我々は、この方法では、原稿やビデオの開発における彼らの支援のためのジェシカ·ジョーンズ、エリカ·クラーク、コンスタンスDucar、ドナ·スミス、ロイ·ルイス、リリーApedaile、ジョアンウィーズナー、デヴィン·アダムス、コーリー·マクベインとスティーブンVoghtに感謝します。 この作品は、ビル·アンド·メリンダ·ゲイツ財団の助成金CAVD 38645（MJM）と健康の補助金の国立研究所UM1 AI068618とAI069481 U01（MJM）によってサポートされていました。 EA-N。 NIHグラントT32 AI007140によってサポートされています。我々は彼らの寛大な機器の寄付のためにジェームズ·B·ペンドルトン公益信託に感謝します。

注：光から蛍光色素標識抗体を保護するために、光をオフにしてバイオ安全キャビネット内のすべての手順を実行します。 1。抗体染色パネルの準備<ol><li>抗体染色パネルが表1に記載されています。抗体濃度は全血で滴定することにより定​​義され、染色された表現型のサンプルを取得するために使用されるのと同じフローサイトメトリー機器および手順を使用する必要があります。 </li><li>適切な染色力価が決定されると、ロックキャップチューブ内の単一の混合物の中にすべての抗体を結合します。 100μlに総量をもたらすために、フローの洗浄バッファー（2％熱不活性化ウシ胎児血清を含むダルベッコPBS）を追加します。染色されたサンプル数の混合物を拡大縮小します。この混合物を、最大8週間まで4℃で保存することができます。 </li></ol> 2。染色<ol><li>採取した血液は、広告内に他の目的に使用する場合もっと時間に敏感な手続きが残っている血液に行っている間、このアッセイにditionはさておき、アリコートを設定します。アリコートは有意な細胞を損失することなく静脈穿刺後4時間まで室温で保存することができます。 </li><li>無傷ビーズTrucountチューブの底にペレットとラベルが染色されたサンプルを識別するためのチューブがあることを確認します。 HVTN臨床試験では、実験室データマネジメントシステム（フロンティア科学技術研究財団、アマースト、ニューヨーク州）は、染色したサンプルにラベルを付けて追跡するために使用されます。 </li><li>すべての試薬のロット番号と有効期限を記録します。チューブの袋の上にメーカーから提供されTrucountチューブビーズカウント数を記録し、袋の上にロット番号がチューブにロット番号と一致していることを確認してください。 </li><li>正確にちょうど金属リテーナの上、Trucount管に全血100μlをピペットにピペット逆に使用します。チューブの側面に塗沫血を避けてください。 </li><li&gt; Trucount管に通常の（前方）ペッティング技術、混合抗体染色パネルの各々 100μL添加します（ 表1を参照）を使用する。混ぜて約15秒間低速でチューブと渦をキャップ。視覚的にビーズペレットが完全に溶解していることを確認するためにチューブを点検してください。 </li><li>暗所で室温（15-30℃）で15分間Trucountチューブをインキュベートする。 </li><li>必要に応じて、DIH 2 Oを用いて1Xに10のアリコート×FACS溶解溶液を希釈900μlの1×チューブにFACS溶解溶液を追加します。 </li><li>混ぜて約15秒間低速で徹底的に管と渦をキャップ。実験室でのフィルムでチューブおよびシールの位置をロックにしっかりとキャップを押し下げます。 </li><li> -65℃チューブを-95℃で保存°Cのサンプルは、中央分析室への出荷のためか、家の中で分析のための準備が整うまで。サンプルは、少なくとも4週間は、この段階では安定しています。直ちにを出荷またはアッセイする場合diately、この工程を省略することができる。 </li></ol> 3。出荷 注：以下の手順は、特に国際航空及び運送協会（IATA）の規則に従って船積みカテゴリB免除生物学的物質用に設計されたSAF-T-Pakは、（株）から絶縁された出荷システムを利用しています。染色が発生した場所と同じ場所にサンプルを分析する場合は、セクション4に進みます。 <ol><li>サンプルは直ちに染色後出荷することができるか、一度それらが-65℃に凍結さ℃〜-95℃まで染色標本ボックスに箔と場所で完全に各チューブを巻き付けます。吸収物質で漏れ防止ポリ袋の中に染色標本ボックスを配置します。 </li><li>袋の中でできるだけ少ない空気のようにタイベックの袋とシールに漏れ防止ポリ袋と中身を置きます。 </li><li>内側の茶色のボックス内の標本パッケージ（二次包装内部の標本）を配置します。 </li><li>インナーブラウンBを配置シフトを防ぐために、インデントにそれを固定している発泡スチロールの胸部内の牛、。 </li><li>ドライアイス（約8キロ）で発泡スチロールの箱を記入し、胸にしっかりと蓋を置く。 </li><li>適切なドライアイス（UN1845）のマーキングとしっかりとテープ生物由来物質として梱包箱や船、カテゴリBを（UN3373）のIATA PI-650の指示に従ってください。 </li><li>受信すると、サンプルは-65℃で保存するC -95°Cまで、彼らが分析されるまで。 </li></ol> 4。解凍およびフローサイトメトリー分析<ol><li>フローサイトメーターで収集する前に、暗闇の中で、室温で解凍し、冷凍庫から染色されたサンプルを削除します。複数のサンプルについてのデータを収集する場合は、チューブが1時間以上各室温で座ってされないように解凍ずらすことによって、すべてのチューブのためのプロセスを標準化しています。 </li><li>サンプルは、例えばBD LSRとして適切なフィルターを装備して4つのレーザーフローサイトメーターを用いて取得しておく必要がありますⅡ。データ収集のための標準的な4メーターのキャリブレーションおよび蛍光補正メソッドを使用します。 </li></ol> 注意：コレクション5時に前方散乱または側方散乱のしきい値を設定しないでください。 Trucountビーズは、分析中に計上されないように、ビーズのサブセットを引き起こし、これらのパラメータの可能な限り低いしきい値の設定値を下回ることができます。しきい値を設定する機器によって必要な場合は、可能な限り低いアムシアンチャネルのしきい値を設定します。パネルで染色し、CD45 +白血球もアムシアンチャンネルで蛍光を発するアムシアン陽性、Trucountビーズとなりますので、これは適切に収集することができるため、関連するすべてのデータを考慮するべきです。 <ol start="3"><li>フローサイトメーター上でロードする前に、5秒間ボルテックスサンプルチューブを。 Trucountビーズは多くのチャンネルで高い蛍光を発する。ゲートPE Cy5およびAPC（2 coloのために非常に重陽性である人口を見つけることによって、収集時にビーズ最も容易に細胞からビーズを区別するRS）は、インスツルメンテーションによって異なります。少なくとも20,000ビーズが取得されるまで、停止ゲートとしてビーズゲートを選択し、レコードデータ。 </li><li>そのようなFlowJo（Treestar;オレゴン州アッシュランド）のような適切なソフトウェアを使用してデータを分析します。 図1は代表的な制御採血異なる白血球集団の分析のために利用ゲーティング方式を示しています。 </li></ol> 5。 Trucount計算<ol><li>各Trucount管は蛍光ビーズの凍結乾燥ペレットを含んでいます。チューブに液を添加し、ボルテックスした後、ビーズが均等にサンプル全体に分散になる必要があります。ペレットのビーズの数はロット番号により若干異なりますが、チューブ用の収納袋で見つけることができます。 </li><li>ゲートTrucountビーズと細胞集団を各集団のイベントカウントを決定するために、図1に示すように。イベントiの数を比較するn個のチューブ内に元々ビーズの総数にビーズゲートでは、各人口の絶対濃度（ すなわち 、細胞/μl）を決定するために使用できるサンプル回収し、の比率を決定することができます。細胞濃度（＃細胞/μlの全血）= [/＃人口イベント（＃ビーズイベント/＃ペレットの総ビーズ）] /100μlの次の式は、この目的のために使用することができます。 </li></ol> 6。代表的な結果<img src="/files/ftp_upload/4302/4302fig1.jpg" alt="図1" fo:content-width="5.5in" fo:src="/files/ftp_upload/4302/4302fig1highres.jpg" /> 図1ゲーティング方式は、健康なボランティアからの代表的なデータを示す主要な白血球集団の分析のために利用した。 A）はTrucountビーズは、（i）gatedおよび細胞から排除されています。顆粒球（ii）が線引きされており、lympohcytesと単球は3 popluationsに分かれています：CD14負のリンパ球（iii）では、すべてのCD14陰性細胞（IV）、および非リンパ球​​（v）である。 B）は、CD14陰性のリンパ球は、CD4 + T細胞（VI）、CD8 + T細胞（VII）、B細胞（ⅷ）、CD56明るいNK細胞（IX）、CD56薄暗いNK細胞（x）、およびCD56陰性NK細胞を区別するためにゲートさ（XI）。 c）すべてのCD14陰性細胞は、骨髄（XII）と形質（XIII）樹状細胞を区別するゲートさ。 d）非lympocytesは非古典（XIV）、中間（XV）、古典（XVI）の単球を区別するためにゲートされる。 <a href="http://www.jove.com/files/ftp_upload/4302/4302fig1large.jpg" target="_blank">拡大図を表示するには、ここをクリックしてください</a> 。 （ 例えば 、NKT細胞または好中球）、図1に示されていない、より具体的な細胞サブセットはまた、我々が提示するパネルを使用して区別することができ、かつゲーティング方式は展開したり、特定の研究ニーズに合わせて変更することができます。示される特定のゲーティングの手順では、このメソッドに固有のものです。特に注目すべきは、インクルージョンのゲートと除外ゲートがTrucountビーズの周りに描かれ、互いに、カウントするためのゲートビーズを1つ、および細胞分析（ 図1A）からビーズを除外する1つの上に置かれています。また、（ 図1A）CD45発現および側方散乱を用いて、これらの細胞はしばしば必要であるゲー、彼らは前方散乱および側方散乱による末梢血単核細胞にあるようにリンパ球、単球、顆粒球は、同じように簡単に全血中で区別されていないためです。 CD45および側方散乱を用いたリンパ球と単球から分離することができませんでした汚染顆粒球（円内）が高いCD16発現（ 図1Cおよび図1D）によっていくつかのプロットで区別することができる。汚染顆粒球の数は通常小さく、それらは単球とNK細胞ゲーティングを妨げることはありません。

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特別な利害関係は宣言されません。

本稿では、フローサイトメトリーによって新鮮全血中の白血球集団を列挙するためのビーズをベースにした方法を提示し、一元的なサンプル分析による多施設臨床試験での使用に必要なパラメータをカバーしています。この方法は、上に構築し、BD Trucountプロトコルを最適化し、多施設臨床試験の設定で、その信頼性の高い使用を可能にします。染色アッセイはシンプルであり、それは血液?...

<table border="1"><tbody><tr><td> 試薬名 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td>絶対計数管をTrucount </td><td> BD Biosciences社</td><td> 340334 </td></tr><tr><td> Solutionを溶解10X FACS </td><td> BD Biosciences社</td><td> 349202 </td></tr><tr><td>カテゴリーB＆免除配送システム、絶縁</td><td> SAF-T-Pakの</td><td> STP-320 </td></tr><tr><td> CD45モノクローナル抗体AmCyan </td><td> BD Biosciences社</td><td> 339192 </td></tr><tr><td> CD3 FITCモノクローナル抗体</td><td> BD Biosciences社</td><td> 349201 </td></tr><tr><td> CD8も、PerCP-Cyは5.5モノクローナル抗体</td><td> BD Biosciences社</td><td> 341051 </td></tr><tr><td> CD4のAlexa Fluor 700モノクローナルantiboDY </td><td> BD Biosciences社</td><td> 557922 </td></tr><tr><td> HLA-DRのECDのモノクローナル抗体</td><td>ベックマン·コールター</td><td> IM3636 </td></tr><tr><td> CD14モノクローナル抗体V450 </td><td> BD Biosciences社</td><td> 560349 </td></tr><tr><td> CD19モノクローナル抗体、PE </td><td> BD Biosciences社</td><td> 555413 </td></tr><tr><td> CD16 APC-H7型モノクローナル抗体</td><td> BD Biosciences社</td><td> 560195 </td></tr><tr><td> CD56 PE-Cy7モノクローナル抗体</td><td> BD Biosciences社</td><td> 335791 </td></tr><tr><td> CD11cはAPCのモノクローナル抗体</td><td> BD Biosciences社</td><td> 559877 </td></tr><tr><td> CD123、PE-Cy5のモノクローナル抗体</td><td> BD Biosciences社</td><td> 551065 </td></tr></tbody></table>

enumeration of major peripheral blood leukocyte populations for multicenter clinical trials using a whole blood phenotyping assay

末梢血白血球の凍結保存は、広く臨床試験において免疫応答評価のための細胞を保持するために使用され、免疫学的な評価のしやすさと標準化のための多くの利点を提供していますが、このプロセスの有害な影響は、顆粒球、B細胞などの一部の細胞サブセットで観察されているされている、および樹状細胞1-3。新鮮な白血球をアッセイする細胞の in vivo の状態でのより正確な画像が得られますが、大規模臨床試験のコンテキストで実行することはしばしば困難である。新鮮な細胞アッセイは、時間がかかる場合には、それらのアプリケーションが原因で検査室の職員に求められる労働時間に非現実的なことができ、ボランティアのコミットメントと時間枠に依存していると。また、治験が複数の施設で実施された場合、アッセイを行うために必要なリソースと訓練研究所は臨床現場に十分に近接して配置されていない可能性があります。これらの問題に対処するために、我々は持っている開発表現型と末梢血内に主要な白血球集団を列挙するなど、アッセイよりも堅牢な細胞型に特異的な情報を得る工程; Trucountチューブ（サンノゼ、カリフォルニア州Becton Dickinson）を用いて使用することができる11色の抗体染色パネルをOPED全血球算定（CBC）または少数の細胞型のため染色Trucountチューブ用に設計された市販のパネルとアッセイ。染色手順は簡単ですが、新鮮な全血のみを100μlを必要とし、約45分かかり、それが実現可能な標準的な血液処理ラボが行うようにすること。それは、BD Trucount管技術データシート（から適合さ<a href="http://www.bdbiosciences.com/external_files/is/doc/tds/Package_Inserts_CE/live/web_enabled/23-3483-06_PI_Trucount tubes_CE_EN.pdf" target="_blank">バージョン2010分の8</a> ）。染色抗体カクテルは、中央アッセイ研究室で一括して事前に準備し、現場での加工ラボに出荷することができます。ステンドチューブを固定することができと多色フローサイトメトリー分析のための中央検査所への出荷のために凍結した。この染色パネルから生成されたデータは、介入との関係で時間をかけて白血球濃度の変化を追跡するために使用することができ、簡単にさらに関心のある特定の種類の細胞の活性化状態を評価するために開発される可能性がある。本稿では、新鮮な全血と多施設臨床試験を支える中心的なアッセイ研究室でこれらの染色されたサンプルを分析するための手順に染色を実行するために血液処理ラボの技術者が使用するプロシージャを示しています。それがHIVワクチン治験ネットワーク（HVTN）における臨床試験採血のコンテキストで実行されるビデオの詳細手順。

Watch this Scientific Journal Video about Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay at JoVE.com

Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay

本稿では、大規模な自然免疫と適応白血球集団を列挙するために新鮮な全血で行わ表現型アッセイの染色と分析の手順を紹介します。我々は、多施設臨床試験のコンテキストでこれらの手順を実行する際の考慮事項を強調している。

全血の表現型アッセイを用いて多施設臨床試験のための主要な末梢血白血球集団の列挙

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many ...

enumeration-major-peripheral-blood-leukocyte-populations-for

Research

JoVE Journal

Immunology and Infection

14.9K ビュー.  Fred Hutchinson Cancer Research Center. 本稿では、大規模な自然免疫と適応白血球集団を列挙するために新鮮な全血で行わ表現型アッセイの染色と分析の手順を紹介します。我々は、多施設臨床試験のコンテキストでこれらの手順を実行する際の考慮事項を強調している。

ビデオ: Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

補体の枯渇により特定の細胞集団の枯渇

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

免疫・感染学

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known phenotype.  Other bulk methods of purification include panning, complement depletion and magnetic bead separation.  However, FACS has several advantages over other available methods.  FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density.  In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker.  FACS allows the purification of individual cells based on size, granularity and fluorescence.  In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1).  Negative selection of unstained cells is also possible.  FACS purification requires a flow cytometer with sorting capacity and the appropriate software.  For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser.  The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells.  The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2).  The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb.  Sorting parameters can be adjusted depending on the requirement of purity and yield.  Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.

蛍光活性化細胞選別（FACS）によって特定の細胞集団の精製

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

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

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

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

拡大、精製、及びヒト末梢血NK細胞の機能的評価

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

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 ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

細菌付着の研究ではせん断応力を導入する

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb (BCG lux/M.Tb lux)

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal titers. Studies of serum bactericidal titers in response to childhood vaccines have enabled us to develop and validate cut-off levels for protective immune responses and such cut-offs are in routine use. No such assays have been taken forward into the routine assessment of vaccines that induce primarily cell-mediated immunity in the form of effector T cell responses, such as TB vaccines. In the animal model, the performance of a given vaccine candidate is routinely evaluated in standardized bactericidal assays, and all current novel TB-vaccine candidates have been subjected to this step in their evaluation prior to phase 1 human trials. The assessment of immunogenicity and therefore likelihood of protective efficacy of novel anti-TB vaccines should ideally undergo a similar step-wise evaluation in the human models now, including measurements in bactericidal assays.
Bactericidal assays in the context of tuberculosis vaccine research are already well established in the animal models, where they are applied to screen potentially promising vaccine candidates. Reduction of bacterial load in various organs functions as the main read-out of immunogenicity. However, no such assays have been incorporated into clinical trials for novel anti-TB vaccines to date.
Although there is still uncertainty about the exact mechanisms that lead to killing of mycobacteria inside human macrophages, the interaction of macrophages and T cells with mycobacteria is clearly required. The assay described in this paper represents a novel generation of bactericidal assays that enables studies of such key cellular components with all other cellular and humoral factors present in whole blood without making assumptions about their relative individual contribution. The assay described by our group uses small volumes of whole blood and has already been employed in studies of adults and children in TB-endemic settings. We have shown immunogenicity of the BCG vaccine, increased growth of mycobacteria in HIV-positive patients, as well as the effect of anti-retroviral therapy and Vitamin D on mycobacterial survival in vitro. Here we summarise the methodology, and present our reproducibility data using this relatively simple, low-cost and field-friendly model.
Note: Definitions/Abbreviations
BCG lux = M. bovis BCG, Montreal strain, transformed with shuttle plasmid pSMT1 carrying the luxAB genes from Vibrio harveyi, under the control of the mycobacterial GroEL (hsp60) promoter. 
CFU = Colony Forming Unit (a measure of mycobacterial viability).

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux

We describe an alternative approach to the enumeration of mycobacteria in vitro, which uses reporter-gene tagged mycobacteria instead of colony-forming units (CFU). &#x201C;Survival&#x201D; of organisms as well as host response-markers are measured simultaneously, providing a low-cost, versatile and functional system for studies of host/pathogen interactions in the context of tuberculosis.

BCGまたはM. TB（BCGタグレポーター遺伝子を用いて、抗酸菌の生存率を測定するための機能全血アッセイルクス / M. Tbをルクス）

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal ...

Fast and Specific Assessment of the Halogenating Peroxidase Activity in Leukocyte-enriched Blood Samples

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on the hypotonic lysis of erythrocytes and can be applied to human samples as well as to blood of non-human origin. The small initial sample volume of about 50 to 100 &#181;l makes this method applicable to recurrent blood sampling from small laboratory animals. Moreover, leukocyte enrichment is achieved within minutes and with low material efforts regarding chemicals and instrumentation, making this method applicable in multiple laboratory environments.
Standardized purification of leukocytes is combined with a highly selective staining method to evaluate halogenating peroxidase activity of the heme peroxidases, myeloperoxidase (MPO) and eosinophil peroxidase (EPO), i.e., the formation of hypochlorous and hypobromous acid (HOCl and HOBr). While MPO is strongly expressed in neutrophils, the most abundant immune cell type in human blood as well as in monocytes, the related enzyme EPO is exclusively expressed in eosinophils. The halogenating activity of these enzymes is addressed by using the almost HOCl- and HOBr-specific dye aminophenyl fluorescein (APF) and the primary peroxidase substrate hydrogen peroxide. Upon subsequent flow cytometry analysis all peroxidase-positive cells (neutrophils, monocytes, eosinophils) are distinguishable and their halogenating peroxidase activity can be quantified. Since APF staining may be combined with the application of cell surface markers, this protocol can be extended to specifically address leukocyte sub-fractions. The method is applicable to detect HOCl and HOBr production both in human and in rodent leukocytes.
Given the widely and diversely discussed immunological role of these enzymatic products in chronic inflammatory diseases, this protocol may contribute to a better understanding of the immunological relevance of leukocyte-derived heme peroxidases.

This protocol describes the quick enrichment of leukocytes from small blood samples for a subsequent specific determination of the halogenating peroxidase activity within the cells. The method can be applied to human and non-human material and may contribute to the evaluation of new inflammatory markers.

白血球に富んだ血液サンプル中のハロゲンペルオキシダーゼ活性の迅速かつ具体的な評価

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

Use of a Monocyte Monolayer Assay to Evaluate Fc&amp;#947; Receptor-mediated Phagocytosis

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

Fcγ受容体媒介食作用を評価するために、単球単層アッセイの使用

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

デキストランは、マウスとヒト NK 細胞のレンチ ウイルスの伝達効率を向上します。

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies in an attempt to understand the precise mechanisms that lead to the breakdown of self-tolerance and subsequent autoimmunity. Approaches thus far have been based on the study of one specific aspect of the immune system (a single or few cell types or cytokines), and do not offer a global assessment of complex autoimmune disease. While patient sera-based studies have afforded important insights into autoimmunity, they do not provide the specific cellular source of the dysregulated cytokines detected. A comprehensive single-cell approach to evaluate cytokine production in multiple immune cell subsets, within the context of &#34;intrinsic&#34; patient-specific plasma circulating factors, is described here. This approach enables monitoring of the patient-specific immune phenotype (surface markers) and function (cytokines), either in its native &#34;intrinsic pathogenic&#34; disease state, or in the presence of therapeutic agents (in vivo or ex vivo).

Here we describe a single-cell proteomic approach to evaluate immune phenotypic and functional (intracellular cytokine induction) alterations in peripheral whole blood samples, analyzed via mass cytometry.

イムノフェノ タイプと大量のフローサイトメトリーによる末梢血中のサイトカイン産生の単一細胞解析

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies ...