Name: streamlined single cell tcr isolation and generation of retroviral vectors for in vitro and in vivo expression of human tcrs
Uploaded: 2017-09-10T13:00:00.000+00:00
Duration: 11 min 21 s
Description: Watch this Scientific Journal Video about Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs at JoVE.com

本研究は、JDRF 1-FAC-2014-243-A-N、ADA 7-14-ユニデン-07、NIH 5 P30 DK079638 05 パイロット PJ と、ロバートとジャニス ・ マクネイア財団によって賄われていた。
患者の募集、テクニカル サポート、博士ジョージ Makedonas 抗体とコントロール PCR の最適化に使用される DNA のギフトのためにサミュエル ・ ブラム、サンドラ ペーニャと Andrene マクドナルドに感謝します。

<ol>
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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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 (5), 589-594 (2004).</li><li>Szymczak, A. L., Vignali, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+2A+peptide-based+strategies+in+the+design+of+multicistronic+vectors.">Development of 2A peptide-based strategies in the design of multicistronic vectors.</a> Expert Opin Biol Ther. 5 (5), 627-638 (2005).</li><li>Bettini, M. L., Bettini, M., Vignali, D. 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I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive+method+for+detecting+proliferation+of+rare+autoantigen-specific+human+T+cells.">A sensitive method for detecting proliferation of rare autoantigen-specific human T cells.</a> J Immunol Methods. 283 (1-2), 173-183 (2003).</li><li>James, E. A., LaFond, R., Durinovic-Bello, I., Kwok, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+antigen+specific+CD4++T+cells+using+MHC+class+II+tetramers.">Visualizing antigen specific CD4+ T cells using MHC class II tetramers.</a> J Vis Exp. (25), (2009).</li><li>Corthay, A., Nandakumar, K. S., Holmdahl, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+percentage+of+peripheral+T+cells+with+two+different+T+cell+receptor+alpha-chains+and+of+their+potential+role+in+autoimmunity.">Evaluation of the percentage of peripheral T cells with two different T cell receptor alpha-chains and of their potential role in autoimmunity.</a> J Autoimmun. 16 (4), 423-429 (2001).</li><li>Lee, T., Shevchenko, I., Sprouse, M. L., Bettini, M., Bettini, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retroviral+Transduction+of+Bone+Marrow+Progenitor+Cells+to+Generate+T-cell+Receptor+Retrogenic+Mice.">Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice.</a> J Vis Exp. (113), (2016).</li><li>Bettini, M. L., Bettini, M., Nakayama, M., Guy, C. S., Vignali, D. A. <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:+improved+retroviral-mediated+stem+cell+gene+transfer.">Generation of T cell receptor-retrogenic mice: improved retroviral-mediated stem cell gene transfer.</a> Nat Protoc. 8 (10), 1837-1840 (2013).</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 (17), 8878-8886 (2006).</li></ol>

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現在のプロトコルの単一セルの TCR 増幅特性とそれに続く補助的クローニング ペアの TCR アルファとベータ鎖のテンプレート レトロ ウイルス発現ベクターに効率的な方法をについて説明します。単一セル PCR のいくつかのプロトコルが開発されているが、どれもはこれまで即時サブ表現のベクトルへのクローニングと互換性があるされています。ほとんどの場合、可変領域を推定する十分な?...

T 細胞受容体 (TCR) は、開発、定常状態/恒常性やその周囲1,2,3の抗原的な刺激の中に T 細胞の運命の決定を決定します。深い技術の最近の展開は、抗原特異的 T 細胞応答内以前過小評価 TCR の多様性を発見しました。TCR の多様性では、機能的な T 細胞の反応の可能性を示唆しています。実験的システムと生体内でのモデル選択の後続機能解析のために利用に対応する TCR 機能アッセイと TCR シーケンス レパートリー解析を統合するためにシーケンスのアプローチを設計する必要があります。Tcr。人間の TCR シーケンスの分離のための効率的な方法を開発し、HLA ヒト化マウス4TCR 式と互換性のあるキメラマウス人間 TCR テンプレートのベクターへのクローニングはサブを合理化します。ヘテロ二量体型 Tcr の対応するアルファとベータ鎖の分離には、単一のセルから両方の鎖の PCR 増幅が必要です。とはいえ、いくつかの単一セル TCR のクローニング プロトコルが開発され利用どれもこれまでのところされている高スループット合理化された直接クローニングによる未知の Vα/Vβ Tcr の retroviral ベクトル再式の生体内での必要に簡単に互換性のあります。 4,5,6,7。以前の研究は、シーケンスを外挿法で推定したり、全体 TCR シーケンス4,5,6を増幅するための十分な TCR の限られた部分を選択的に増幅する 2 つの主要なアプローチを活用して,7します。 最初のアプローチを介して TCR 系列の下流の機能解析が必要です高価なインシリコアセンブリおよびde novoの建設、TCR。2 番目のアプローチでは、TCR の完全な配列を提供しています、人間の一定の領域はマウス モデルでクローンの Tcr の体内式と互換性ありません。我々 のアプローチは生体内機能解析 Tcr のマウスのモデルに対応する設計します。我々 は、効率的で合理化された単一電池直接補助的クローニング PCR のフラグメントのテンプレートの発現ベクターには、PCR のプロトコルを開発しました。
我々 のアプローチは、2 つの手順で実行される高感度多重入れ子に PCR 反応を利用しています。最初の多重反応 40 プライマーすべての V β 鎖の特定のプールと 44 プライマー シーケンス (表 1) の事前知識がなくて TCR アルファまたは TCR β を増幅するための V α 鎖すべての特定のプールをステップします。前方のプライマーが 5' の PCR の製品に組み込まれているアダプターのシーケンスです。逆のプライマーは、TCR の一定した領域に基づいています。もとは、ユニークな制限のサイト人間変数またはジャンクション TCR 地域から欠席し、新しく再設計された TCRα と TCRβ のプライマー (図 1) にこれらを組み込みます。2 番目の入れ子になった反応アダプター シーケンスの特定のプライマーおよび一定した領域内で入れ子になった逆プライマーを使用して高められた特定性 (表 1、図 1) と TCR チェーンをさらに増幅します。単一細胞の分離後、2 つの PCR のラウンド (Vα と Vβ の両方のプライマーのプールと 2 番目アダプター プライマーの最初の反応) 直接補助的クローン作成できるテンプレート retroviral ベクトルに PCR の製品の結果します。1 つのオープンリーディング フレーム (ORF)、'自己切断' 蛋白質シーケンスによって接続されているマウスの一定領域と組み合わせて人間の可変領域で、TCR の最終的な構成要素をエンコード P2A (hVα-mCα-P2A-hVβ-mCβ)8。P2A シーケンスは、複数のシステムで使用されている、Tcr8,9,10,11の表現のために具体的には広範囲にテストされています。とはいえ、P2A シーケンスの残物のほとんどに接続されている後の翻訳ベータ鎖信号シーケンスが追加プロリン、この変更適用範囲が広いリンカーによって接続されているアルファ鎖の C 末端に TCR 機能に有害な影響がないです。マウスの一定の領域が下流のシグナル伝達コンポーネント潜在的な変更された相互作用を避けるために人間ではなく構成要素で使用が場合再マウスの細胞で発現します。単一 ORF は、アルファおよびベータ鎖9,11の化学量論的個別式になります。現在のプロトコルは試薬および広く利用可能、アプローチに基づいており、合理的で効率的な方法で実行するように設計。このプロトコルは同定と人間 Tcr の特定の機能評価に広く適用することができますが、特に自己免疫性糖尿病に関与する自己反応性 T 細胞の Tcr を試金するこのテクニックを使いました、見込んでください。自己免疫性エピトープ、がん抗原や病原体とワクチンへの応答。

<table><tbody><tr><td>CFSE&amp;nbsp;</td><td>eBioscience&amp;nbsp;</td><td>65-0850-84</td><td>5 mM</td></tr><tr><td>anti-human CD4 (OKT4) BV421</td><td>Biolegend&amp;nbsp;</td><td>317433</td><td>2 mg/mL</td></tr><tr><td>anti-human CD3 (OKT3) PerCP/Cy5.5</td><td>Biolegend&amp;nbsp;</td><td>317336</td><td>2 mg/mL</td></tr><tr><td>anti-mouse CD3 AF647</td><td>Biolegend&amp;nbsp;</td><td>100322</td><td>2.5 mg/mL</td></tr><tr><td>Recombinant RNAse inhibitor biotech grd 2.5KU&amp;nbsp;&amp;nbsp;</td><td>VWR</td><td>97068-158</td><td>0.7 U</td></tr><tr><td>Applied Biosystems High capaity cDNA RT kit&amp;nbsp;</td><td>Invirtogen&amp;nbsp;</td><td>4368814</td><td>9 U (RT enzyme)&amp;nbsp;</td></tr><tr><td>NF-H2O: Nuclease-free water 1000mL</td><td>Invirtogen&amp;nbsp;</td><td>AM9932</td><td></td></tr><tr><td>Gotaq DNA polymerase 2500U</td><td>VWR</td><td>PAM3008</td><td>1 U</td></tr><tr><td>96 Well Half Skirt PCR Plate</td><td>Phenix</td><td>MPX-96M2</td><td></td></tr><tr><td>MicroAmp Clear Adhesive Film&amp;nbsp;</td><td>ThermoFisher&amp;nbsp;</td><td>4306311</td><td></td></tr><tr><td>UltraPure Agarose&amp;nbsp;</td><td>Invirtogen&amp;nbsp;</td><td>15510-027</td><td></td></tr><tr><td>SnaBI</td><td>New England Biolabs&amp;nbsp;</td><td>R0130</td><td>15 U (insert) 30 U (vector)</td></tr><tr><td>SacII</td><td>New England Biolabs&amp;nbsp;</td><td>R0157</td><td>40 U (insert) 80 U (vector)</td></tr><tr><td>MfeI</td><td>New England Biolabs&amp;nbsp;</td><td>R3589S</td><td>40 U (insert) 80 U (vector)</td></tr><tr><td>BstBI</td><td>New England Biolabs&amp;nbsp;</td><td>R0519</td><td>40 U (insert) 80 U (vector)</td></tr><tr><td>Calf Intestinal Phosphatase (CIP)</td><td>New England Biolabs&amp;nbsp;</td><td>M0290S</td><td>10 U</td></tr><tr><td>Quick ligase&amp;nbsp;</td><td>New England Biolabs&amp;nbsp;</td><td>M2200S</td><td></td></tr><tr><td>Wizard Plus minipreps DNA purification systems&amp;nbsp;</td><td>Promega&amp;nbsp;</td><td>A1460</td><td></td></tr><tr><td>Wizard Plus midipreps DNA purification systems&amp;nbsp;</td><td>Promega&amp;nbsp;</td><td>A7640</td><td></td></tr><tr><td>DNA clean &amp;amp; concentrator-25 kit</td><td>Genesee Scientific&amp;nbsp;</td><td>11-304C</td><td></td></tr><tr><td>ZR-96 DNA clean &amp;amp; concentrator-5 kit</td><td>Genesee Scientific&amp;nbsp;</td><td>11-306A</td><td></td></tr><tr><td>QIAquick gel extraction kit</td><td>Qiagen</td><td>28704</td><td></td></tr><tr><td>QIAquick PCR purification kit</td><td>Qiagen</td><td>28104</td><td></td></tr><tr><td>Subcloning Efficiency DH5a Competent Cells&amp;nbsp;</td><td>ThermoFisher&amp;nbsp;</td><td>18265017</td><td></td></tr><tr><td>Transit LT-1 Reagent 1mL (transfection reagent)</td><td>Mirus&amp;nbsp;</td><td>MIR2300</td><td></td></tr><tr><td>BD FACS Aria II (sorter)</td><td>BD</td><td></td><td></td></tr><tr><td>BD Fortessa (flow cytometer)</td><td>BD&amp;nbsp;</td><td></td><td></td></tr></tbody></table>

streamlined single cell tcr isolation and generation of retroviral vectors for in vitro and in vivo expression of human tcrs

1 つの T 細胞からペアの T 細胞受容体 (TCR) アルファとベータ鎖の配列のためのいくつかの方法が開発されているが、どれもはこれまで TCR ヘテロの下流に資する生体内機能解析をされています。人間の TCR アルファとベータ鎖の可変領域を全体にまたがる PCR の製品の結果を持つ多重ネストの 2 段階 PCR に基づく改良されたプロトコルを開発しました。ユニークな制限のサイトを識別し、PCR のプライマーに組み込む、によって行った PCR の製品テンプレート レトロウイルスベクターに直接補助的クローニングと互換性があります。結果としてレトロ ウイルス構築キメラ人間/TCR を持つマウス マウス細胞やマウスモデルの生体内での機能はマウスの細胞内ドメインをエンコードします。全体的に、ここで説明されているプロトコルは生体外でそして生体内の TCR 式のレトロウイルスベクター容易に適応できるの合理化された世代対単一細胞 TCR アルファとベータ チェーン識別を組み合わせたものです。ビデオとそれに伴う材料は、重要なステップは続くと潜在的な落とし穴を回避できるように、単一セル PCR の非常に詳細な説明を与えるに設計されています。また、発現ベクターを生成に必要なクローン作成の手順の詳細な説明を提供します。一度マスター、TCR 式に並べ替え 1 つのセルから全体の手順は 2 週間の短期間で実行でした。

多重ネスト PCR の反作用の効率は agarose のゲルの 5µL 2 番目の反応のうち実行している 3.2.7 (図 2) の手順でチェックします。TCR アルファ反応の効率は通常より低い、約 50% に 80% を前後になると TCR β 増幅の効率の予想平均4。TCR 式だけペア TCR アルファと TCR β 鎖使えますただし、すべての PCR の製品は、TCR シーケンスおよびレ?...

Watch this Scientific Journal Video about Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs at JoVE.com

Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs

単一のセル対人間 TCR アルファとベータ鎖シーケンスで現在のプロトコル結合合理化レトロウイルスベクターの in vitroとin vivoの TCR 式と互換性の世代です。

合理化単細胞 TCR 分離し体外の Retroviral ベクトルの生成、人間 Tcr の生体内で発現

Although, several methods for sequencing of paired T cell receptor (TCR) alpha and beta chains from single T cells have been developed, none so far have ...

streamlined-single-cell-tcr-isolation-generation-retroviral-vectors

Research

JoVE Journal

Immunology and Infection

Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs

9.4K ビュー.  Baylor College of Medicine, Texas Children's Hospital. 単一のセル対人間 TCR アルファとベータ鎖シーケンスで現在のプロトコル結合合理化レトロウイルスベクターの in vitroとin vivoの TCR 式と互換性の世代です。

ビデオ: Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs

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

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 presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

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 ...

免疫・感染学

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 ...

Generation of Human Regulatory T Cell Clones

Human regulatory T cells (Treg) are notoriously difficult to isolate in high purity given the current methods of Treg enrichment. These methods are based on the identification of Treg through several activation-dependent cellular surface markers with varying expression levels in different physiologic and pathologic conditions. Populations isolated as &#8220;Treg&#8221; therefore often contain considerable numbers of non-Treg effector cells (i.e., Teff) which hamper the precise phenotypic and functional characterization of these cells, their genomic and proteomic characterization, their reliable enumeration in different states of health and disease, as well as their isolation and expansion for therapeutic purposes. The latter, in particular, remains a major hurdle, as the inadvertent expansion of effector cells homing in Treg-relevant cellular compartments (e.g., CD4+CD25+ T cells) may render Treg-based immunotherapy ineffective, or even harmful. This work presents a method that circumvents the problems associated with population-based isolation and expansion of Treg and shows that the generation of Treg candidate clones with the subsequent selection, culture, and expansion of only carefully vetted, monoclonal cells, enables the generation of an ultrapure Treg cell product that can be kept in culture for many months, enabling downstream investigation of these cells, including for possible therapeutic applications.

This protocol describes the cloning and expansion of human regulatory T cells for the generation of ultra-high purity viable human Treg with stable demethylation at the Treg-specific demethylated region (TSDR) and Treg-specific phenotypic features.

Human regulatory T cells (Treg) are notoriously difficult to isolate in high purity given the current methods of Treg enrichment. These methods are based ...

Production of Human CRISPR-Engineered CAR-T Cells

Adoptive cell therapies using chimeric antigen receptor T cells (CAR-T cells) have demonstrated remarkable clinical efficacy in patients with hematological malignancies and are currently being investigated for various solid tumors. CAR-T cells are generated by removing T cells from a patient's blood and engineering them to express a synthetic immune receptor that redirects the T-cells to recognize and eliminate target tumor cells. Gene editing of CAR-T cells has the potential to improve safety of current CAR-T cell therapies and further increase the efficacy of CAR-T cells. Here, we describe methods for the activation, expansion, and characterization of human CRISPR-engineered CD19 directed CAR-T cells. This comprises transduction of the CAR lentiviral vector and use of single guide RNA (sgRNA) and Cas9 endonuclease to target genes of interest in T cells. The methods described in this protocol can be universally applied to other CAR constructs and target genes beyond the ones used for this study. Furthermore, this protocol discusses strategies for gRNA design, lead gRNA selection and target gene knockout validation to reproducibly achieve high-efficiency, multiplex CRISPR-Cas9 engineering of clinical grade human T cells.

Here, we present a protocol for gene editing in primary human T cells using CRISPR Cas Technology to modify CAR-T cells.

ヒトCRISPR設計CAR-T細胞の製造

Adoptive cell therapies using chimeric antigen receptor T cells (CAR-T cells) have demonstrated remarkable clinical efficacy in patients with ...

同系モデルにおけるマウスCAR-T細胞生成のための合理化されたプロトコール

Efficient Generation of Murine Chimeric Antigen Receptor (CAR)-T Cells

Engineered cell therapies utilizing chimeric antigen receptor (CAR)-T cells have achieved remarkable effectiveness in individuals with hematological malignancies and are presently undergoing development for the treatment of diverse solid tumors. So far, the preliminary evaluation of novel CAR-T cell products has predominantly taken place in xenograft tumor models using immunodeficient mice. This approach is chosen to facilitate the successful engraftment of human CAR-T cells in the experimental setting. However, syngeneic mouse models, in which tumors and CAR-T cells are derived from the same mouse strain, allow evaluation of new CAR technologies in the context of a functional immune system and comprehensive tumor microenvironment (TME). The protocol described here aims to streamline the process of mouse CAR-T cell generation by presenting standardized methods for retroviral transduction and ex vivo T cell culture. The methods described in this protocol can be applied to other CAR constructs beyond the ones used in this study to enable routine evaluation of new CAR technologies in immune-competent systems.

著者スポットライト:同系腫瘍モデルにおけるCAR-T細胞機能の増強 

This protocol streamlines retroviral vector production and murine T cell transduction, facilitating the efficient generation of mouse CAR-T cells.

マウスキメラ抗原受容体(CAR)-T細胞の効率的な作製

Engineered cell therapies utilizing chimeric antigen receptor (CAR)-T cells have achieved remarkable effectiveness in individuals with hematological ...

生物工学

<meta charset="utf8"></head><body>Th17分化における遺伝子編集のためのガイドRNAのレトロウイルス形質導入

Retroviral CRISPR/Cas9-Mediated Gene Targeting for the Study of Th17 Differentiation in Vitro

T helper cells that produce IL-17A, known as Th17 cells, play a critical role in immune defense and are implicated in autoimmune disorders. CD4 T cells can be stimulated with antigens and well-defined cytokine cocktails in vitro to mimic Th17 cell differentiation in vivo. Research has been conducted extensively on the Th17 differentiation regulation mechanisms using the in vitro Th17 polarization assay.
Conventional Th17 polarization methods typically involve obtaining na&#239;ve CD4 T cells from genetically modified mice to study the effects of specific genes on Th17 differentiation and function. These methods can be time-consuming and costly and may be influenced by cell-extrinsic factors from the knockout animals. Thus, a protocol using retroviral transduction of guide RNA to introduce gene knockout in CRISPR/Cas9 knockin primary mouse T cells serves as a very useful alternative approach. This paper presents a protocol to differentiate na&#239;ve primary T cells into Th17 cells following retroviral-mediated gene targeting, as well as the subsequent flow cytometry analysis methods for assaying infection and differentiation efficiency.

<meta charset="utf8"></head><body>in vitroでのTh17分化の研究のためのレトロウイルスCRISPR/Cas9媒介遺伝子ターゲティング

We present a protocol for retroviral transduction of guide RNA into primary T cells from Cas9 transgenic mice, providing an efficient alternative for gene editing in studying Th17 differentiation.

in vitroでのTh17分化の研究のためのレトロウイルスCRISPR/Cas9媒介遺伝子ターゲティング

T helper cells that produce IL-17A, known as Th17 cells, play a critical role in immune defense and are implicated in autoimmune disorders. CD4 T cells ...

合理化単細胞 TCR 分離し体外の Retroviral ベクトルの生成、人間 Tcr の生体内で発現

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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

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、

生成

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

Generation of Human Regulatory T Cell Clones

ヒトCRISPR設計CAR-T細胞の製造

マウスキメラ抗原受容体(CAR)-T細胞の効率的な作製

マウスキメラ抗原受容体(CAR)-T細胞の効率的な作製

マウスキメラ抗原受容体(CAR)-T細胞の効率的な作製

in vitroでのTh17分化の研究のためのレトロウイルスCRISPR/Cas9媒介遺伝子ターゲティング