SSはTBMMプログラムを通じて卒業生のトレーニンググラントにHHMIメッドによって部分的にサポートされています。厚生省は、退役軍人省からキャリア開発賞と博士夫妻ハロルド·M·Selzmanの寛大な支援によって部分的にサポートされています。この作品はまた、NIHリンパ腫SPORE助成P50CA126752とNIH R01 DK093660によって部分的にサポートされていました。

0日目 1。ヒト血液由来のPBMCの分離<ol><li> Na型ヘパリンバキュテイナチューブに静脈穿刺を用いて新鮮なヒト血液20mlを採取します。 </li><li> 1:1（v / v）の割合でミックス血液およびAdvanced RPMI 1640。 </li><li> 50ml遠心チューブ（25℃）に20ミリリットルlymphoprep媒体を追加します。 lymphoprepの上に血のRPMI 1640ミックスのゆっくり層25〜30ミリリットル。 </li><li>ブレーキなしで40分間、400×gで遠心分離します。 </li><li> 1X PBSを10ml（25℃）に使い捨てのピペットを用いてはっきりとファジー層の両方を収集し、1×PBSで50mlにボリュームを上げます。 </li><li> 10分間、450×gで遠心分離します。 </li><li>完全に上清を吸引除去し、高度なRPMI 1640 20mlを加える。 </li><li> 5分間400×gで遠心分離します。 </li><li>上清を吸引します。 5 ng / mlでのrhIL-15を補充した完全なT細胞の培地10mlを加え、37℃に温め</li><li> 24ウェルの組織培養コート内の細胞とプレートの数を数える5 ng / mlでのrhIL-15周囲のウェルに滅菌水を追加で補充完了し、T細胞培地で2×10 6細胞/ウェルでEDプレート。 37℃加湿インキュベーターで一晩インキュベート℃、5％CO 2で 。 </li></ol> 1日目 2。 T細胞を刺激するための抗CD28および抗CD3抗体でコーティングプレート<ol><li> 1μg/ mlの各濃度の滅菌水に抗CD28および抗CD3抗体を希釈します。 </li><li>非コート組織培養24ウェルプレートの5とマークウェルへ500μlの各抗体溶液を追加します。 </li><li>井戸の残りの部分に滅菌水を追加します。 4℃の冷蔵庫でシュリンクラップと場所でプレートを包みます。 </li></ol> 3。未刺激T細胞のヌクレオ<ol><li>のrhIL-15の5 ng / mlで37℃のとサプリメントでPrewarm T細胞メディア。ヌクレオの溶液2.25ミリリットルにヌクレオ補足1 500μLを加えることによって、完全なヌクレオ溶液を調製します。 </li><li> Aliquot5μgのトランスポゾン（ZEO-PT-CMV-EGFP）と1.5 mlのマイクロチューブ内のトランスポザーゼ（のpCMV-piggyBac）の各注：それが最適な遺伝子送達のためのトランスポザーゼ及びトランスポゾンDNA量を最適化し、最小限にする必要があるかもしれません細胞毒性。プラスミドは、要求によって著者から入手することができます。 </li><li> 50 mlチューブに、24ウェル組織培養プレートから収穫のPBMCs。細胞数をカウントします。フローサイトメトリー中にコントロールとして使用するために2×10 6個の細胞を保存します。 </li><li> 400×gで15 mlのチューブと遠心分離機に7から10×10 6個の細胞を、5分間、上澄みと指フリックを吸引し、ペレットを追加します。 </li><li>緩んで細胞ペレットにT細胞の完全なヌクレオ溶液100μlを添加します。 </li><li>プラスミドを含むチューブに溶液 - 細胞混合物を追加します。 </li><li>クレオキュベットの底に溶液セルプラスミド混合物を追加します。任意の気泡を導入しないように注意してください。 </li><li>プロを用いて細胞をNucleofectグラム、U-014（未刺激T細胞、ヒト、 <a href="http://bio.lonza.com/resources/product-instructions/protocols/" target="_blank">http://bio.lonza.com/resources/product-instructions/protocols/</a> ）。 </li><li>すぐにキュベットにのrhIL-15で温めておいたメディアの500μlを添加する。 rhIL15で温めておいた培地1.5mlで24ウェルプレートの各ウェルに細胞を移します。 </li><li> 37℃加湿インキュベーターで一晩インキュベート℃、5％CO 2で 。 </li></ol> 2日目 4。 T細胞の非特異的刺激<ol><li>細胞を回収し、細胞数を決定します。 GFP陽性細胞の頻度を決定するために、フローサイトメトリー用の0.5×10 6個の細胞を脇に置きます。コントロールとして、非トランスフェクトPBMCを使用します。 </li><li>非組織培養コーティングされたプレートからCD3/28抗体溶液を吸引除去し、T細胞のメディアで各ウェルを洗浄してください。 </li><li>完全なCTLのメディアでミリリットルあたり0.5×10 6個の細胞でnucleofected細胞を再懸濁5 ng / mlでのrhIL-15を補充した。 2.0ミリリットルの非組織培養プレートの4ウェル中のnucleofectedの各セルを追加します。よく5 回目に0.5×10 6非nucleofectedセルを追加します。 </li><li> 37℃加湿インキュベーターで3日間インキュベート℃、5％CO 2で 。 </li></ol> 5日目<ol><li>非コート組織培養プレートから刺激されたT細胞を採取。 </li><li> 5 ng / mlのIL-15とT細胞培地で0.7×10 6細胞/ mlで24ウェルコート組織培養プレート中の細胞を数え、replate。 </li></ol> 7日目 7。遺伝子発現の分析<ol><li>細胞を回収し、抗ヒトCD8抗体（抗CD3および抗CD4を使用することができます）で染色し、％GFP発現についてフローサイトメトリーで分析する。 </li></ol> 8日目（オプション） 8。 T細胞の増殖<ol><li> トランスフェクション後8日目に、T細胞は、Tで再播種することができますセルの更なる拡大のために16ウェル当たり0.7×10 6細胞の密度で、IL-15を持つメディア。 </li></ol>

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特別な利害関係は宣言されません。

本明細書に記載の方法は、一次ヒトTリンパ球の安定した導入遺伝子の改変を可能にします。我々は以前に、レポーター遺伝子（4週間以上）、非免疫原性の自殺遺伝子、養子免疫療法のためのキメラ抗原受容体（100日以上）を発現するT細胞を変更するpiggyBacトランスポゾンシステムの使用をテストしたと免疫抑制薬7,13-15に対する耐性をエンジニアに。養子免疫療法と他のアプ?...

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（オプション） </td></tr><tr><td> Lympholyte </td><td> Cedarlane </td><td> CL5015 </td><td></td></tr><tr><td>高度なRPMI 1640 </td><td> LifeTechnologies </td><td> 12633020 </td><td></td></tr><tr><td>ハイクローンウシ胎児血清</td><td>フィッシャー·サイエンティフィック</td><td> SH3008803 </td><td></td></tr><tr><td>グルタマックス-Iサプリメント</td><td> LifeTechnologies </td><td> 35050-061 </td><td></td></tr><tr><td>ヒトIL-15組換えタンパク質</td><td> eBioscience社</td><td> 14から8159 </td><td></td></tr><tr><td> EndoFreeプラスミドマキシキット</td><td>キアゲン</td><td> 12362 </td><td></td></tr><tr><td> Amaxaヌクレオ</td><td>ロンザ</td><td> AAD-1001S </td><td> ＆NBSPは、 </td></tr><tr><td>ヒトT細胞ヌクレオキット</td><td>ロンザ</td><td> VPA-1002 </td><td></td></tr><tr><td> CD8-APC </td><td>サザンバイオテクノロジー</td><td> 9536から11 </td><td></td></tr><tr><td>抗ヒトCD3 </td><td> eBioscience社</td><td> 16-0037-81 </td><td></td></tr><tr><td>抗ヒトCD28 </td><td> BDファーミンジェン</td><td> 555725 </td><td></td></tr><tr><td> 24ウェル組織培養処理プレート</td><td> BDファルコン</td><td> 353047 </td><td></td></tr><tr><td>プレート処理された24ウェル非組織培養</td><td> BDファルコン</td><td> 351147 </td><td></td></tr><tr><td></td><td></td><td></td><td> 完全なT細胞培地組成 1X高度RPMI 1640 5％の熱不活性化ウシ胎児血清 2mMのGlutamaxIM-I </td></tr></tbody></table>

piggybac transposon system modification of primary human t cells

piggyBacトランスポゾンシステムはもともとキャベツルーパー蛾1,2から派生した、自然にアクティブになります。この非ウイルス系は、最も一般的に1はpiggyBacトランスポサーゼ酵素と遺伝子導入活性に必要な逆方向反復要素の間に目的の遺伝子（複数可）を保有するプラスミド、トランスポゾンを発現する2つのプラスミドを利用して、ベースのプラスミドである。PiggyBacを媒介遺伝子転移は通っトランスポザーゼは、目的の標的細胞（S）のゲノムにトランスポゾンセグメントを統合することにより&quot;カット＆ペースト&quot;メカニズムは。PiggyBacは 3-5哺乳類昆虫1,2、多種多様で効率的な遺伝子送達活性を示した、としているプライマリーヒトT細胞を含むヒトの7,8 cells6。最近では、非常に活発piggyBacのトランスポザーゼは、遺伝子導入効率を向上させる9,10生成されました。 ヒトTリンパ球は、臨床intereであるがん11の養子免疫療法のst。注目すべきは、 眠れる森の美女のトランスポゾンシステムを用いて、ヒトT細胞のトランスポゾンの修正を含む最初の臨床試験は、12を承認されています。我々は以前に、ヒトT細胞の遺伝子改変のための非ウイルス性の方法論としてpiggyBacの有用性を評価した。我々は、レポーター遺伝子と非免疫原性誘導自殺遺伝子7とヒトT細胞の遺伝子改変で効率的であることがpiggyBac発見。ゲノム組み込み部位の解析では、既知の癌原遺伝子13内又はその付近の統合のための好みの欠如を明らかにした。我々は、腫瘍抗原、HER2に対して向けられたキメラ抗原受容体を運ぶために細胞傷害性Tリンパ球の遺伝子変更するpiggyBac使用され、遺伝子改変T細胞は、同所性マウスモデルにおいて、in vitroおよび in vivo でのHER2陽性腫瘍細胞の死滅標的媒介ことがわかった14。我々はまた、piggyBacを使用していたラパマイシンは15を利用されている癌治療に有用であるはずであるラパマイシンに耐性のヒトT細胞を生成します。 ここで、我々は、遺伝的に一次ヒトT細胞を変更するpiggyBacを使用するための方法を説明します。これは、文化、遺伝子改変し、T細胞の活性化に続いてヒト血液から末梢血単核細胞（PBMC）の単離を含む。このレポートの目的は、T細胞は、フローサイトメトリーによる遺伝子発現の解析と定量化のためのレポーター遺伝子（EGFP）で修飾した。 PiggyBacは、関心のある遺伝子の様々なヒトT細胞を変更するために使用することができます。我々は腫瘍抗原14〜T細胞に指示するpiggyBac使用しましたが、我々はまた、7を必要に応じて遺伝子改変された細胞を排除するために、誘導の安全スイッチを追加するpiggyBac使用しています。 piggyBacの大型貨物の容量も遺伝子導入が可能になりました大ラパマイシン耐性のmTOR分子（15キロバイト）15。したがって、我々は様々な目的のための主要なヒトT細胞の安定した遺伝子改変のための非ウイルス性の方法論を提示する。

遺伝的にレポーター遺伝子（EGFP）を有するヒトTリンパ球を変更するの手順を示す模式図を図1に示します。これらのプラスミドは、著者からのリクエストも承ります。レポーター遺伝子（EGFP）を遺伝的に改変されたヒトTリンパ球の手順を実演schemticは、 図2のshowinです。それは彼らが、分割展開し、文化に伝播するように取得するために、T細胞を活性化...

Watch this Scientific Journal Video about piggyBac Transposon System Modification of Primary Human T Cells at JoVE.com

piggyBac Transposon System Modification of Primary Human T Cells

我々は、遺伝的に非ウイルスを用いて導入遺伝子を持つプライマリヒトT細胞を変更する方法を説明します<em&gt; piggyBac</em&gt;トランスポゾンシステム。 T細胞は、使用して変更された<em&gt; piggyBac</em&gt;トランスポゾンシステムは安定した導入遺伝子の発現を示す。

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 ...

piggybac-transposon-system-modification-of-primary-human-t-cells

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JoVE Journal

Immunology and Infection

piggyBac Transposon System Modification of Primary Human T Cells

17.1K ビュー.  Baylor College of Medicine. 我々は、遺伝的に非ウイルスを用いて導入遺伝子を持つプライマリヒトT細胞を変更する方法を説明します piggyBacトランスポゾンシステム。 T細胞は、使用して変更された piggyBacトランスポゾンシステムは安定した導入遺伝子の発現を示す。

ビデオ: piggyBac Transposon System Modification of Primary Human T Cells

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

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

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

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

ゾレドロネートを使用してヒト末梢血γδT細胞の拡大

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

免疫・感染学

Determining Optimal Cytotoxic Activity of Human Her2neu Specific CD8 T cells by Comparing the Cr51 Release Assay to the xCELLigence System

Cytotoxic CD8 T cells constitute a subgroup of T cells that are capable of inducing the death of infected or malignant host cells1. These cells express a specialized receptor, called the T cell receptor (TCR), which can recognize a specific antigenic peptide bound to HLA class I molecules2. Engagement of infected cells or tumor cells through their HLA class I molecule results in production of lytic molecules such as granzymes and perforin resulting in target cell death. While it is useful to determine frequencies of antigen-specific CD8 T cells using assays such as the ELIspot or flow cytometry, it is also helpful to ascertain the strength of CD8 T cell responses using cytotoxicity assays3. The most recognizable assay for assessing cytotoxic function is the Chromium Release Assay (CRA), which is considered a standard assay 4. The CRA has several limitations, including exposure of cells to gamma radiation, lack of reproducibility, and a requirement for large numbers of cells. Over the past decade, there has been interest in adopting new strategies to overcome these limitations. Newer approaches include those that measure caspase 
release 4, BLT esterase activity 5 and surface expression of CD107 6. The impedance-based assay, using the Roche xCelligence system, was examined in the present paper for its potential as an alternative to the CRA. Impedance or opposition to an electric current occurs when adherent tumor cells bind to electrode plates. Tumor cells detach following killing and electrical impedance is reduced which can be measured by the xCelligence system. The ability to adapt the impedance-based approach to assess cell-mediated killing rests on the observation that T cells do not adhere tightly to most surfaces and do not appear to have much impact on impedance thus diminishing any concern of direct interference of the T cells with the measurement. Results show that the impedance-based assay can detect changes in the levels of antigen-specific cytotoxic CD8 T cells with increased sensitivity relative to the standard CRA. Based on these results, impedance-based approaches may be good alternatives to CRAs or other approaches that aim to measure cytotoxic CD8 T cell functionality.

The chromium release assay, a common assay for detecting cytotoxic T cell activity, has several limitations. Using antigen-specific CD8 T cells and the human breast cancer tumor line, SKBR3, in the present article, an impedance-based approach was examined for the capability of detecting cell killing.

xCELLigenceシステムにCR51リリースアッセイを比較することによって、人間Her2neu特異的CD8 T細胞の最適な細胞傷害活性を決定する

Cytotoxic CD8 T cells constitute a subgroup of T  cells that are capable of inducing the death of infected or malignant host  cells1. These cells express ...

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

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

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

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

初代ヒトナイーブおよびメモリーT細胞から誘導した調節性T細胞の生成

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

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

HIV-1感染CD4の生得センシングを評価<sup&gt; +</sup&gt;使用して形質細胞様樹状細胞によるT細胞<em&gt;エクスビボ</em&gt;共培養系。

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

Pseudomonas aeruginosa is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa is an opportunistic bacterial pathogen which establishes chronic pulmonary infections in patients with cystic fibrosis (CF). The overproduction of a capsular polysaccharide called alginate, also known as mucoidy, promotes the formation of mucoid biofilms which are more resistant than planktonic cells to antibiotic chemotherapy and host defenses. Additionally, the conversion from the nonmucoid to mucoid phenotype is a clinical marker for the onset of chronic infection in CF. Alginate overproduction by P. aeruginosa is an endergonic process which heavily taxes cellular energy. Therefore, alginate production is highly regulated in P. aeruginosa. To better understand alginate regulation, we describe a protocol using the mini-himar1 transposon mutagenesis for the identification of novel alginate regulators in a prototypic strain PAO1. The procedure consists of two basic steps. First, we transferred the mini-himar1 transposon (pFAC) from host E. coli SM10/&#955;pir into recipient P. aeruginosa PAO1 via biparental conjugation to create a high-density insertion mutant library, which were selected on Pseudomonas isolation agar plates supplemented with gentamycin. Secondly, we screened and isolated the mucoid colonies to map the insertion site through inverse PCR using DNA primers pointing outward from the gentamycin cassette and DNA sequencing. Using this protocol, we have identified two novel alginate regulators, mucE (PA4033) and kinB (PA5484), in strain PAO1 with a wild-type mucA encoding the anti-sigma factor MucA for the master alginate regulator AlgU (AlgT, &#963;22). This high-throughput mutagenesis protocol can be modified for the identification of other virulence-related genes causing change in colony morphology.

Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

Here we describe a protocol using the mini-himar1 mariner transposon-mediated mutagenesis for generating a high-density insertion mutant library to screen, isolate and identify novel alginate regulators in the prototypic Pseudomonas aeruginosa strain PAO1.

アルギン酸塩産生に関連する新規遺伝子の同定<em&gt;緑膿菌</em使用&gt;ミニ<em&gt; himar1</em&gt;マリナートランスポゾンによる突然変異誘発

Pseudomonas aeruginosa is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa is an opportunistic bacterial ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

末梢血からのヒトアロ抗原特異的T細胞の生成

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

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

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

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

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

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

Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von Willebrand Factor (VWF). This glycoprotein changes its molecular conformation in response to shear stress, thereby exposing binding sites for a broad spectrum of host-ligand interactions. In general, culturing of primary endothelial cells under a defined shear flow is known to promote the specific cellular differentiation and the formation of a stable and tightly linked endothelial layer resembling the physiology of the inner lining of a blood vessel. Thus, the functional analysis of interactions between bacterial pathogens and the host vasculature involving mechanosensitive proteins requires the establishment of pump systems that can simulate the physiological flow forces known to affect the surface of vascular cells.
The microfluidic device used in this study enables a continuous and pulseless recirculation of fluids with a defined flow rate. The computer-controlled air-pressure pump system applies a defined shear stress on endothelial cell surfaces by generating a continuous, unidirectional, and controlled medium flow. Morphological changes of the cells and bacterial attachment can be microscopically monitored and quantified in the flow by using special channel slides that are designed for microscopic visualization. In contrast to static cell culture infection, which in general requires a sample fixation prior to immune labeling and microscopic analyses, the microfluidic slides enable both the fluorescence-based detection of proteins, bacteria, and cellular components after sample fixation; serial immunofluorescence staining; and direct fluorescence-based detection in real time. In combination with fluorescent bacteria and specific fluorescence-labeled antibodies, this infection procedure provides an efficient multiple component visualization system for a huge spectrum of scientific applications related to vascular processes.

This study describes the microscopic monitoring of pneumococcus adherence to von Willebrand factor strings produced on the surface of differentiated human primary endothelial cells under shear stress in defined flow conditions. This protocol can be extended to detailed visualization of specific cell structures and quantification of bacteria by applying differential immunostaining procedures.

一定の流れの原発性ヒト内皮細胞の肺炎球菌感染

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von ...

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 ...

Real-time Monitoring of Mitochondrial Respiration in Cytokine-differentiated Human Primary T Cells

During activation, the metabolism of T cells adapts to changes that impact their fate. An increase in mitochondrial oxidative phosphorylation is indispensable for T cell activation, and the survival of memory T cells is dependent on mitochondrial remodeling. Consequently, this affects the long-term clinical outcome of cancer immunotherapies. Changes in T cell quality are often studied by flow cytometry using well-known surface markers and not directly by their metabolic state. This is an optimized protocol for measuring real-time mitochondrial respiration of primary human T cells using an Extracellular Flux Analyzer and the cytokines IL-2 and IL-15, which differently affect T cell metabolism. It is shown that the metabolic state of T cells can clearly be distinguished by measuring the oxygen consumption when inhibiting key complexes in the metabolic pathway and that the accuracy of these measurements is highly dependent on optimal inhibitor concentration and inhibitor injection strategy. This standardized protocol will help implement mitochondrial respiration as a standard for T cell fitness in monitoring and studying cancer immunotherapies.

Metabolic adaptation is fundamental for T cells as it dictates differentiation, persistence, and cytotoxicity. Here, an optimized protocol for monitoring mitochondrial respiration in ex vivo cytokine-differentiated human primary T cells is presented.