は、事前に冷却し、氷上に保持する必要があります。扁桃組織および単離した細胞を保持し、氷上で処理する必要があります。 <ol><li>扁桃摘出やへんとう切除（図2）を受けた患者からの新鮮な扁桃腺を取得します。組織clumプランジ氷冷滅菌ハンクス平衡塩溶液を充填した50 ml遠心管（HBSS）、5％ウシ胎児血清（FBS）を補充し、10 mMのグルタミン、0.05 mg / mlのゲンタマイシンおよび1％抗生物質 - 抗真菌ミックス（ペニシリン、にPSストレプトマイシン及びアンホテリシンB）。 </li><li>氷の上に常時水没扁桃組織サンプルを用いてチューブを保管してください。できるだけ早く扁桃腺を処理しようとせず、遅くとも3時間よりも外科的除去後。 </li><li>氷上で60ミリメートルのプラスチック細胞培養プレートに扁桃腺を置き、HBSSで湿らせたティッシュを維持します。扁桃表面からのきれいなピンセットで目に見える血液凝固、脂肪および結合組織を除去します。 </li><li> 5ミリリットルのHBSSを含む新しい60ミリメートルの細胞培養プレートを使用してください。はさみおよび/またはメスの無菌のペアを使用して3〜10ミリメートルの断片に扁桃組織の塊をカットします。 </li><li> HBSS 10mlのを含む新しい60ミリメートルの細胞培養プレートを準備し、HBSS溶液中に100μmのプラスチック製セルストレーナーを配置します。 </li><li>ディス転送滅菌ピンセットを用いて細胞ストレーナーに扁桃腺フラグメントをected。プラスチックシリンジのプランジャの端を使用して、スムーズにセルストレーナーを介して組織片を絞ります。扁桃腺断片が完全にHBSS中に浸漬されていることを確認します。 </li><li>組織が残存して細胞ストレーナーを廃棄し、50mlプラスチック遠心管に60 mmの細胞培養プレートから得られた細胞懸濁液を移します。ステップ1.8および1.9を進めながら、氷上でチューブを残します。 </li><li>大きさが2cm（ 図2）よりも大きい扁桃腺の場合には、ストレーナメッシュの目詰まりを回避するために、2つのセルストレーナーで扁桃腺断片を絞ります。 </li><li>細胞懸濁液を35 mlの最終容積を得ます。 注：このステップでは、凍結保存（セクション2を参照）の細胞懸濁液を調製することが可能です。 </li><li>新しい50ml遠心管にフィコール等密度勾配溶液10mlを加えます。静か細胞suspensをオーバーレイイオン密度勾配液の上に（ステップ1.9）。細胞懸濁液および密度勾配溶液の混合を避けます。 注：密度勾配溶液を室温まで加温しなければなりません。 </li><li> RTで20分間、700×gで、スイングアウトローター中で細胞懸濁液を遠心。勾配が中断される可能性があり、高速ブレーキとして遠心分離機のブレーキ機能をアクティブにしないでください。また、遠心分離器で利用可能な最低の加速関数を使用します。 注：この遠心分離工程の後、チューブの底に沈殿物として界面でふわふわした白色層、および赤血球（RBC）、線維芽細胞および細胞残屑などのMNCを観察します。 </li><li>慎重に10ミリリットルピペットを用いて、多国籍企業層を収集します。新しい50ミリリットルの遠心管に細胞懸濁液を置きます。 </li><li>細胞懸濁液に氷冷PBS 25 mlを加え、4℃でスイングアウトローター中で5分間300×gでチューブをスピン。 </li><li> 25ミリリットルピペットを用いて上清を削除細胞ペレットの洗浄工程をさらに2回繰り返します。最後のPBS 15mlに細胞ペレットを再懸濁します。 注：最後の洗浄工程の後に、（セクション2を参照）の細胞懸濁液を凍結保存することが可能です。 </li><li>血球計数器細胞計数室に10μlの細胞懸濁液を適用し、顕微鏡下で細胞数をカウントします。後続の磁気ビーズ分離に15 mlの遠心管中の細胞の適切な量（例えば、2-3×10 7）を分注します。ステップ3.2まで氷上にこのアリコートを保管してください。 </li></ol>扁桃細胞の凍結保存2. <ol><li>凍結培地（90％FBSおよび10％DMSO）を準備し、氷の上に保管してください。長期保存のために-20℃で凍結メディアを保持します。 </li><li>血球計数器の細胞計数室（ステップ1.15）を用いて細胞の総数を決定します。希望の凍結細胞密度に応じて凍結媒体の必要量を計算する（例えば、10 7細胞凍結培地のS / mlで）。 </li><li> 5分間300×gで細胞懸濁液を遠心。細胞ペレットを乱すことなく上清を除去し、（ステップ2.1から）を氷冷凍結培地中で細胞ペレットを再懸濁します。 </li><li>液体窒素中で長期保存のために設計され、滅菌バイアル中に細胞懸濁液1mlのアリコートを分配します。イソプロパノール室でバイアルを凍結し、-80°のCO / Nでそれらを格納します。長期保存のために貯蔵タンクまたは-140℃の細胞冷凍庫を含む液体窒素中にバイアルを転送します。 </li><li>凍結細胞とのバイアルを解凍し、37℃の水浴中で急速にそれらを暖めます。解凍した直後と、15mlの遠心管中で（サプリメント、ステップ1.1）、予め温めておいたHBSS 10mlの中に細胞懸濁液を分散させます。 、5分間300×gでチューブをスピンHBSSを除去し、（サプリメントと、ステップ1.1）HBSS中の所望の細胞密度で細胞ペレットを再懸濁。 注意：多国籍企業AFの実行可能性をター解凍は死細胞の存在は、精製されたBおよびTリンパ球の最終収率が低下するので、重要です。 注：細胞単離の効率を低下させる、細胞生存率が80％、次に我々の経験によります。 </li></ol>扁桃多国籍企業からのTリンパ球の人口の3ポジティブ選択注1：このプロトコルは、CD3抗体に結合された磁気ビーズを使用して、扁桃多国籍企業からのヒトCD3 + Tリンパ球の正の選択に基づいています。これは、新鮮な（セクション1）又は冷凍（セクション2）多国籍企業から、このセクションを開始することが可能となります。 注2：3×10 7の多国籍企業から開始します。分離カラムが詰まることがあり、これは、分離効率が低下するので、この細胞数を超えないようにしてください。より多くの細胞が処理される場合は、より大きな列を使用します。このプロトコルで使用されるボリュームは、実験的に細胞たちの数のために最適化されています私たちの実験で編。 <ol><li>分離緩衝液[PBS（pH7.2）中、0.5％ウシ血清アルブミン（BSA）および2mM EDTA]を準備します。 4℃で0.45μmフィルターとストアを介して分離バッファをフィルタリングします。 </li><li> 4℃で5分間、300×gでのMNC懸濁液（ステップ1.14）をスピン。氷冷分離緩衝液の240μlの（10 7個の細胞あたり80μl）をで得られた細胞ペレットを再懸濁します。無菌の2mlチューブに細胞懸濁液を転送します。 </li><li>細胞溶液にCD3磁気抗体20μlのを追加します。懸濁液中の細胞を維持するために連続的に穏やかに混合しながら4℃で1時間、チューブをインキュベートします。 </li><li>細胞を洗浄し、5 mlの氷冷分離緩衝液を含む15 mlのチューブに細胞懸濁液の全てを転送します。 </li><li>スイングアウトローターで4℃で10分間300×gでチューブを遠心。 </li><li>一方、磁気分離と列を設定します。スタンドに磁気分離器を取り付け、SEPARで列を配置レータ。氷冷分離緩衝液500μlを適用することにより、カラムを洗浄。フロースルーを捨てます。 </li><li>列の下に新しい15ミリリットルのコレクションチューブを置きます。氷の上にコレクションチューブを保管してください。 </li><li>ピペットで上清（ステップ3.5）を破棄し、ゆっくりと500μlの分離バッファで細胞ペレットを再懸濁します。予め洗浄したカラムの上に細胞懸濁液を適用し、それを介して、実行してみましょう。 注：細胞塊は、カラムを詰まらせるので、流量を減少させることができます。この問題を回避するには、列の上に40μmのプラスチック製セルストレーナーを配置します。このメッシュを通して細胞を渡すと、非常にこの方法の効率を改善します。 </li><li>分離緩衝液1.5mlの（10 7個の細胞のための500μl）を用いてカラムを4回洗浄します。次の洗浄工程を適用する前に、（液体が列で観察されるべきではない）空に列リザーバのを待ちます。 注：Bリンパ球画分としてみなさCD3非標識細胞を得る、そのままコレクションチューブに溶出しました。 </li><li>新しい15ミリリットルのコレクションチューブにセパレーターと場所から列を削除します。ピペットカラムに分離緩衝液2mlの。 Tリンパ球画分としてみなされる正に選択されたTリンパ球を、溶出カラム付属のプランジャーを使用して。 </li><li>必要に応じて精製された細胞（ステップ1.15）の数を決定します。細胞懸濁液は現在、下流の実験の準備ができています。 </li></ol>単離した扁桃BおよびTリンパ球の4フローサイトメトリー分析注意：パラホルムアルデヒドソリューションは、刺激物と疑われる発がん性物質です。適切な保護衣、手袋、眼/顔面用の保護具を着用します。 注1：このプロトコルは、（FACS）分析を蛍光活性化細胞選別によって分離されたB細胞およびT細胞を直接染色する方法が記載されています。この工程の主な目的は、単離された細胞集団のAFの純度を評価することですターCD3抗体磁気分離。 BおよびT細胞マーカー、フルオロフォア結合モノクローナル抗体CD20-FITCおよびCD2-APC確立し、この目的のために、使用されています。また、アイソタイプコントロール抗体を含めることが非常にCD2とCD20抗体（ステップ4.8を参照）の非特異的結合 &quot;背景&quot;を区別することをお勧めします。 注2：これは、染色時まで4℃で暗所で1％パラホルムアルデヒド（PFA）溶液に精製された細胞画分を維持することが可能である（例えば、次の日。）が。染色およびFACS分析の間の時間間隔がある場合にも同じ手順が適用可能です。どちらの場合も、細胞が染色前またはFACS分析にPBSA（0.2％BSAを含有するPBS）緩衝液で適切に洗浄する必要があることを覚えておいてください。 <ol><li>ステップ1.15からの多国籍企業の6×10 6個の細胞 （前の列に適用する）、ステップ3.9およびステップ3.10からのTリンパ球画分からのBリンパ球画分を取り出し。 </li><li>細胞をスピンスイングアウトローターを用いて4℃で5分間、300×gで懸濁液。 </li><li>ピペットで上清を除去し、氷冷PBSA 1mlで一度細胞ペレットを洗浄します。 4℃で5分間、300×gで細胞懸濁液をスピン。ピペットで上清を除去し、再懸濁 氷冷PBSAバッファー600μlの細胞。 </li><li> FACSチューブの底に100μlの細胞懸濁液を追加します。 </li><li>各チューブに、10％の熱不活性化ヒト血清を含む100μlのPBSを加え、よく混ぜ、氷上に〜RTで1分または20分間インキュベートします。 注：Bリンパ球は、Fc受容体を運びます。 Fcをブロックするために精製された細胞画分を、10％熱不活性化ヒト血清と共にインキュベートした受容体。ヒト血清を1時間56℃でインキュベーションすることにより熱不活性化です。 -20℃で凍結小分けとストアへの熱不活性化血清を分割します。 </li><li> SWで4℃で5分間、300×gで細胞懸濁液を遠心るアウトローター。 </li><li>ピペットで上清を除去し、1mlのPBSAで細胞ペレットを洗浄します。遠心分離（4℃で5分間、300×gで）を繰り返します。 </li><li>氷の上に各チューブに100μlのPBSAを追加します。製造業者の推奨に従って、フルオロフォアコンジュゲートモノクローナル抗体の適切な量を追加します（例えば、抗CD2020μlのおよび/ ​​またはPBSA100μlの抗CD2抗体を5μl）。注意：FACS分析のために十分な制御管を割り当てることを忘れないでください。このプロトコルでは、次のコントロールが含まれるべきである：1）細胞は、個々に、抗CD2および抗CD20抗体で染色し、抗CD2で染色した2）細胞と抗CD20アイソタイプ対照抗体単独で及び、3）細胞を添加していません抗体の。 </li><li>簡単に説明すると、チューブをボルテックスし、暗所で4℃で30分間インキュベートします。 </li><li> PBSAで2回洗浄します。サンプルに1％PFA溶液2mlを追加します。細胞は4でPFA溶液中に残ってみよう76; C暗所でのFACS分析時まで。 </li><li>直ちにFACS機でサンプルを実行する前に、（4℃で5分間、300×gで）をPBSAで細胞を2回洗浄します。 1mlのPBSと再懸濁サンプルを前に、フローサイトメーター上で分離し、光から保護し、4℃（または氷上）で保ちます。 </li><li>メーカーフローサイトメーターからのプロトコルに従って、FACSソーティングを行います。 </li></ol>単離した扁桃BおよびTリンパ球におけるアデノウイルスDNAの5 PCR検出注1：アデノウイルスヘキソン遺伝子プライマーはAdRJC1（5&#39;-GACATGACTTTCGAGGTCGATCCCATGGA-3 &#39;）及びAdRJC2（5&#39;-CCGGCTGAGAAGGGTGTGCGCAGGTA-3&#39;）11です。これらのプライマーは、139塩基対のアンプリコンを生成します。ホスト18S rRNA遺伝子を、プライマーtp206（5&#39;-CCCCTCGATGCTCTTAGCTG-3 &#39;）及びtp207（5&#39;-TCGTCTTCGAACCTCCGACT-3&#39;）を用いて検出することができます。予想されるアンプリコンサイズは300 bpです。 注2：すべてのPCRランは、陰性対照（蒸留H 2 O）およびヒトアデノウイルス5型12に感染したヒトB細胞株（BJAB）から得られた陽性DNAコントロールを含んでいます。 <ol><li>シリカ膜ベースのカラム精製法を用いて、（少なくとも1×10 6個の細胞 、ステップ3.11から）のDNAを抽出します。フェノールとグアニジンイソチオシアネート液13を用いてRNAを抽出します。 </li><li> 20μlの総容量で、1×HF緩衝液を含む反応混合物にBおよびTリンパ球からの100ngのDNAを加えるデオキシヌクレオチド三リン酸混合物（のdNTP）の0.2mMの各プライマーの0.25μMと高忠実度DNAポリメラーゼの1 U。 </li><li>以下のサイクル条件でPCR増幅を実行します。30秒、72℃10秒、67℃（ヘキソン）または58℃（18S rRNAの）98℃の30サイクル、続いて98℃で30秒の変性、 30秒間。 PCR反応は、72℃で7分間の最終伸長工程で終了しました。 </li><li>得られたPCR amplifを分離1×TBE中の1.5％アガロースゲル上でication製品は、緩衝液及び核酸染色で染色することによって期待されるバンドを可視化します。 </li></ol>

<ol>
	<li>Nave, H., Gebert, A., Pabst, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphology+and+immunology+of+the+human+palatine+tonsil.">Morphology and immunology of the human palatine tonsil.</a> Anat Embryol (Berl). 204, 367-373 (2001).</li><li>Perry, M., Whyte, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunology+of+the+tonsils.">Immunology of the tonsils.</a> Immunology today. 19, 414-421 (1998).</li><li>Alkhalaf, M. A., Guiver, M., Cooper, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+and+quantitation+of+adenovirus+DNA+from+human+tonsil+and+adenoid+tissues.">Prevalence and quantitation of adenovirus DNA from human tonsil and adenoid tissues.</a> J Med Virol. 85, 1947-1954 (2013).</li><li>Brandtzaeg, P., Halstensen, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunology+and+immunopathology+of+tonsils.">Immunology and immunopathology of tonsils.</a> Adv Otorhinolaryngol. 47, 64-75 (1992).</li><li>Imai, 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=Epstein-Barr+virus+(EBV)-carrying+and+-expressing+T-cell+lines+established+from+severe+chronic+active+EBV+infection.">Epstein-Barr virus (EBV)-carrying and -expressing T-cell lines established from severe chronic active EBV infection.</a> Blood. 87, 1446-1457 (1996).</li><li>Veen, J., Lambriex, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+of+adenovirus+to+lymphocytes+in+naturally+infected+human+tonsils+and+adenoids.">Relationship of adenovirus to lymphocytes in naturally infected human tonsils and adenoids.</a> Infect Immun. 7, 604-609 (1973).</li><li>Friedman, M., Wilson, M., Lin, H. C., Chang, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Updated+systematic+review+of+tonsillectomy+and+adenoidectomy+for+treatment+of+pediatric+obstructive+sleep+apnea/hypopnea+syndrome.">Updated systematic review of tonsillectomy and adenoidectomy for treatment of pediatric obstructive sleep apnea/hypopnea syndrome.</a> Otolaryngol Head Neck Surg. 140, 800-808 (2009).</li><li>Garnett, C. T., 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=Latent+species+C+adenoviruses+in+human+tonsil+tissues.">Latent species C adenoviruses in human tonsil tissues.</a> J Virol. 83, 2417-2428 (2009).</li><li>Garnett, C. T., Erdman, D., Xu, W., Gooding, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+and+quantitation+of+species+C+adenovirus+DNA+in+human+mucosal+lymphocytes.">Prevalence and quantitation of species C adenovirus DNA in human mucosal lymphocytes.</a> J Virol. 76, 10608-10616 (2002).</li><li>Perez, M. E., Billordo, L. A., Baz, P., Fainboim, L., Arana, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+memory+B+cells+isolated+from+blood+and+tonsils+are+functionally+distinctive.">Human memory B cells isolated from blood and tonsils are functionally distinctive.</a> Immunol Cell Biol. 92, 882-887 (2014).</li><li>Cooper, R. J., Yeo, A. C., Bailey, A. S., Tullo, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus+polymerase+chain+reaction+assay+for+rapid+diagnosis+of+conjunctivitis.">Adenovirus polymerase chain reaction assay for rapid diagnosis of conjunctivitis.</a> Invest Ophthalmol Vis Sci. 40, 90-95 (1999).</li><li>Zhang, Y., Huang, W., Ornelles, D. A., Gooding, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+adenovirus+latency+in+human+lymphocyte+cell+lines.">Modeling adenovirus latency in human lymphocyte cell lines.</a> J Virol. 84, 8799-8810 (2010).</li><li>Chomczynski, P., Sacchi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-step+method+of+RNA+isolation+by+acid+guanidinium+thiocyanate-phenol-chloroform+extraction.">Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.</a> Anal. biochem. 162, 156-159 (1987).</li><li>Watanabe, T., Yoshizaki, K., Yagura, T., Yamamura, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+antibody+formation+by+human+tonsil+lymphocytes.">In vitro antibody formation by human tonsil lymphocytes.</a> J Immunol. 113, 608-616 (1974).</li></ol>

The authors have nothing to disclose.

このプロトコルの結果に影響を与える最も重要な要因の一つは、出発原料として、新鮮な扁桃体の使用です。したがって、扁桃サンプルは手術後3時間以内に処理しなければなりません。扁桃腺は、大人と子供の両方から得ることができました。子供から扁桃物質が原因で扁桃腺（へんとう切除）の部分的な外科的除去に通常小さいです。そのため、多国籍企業の数が少ない扁桃摘出サンプル...

Tonsils are collections of incompletely encapsulated lymphoid tissues that lie under, and in contact with, the epithelium in the upper aero-digestive tract. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. The paired palatine tonsils together with the nasopharyngeal tonsil (adenoid), paired tubal tonsils and lingual tonsils constitute the so-called &#8220;Waldeyer&#180;s ring&#8221;. The latter is responsible for the initial contact between inhaled or ingested pathogens and the lymphoid tissues of the aerodigestive tract1,2. Indeed, numerous reports have shown that both bacterial and viral antigens can be detected in palatine tonsil tissue samples2-6.
The palatine tonsils are composed of dense lymphoid tissue covered by a stratified squamous non-keratinising epithelium. The tonsils have numerous crypts, epithelial invaginations, which penetrate the parenchyma increasing the surface area. Histologically, the palatine tonsils contain numerous lymphoid follicles with germinal centers, which are the sites for B cell maturation and differentiation (B-cell areas). Likewise, the palatine tonsils encompass T cells, which are mainly located in the extrafollicular regions (T-cell areas). In addition to the B and T cells, also various follicular dendritic cells can be detected in palatine tonsils1,2. 
Due to their anatomic location, the palatine tonsils are easily accessible by surgical interventions. For example, surgical removal of tonsils, known as tonsillectomy, is routinely carried out worldwide7. In children with tonsillar hyperplasia, a partial surgical removal of the tonsils (tonsillotomy) is sometimes used, causing less postoperative pain to the patients. Considering the accumulation of various pathogens in tonsils, surgically removed tonsils provide a unique opportunity to study the influence of viral and bacterial agents on tonsillar lymphocyte functions2,8. Furthermore it is possible to study if some pathogens prefer to reside in specific cell subpopulations9. In addition, as the tonsils are rich source of B lymphocytes, isolated tonsillar B lymphocytes can be efficiently used to study the activity of different B cell subpopulations10. However, as the palatine tonsils contain a mixture of cell types an efficient method to separate the different cell subpopulations is needed. 
Here, we describe a simple method for efficient and rapid isolation of tonsillar B and T cell populations from human palatine tonsils by using a magnetic-activated cell separation technique (Figure 1). The method described here is useful for scientists who want to assess the role of different infectious agents in human lymphoid organs such as palatine tonsils.

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			<td>Hanks balanced salt solution (HBSS)&#160;</td>
			<td>Gibco</td>
			<td>14175-053</td>
			<td>Contains 5% fetal bovine serum (FBS), 10 mM Glutamine,&#160;</td>
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		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>&#160;0.05 mg/ml Gentamicin and 1% Antibiotic-Antimycotic mix</td>
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		<tr>
			<td>Freezing medium</td>
			<td></td>
			<td></td>
			<td>90% FBS and 10% DMSO</td>
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		<tr>
			<td>MACS buffer</td>
			<td></td>
			<td></td>
			<td>PBS (pH 7.2), 0.5% BSA and 2mM EDTA</td>
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		<tr>
			<td>PBSA&#160;</td>
			<td></td>
			<td></td>
			<td>PBS containing 0.2% BSA</td>
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		<tr>
			<td>PFA</td>
			<td></td>
			<td></td>
			<td>PBS containing 1% paraformaldehyde (PFA). Make fresh.</td>
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		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>PFA is suspected carcinogen. Wear gloves and goggles.</td>
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			<td></td>
			<td></td>
			<td></td>
			<td></td>
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		<tr>
			<td>Antibiotic-Antimycotic mix</td>
			<td>Gibco</td>
			<td>15240-062</td>
			<td></td>
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		<tr>
			<td>100-mm petridish</td>
			<td>Nunc</td>
			<td>172958</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting foreceps</td>
			<td>Fisher Scientific</td>
			<td>1381241</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight iris scissors&#160;</td>
			<td>Fisher Scientific</td>
			<td>12912055</td>
			<td></td>
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		<tr>
			<td>disposable scalpels</td>
			<td>Swann-Morton</td>
			<td>REF 0501</td>
			<td></td>
		</tr>
		<tr>
			<td>100&#956;m plastic cell strainer</td>
			<td>Corning Life Sciences</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>40&#956;m plastic cell strainers&#160;</td>
			<td>Corning Life Sciences</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>2-ml plastic syringe</td>
			<td>BD Biosciences&#160;</td>
			<td>300185</td>
			<td></td>
		</tr>
		<tr>
			<td>15-ml conical centrifuge tubes</td>
			<td>SARSTEDT</td>
			<td>62554502</td>
			<td></td>
		</tr>
		<tr>
			<td>50-ml conical centrifuge tubes</td>
			<td>SARSTEDT</td>
			<td>62547254</td>
			<td></td>
		</tr>
		<tr>
			<td>Low-speed centrifuge with fixed-angle or swinging-bucket rotor</td>
			<td colspan="2">Thermo Scientific Heraeus Megafuge 16R</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll&#8211;Hypaque</td>
			<td>Sigma-Aldrich</td>
			<td>F5415-50ML</td>
			<td>Ficoll solution</td>
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		<tr>
			<td>Fetal calf serum</td>
			<td>Biological industries</td>
			<td>040071A</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulphoxide (DMSO)</td>
			<td>SIGMA</td>
			<td colspan="2">D2650-5X5ML</td>
		</tr>
		<tr>
			<td>MACS MS columns&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS human CD3 MicroBeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-092-881</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS separator (Octo MACS)</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-109</td>
			<td></td>
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		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Merck Millipore</td>
			<td>1120180100</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>AnalaR NORMAPUR</td>
			<td>20302.293</td>
			<td></td>
		</tr>
		<tr>
			<td>CD2 conjugated to allophycocyanin (CD2-APC)&#160;</td>
			<td>BD Biosciences&#160;</td>
			<td>560642</td>
			<td></td>
		</tr>
		<tr>
			<td>CD20 conjugated to fluorescein isothiocyanate (CD20-FITC)&#160;</td>
			<td>BD Biosciences&#160;</td>
			<td>556632</td>
			<td></td>
		</tr>
		<tr>
			<td>Human serum&#160;</td>
			<td>Rockland Immunochemicals</td>
			<td>D119-0100</td>
			<td></td>
		</tr>
		<tr>
			<td>Phusion High-Fidelity DNA Polymerase</td>
			<td>Thermo Scientific</td>
			<td>F-530L</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS tubes</td>
			<td>BD Falcon</td>
			<td>352003</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryotube</td>
			<td>SARSTEDT</td>
			<td>72379</td>
			<td></td>
		</tr>
		<tr>
			<td>BD LSRII flowcytometer</td>
			<td>BD Biosciences&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSDiva 4.1 software</td>
			<td>BD Biosciences&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nucleospin Blood&#160;</td>
			<td>Macherey-Nagel</td>
			<td>740951.50</td>
			<td>DNA isolation kit</td>
		</tr>
		<tr>
			<td>TRIzol&#160; reagent</td>
			<td>Life technologies</td>
			<td>15596</td>
			<td>RNA isolation reagent</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>The Paul Marienfeld GmbH &#38; Co. KG</td>
			<td>0610030</td>
			<td>cell counter device</td>
		</tr>
		<tr>
			<td>GelRed</td>
			<td>Biotium</td>
			<td align="right">41003</td>
			<td>Nucleic acid gel stain&#160;</td>
		</tr>
	</tbody>
</table>

efficient isolation protocol for b and t lymphocytes from human palatine tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

BおよびTリンパ球の高度に精製された亜集団における扁桃のMNC結果の効率的な分離。これは、（ 図3A）は、それぞれ、BおよびTリンパ球集団を検出するために、抗CD20および抗CD2抗体を用いたFACS分析によって確認しました。 BおよびTリンパ球の亜集団（ 図3B）の両方からの細胞の混合物である細胞画分中のMNCの結果とは対照的に、非効率的な分離。 <p class="jove_conten...

Watch this Scientific Journal Video about Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils at JoVE.com

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

ヒトパラティーノ扁桃腺からBおよびTリンパ球の効率的な単離プロトコール

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

efficient-isolation-protocol-for-b-t-lymphocytes-from-human-palatine

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Immunology and Infection

Unit 1

Upper Respiratory Tract Infections

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15.7K ビュー.  Uppsala Biomedical Center, Uppsala University. Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

ビデオ: Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

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

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where the CB does not contain appreciable numbers of virus-experienced T cells which can protect the recipient from infection.1-4 We and others have shown that virus-specific CTL generated from seropositive donors and infused to the recipient are safe and protective.5-8 However, until recently, virus-specific T cells could not be generated from cord blood, likely due to the absence of virus-specific memory T cells. 
In an effort to better mimic the in vivo priming conditions of na&iuml;ve T cells, we established a method that used CB-derived dendritic cells (DC) transduced with an adenoviral vector (Ad5f35pp65) containing the immunodominant CMV antigen pp65, hence driving T cell specificity towards CMV and adenovirus.9 At initiation, we use these matured DCs as well as CB-derived T cells in the presence of the cytokines IL-7, IL-12, and IL-15.10 At the second stimulation we used EBV-transformed B cells, or EBV-LCL, which express both latent and lytic EBV antigens. Ad5f35pp65-transduced EBV-LCL are used to stimulate the T cells in the presence of IL-15 at the second stimulation. Subsequent stimulations use Ad5f35pp65-transduced EBV-LCL and IL-2. 
From 50x106 CB mononuclear cells we are able to generate upwards of 150 x 106 virus-specific T cells that lyse antigen-pulsed targets and release cytokines in response to antigenic stimulation.11 These cells were manufactured in a GMP-compliant manner using only the 20% fraction of a fractionated cord blood unit and have been translated for clinical use.

Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly na&#238;ve T cells.

臍帯血から細胞傷害性Tリンパ球の拡大をそのターゲットサイトメガロウイルス、エプスタイン - バーウイルス、およびアデノウイルス

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

マウス生殖管粘膜からのリンパ球の単離

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

臍帯血から前駆B細胞サブセットの分離

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&amp;#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

離とナイーブCD4 Tリンパ球のTh17分化

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

PNA-FISHによるマウスのBリンパ球における染色体異常の迅速分析

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

ヌクレオによるヒトTHP-1マクロファージの高効率トランスフェクション

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

簡単かつ迅速なプロトコル非酵素的に浸潤リンパ球の分析のための新鮮なヒト組織を解離

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

不死化ソートB細胞からの組換えヒトIgGモノクローナル抗体の作製

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Na&#239;ve CD4+ T Cells Using a TGF-&#946;-containing Protocol

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing autoimmune diseases. Clinical approaches to adoptively transfer Tregs, or to deplete Tregs in cancer, are underway with promising first outcomes. 
Because the number of naturally occurring Tregs (nTregs) is very limited, studying certain Treg features using in vitro induced Tregs (iTregs) can be advantageous. To date, the best although not absolutely specific protein marker to delineate Tregs is the transcription factor FOXP3. Despite the importance of Tregs including non-redundant roles of peripherally induced Tregs, the protocols to generate iTregs are currently controversial, particularly for human cells. This protocol therefore describes the in vitro differentiation of human CD4+FOXP3+ iTregs from human na&#239;ve T cells using a range of Treg-inducing factors (TGF-&#946; plus IL-2 only, or their combination with retinoic acid, rapamycin or butyrate) in parallel. It also describes the phenotyping of these cells by flow cytometry and qRT-PCR. 
These protocols result in reproducible expression of FOXP3 and other Treg signature genes and enable the study of general FOXP3-regulatory mechanisms as well as protocol-specific effects to delineate the impact of certain factors. iTregs can be utilized to study various phenotypic aspects as well as molecular mechanisms of Treg induction. Detailed molecular studies are facilitated by relatively large cell numbers that can be obtained. 
A limitation for the application of iTregs is the relative instability of FOXP3 expression in these cells compared to nTregs. iTregs generated by these protocols can also be used for functional assays such as studying their suppressive function, in which iTregs induced by TGF-&#946; plus retinoic acid and rapamycin display superior suppressive activity. However, the suppressive capacity of iTregs can differ from nTregs and the use of appropriate controls is crucial.

&lt;em&gt;In Vitro&lt;/em&gt; Differentiation of Human CD4&lt;sup&gt;+&lt;/sup&gt;FOXP3&lt;sup&gt;+&lt;/sup&gt; Induced Regulatory T Cells (iTregs) from Na&amp;#239;ve CD4&lt;sup&gt;+&lt;/sup&gt; T Cells Using a TGF-&amp;#946;-containing Protocol

This protocol describes the reproducible generation and phenotyping of human induced regulatory T cells (iTregs) from na&#239;ve CD4+ T cells in vitro. Different protocols for FOXP3 induction allow for the study of specific iTreg phenotypes obtained with respective protocols.