著者らは、このプロトコルの開発で助けジェンキンスラボのアンドレ漢とローレンス·イェン技術上の支援のため、メンバーに感謝したいと思います。

1。リンパ組織からの細胞の単離<ol><li>の小さな正方形を含む60mmの培養皿に、氷冷cEHAA（EHAA +10％FBS、ペニシリン/ストレプトマイシン、ゲンタマイシン、2mM L-グルタミン、55 mM 2 - メルカプトエタノール）または他の同等のT細胞培地1mlを追加氷の上に100μmのナイロンメッシュと場所。 </li><li>マウスを安楽死させる。 </li><li>脾臓、できるだけ多くの簡単にアクセスできるリンパ節を切除する。これらは少なくとも鼠径部、腋窩、上腕、頸部、および腸間膜リンパ節を含める必要があります。培養皿にナイロンメッシュの上に置きます。 </li><li>ナイロンメッシュ上のリンパ組織はリンパ球を解放するために、閉じた1.5 mlのマイクロチューブのフラットトップを使って、 優しくマッシュ。懸濁液中に細胞を動作するように上下cEHAAとピペットの別の1ミリリットルを追加します。 15 mlのポリプロピレン製遠心分離管の上に配置されたナイロンメッシュの別の部分を介して細胞を移す。氷冷cEHAA、POの別の1ミリリットルと皿とメッシュをすすぐ同じチューブにボリュームをoling。 4ミリリットルの最終細胞懸濁液の容積を達成するために繰り返します。 </li><li> 15ミリリットルの最終容量に冷たいソーター酸緩衝液（PBS + 2％FBS、0.05％アジ化ナトリウム）を追加し、300×gで5分間遠心分離管を、4℃ </li><li>慎重に液体が滴がチューブの両側に残っていないことを確認し、上清を吸引除去する。約2倍のペレット自体のそれに等しい最終容量へのFcブロック（ソーターバッファ+ 2.4G2抗体）で細胞ペレットを再懸濁します。例えば、ナイーブマウスの脾臓およびリンパ節は通常、約100μlの細胞ペレットを生成します。この場合は、200μlにボリュームを持って来るためのFcブロックの100μlを添加する。細胞凝集度が大きいが発生した場合は、ピペットチップを用いて、この時点で細胞塊を除去します。 </li></ol> 2。四量体染色<ol><li> 10 NM（または経験的に最適化された濃度）の最終濃度にPEまたはAPC標識pMHC複合テトラマーを追加します。 </li><li>ミックスとインキュベーション室温で1時間TE（または経験的に最適化された時間と温度）。 </li><li> 15ミリリットルの容積に冷たいソーター·バッファを追加し、300×gで、4℃で5分間、チューブを遠心氷の上または°C今から4でサンプルを保管してください。 </li><li>慎重に液体が滴がチューブの両側に残っていないことを確認し、上清を吸引除去する。 200μlの最終容量にソーター·バッファ内の細胞ペレットを再懸濁します。二量体の濃縮​​は、150μlの最終容量に再懸濁します。 </li></ol> 3。磁気濃縮<ol><li>ミルテニー抗PEまたは抗APCマイクロビーズの50μlを添加する。二量体の濃縮​​のために、両方の50μlを添加する。 </li><li> 4で混ぜ合わせ、20分間インキュベート℃、 </li><li> 15ミリリットルの容積に冷たいソーター·バッファを追加し、300×gで、4℃で5分間、チューブを遠心</li><li>待っている間、またはMidiMACS QuadroMACS磁石にミルテニーLSの磁気カラムを設定します。上の開いている15mlのポリプロピレン遠心管を置き列の真下ラック。 </li><li>それは15 mlのチューブに排出できるように、列の先頭にソーター緩衝液3mlを加える。 </li><li>列の上に100μmのナイロンメッシュ正方形を置きます。 </li><li>細胞が回転し終わったら、慎重に上清を吸引し、ソーター緩衝液3mlでペレットを再懸濁します。 </li><li>カラムの上にナイロンメッシュを介して細胞懸濁液を移す。 </li><li>細胞懸濁液が完全にカラムに排水されている場合、プロセス内でメッシュをすすぎ、ソータバッファの別の3mlでオリジナルのチューブを洗浄し、カラムにナイロンメッシュを介してバッファを転送します。ナイロンメッシュを破棄します。 </li><li>バッファが完全にカラムに排水している場合、その列にソーター·バッファの別の3ミリリットルを追加します。 </li><li> 3×3 mlの洗浄の合計に対して、ステップ10を繰り返します。 </li><li>新しい15 mlのポリプロピレン製遠心管を介して磁石と場所からカラムを取り外します。 </li><li> tにソーター緩衝液5mlを追加彼列。 </li><li>すぐに列の上部にプランジャーを挿入し、一つの連続した​​動きで、チューブにカラムOUTバッファを強制的に、プランジャーのすべての方法を下に押します。 </li><li> 300×gで5分間溶出結合画分およびフロースルー非結合画分、4℃を含むチューブを遠心</li><li>慎重に液体が滴がチューブの両側に残っていないことを確認しながら、結合画分から上清を吸引除去する。正確に95μlの最終容量にソーター·バッファ内の細胞ペレットを再懸濁します。 2ミリリットルの最終容量に非結合画分を吸引除去し、再懸濁します。 </li></ol> 4。フローサイトメトリー<ol><li>各画分については、5μlを削除し、5ミリリットルのFACS管で計数ビーズを200μl（ソーター·バッファ内の20万個/ mlの濃度に調整）に追加します。 4で取っておく°後の分析のためのC。時間を節約するために、ビーズは、実験開始前にラベルしたチューブにあらかじめ等分することができます。 </li><li>午前の準備ASTERは、セル（ 表1）の表面マーカーを染色する抗体のミックス。時間を節約するために、これは実験の開始前に行うことができます。 </li><li>結合分画は、チューブ内の細胞の約90μlに直接抗体カクテルの用量を追加します。非結合画分の分析が必要な場合は、5ミリリットルのFACSチューブに細胞の90μlを転送し、抗体カクテルの用量を追加します。 </li><li>フローサイトメーター用の単一色補正のコントロールパネルを設定します。使用する各蛍光色素の場合は、対応する蛍光色素に結合した抗CD4抗体の1μlを5ミリリットルのFACSチューブ内に残った非結合分画細胞の50μlを混合します。同様に染色されていないコントロールを設定することを忘れないでください。 </li><li>ボルテックスし、4℃で30分間、すべてのサンプルをインキュベート℃に</li><li> 300×gで5分間、4℃で各チューブと遠心分離機にソーター緩衝液5mlを追加</li><li>結合画分については、慎重に上清を吸引し、ソーターbufの200μlの細胞を再懸濁FER。 1.2ミリリットルのFACSマイクロチューブに細胞を移します。同じマイクロチューブにソーター·バッファーおよびプールの別の200μlで管を洗浄します。細胞塊が明らかである場合には、50μmのフィルターを介して細胞を渡す。 </li><li>非結合画分と補償コントロール、またはデカントで上澄みを再懸濁し、2mlのソーターバッファを吸引するための。 </li><li>単一色補正コントロールを使用して、フローサイトメーターを設定します。記録する追加パラメータとして、側方散乱幅（SSC-W）を選択します。 </li><li> 図1および図2に示すように、CD4 +またはCD8 + T細胞を同定するために連続したインクルージョンゲートのシーケンスを使用して染色したサンプルを分析します。結合画分については、最大2,000,000の合計の最大イベントにできるだけ多くの細胞を収集しています。非結合分画、1,000,000合計イベントを収集します。買収レートを1秒あたりのイベントで、または3000以下に保つ。 </li><li>同じマシンの設定を使用して、数えるビーズサンプルを分析する。万合計イベントを収集します&lt;/ LI&gt; <li> FCSのファイルなど、すべてのデータを保存します。 </li></ol> 5。データ解析<ol><li> FlowJoソフトウェアを使用してカウントビーズサンプル用のFCSデータファイルを分析します。前方にプロットFITCによって散乱および（ 図1および図2を参照）をカウントするビーズをaroungゲートを設定します。各試料で検出されたビーズをカウントの合計数を決定する。採取した細胞イベントの数を決定するために収集されたイベントの合計数から減算します。 </li><li>ボックス1で概説方程式を用いて、各サンプル中のすべてのセルの総数を計算します。 </li><li>ステンドバインドおよび非バインド分数サンプル用のFCSデータファイルを分析します。リンパ+サイド散布幅LO、ダンプ、CD3 +、CD4 +またはCD8 +、テトラマー、図1および図2に示すように、各試料中の+ T細胞。を識別するために、連続的なインクルージョンゲートのシーケンスを設定</li><li> CD4を定義するために使用される包接ゲートの各々の周波数によって、サンプル中の細胞の総数を掛け+テトラマー+またはCD8 +テトラマー+細胞エピトープ特異的T細胞（ボックス1）の合計数を計算する。 </li></ol>

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特別な利害関係は宣言されません。

このプロトコルによって提示pMHC複合テトラマー基づく細胞濃縮法は、内因性のT細胞レパートリーからエピトープ特異的T細胞を研究するための強力なツールです。 pMHC複合テトラマーの使用は、同族のpMHC複合リガンドと結合するTCRの能力に直接基づくエピトープ特異的T細胞の検出を可能にします。濃縮は、抗原特異的T細胞の非常にまれな集団が彼らの遺伝子構造または前駆周波数の任意の?...

<table border="1"&gt;<tbody&gt;<tr&gt;<td<strong&gt;試薬</strong</td&gt;<td<strong&gt;ベンダー</strong</td&gt;<td<strong&gt;カタログ番号</strong</td&gt;</tr&gt;<tr&gt;<td&gt; PEまたはAPCの共役pMHC複合テトラマー（またはマルチマー）</td&gt;<td&gt; NIHのテトラマーコアから得られた、研究者によって作られた、または商業的供給源から購入</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;アンチPE結合磁性マイクロビーズ</td&gt;<td&gt;ミルテニー</td&gt;<td&gt; 130-048-801</td&gt;</tr&gt;<tr&gt;<td&gt;アンチAPC共役磁性マイクロビーズ</td&gt;<td&gt;ミルテニー</td&gt;<td&gt; 130-090-855</td&gt;</tr&gt;<tr&gt;<td&gt; LS磁気列</td&gt;<td&gt;ミルテニー</td&gt;<td&gt; 130-042-401</td&gt;</tr&gt;<tr&gt;<td&gt; MidiMACSまたはQuadroMACS磁石</td&gt;<td&gt;ミルテニー</td&gt;<td&gt; 130-042-302または130-090-976</td&gt;</tr&gt;<tr&gt;<td&gt;細胞計数ビーズ</td&gt;<td&gt;ライフテクノロジーズ</td&gt;<td&gt; PCB-100</td&gt;</tr&gt;</tbody&gt;</table

peptide:mhc tetramer-based enrichment of epitope-specific t cells

in vivo実験モデルでと適応免疫を勉強研究者のための基本的な必要性は、それらのT細胞抗原受容体（TCR）の特異性に基づいてT細胞を識別する能力である。 T細胞のバルク集団が特定の抗原およびエピトープ特異的T細胞をin vitro で刺激されている多くの間接的な方法が利用可能です、増殖、サイトカイン産生や活性化マーカー1の発現などの機能的応答の測定によって識別されます。しかし、これらの方法では、唯一の多くの可能な機能の一つを示すエピトープ特異的T細胞を同定し、そして、彼らは素朴な前駆周波数でエピトープ特異的T細胞を検出するのに十分な小文字は区別されません。人気のある選択肢は、TCRトランスジェニック養子移入モデルであるTCRトランスジェニックマウス由来モノクローナルT細胞はEASIできるエピトープ特異的T細胞の大前駆体細胞集団を作成するために組織適合ホストに播種するLyはコンジェニックマーカー抗体2,3の使用で追跡。パワフルながら、この方法では、4,5の単一エピトープに対して特異性を有するT細胞の非生理的な周波数に関連付けられている実験的な成果物に苦しんでいる。また、本システムは、ポリクローナル集団内のエピトープ特異的T細胞クローンの機能的な異質性を調査するために使用することはできません。 MHC（pMHC複合）錯体：適応免疫を研究するための理想的な方法は、単にその同族ペプチドに結合することにより、TCRの特異性を区別する方法を用いて内因性のT細胞レパートリーからのエピトープ特異的T細胞の直接検出を関与させるべきである。 pMHC複合テトラマーおよびフローサイトメトリーの使用は、この6を達成しますが、のみ、次の抗原誘発クローン増殖を発見したエピトープ特異的T細胞の高頻度の個体群の検出に限定されています。このプロトコルでは、pMHC複合テトラマーの使用を調整する方法を説明し、マグネチック細胞濃縮技術は、マウスリンパ組織3,7から超低周波エピトープ特異的T細胞の検出を可能にします。この手法では、1は、包括的に免疫応答の全ての段階で、マウスにおける内因性T細胞の全体のエピトープ特異的集団を追跡することができます。

図2は、以前に関連するペプチド+ CFAで免疫したマウスについての代表的なデータを示しながら、 図1は 、ナイーブマウスからpMHCIIテトラマー濃縮脾臓及びリンパ節試料の代表的なフローサイトメトリープロットを描いている。シリアル·ゲーティングは、CD4の分析+ T細胞集団からの自家蛍光およびその他の不要なイベントを削除します。 CD8 + T細胞集団?...

Watch this Scientific Journal Video about Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells at JoVE.com

Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

エピトープ特異的T細胞の低周波の集団を区別し、フローサイトメトリーによって、それらを分析するためのMHCテトラマーと磁性マイクロビーズ：このプロトコルは、ペプチドの使用について説明します。この方法は、から対象の内因性T細胞集団の直接調査を可能に<em生体内で&gt;</em&gt;実験システム。

ペプチドエピトープ特異的T細胞のMHCテトラマーベースの濃縮

A  basic necessity for researchers studying adaptive immunity with in vivo experimental models is an  ability to identify T cells based on their T cell ...

peptidemhc-tetramer-based-enrichment-of-epitope-specific-t-cells

Research

JoVE Journal

Immunology and Infection

17.8K ビュー.  Massachusetts General Hospital and Harvard Medical School. エピトープ特異的T細胞の低周波の集団を区別し、フローサイトメトリーによって、それらを分析するためのMHCテトラマーと磁性マイクロビーズ：このプロトコルは、ペプチドの使用について説明します。この方法は、から対象の内因性T細胞集団の直接調査を可能に実験システム。

ビデオ: Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

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

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

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

マウスT細胞におけるT細胞受容体のレトロウイルス導入

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

免疫・感染学

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&#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 ...

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies 1. CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines 2-7. However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies.
TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases 8-10. However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic 11-13, HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture 14-16. Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs.
Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.

Generation of T lymphocytes from induced pluripotent stem (iPS) cells gives an alternative approach of using embryonic stem cells for T cell-based immunotherapy. The method shows that by utilizing either in vitro or in vivo induction system, iPS cells are able to differentiate into both conventional and antigen-specific T lymphocytes.

Tリンパ球に向かって誘導多能性幹細胞の分化の指示

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

piggyBac Transposon System Modification of Primary Human T Cells

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

piggyBac Transposon System Modification of Primary Human T Cells

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

プライマリーヒトT細胞のpiggyBacトランスポゾンシステムの変更

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

Isolation of Double Negative &#945;&#946; T Cells from the Kidney

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in various immune responses. DN T cells are a unique immune cell type that has been implicated in regulating immune and autoimmune responses and tolerance to allotransplants1-6. DN T cells are, however, rare in peripheral blood and secondary lymphoid organs (spleen and lymph nodes), but are major residents of the normal kidney. Very little is known about their pathophysiologic function7 due to their paucity in the periphery. We recently described a comprehensive phenotypic and functional analysis of this population in the kidney8 in steady state and during ischemia reperfusion injury. Analysis of DN T cell function will be greatly enhanced by developing a protocol for their isolation from the kidney.
Here, we describe a novel protocol that allows isolation of highly pure ab CD4+ CD8+ T cells and DN T cells from the murine kidney. Briefly, we digest kidney tissue using collagenase and isolate kidney mononuclear cells (KMNC) by density gradient. This is followed by two steps to enrich hematopoietic T cells from 3% to 70% from KMNC. The first step consists of a positive selection of hematopoietic cells using a CD45+ isolation kit. In the second step, DN T cells are negatively isolated by removal of non-desired cells using CD4, CD8, and MHC class II monoclonal antibodies and CD1d &#945;-galcer tetramer. This strategy leads to a population of more than 90% pure DN T cells. Surface staining with the above mentioned antibodies followed by FACs analysis is used to confirm purity.

DNabT cells are rare among peripheral T cells; however, they are abundant in certain non-lymphoid tissues. Difficulty of isolating DN T cells from non-lymphoid tissue hinders their functional analysis despite increasing recognized pathophysiologic significance. We describe a novel protocol for isolation of highly purified DN T cells from murine kidney.

腎臓からの二重否定αβT細胞の分離

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

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

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

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

T細胞の導出<em&gt;体外</em&gt;マウス胚性幹細胞からの

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

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 ...

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-&#947;). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.

The goal of this protocol is to manufacture pathogen-specific clinical-grade T cells using a bench-top, automated, second generation cell enrichment device that incorporates a closed cytokine capture system and does not require dedicated staff or use of a GMP facility. The cytomegalovirus pp65-specific-T cells generated can be directly administered to patients.

サイトカインキャプチャシステムを用いた臨床応用のためのサイトメガロウイルス特異的T細胞の自動化された細胞濃縮

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Here a protocol is presented to measure bacterial capture by CD4+ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

T細胞は、樹状細胞からTransinfectionによって細菌をキャプチャ

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

表現型および機能解析のためのレア抗原特異的B細胞の検出および濃縮

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...