この作品は、NIH（5U01CA137070）からの助成金と米国癌学会（RSG - 09から070 - 01 - LIB）、がん研究所の研究者賞（MK）、米国心臓協会の事前博士フェローシップ（KM）とによってサポートされていました教育と科学フェローシップ（APG）のスペイン省。 我々は、プロトコルに有用な提案に感謝博士ニコラスRestifoと博士致雅ゆう（NIH / NCI）をしたいと思います。

1。レトロウイルスコンストラクトを準備<ol><li>レトロウイルスベクター（図1、7 PMSG例のベクトル、pMIGII 5,8、CellbiolabsからpMXs）に興味のあるT細胞受容体（TCR）遺伝子をサブクローン。 TCRαおよびβ鎖遺伝子は同じ発現を保証するために、同じプロモーターの制御下にある同じベクトル上に存在する必要があります。興味のTCRが人間である場合、ヒト定常ドメインは、マウスの定常領域9によって置き換える必要があります。私たちの観察は、その全長ヒトTCRのが安定してマウスのT細胞に発現しないです。 </li><li>キアゲンマキシプレップキットまたは類似の製品を使用して高品質のプラスミドDNAを準備します。プラスミドの濃度は約1μg/μLのになっているはず。 </li></ol> 2。トランスフェクションとT細胞の分離デイ-1（プレートのパッケージング細胞） <ol><li>トリプシン- EDTAでプラチナ- E（PLAT - E、Cellbiolabs）レトロウイルスパッケージング細胞を取り除くとDMEM培地で中和する。 </li><li> 5分間1,000 × gで細胞を遠心分離します。上清を吸引除去し、DMEM培地で細胞ペレットを再懸濁します。 </li><li>細胞数を決定し、0.6x10 6 / mLで細胞を希釈する。プレート10 10ミリメートルポリリジンコートした組織培養プレートに細胞懸濁液の添加および37℃で細胞インキュベーターで一晩増殖° C、5％CO 2。 </li></ol> 0日目（トランスフェクション） <ol start="4"><li>翌朝、光学顕微鏡下で細胞を調べます。細胞は約80％コンフルエントにする必要があります。 </li><li>静かにプレートからメディアを取り外します。 1 × PBSで細胞を1回洗浄し、ペニシリン - ストレプトマイシン（ペン/連鎖球菌）なしで10 mLを予め温めておいた、新鮮なDMEM培地を加える。 （注：ペン/咽頭トランスフェクションに干渉することができます） </li><li>トランスフェクション複合体を準備する。 次のようにAとBを混ぜる準備します。 <table border="0"><tbody><tr><td>プラスミドDNA：ミックス</td><td> 9μgの</td></tr><tr><td>プラスミドPCL -エコヘルパー</td><td> 6.3μgの</td></tr><tr><td> OPTIMEM </td><td> 1.5 mLの</td></tr><tr><td colspan="2"></td></tr><tr><td>ミックスB：Lipopfectime 2000 </td><td> 60μL </td></tr><tr><td> OPTIMEM </td><td> 1.5 mLの</td></tr></tbody></table> 室温で5分間別々にAとBをインキュベート 優しく一緒にAとBを混ぜ合わせ、トランスフェクション複合体を形成するために、室温で20分間インキュベートする。 </li><li>ゆっくりPLAT - E細胞に混合物（合計3 mL）を滴下。ゆっくりと均等にトランスフェクション混合物を配布するために、プレートを前後揺らし。 </li><li> 37細胞をインキュベート° C / 5％セルインキュベーターのCO 2。 </li><li> 6-8時間後に、ウイルス産生のための新鮮なDMEM培地（ペン/連鎖球菌を含む）の10mLで培地を交換してください。 </li></ol>抗体でコーティングプレート<ol start="10"><li>マウスT細胞はウイルスの形質導入の前にアクティブにする必要があります。 T -細胞を活性化するために、さまざまの方法があります。ここでは、プレート結合抗CD3eと抗CD28抗体を使用してください。抗CD3eと滅菌PBSで抗CD28の2μg/ mLの1μg/ mLの抗体の混合物を準備します。 </li><li> 24ウェル組織培養プレートの各ウェルに抗体の混合物の250μLを分注します。プレートは4℃で一晩コーティングすることができます℃または37℃で2時間を</li></ol>マウスの脾臓からの単一細胞懸濁液の調製<ol start="12"><li>収穫のマウスの脾臓およびRPMI培地への転送。 </li><li>セルストレイナーで脾臓を置きます。マッシュ5 cmの組織培養プレートにシリンジのプランジャーと脾臓。滅菌PBSで細胞ストレーナーから細胞をすすいでください。 </li><li> 1000年XG、5分間遠心します。 </li><li>上清を捨てる。 ACK溶解緩衝液で再懸濁し、細胞ペレット（脾臓あたり2 mL）で。室温で2〜3分インキュベートします。 20 mLのにRPMI培地を加え、5分で1000 × gで遠心する。 </li><li>上清を捨てる。滅菌PBSで再懸濁し、細胞ペレット。細胞数を決定し、4℃で5分間1000 × gでの遠心℃にT細胞の分離に進んでください。 </li></ol>マウスT細胞の単離と活性化<ol start="17"><li>磁気製品の取扱説明書（Miltenyi Biotec社からCD8 + T細胞単離キット）にしたがって細胞を標識する。 </li><li> mのLSカラム（Miltenyi Biotec社）を配置agneticフィールド。カラムに標識細胞懸濁液を適用する。 （マウスCD8 + T細胞を豊かに）、フロースルーを収集する。製品のマニュアルにしたがって3 mLのバッファーでカラムを3回洗浄し、フロースルー前で組み合わせる。 </li><li>抗CD3eと抗CD8a抗体で細胞を染色し、フローサイトメトリー（図2a）で分析する。典型的な分離のために、全細胞の〜80〜10パーセントを単離することができる。 〜単離された細胞の90％はCD8 + T細胞です。 </li><li> 1000年XG、5分で細胞を遠心分離します。上清を捨てる。 1 × 10 6 / mLで組換えヒトIL - 2（rhIL2、20 ng / mL）を含むRPMI培地中で細胞ペレットを再懸濁します。 </li><li>単にセルを追加する前に、プレート（2.11で事前に準備された）から抗体溶液を除去。滅菌PBSと削除PBSで各ウェルを洗浄します。 </li><li>各ウェルに細胞懸濁液1 mLを追加します。 37℃/ 5％セルインキュベーターのCO 2でプレートを置く。 </li></ol> 3。活性化T細胞の（2日目と3日目）感染<ol><li>ウイルス産生の2日後、PLAT - E細胞プレートの培地が黄色になるはず。 15 mLコニカルチューブにウイルス上清を移す。さらにウイルス産生（オプション）用のプレートに、新鮮なDMEM培地10mLを追加します。 </li><li>細胞片を除去するために5分間1,000 × gでウイルス上清を遠心してください。慎重に新しいチューブにウイルス上清を移す。チューブの底にある液体のままにして、細胞の残骸を乱すことがありません。 </li><li>プレートから50 mLコニカルチューブにT細胞活性化集める。細胞数を決定します。ネガティブ（非形質導入）コントロールとして使用される別々のチューブにいくつかのセルを保存。 5分間1,000 × gで遠心すると上清を捨てる。 </li><li>硫酸プロタミンの20 ng / mLのrhIL2および10μg/ mLの1 mL当たり10 6個の細胞でウイルス上清中の細胞ペレットを再懸濁します。 24ウェルプレートの各ウェルに細胞懸濁液1 mLを追加します。 rhIL2と硫酸プロタミンの同量で同じ細胞密度でのRPMI培地でのコントロールのチューブに細胞を再懸濁します。同じプレートにセルを追加。 </li><li>プラスチックフィルムとプレートを包みます。ブレーキなしで32℃で、2000 × gで90分間遠心します。 </li><li>遠心分離後、プラスチックフィルムを取り外します。各ウェルに20 ng / mLのrhIL2と新鮮RPMI培地1 mLを追加します。インキュベーターにバックプレートを置く。 </li><li> （省略可能）TCRの高い発現レベルは、ウイルス上清を収集し、3日目に感染症の手順を繰り返すことによって達成することができます。 </li></ol> 4。細胞の増殖と形質導入効率の評価<ol><li>毎日T -細胞形質を調べます。 1時03分比、必要なときRPMI培地rhIL2の時にセルを分割する。細胞の生い茂るを（培地が黄色に変わる）ようにしてください。 </li><li> 6日目または7日目では、抗体とT細胞の表面上のTCRの発現を評価するためにTCRのための特定のMHCテトラマーで細胞を染色。使用してコントロールとしてT細胞（図2c）非形質導入。 </li><li> T -細胞形質導入は、さらに下流のアプリケーションのための準備が整いました。 </li></ol> 5。代表的な結果脾細胞を精製せずに形質導入​​することができますが、それは伝達の前にT細胞サブセットを精製することがしばしば有利である。高純度のT細胞サブセットは、商業的な磁気ビーズを（図2a）を使用して取得できます。 T細胞上のTCRの発現レベルを評価するために、TCRのαまたはβ鎖に特異的な抗体を使用することができます。一般的に細胞の30％〜80％が発現することができるTCR遺伝子とウイルスの力価に応じて、TCR（図2b）を導入した。 TCRのためのMHCテトラマー特定はさらにTCRの機能的発現（図2c）を確認するために使用することができます。 <img src="/files/ftp_upload/2307/2307fig1.jpg" alt="図1" /> 図1。 T細胞受容体遺伝子の例は、構築するサブクローン化レトロウイルスベクターに。全長αおよびβ鎖の遺伝子は、自己開裂可能な2Aのペプチドリンカー8によってリンクされています。 <img src="/files/ftp_upload/2307/2307fig2.jpg" alt="図2" /> 図2。マウスのT細胞の正常な分離と伝達の例は、。（）CD8 T細胞は、CD8a + T Cellアイソレーションキット（Miltenyi Biotec社）と抗CD3eと抗CD8a抗体で染色を用いて脾細胞から単離した。抗ヒトVbeta 8抗体（B）およびHLA - A2kb gp100四量体（c）と209 -217 4ペプチドとステンドグラス：CD8 T細胞の単離されたヒトgp100のためのR6C12 TCR特異的に導入した。

<ol>
	<li>Krogsgaard, M., 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=How+T+cells+'see'+antigen.">How T cells 'see' antigen.</a> Nat. Immunol. 6, 239-245 (2005).</li><li>Berg, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen/MHC-specific+T+cells+are+preferentially+exported+from+the+thymus+in+the+presence+of+their+MHC+ligand.">Antigen/MHC-specific T cells are preferentially exported from the thymus in the presence of their MHC ligand.</a> Cell. 58, 1035-1046 (1989).</li><li>Hogquist, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+receptor+antagonist+peptides+induce+positive+selection.">T cell receptor antagonist peptides induce positive selection.</a> Cell. 76, 17-27 (1994).</li><li>Yu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poor+immunogenicity+of+a+self/tumor+antigen+derives+from+peptide-MHC-I+instability+and+is+independent+of+tolerance.">Poor immunogenicity of a self/tumor antigen derives from peptide-MHC-I instability and is independent of tolerance.</a> J. Clin. Invest. 114, 551-559 (2004).</li><li>Holst, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+T-cell+receptor+retrogenic+mice.">Generation of T-cell receptor retrogenic mice.</a> Nat Protoc. 1, 406-417 (2006).</li><li>Morgan, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+regression+in+patients+after+transfer+of+genetically+engineered+lymphocytes.">Cancer regression in patients after transfer of genetically engineered lymphocytes.</a> Science. 314, 126-129 (2006).</li><li>Hawley, R. G., Lieu, F. H., Fong, A. Z., Hawley, 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=Versatile+retroviral+vectors+for+potential+use+in+gene+therapy.">Versatile retroviral vectors for potential use in gene therapy.</a> Gene Ther. 1, 136-138 (1994).</li><li>Szymczak, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correction+of+multi-gene+deficiency+in+vivo+using+a+single+'self-cleaving'+2A+peptide-based+retroviral+vector.">Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide-based retroviral vector.</a> Nat. Biotechnol. 22, 589-594 (2004).</li><li>Cohen, C. J., Zhao, Y., Zheng, Z., Rosenberg, S. A., Morgan, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+antitumor+activity+of+murine-human+hybrid+T-cell+receptor+(TCR)+in+human+lymphocytes+is+associated+with+improved+pairing+and+TCR/CD3+stability.">Enhanced antitumor activity of murine-human hybrid T-cell receptor (TCR) in human lymphocytes is associated with improved pairing and TCR/CD3 stability.</a> Cancer Res. 66, 8878-8886 (2006).</li><li>Letourneur, F., Malissen, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+a+T+cell+hybridoma+variant+deprived+of+functional+T+cell+receptor+alpha+and+beta+chain+transcripts+reveals+a+nonfunctional+alpha-mRNA+of+BW5147+origin.">Derivation of a T cell hybridoma variant deprived of functional T cell receptor alpha and beta chain transcripts reveals a nonfunctional alpha-mRNA of BW5147 origin.</a> Eur. J. Immunol. 19, 2269-2274 (1989).</li><li>Bendle, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lethal+graft-versus-host+disease+in+mouse+models+of+T+cell+receptor+gene+therapy.">Lethal graft-versus-host disease in mouse models of T cell receptor gene therapy.</a> Nat. Med. 16, 565-570 (2010).</li></ol>

利害の衝突は宣言されません。

いくつかのステップは、最適な結果を達成するために重要です。 PLAT - Eパッケージング細胞株は、健康な状態に維持する必要があります。必要に応じて、細胞はレトロウイルス構造タンパク質の発現の消失を防ぐために、1μg/ mlのピ​​ューロマイシン、10μg/ mlのブラストサイジンを含むDMEM培地中で数回継代することができます。トランスフェクション当日に、細胞が〜80％コンフルエントにする必要があります。少なすぎるまたは多すぎる細胞は、ウイルスの力価が低下する可能性があります。ウイルスの品質を容易に形質導入​​することができる細胞株でのテストで確認することができます。 TCRのはCD3複合体が細胞表面に発現することが必要とするため、我々は日常的に使用58α-/β-ハイブリドーマ細胞10（内因性TCRαまたはβ鎖を発現しますが、速達CD3複合体を行いますしない細胞株）。ウイルス上清1mlで細胞を形質導入するときに我々は、ハイブリドーマ細胞のほぼ100％の導入効率を得る。レトロウイルスだけ積極的にセルを分割感染することができますので、最後に、T -細胞が活性化し増殖する必要があります。 抗原特異的T -細胞を産生するためにここで紹介する方法はいくつかの制限があります。最初に、それは一次マウスT細胞のために100％の導入効率を達成することは困難です。セルの部分は、金利のTCRを発現しない。第二に、TCRαおよびβ鎖は関心のTCRの発現レベルを減少させる可能性がある、内在性TCRは9に誤対合できる形質。さらに、mispaired TCRは生体 11 の自己免疫を引き起こす可能性があります。最後に、限られた時間枠T -細胞を使用することができる形質導入は、唯一の存在です。約1週間後、細胞を再刺激されている場合を除きアポトーシス受ける。しかし、細胞が枯渇し、反復的な再刺激と機能を失う可能性があります。

メディア： DMEM培地（DMEM +10％の熱不活性化FBS +ペン/咽頭+ L - Gluの+ナトリウムピルビン酸+非必須アミノ酸）。 ペン/咽頭のないDMEM培地（DMEM培地と同じですが、ペン/連鎖球菌を追加せずに）。 RPMI培地（RPMI +10％の熱不活性化FBS + L - Gluの+非必須アミノ酸+β- Mercaphoethanol +ペン/咽頭+ピルビン酸ナトリウム）。

retroviral transduction of t-cell receptors in mouse t-cells

T細胞受容体（TCR）は、免疫系で中心的な役割を果たします。 T細胞表面上のTCRは特に抗原提示細胞（APC）1によって提示されたペプチド抗原を認識することができます。この認識は、T細胞の活性化と機能的転帰（例えば、サイトカイン産生、標的細胞の殺傷）のシリーズにつながる。 TCRの機能的役割を理解することは、免疫関連疾患（例えば、がんや自己免疫）の様々な治療に免疫システムのパワーを活用することが重要です。 それはいくつかの方法で達成することができるマウスモデルにおけるTCRは、勉強すると便利です。 TCRトランスジェニックマウスモデルを作ることはコストと時間がかかり、現在入手可能なそれらの2-4の限られた数があります。また、T細胞抗原特異的とマウスはキメラ骨髄で生成することができます。また、このメソッドは数週間かかると専門知識5が必要です。 in vitroで活性化したマウスにおける T -細胞にTCRのレトロウイルス導入は、所望のペプチド- MHC特異性のT細胞を得るために迅速かつ比較的簡単な方法です。抗原特異的T -細胞は、1週間で生成され、任意のダウンストリームアプリケーションで使用することができます。 T -細胞形質勉強することも、人間の免疫療法に直接アプリケーションを持っている、ヒトT細胞の養子移入として抗原特異的TCRの形質導入は、がんの治療6新たな戦略です。 ここでは、レトロウイルスin vitroで活性化したマウスのT細胞の中にするTCRを形質導入するプロトコルを提示する。ヒトとマウスのTCR遺伝子の両方を使用することができます。特定のTCR遺伝子を有するレトロウイルスが生成され、マウス抗- CD3および抗CD28抗体で活性化T細胞に感染するために使用されます。 in vitroでの展開で 、形質導入の後にT細胞をフローサイトメトリーによって分析されています。

Watch this Scientific Journal Video about Retroviral Transduction of T-cell Receptors in Mouse T-cells at JoVE.com

Retroviral Transduction of T-cell Receptors in Mouse T-cells

我々はレトロウイルス導入を用いて抗原特異的マウスT細胞を生成するためにプロトコルを提示

マウスT細胞におけるT細胞受容体のレトロウイルス導入

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

retroviral-transduction-of-t-cell-receptors-in-mouse-t-cells

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JoVE Journal

Immunology and Infection

34.6K ビュー.  New York University School of Medicine. 我々はレトロウイルス導入を用いて抗原特異的マウスT細胞を生成するためにプロトコルを提示

ビデオ: Retroviral Transduction of T-cell Receptors in Mouse T-cells

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

プライマリーヒトT細胞のpiggyBacトランスポゾンシステムの変更

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

免疫・感染学

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGF&beta; and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown.
Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44low, CD62Lhigh) and resting (CD25-, CD69-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.

Adenoviral gene transfer into naive CD4 T cells with transgenic expression of the Coxsackie adenovirus receptor enables the molecular analysis of regulatory T cell differentiation in vitro.

ナイーブCD4のアデノウイルス形質導入Tregの分化を研究するためにT細胞

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

T細胞の導出<em&gt;体外</em&gt;マウス胚性幹細胞からの

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&amp;#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

マウスナイーブCD4<sup&gt; +</sup&gt; T細胞の単離と<em&gt;インビトロ</em&gt; T細胞サブセットへの分化

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

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 ...

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, cloning TCRs specific for various tumor-associated antigens has been the goal of many studies. To elicit an effective T cell response, the TCR must recognize the target antigen with optimal affinity. However, cloning such TCRs has been a challenge and many available TCRs possess sub-optimal affinity for the cognate antigen. In this protocol, we describe a method of cloning de novo high affinity antigen-specific TCRs using existing TCRs by exploiting hemichain centricity. It is known that for some TCRs, each TCR&#945; or TCR&#946; hemichain do not contribute equally to antigen recognition, and the dominant hemichain is referred to as the centric hemichain. We have shown that by pairing the centric hemichain with counter-chains differing from the original counter-chain, we are able to maintain the antigen specificity, while modulating its interaction strength for the cognate antigen. Thus, the therapeutic potential of a given TCR can be improved by optimizing the pairing between the centric and counter hemichains.

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Herein we describe a novel method to generate antigen-specific T cell receptors (TCRs) by pairing the TCR&#945; or TCR&#946; of an existing TCR, possessing the antigen-specificity of interest, with complementary hemichain of the peripheral T cell receptor repertoire. The de novo generated TCRs retain antigen-specificity with varying affinity.

生成<em&gt;デ・ノボ</em&gt;センHemichainのレトロウイルス形質導入によって抗原特異的ヒトT細胞受容体

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity. Many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper T cell development and function. However, studies using mouse models have proven to be highly informative due to the availability of genetic, cellular, and biochemical systems. One genetic approach in mice used by many labs involves retroviral transduction of primary helper T cells. This is a powerful approach due to its relative ease, making it accessible to almost any laboratory with basic skills in molecular biology and immunology. Therefore, multiple genes in wild type or mutant forms can readily be tested for function in helper T cells to understand their importance and mechanisms of action. We have optimized this approach and describe here the protocols for production of high titer retroviruses, isolation of primary murine helper T cells, and their transduction by retroviruses and differentiation toward the different helper subsets. Finally, the use of this approach is described in uncovering mechanisms utilized by microRNAs (miRNAs) to regulate pathways controlling helper T cell development and function.

Many experimental systems have been utilized to understand the mechanisms regulating T cell development and function in an immune response. Here a genetic approach using retroviral transduction is described, which is economic, time efficient, and most importantly, highly informative in identifying regulatory pathways.

遺伝的分化を制御するメカニズムを研究するためのアプローチと機能などのヘルパーT細胞のレトロウイルス形質導入

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...

Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis

Tissue-resident memory T cell (TRM) is a rapidly expanding field of immunology research. Isolating T cells from various non-lymphoid tissues is one of the key steps to investigate TRMs. There are slight variations in lymphocyte isolation protocols for different organs. Kidney is an essential non-lymphoid organ with numerous immune cell infiltration especially after pathogen exposure or autoimmune activation. In recent years, multiple labs including our own have started characterizing kidney resident CD8+ T cells in various physiological and pathological settings in both mouse and human. Due to the abundance of T lymphocytes, kidney represents an attractive model organ to study TRMs in non-mucosal or non-barrier tissues. Here, we will describe a protocol commonly used in TRM-focused labs to isolate CD8+ T cells from mouse kidneys following systemic viral infection. Briefly, using an acute lymphocytic choriomeningitis virus (LCMV) infection model in C57BL/6 mice, we demonstrate intravascular CD8+ T cell labeling, enzymatic digestion, and density gradient centrifugation to isolate and enrich lymphocytes from mouse kidneys to make samples ready for the subsequent flow cytometry analysis.

Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis

Following viral infection, kidney harbors a relatively large number of CD8+ T cells and offers an opportunity to study non-mucosal TRM cells. Here, we describe a protocol to isolate mouse kidney lymphocytes for flow cytometry analysis.

フローサイトメトリー解析のためのマウス腎臓レジデントCD8+T 細胞の分離

Tissue-resident memory T cell (TRM) is a rapidly expanding field of immunology research. Isolating T cells from various non-lymphoid tissues is one of the ...

Purification and Expansion of Mouse Invariant Natural Killer T Cells for in vitro and in vivo Studies

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or microbial lipid antigens presented by the non-polymorphic MHC class I-related molecule CD1d. Preclinical and clinical studies support a role for iNKT cells in cancer, autoimmunity and infectious diseases. iNKT cells are very conserved throughout species and their investigation has been facilitated by mouse models, including CD1d-deficient or iNKT-deficient mice, and the possibility to unequivocally detect them in mice and men with CD1d tetramers or mAbs specific for the semi-invariant TCR. However, iNKT cells are rare and they need to be expanded to reach manageable numbers for any study. Because the generation of primary mouse iNKT cell line in vitro has proven difficult, we have set up a robust protocol to purify and expand splenic iNKT cells from the iV&#945;14-J&#945;18 transgenic mice (iV&#945;14Tg), in which iNKT cells are 30 times more frequent. We show here that primary splenic iV&#945;14Tg iNKT cells can be enriched through an immunomagnetic separation process, yielding about 95-98% pure iNKT cells. The purified iNKT cells are stimulated by anti-CD3/CD28 beads plus IL-2 and IL-7, resulting in 30-fold expansion by day +14 of the culture with 85-99% purity. The expanded iNKT cells can be easily genetically manipulated, providing an invaluable tool to dissect mechanisms of activation and function in vitro and, more importantly, also upon adoptive transfer in vivo.

We describe a rapid and robust protocol to enrich invariant natural killer T (iNKT) cells from mouse spleen and expand them in vitro to suitable numbers for in vitro and in vivo studies.

インビトロおよびインビボ研究のためのマウス不変性ナチュラルキラーT細胞の精製と拡大

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or ...

Analysis of T-cell Receptor-Induced Calcium Influx in Primary Murine T-cells by Full Spectrum Flow Cytometry

Calcium influx in response to T-cell receptor stimulation is a common measure of T-cell signaling. Several calcium indicator dyes have been developed to assess calcium signaling by band-pass flow cytometry. This protocol is designed to measure calcium responses in primary murine T-cells using full spectrum flow cytometry. Total splenocytes are labeled with the ratiometric calcium indicator dye Indo-1, along with a panel of fluorochrome-conjugated antibodies to cell surface molecules. Leveraging the capabilities of full spectrum flow cytometry provides a platform for utilizing a wide array of cell surface stains in combination with Indo-1. Cells are then analyzed in real-time at 37 &#176;C before and after the addition of an anti-CD3 antibody to stimulate the T-cell receptor. After unmixing the spectral signals, the ratio of calcium-bound to calcium-free Indo-1 is calculated and can be visualized over time for each gated population of splenocytes. This technique can allow for the simultaneous analysis of calcium responses in multiple cell populations.

Calcium influx, a measure of T-cell signaling, is an effective way to analyze responses to T-cell receptor stimulation. This protocol for multiplexing Indo-1 with panels of antibodies directed at cell surface molecules takes advantage of the highly flexible capabilities of full spectrum flow cytometry.

フルスペクトルフローサイトメトリーによる初代マウスT細胞におけるT細胞受容体誘導性カルシウム流入の解析

Calcium influx in response to T-cell receptor stimulation is a common measure of T-cell signaling. Several calcium indicator dyes have been developed to ...