この作品は、ダン·L·ダンカン共同研究助成金（CMBとEJS）、国立心肺血液研究所（US4HL081007）、白血病とリンパ腫協会臨床研究奨学生賞（CMB）、および国立癌研究所によってサポートされていました（RO1 CA06150816; EJS）。

1。単核細胞分離（0日目） <ol><li>臍帯血ユニットの20％分の出口ポートにメスルアーアダプタのスパイクを挿入し、シリンジを接続して、血液を除去する。 50 mLの遠心分離管中で温めたRPMI 20mLに解凍した血液を転送します。 RPMI 5mLで臍帯血袋を洗浄し、同じ遠心チューブに移す。 </li><li> 400 x gで10分間細胞を遠心分離します。上清を吸引します。 </li><li>暖かいRPMI 20mlに細胞を再懸濁する。 50 mLの遠心分離管でLymphoprep 15mLの上に層細胞。 40分@ 400×gで遠心分離します。 </li><li>単核細胞を含むインターフェイスを収穫し、RPMI 20mlに洗う。 10分@ 450×gで遠心します。 </li><li>上清を吸引し、RPMI 20mlに洗う。 5分@ 400×gでのための細胞とスピンを数える。 </li><li> Cellgenix直流メディア（無血清）/ mlの5×10 6個の単核細胞上清に再懸濁した細胞を吸引します。 EBV-LCLの世代のために1 mLを削除し、15 mLのCEに入れチューブをntrifuge。 </li></ol> 2。樹状細胞の生成（day0から） <ol><li> 6ウェルプレートにウェルあたり細胞（DCメディアの/ mLを5×10 6個の細胞に既にある）のプレートを2mL。 1-2時間インキュベーター内にしておきます。 </li><li> 1-2時間後、PBSで3〜4回洗浄することにより、非接着細胞を洗い流してください。非接着細胞と凍結を収集します。を2mL /ウェルの1000 U / mlのIL-4のと800 U / mLのGM-CSFを含む直流メディアを追加します。 </li><li> DCの開始後3〜4日、1000U/mL IL-4および800 U / mLのGM-CSFの最終濃度を含有する培地を100μL/ウェルを添加することにより、IL-4およびGM-CSFを補給してください。 </li><li>日5-6、収穫ホールピペットでよくを削ってDC上。 5分@ 400×gでの大きな細胞や遠心分離機を数える。 2×10 6細胞/ DCのメディアのmLの吸引メディアを再懸濁し、DC。 24ウェルプレートの0.5mL /ウェルを追加します。 </li><li> 10感染単位のMOIで細胞あたりでAd5f35pp65ベクトルを使用してDCを伝達する。 vectoを追加24ウェルプレートの各ウェルにR。 1.5時間細胞をインキュベートします。 1.5時間後、含むDCメディアの1.5mL /ウェルを追加します：1000 U / mlのIL-4、800 U / mLのGM-CSFを、10 ng / mLで、TNF-α、1μg/ mLのPGE-1、100 ng / mLのIL-6、および10 ng / mlのIL-1β。これらのサイトカインは、樹状細胞を成熟させる。 </li></ol> 3。 EBV-LCLの生成（day0から） <ol><li> 5×10 6個の単核細胞の入ったチューブを遠心します。濃縮されたEBV B95.8清との完全培地1.8 mLの培地（RPMI +10％FBS + 2mM L-グルタミン）を含有するシクロスポリン（1μg/ ml）を200μLを加える。 </li><li> 96ウェルプレートと5のウェルに200μLの10ウェルに細胞のアリコートを100μL添加します。完全培地+細胞の唯一の100μLを含むウェルにシクロスポリンの100μLを加える。滅菌水で残りのウェルを埋める。 </li><li> LCL毎週を養うために、約2週間後、24ウェルプレートに96ウェルプレートから展開します。もう一週間後に、T25フラスコに乗り換え、後に週に転送でT75フラスコ。この段階では、それはあなたがLCLのアリコートを凍結することをお勧めします。それは一般的にLCLの十分な数を生成するために1ヶ月位かかります。 </li></ol> 4。 CTLを開始 - 7日DCの開始後<ol><li> 30 Gyで収穫DCおよび照射。 4×、カウントを洗ってください。ヒト血清を含むT細胞培地に再懸濁し、DC（45％RPMI、45％のCLICK、10％ヒト血清、2mM L-グルタミン）@ 1×10 5個のDC / mLであった。 </li><li>ステップ2.2から非接着細胞を解凍します。細胞を洗浄し、カウント。ヒト血清を含むT細胞培地で2×10 6個の非接着細胞/ mLで再懸濁する。 10 ng / mLのIL-7およびIL-12、および5 ng / mLのIL-15を追加します。 24ウェルプレートのウェルあたりの細胞の1 mLを加える。ウェルあたりのDCの1 mLを加える。滅菌水で24ウェルプレートの空のウェルを埋める。 </li><li> 7日T細胞開始後、飼料および/またはヒト血清を含むT細胞培地で細胞を分割します。 </li><li> LCLは準備が整うまで、9から12日T細胞開始後、T細胞を凍結します。 </li><li> OLCLが拡大し、T75フラスコに継代されるNCE、5分@ 400×gで遠心分離することにより、LCLを伝達する。 MOIのセル当たり100感染単位ので細胞ペレットにベクトルを追加します。 1.5時間インキュベートします。 1.5時間後、5×10 5 LCL / mLの完全培地で再懸濁し、24ウェルプレートにウェル当たり2 mLを加え12 </li><li> 2日後、40 GyでLCLおよび照射を収穫。 4×、カウントを洗ってください。 2.5×10 5細胞/ mLでヒト血清を含むT細胞の培地中でLCLを再懸濁します。 LCLの1 mL当たり24ウェルプレートを追加します。また、Grex40培養装置だけでなく、5 ng / mLのIL-15から5×10 6 LCLを追加します13 </li><li> T細胞を収穫します。 @ 400×gで5分間遠心し、上清を吸引し、1×10 6のT細胞/ mLのヒト血清を含むT細胞培地に再懸濁します。 5 ng / mLのIL-15を追加し、24ウェルプレートに既に登録されているLCLに1ml /ウェルのT細胞（1×10 6 T細胞の合計）を追加します。 Grex40では、持参最大30 mLにメディアの総容量。 </li><li>細胞がコンフルエントにある場合刺激後3-4日で、その後、（プレートの）1:1それらを分割し、50 U / mLのIL-2を含む新鮮な培地を追加します。コンフルエントにない場合は、単に半培地を吸引し、50 U / mLのIL-2を含む培地と交換してください。 Grex40では、ハーフメディアを吸引し、新鮮な培地と交換し、50 U / mLのIL-2を追加します。 </li><li> 7日目に、ステップ4.6のように繰り返したが、IL-15は、刺激の日にIL-2を使用しない。 </li></ol> 5。代表的な結果 GMP準拠のFDAの承認を受けた製造プロトコルの概略図を図1に描かれている。プロセスは約50日かかります。しかし、臍帯血は、ウイルス経験豊富なT細胞を欠いており、したがって、私たちはプライムナイーブT細胞をex vivoで移動する必要があります。ウイルス特異的T細胞が既存のメモリーT細胞を増殖生成する典型的な方法。そうするために、我々は、IL-12、IL-15、樹状細胞ならびにサイトカインIL-7を使用するウイルスの特異性を発生させるために必要。 3刺激した後、収量は100×6セルの上でなければなりません。十分な細胞が利用できない場合は、CTLの希望数が利用可能になるまで、追加の刺激を行うことができます。これらの細胞の大部分は、CD4 +およびCD8 + T細胞の混合物と、CD3 +である必要があります。 15％未満のNK細胞（CD3-/CD56 +）と1％未満のCD19 + B細胞（ 図2）があるはずです。 拡大CTLは、アデノウイルスから、CMV、ヘキソンとペントンから抗原pp65抗原を認識するだけでなく、EBV-LCLに発現している多数のEBV抗原があります。 ELISPOTアッセイで試験した場合、CTLが分泌しなければなら以上のIFN-？これらの抗原に反応して無関係な抗原（ 図3）より。 CTLはまた、ウイルスペプチドパルス目標などPHA芽を溶解する必要があります。 51 Cr放出アッセイでは、CTLはCMVpp65·パルスおよびアデノウイルス、LCLを溶解させる必要がありますヘキソンおよび/ペントンパルスしたターゲットが、無関係のペプチド（ 図4）をパルスしていないターゲットや。 <img src="/files/ftp_upload/3627/3627fig1.jpg" alt="図1。"/> 図1臍帯血から多ウイルス特異的CTLの生成 。臍帯血からCTL製造の全過程を模式的に示す。最初の臍帯血単核球を分画した臍帯血ユニットの20％画分から分離されています。 LCLの世代のために保存されている5×10 6個の細胞を除いて、全ての細胞は、その後、非接着細胞を回収し、凍結された時点で、1〜2時間、樹状細胞の培地に播種されています。 DCはその後、IL-4およびGM-CSFを含む供給のDCメディアです。培養5日後に、DCが熟成されており、免疫CMV抗原pp65抗原を含むアデノウイルスベクターを用いて形質導入。開始時に、これらのDCは、非付着細胞ならびにサイトカインIL-7、IL-12と結合されおよびIL-15。後続の刺激で、同じアデノウイルスベクターは、抗原提示細胞として使用され、EBV-LCLを形質導入するために使用されます。 IL-15は、その後、第2の刺激とIL-2で使用されています。 <img src="/files/ftp_upload/3627/3627fig2.jpg" alt="図2。"/> 図2に得られたT細胞の表現型 。リンパ球ゲートに含まれる生細胞の割合が示されている。 CTLは、CD3 +およびCD4 +またはCD8 +が、大部分はCD3-/CD56-とアールCD19-。 <img src="/files/ftp_upload/3627/3627fig3.jpg" alt="図3。"/> 図3：T細胞の機能を提供します 。 T細胞の特異性は、IFN-γELISPOTにより試験した。 T細胞は、ヘキソン、ペントン、pp65を、そして無関係の抗原のIE-1のタンパク質全体にまたがる重複ペプチドをパルスした。単独でCTLが単独でメディアを示します。自家LCLは照射して1×10 5 /ウェルで添加した。セルCを形成し、平均点が示されている3ウェルのount。 <img src="/files/ftp_upload/3627/3627fig4.jpg" alt="図4。"/> 図4 CTLの細胞傷害活性 。ウイルス抗原を発現している標的を溶解する結果、CTLの能力を51 Crアッセイで試験した51 Cr標識自家PHA芽がLCLの自家Cr標識全体抗原にまたがる重複ペプチドをパルスしたり51したCTLの共培養を行った。 4時間後、ガンマリリースをガンマカウンターで計数した。

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著者らは、開示することは何もない。

<html> CBTの後にウイルス感染を制御することを目的と現在の戦略が効果的ですが、彼らはかなりの毒性に関連付けられている、高価なもので、その後の感染症に対する長期の保護を与えない。実際には、いくつかの抗ウイルス薬の使用は、他の方法で保護されるであろうウイルス特異的T細胞の増殖を制限することがあります。14もう一つのオプションは、ドナー由来のウイルス特異的T...

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td> RPMI 1640 </td><td>インビトロジェン</td><td> 21870-076 </td></tr><tr><td> DCのメディア</td><td> CellGenix </td><td> 20801-0500 </td></tr><tr><td> EHAA（クリックのミディアム） </td><td>アーバイン·サイエンティフィック</td><td> 9195 </td></tr><tr><td>ヒト血清</td><td>ジェミニバイオ製品</td><td> 100から110 </td></tr><tr><td>ガス透過性Cultureware 18 </td><td>ウィルソン - ウルフ</td><td> 80040S </td></tr><tr><td> IL-2の</td><td>カイロン（TCH薬局） </td><td></td></tr><tr><td> IL-12の</td><td> NCI / CTEP </td><td></td></tr><tr><td> IL-15の</td><td> CellGenix </td><td> 1013-050 </td></tr><tr><td> IL-7の</td><td>研究開発</td><td> AFL207 </td></tr><tr><td> IL-1βの</td><td>研究開発</td><td> AFL201 </td></tr><tr><td> IL-6の</td><td>セルGenix </td><td> 1004-050 </td></tr><tr><td> GM-CSF </td><td> TCH薬局</td><td></td></tr><tr><td> IL-4 </td><td>研究開発</td><td> <a href="http://www.rndsystems.com/pdf/AFL204.pdf" target="_blank">AFL204</a> </td></tr><tr><td> TNF-α </td><td>研究開発</td><td> AFL210 </td></tr><tr><td> Ad5f35pp65 </td><td> BCM CAGTベクトル製造施設</td><td></td></tr><tr><td>プラズマ転送が雌型ルアーアダプターを使用して設定</td><td>チャーターメディカル</td><td> 89から550-66j </td></tr><tr><td> Lymphoprep </td><td>ナイコメッド</td><td> 1114550 </td></tr></tbody></table>

expanding cytotoxic t lymphocytes from umbilical cord blood that target cytomegalovirus, epstein-barr virus, and adenovirus

幹細胞移植後のウイルス感染は、特にCBは感染から受信者を保護することができ、ウイルス経験豊富なT細胞のかなりの数字を含まない臍帯血（CB）移植（CBT）の後に、死の最も一般的な原因の一つである1 -我々や他のウイルス特異的CTL抗体陽性ドナーから生成され、受信者に注入が安全かつ保護的であることが示されている。5-8しかし、最近まで、ウイルス特異的T細胞が臍帯血から生成することができませんでした、おそらくのために4ウイルス特異的メモリーT細胞が存在しない。 良いナイーブT細胞の in vivoでのプライミング条件で模倣するための努力で、我々は、したがってT細胞特異性を駆動する、CB由来樹状細胞（DC）は免疫CMV抗原pp65抗原を含むアデノウイルスベクター（Ad5f35pp65）で形質導入に使用する方法を確立し開始時9。CMV、およびアデノウイルスに向かって、我々はこれらのmaを使うtured樹と同様に、CB由来T細胞サイトカインの存在下で、IL-7、IL-12およびIL-15 10我々は両方を発現するEBV形質転換B細胞、またはEBV-LCLを、使用される第二の刺激で潜伏と溶解EBV抗原。 Ad5f35pp65形質のEBV-LCLは第2の刺激でのIL-15の存在下でT細胞を刺激するために使用されます。後続の刺激は、EBV-LCLおよびIL-2 Ad5f35pp65形質を使用しています。 50x10 6、CB単核細胞から、我々は抗原刺激に応答して、抗原をパルス目標とリリースサイトカインを溶解さ150×10 6のウイルス特異的T細胞の上方に生成することができます。11これらの細胞は、使用して、GMPに準拠した方法で製造されただけ画し臍帯血ユニットの20％画分および臨床使用のために翻訳されています。

Watch this Scientific Journal Video about Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus at JoVE.com

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

ここでは、最初の良い製造基準（GMP）に準拠した臍帯血、主にナイーブT細胞のソースからウイルス特異的細胞傷害性Tリンパ球（CTL）を製造する方法を説明します。

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

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

expanding-cytotoxic-t-lymphocytes-from-umbilical-cord-blood-that

Research

JoVE Journal

Immunology and Infection

12.7K ビュー.  Baylor College of Medicine. ここでは、最初の良い製造基準（GMP）に準拠した臍帯血、主にナイーブT細胞のソースからウイルス特異的細胞傷害性Tリンパ球（CTL）を製造する方法を説明します。

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

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

ナチュラルキラー細胞傷害性アッセイにおけるHIV - 1感染プライマリCD4 + T細胞を標的細胞としての準備と使用

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

免疫・感染学

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as 
insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during 
the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the 
radionuclide Chromium 51 (51Cr) to label target cells. These targets are then exposed to effector cells and the release of 51Cr from target 
cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of 51Cr.
 As effector cells, we used an activated autoreactive clonal population of CD8+ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with 51Cr labeled target NIT cells for 16 hours, release of 51Cr was recorded to calculate specific lysis 
Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37&deg;C, with 5% CO2, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.
Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.

Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.

傷害性Tリンパ球によって媒介ベータ細胞死を評価するための方法

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

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 ...

Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid cell lines (LCL) in vitro. Since LCL are latently infected with EBV, they provide a model system to investigate EBV latency and virus-driven B cell proliferation and tumorigenesis1. LCL have been used to present antigens in a variety of immunologic assays2, 3. In addition, LCL can be used to generate human monoclonal antibodies4, 5 and provide a potentially unlimited source when access to primary biologic materials is limited6, 7.
A variety of methods have been described to generate LCL. Earlier methods have included the use of mitogens such as phytohemagglutinin, lipopolysaccharide8, and pokeweed mitogen9 to increase the efficiency of EBV-mediated immortalization. More recently, others have used immunosuppressive agents such as cyclosporin A to inhibit T cell-mediated killing of infected B cells7, 10-12.
The considerable length of time from EBV infection to establishment of cell lines drives the requirement for quicker and more reliable methods for EBV-driven B cell growth transformation. Using a combination of high titer EBV and an immunosuppressive agent, we are able to consistently infect, transform, and generate LCL from B cells in peripheral blood. This method uses a small amount of peripheral blood mononuclear cells that are infected in vitroclusters of cells can be demonstrated. The presence of CD23 with EBV in the presence of FK506, a T cell immunosuppressant. Traditionally, outgrowth of proliferating B cells is monitored by visualization of microscopic clusters of cells about a week after infection with EBV. Clumps of LCL can be seen by the naked eye after several weeks. We describe an assay to determine early if EBV-mediated growth transformation is successful even before microscopic clusters of cells can be demonstrated. The presence of CD23hiCD58+ cells observed as early as three days post-infection indicates a successful outcome.

We describe a method for generating transformed B cell lines using Epstein-Barr virus. We also illustrate a novel assay that can identify B cells destined to undergo transformation as early as three days after infection.

Epstein - Barrウイルスの成長形質転換リンパ芽球様細胞株の樹立

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid ...

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

の臨床応用<em&gt;眠れる森の美女</em遺伝的に末梢及び臍帯血由来のT細胞を変更する&gt;と人工抗原提示細胞

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

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

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

臍帯血から前駆B細胞サブセットの分離

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

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

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

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

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

離とナイーブCD4 Tリンパ球のTh17分化

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

Development of an IFN-&#947; ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, antiherpetic prophylaxis is administrated systematically to pediatric UCBT recipients to prevent complications associated with VZV infection, but there is no strong, evidence based consensus that defines its optimal duration. Because T cell mediated immunity is responsible for the control of VZV infection, assessing the reconstitution of VZV specific T cell responses following UCBT could provide indications as to whether prophylaxis should be maintained or can be discontinued. To this end, a VZV specific gamma interferon (IFN-&#947;) enzyme-linked immunospot (ELISpot) assay was developed to characterize IFN-&#947; production by T lymphocytes in response to in vitro stimulation with irradiated live attenuated VZV vaccine. This assay provides a rapid, reproducible and sensitive measurement of VZV specific cell mediated immunity suitable for monitoring the reconstitution of VZV specific immunity in a clinical setting and assessing immune responsiveness to VZV antigens. &#160;

Development of an IFN-&amp;#947; ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

Novel generations of functional assays such as gamma interferon (IFN-&#947;) ELISpot, which detect cytokine production at the single cell level and provide both quantitative and qualitative characterization of T cell responses can be used to assess cell-mediated immune responses directed against varicella zoster virus (VZV).

臍帯血移植後の水痘帯状疱疹ウイルス特異的細胞性免疫を評価するために、IFN-γELISPOTアッセイの開発

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, ...

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 ...

Radial Mobility and Cytotoxic Function of Retroviral Replicating Vector Transduced, Non-adherent Alloresponsive T Lymphocytes

We report a novel adaptation of the Radial Monolayer Cell Migration assay, first reported to measure the radial migration of adherent tumor cells on extracellular matrix proteins, for measuring the motility of fluorescently-labeled, non-adherent human or murine effector immune cells. This technique employs a stainless steel manifold and 10-well Teflon slide to focally deposit non-adherent T cells into wells prepared with either confluent tumor cell monolayers or extracellular matrix proteins. Light and/or multi-channel fluorescence microscopy is used to track the movement and behavior of the effector cells over time. Fluorescent dyes and/or viral vectors that code for fluorescent transgenes are used to differentially label the cell types for imaging. This method is distinct from similar-type in vitro assays that track horizontal or vertical migration/invasion utilizing slide chambers, agar or transwell plates. The assay allows detailed imaging data to be collected with different cell types distinguished by specific fluorescent markers; even specific subpopulations of cells (i.e., transduced/nontransduced) can be monitored. Surface intensity fluorescence plots are generated using specific fluorescence channels that correspond to the migrating cell type. This allows for better visualization of the non-adherent immune cell mobility at specific times. It is possible to gather evidence of other effector cell functions, such as cytotoxicity or transfer of viral vectors from effector to target cells, as well. Thus, the method allows researchers to microscopically document cell-to-cell interactions of differentially-labeled, non-adherent with adherent cells of various types. Such information may be especially relevant in the assessment of biologically-manipulated or activated immune cell types, where visual proof of functionality is desired with tumor target cells before their use for cancer therapy.

We describe a protocol to monitor radial mobility of non-adherent immune cells in vitro using a cell sedimentation manifold/slide apparatus. Cell migration is tracked on monolayers of tumor cells or on extracellular matrix proteins. Examination by light and fluorescence microscopy allows for observation of cell mobility and cytotoxic functionality.

ラジアルモビリティおよびレトロウイルス複製ベクター形質導入、非付着Alloresponsive Tリンパ球の細胞傷害機能

We report a novel adaptation of the Radial Monolayer Cell Migration assay, first reported to measure the radial migration of adherent tumor cells on ...