著者らは、有用な議論をコロンビア大学で微生物学免疫学教室から博士ボリスReizisに感謝したい。我々はまた、フローサイトメトリーおよびビデオ編集の支援のための陸黄との技術援助のために獣医学部の大学、コーネル大学で微生物学免疫学専攻から博士マーガレットバイノー、博士ロッドマンゲッチェルとDeequa Mahamedに感謝したい。 - / -マウス著者らはまた、p53を繁殖し、遺伝子型決定のためにワイス研究所からその重要な原稿や映像制作の見直し、そしてステファニーYazinskiためデュアメルとワイスの研究室のメンバーに感謝したい。動物実験は、コーネル大学動物実験用の委員会が定めるガイドラインおよび規則に従って行った。この研究は米国農務省、協同組合の国家研究、教育、および拡張サービス、動物の健康と疾病の研究プログラム（GEDおよびRSWに）とNIHの助成金R03 HD058220とR01CA108773（RSWまで）で提供される資金によって部分的にサポートされていました。ビデオはデュアメル＆ヴァイス研究所から担当者が社内の設備を用いて作製した。

1。腫瘍の解剖<ol><li>腫瘍切開の場合は、きれいなペーパータオルや使い捨て外科用ドレープは、解剖のパッド上に配置され、70％エタノールを噴霧。 </li><li>このプロトコルのためのマウスは、定期的にCO 2で安楽死されています。解剖パッドで安楽死させたマウスを重ね、エタノールで動物の両面をスプレー。マウスは足を通して挿入し、70％エタノールで十分に噴霧し、4つ以上のプッシュピンを使用して解剖のパッド上に固定されています。 </li><li>正中線上の皮膚が下層にある筋肉を回避し、体腔内に浸透しながら、滅菌済みのハサミを使って、恥骨から頭のベースにカットされます。 </li><li>内臓を損傷しないように注意して滅菌済みのハサミとピンセットの新しいペアと腹腔を開きます。 </li><li>血管を傷つけることなく、腫瘍を明らかにするために胸郭を解剖し、偏向して胸腔を開きます。 </li><li>他の臓器から腫瘍を分離し、冷PBSに合計腫瘍（約最大1.5 × 0.8 × 0.8センチ）の約40〜50％の部分を転送するには、氷上で保存。 </li><li>腫瘍の残りの部分は、他のアッセイのために保存することができ、完全な剖検は、この時点で行うことができます。 </li></ol> 2。培養リンパ腫細胞<ol><li> 70％エタノールとバイオセーフティキャビネットへの転送で切除した腫瘍を含むチューブをスプレーしてください。 </li><li>無菌150ミリメートル2ペトリ皿に培地10mLを追加します。 </li><li>ピペットで穏やかな吸引により、シャーレに腫瘍の一部を譲渡する。 </li><li>斜めの面を外向きtwo滅菌18ゲージの針を曲げます。 </li><li>曲がった針を用いて腫瘍を取り除きます。 </li><li>渦巻料理は、優しくタップして、腫瘍の大部分を回避しながら、側に細胞懸濁液を収集するために斜めに置きます。 </li><li>腫瘍の大部分を回避しながら、50 mLコニカルチューブに細胞懸濁液と転送を吸引除去する。 </li><li>腫瘍の大部分を回避しながら、同じチューブに培地と転送10mLでプレートを洗浄してください。 </li><li>優しくリンパ球とストローマ細胞から成る細胞懸濁液を混合し、T - 75組織培養フラスコにT - 25組織培養フラスコ、10 mLに5 mLを転送する。優しくスワールフラスコを。 </li><li> 2.5mLの最終容量（培養容器のいくつかのタイプを使用して異なる密度で細胞を培養T細胞ラインを確立するの尤度を最大）に2、4、10100、1000倍希釈液を作るために、残りの細胞懸濁液を使用してください。 </li><li> 6ウェルプレートの各ウェルにそれぞれ希釈のプレート2.0 mLを。優しくスワールプレートを。 </li><li>空気中の加湿CO 2雰囲気下で48時間細胞をインキュベートする。 </li></ol> 3。摂食リンパ腫細胞<ol><li>インキュベーションの48時間後、細胞の軽微な障害を持つ壁に沿ってT - 75フラスコに、T - 25フラスコおよび培地10mLに培地5mLを加える。 </li><li>細胞の軽微な障害を持つ壁に沿って6ウェルプレートの各ウェルに培地2 mlを加え。 </li><li>追加の72時間細胞をインキュベート</li></ol> 4。リンパ腫細胞を継代<ol><li>最初の一節<ol><li>オリジナルのフラスコやプレートを（最初の継代の間に約3x106 / mLの濃度で細胞密度を維持するのに成功確立を促進維持しながら、インキュベーション5日目の終わりに、細胞は、1:1の比率で継代されています連続的な細胞株）。 <ol class="lalpha"><li> T - 25フラスコは、培地5 mLを含む新しいフラスコに細胞懸濁液5 mLを転送する。オリジナルのフラスコに培地5mLを追加。 </li><li> T - 75フラスコは、培地10 mLを含む新しいフラスコに細胞懸濁液10mLを転送する。オリジナルのフラスコに培地10mLを追加。 </li><li> 6ウェルプレートの場合は、各ウェルに培地2mLを含む新しいプレートの対応するウェルに各ウェルから細胞懸濁液の2 mLを転送する。オリジナルのウェルの各ウェルに培地2 mlを加え。 </li></ol></li><li>追加の72時間のすべてのフラスコやプレートをインキュベート我々の経験ではほとんどの細胞系は、これらの通路one文化（添付のストローマ細胞上にゆるく付着懸濁細胞凝集体の存在が確立された細胞株の早期の指示である）から確立されています。 </li><li>より少ない、またはまったく緩く接着懸濁細胞凝集体との文化のために、慎重に培地ごとに72時間の約50％を置き換えることにより、フラスコやプレートを供給オリジナルの培養液から細胞を削除しないように、培養容器は、地位、上清培地の穏やかな吸引の前に重力により下部の細胞の沈殿をさせるには、少なくとも5分間静置されています。 </li></ol></li><li>樹立した細胞株の維持<ol><li>樹立した細胞株は、新しいフラスコやプレートに継代されています。 T - 25およびT - 75フラスコのため、オリジナルの5 mLを追加したり、培地5mLに通過oneサスペンション。 6ウェルプレートの場合は、T - 25フラスコに培地7mlの中にオリジナルまたは通過つのセルの懸濁液3 mLを加える。餌と新鮮な培養液で除去量に置き換えることによって、オリジナルまたは通路一つの文化を保持する。 </li><li>通路は、新しいT - 25フラスコに細胞株を確立した。細胞株は、18〜24時間の倍加時間を持っている文化は日常的に1 - 3x106 / mLの密度で維持し、3日毎に継代されています。 10mLに補うために新しい培地に、以前の継代培養の必要量を追加。フィードと独自の文化を保持。 </li><li>いくつかの継代後、非接着細胞は、自己接着性間質細胞なしで維持されると付着細胞のより小さい数は、各通路に引き継がれます。このポイントを超えて、文化は、T - 25フラスコを扱う非組織培養で維持することができます。 </li></ol></li></ol> 5。リンパ腫細胞株の凍結およびリカバリ<ol><li>細胞株は、20％FBS、10％のDMSOを含む新しく調製したRPMI 1640培地中で凍結されています。 </li><li> T - 25フラスコ（約1.5 × 10 6細胞/ mL）で48時間古い文化からの細胞を室温で5分間1500rpmで遠心分離されています。廃棄上清をした後、細胞を冷凍結培地2mlに再懸濁されている。各フラスコから細胞懸濁液の2つの1.0 mLのボリュームは、凍結バイアルに入れ、直ちに-80℃で凍結し、翌日、液体窒素に転送されます。 </li><li>細胞は急速解凍、そして新鮮な培地10mLに内容を再懸濁することにより、液体窒素貯蔵から回収されています。また、新たに解凍した細胞は、新鮮な培養液10mlに再懸濁する前に、DMSOを除去するために暖かい培地で一度洗浄されています。細胞は、その後の継代する前に少なくとも72時間培養する。 </li></ol> 6。代表的な結果 ATMは - / - T細胞ラインこのプロトコルを使用して開発し、指定さDWJ - ATM - 1は抗CD3 - FITC、抗CD4 - APCおよび抗CD8 - PEとフローサイトメトリー分析（図1）で染色することを特徴とした。 T -細胞の90％以上は、CD3、CD4およびCD8三重陽性であった。 <img src="/files/ftp_upload/2598/2598fig1.jpg" alt="図1" /> 図1は、このプロトコルを使用して開発されたT細胞ラインのキャラクタリゼーション。 / - -確立された櫃の細胞表面マーカーDWJ - ATM - 1、指定された細胞株は、抗CD3 - FITC、抗CD4 - APCで染色、抗CD8 - PEとフローサイトメトリー分析によって特徴づけた。簡単に言えば、1 × 10 6、新鮮な細胞は、（回転数1500rpm / 5分）冷PBSで一度洗浄し、4℃でインキュベートした抗体のカクテル（各抗体を1％BSAを含むPBSで1:200希釈で200μLの10分間。新鮮なまたは後の分析用に固定。固定細胞を4％（vol / vol）の最終濃度で加える中性緩衝ホルマリンで準備し、遠心分離により収集30分、（のために4℃でインキュベートしている間、細胞は、即時に取り調べることができる回転数1500rpm / 5分）と1％BSAを含むPBS 200μLで再懸濁し、1 × 10 4細胞の総数は、BD FACSキャリバーアナライザとCellQuestソフトウェア（BD Biosciences社、サンノゼ、CA）を用いて分析した。

<ol>
	<li>Barlow, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atm-deficient+mice:+a+paradigm+of+ataxia+telangiectasia.">Atm-deficient mice: a paradigm of ataxia telangiectasia.</a> Cell. 86, 159-171 (1996).</li><li>Elson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pleiotropic+defects+in+ataxia-telangiectasia+protein-deficient+mice.">Pleiotropic defects in ataxia-telangiectasia protein-deficient mice.</a> Proc Natl Acad Sci U S A. 93, 13084-13089 (1996).</li><li>Kuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+Atm-/-+thymic+lymphoma+cell+proliferation+in+vitro+and+in+vivo+by+dexamethasone.">Control of Atm-/- thymic lymphoma cell proliferation in vitro and in vivo by dexamethasone.</a> Cancer Chemother Pharmacol. 55, 203-212 (2005).</li><li>Browne, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+with+a+catalytic+antioxidant+corrects+the+neurobehavioral+defect+in+ataxia-telangiectasia+mice.">Treatment with a catalytic antioxidant corrects the neurobehavioral defect in ataxia-telangiectasia mice.</a> Free Radic Biol Med. 36, 938-942 (2004).</li><li>Jacks, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+spectrum+analysis+in+p53-mutant+mice.">Tumor spectrum analysis in p53-mutant mice.</a> Curr Biol. 4, 1-7 (1994).</li><li>Chervinsky, D. S., Lam, D. H., Zhao, X. F., Melman, M. P., Aplan, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+T+cell+leukemia+cell+lines+established+from+SCL/LMO1+double+transgenic+mice.">Development and characterization of T cell leukemia cell lines established from SCL/LMO1 double transgenic mice.</a> Leukemia. 15, 141-147 (2001).</li><li>Sharma, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch1+contributes+to+mouse+T-cell+leukemia+by+directly+inducing+the+expression+of+c-myc.">Notch1 contributes to mouse T-cell leukemia by directly inducing the expression of c-myc.</a> Mol Cell Biol. 26, 8022-8031 (2006).</li><li>Maser, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomally+unstable+mouse+tumours+have+genomic+alterations+similar+to+diverse+human+cancers.">Chromosomally unstable mouse tumours have genomic alterations similar to diverse human cancers.</a> Nature. 447, 966-971 (2007).</li></ol>

利害の衝突は宣言されません。

このプロトコルでは、我々は二つの異なるマウスの遺伝子型からマウスT細胞株を樹立するための詳細な手順を提供し、 - / - [FVB/N- 櫃tm1Led / J] と p53 - / -櫃 [129/S6- Trp53 tm1Tyj / J] 。 / - -三（5回の試行のうち）p53の- / -このプロトコルは、6の合計（7回のうち）ATMを使って、マウスのT細胞株は、?...

<ul><li>氷のバケット</li><li> 70％エタノール</li><li>アルミ箔と端子で覆われた発泡体のブロックで構成される解剖パッド</li><li>小さな滅菌済みのハサミとピンセットの二組</li><li>無菌150ミリメートル2シャーレ（サーモフィッシャーサイエンティフィック、ウォルサム、マサチューセッツ州、猫＃08-757-13。） </li><li>滅菌18ゲージの針</li><li> 50 mLコニカルチューブ（コーニング社、ニューヨーク州コーニング、猫＃430829） </li><li> 10％緩衝ホルマリン</li><li>組織培養フラスコ<ul><li> T - 25（TPP、Trasadingen、スイス、猫＃90025） </li><li> T - 75（TPP、Trasadingen、スイス、猫＃90075） </li></ul></li><li> 6ウェルプレート（コーニング社、ニューヨーク州コーニング、猫＃3506） </li><li>滅菌リン酸緩衝食塩水pH7.2の（PBS）を</li><li>培地<ul><li> ：以下を補った、25mMのHEPES、200 mMのL -グルタミン（猫＃12 - 115Fロンザ、ウォーカー、MD）を含むRPMI 1640 <ul><li> 10％の熱不活性化（56℃、30分）ウシ胎児血清（FBS; Hyclone、ローガン、ユタ州、猫＃SH30088.03） </li><li> 1％のペニシリンとストレプトマイシン（Mediatech、マナサス、バージニア州、猫＃30 - 002 - CI） </li><li> 1パーセント非必須アミノ酸（Mediatech、猫＃25 - 025 - CI） </li><li> 55 mmの2β-メルカプトエタノール（シグマ、セントルイス、MO、猫＃M752） </li></ul></li></ul></li><li>ジメチルスルホキシド（DMSO、カルビオケム、ラホーヤ、カリフォルニア州、猫＃317275） </li><li>抗体（eBioscience社、サンディエゴ、カリフォルニア州） <ul><li>抗CD3 - FITC（カタログ番号11-0031-85） </li><li>抗CD4 - APC（カタログ番号17-0041-83） </li><li>抗CD8 - PE（カタログ番号12-081-85） </li></ul></li><li>ウシ血清アルブミン（BSA、ライフテクノロジーズ、グランドアイランド、ニューヨーク;猫＃11018〜017） </li><li>コンカナバリンA（Sigmaカタログ＃C5275） </li><li> 1.8mlの凍結バイアル（Nunc社製、Roskilde、デンマーク、猫＃368632） </li></ul>

derivation of thymic lymphoma t-cell lines from atm-/- and p53-/- mice

確立された細胞株は、研究に実験動物の使用を減らすことができる重要な研究ツールです。特定のATMのような遺伝子改変マウスの系統、 - / - および p53 - / -は一貫して、人生の早い段階1,2の胸 ​​腺リンパ腫を開発し、その結果、マウスのT細胞株の導出のための信頼できるソースとして利用することができます。ここでは、前述のプロトコール1,3で説明されているインターロイキンを追加することなく、確立されたマウス胸腺リンパ腫のT細胞株の開発のための詳細なプロトコールを提示する。腫瘍が3〜6カ月歳のマウスから採取した、猫背の姿勢の観察に基づいて、目に見える腫瘍の最も初期に通知されたときに、貧しいグルーミングおよび感受性株の1,4の浪費、呼吸をこじつけ。 / - - [FVB/N- 櫃tm1Led / J] 2 と p53 - / - [129/S6- Trp53 tm1Tyj / J] 5匹のマウス我々は正常にこのプロトコルを使用していくつかのT -細胞株および ATMの近交系を確立している。我々はさらに確立されたT細胞集団の90％以上がCD3、CD4とCD8を発現していることを示しています。安定的に確立された細胞株と一致し、現在のプロトコルを使用して生成されるT細胞は、一年以上継代されています。

Watch this Scientific Journal Video about Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice at JoVE.com

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

このビデオでは、マウス胸腺リンパ腫細胞株を確立するためのプロトコルを示す。およびp53 - / - - 胸腺リンパ腫を有するマウスはこのプロトコルに従うことによって、我々は成功したいくつかのATM - /からT -細胞株を確立している。

から胸腺リンパ腫のT細胞株の導出 ATM - / -</supと p53の - / -</supマウス

Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified ...

derivation-of-thymic-lymphoma-t-cell-lines-from-atm-and-p53-mice

Research

JoVE Journal

Immunology and Infection

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

12.2K ビュー.  Cornell University. このビデオでは、マウス胸腺リンパ腫細胞株を確立するためのプロトコルを示す。およびp53 - / - - 胸腺リンパ腫を有するマウスはこのプロトコルに従うことによって、我々は成功したいくつかのATM - /からT -細胞株を確立している。

ビデオ: Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged antigen-specific T cell receptor (TCR) have been an indispensable tool to study T cell development and function. However, such TCRs are usually isolated from the relevant T cells after long-term culture often following repeated antigen stimulation, which unavoidably selects for T cells with high affinity. Random genomic integration of the TCR &alpha;- and &beta;-chain and expression from non-endogenous promoters can lead to variations in expression level and kinetics.
Epigenetic reprogramming via somatic cell nuclear transfer provides a tool to generate embryonic stem cells and mice from any cell of interest. Consequently, when SCNT is applied to T cells of known specificity, these genetic V(D)J rearrangements are transferred to the SCNT-embryonic stem cells (ESCs) and the mice derived from them, while epigenetic marks are reset. We have demonstrated that T cells with pre-defined specificities against Toxoplasma gondii can be used to generate mouse models that express the specific TCR from their endogenous loci, without experimentally introduced genetic modification. The relative ease and speed with which such transnuclear models can be obtained holds promise for the construction of other disease models.

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

We demonstrate here that epigenetic reprogramming via Somatic Cell Nuclear Transfer (SCNT) can be used as a tool to generate mouse models with pre-defined T cell receptor (TCR) specificities. These transnuclear mice express the corresponding TCR from their endogenous locus under the control of the endogenous promoter.

に対する事前定義されたT細胞受容体の特異性をもつTransnuclearマウストキソプラズマ SCNTを介して取得

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged ...

免疫・感染学

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

フローサイトメトリーによる胸腺正と負の選択の検討

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

マウスにおける同種のCD4 T細胞応答を研究する尾皮膚の移植

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

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 ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&amp;#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

マウスナイーブCD4<sup&gt; +</sup&gt; T細胞の単離と<em&gt;インビトロ</em&gt; T細胞サブセットへの分化

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCR&#945; and TCR&#946; chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR.

We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice.

骨髄前駆細胞のレトロウイルス形質導入は、T細胞受容体Retrogenicマウスを生成するために、

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed feeder cells, purified NK or T cells with as much as 10 mL of blood, or a high-dose of IL-2. This study presents a new method with a powerful and simple strategy to establish NK and T cell lines by culturing the peripheral blood mononuclear cells (PBMC) with the addition of recombinant human IL-2 (rhIL-2), and uses as little as 2 mL of whole blood. The cells can proliferate quickly in two weeks and be maintained for more than 3 months. With this method, 7 NK or T cell lines have been established with a high success rate. This method is simple, reliable, and applicable to establishing cell lines from more cases of CAEBV or NK/T cell lymphoma.

A simple method for obtaining NK and T cell clones from CAEBV patients was developed with high efficiency, a small amount of peripheral blood, and a low-dose of IL-2.

NK と慢性的なアクティブなエプスタイン ‐ バーウイルス感染症から T 細胞株を確立する効率的かつ簡単な方法

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

ジカ ウイルス特異 Immunoprivileged 臓器感染 Ifnar1-/-マウスの T 細胞応答の評価

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...

Gene Knock-in by CRISPR/Cas9 and Cell Sorting in Macrophage and T Cell Lines

Functional genomics studies of the immune system require genetic manipulations that involve both deletion of target genes and addition of elements to proteins of interest. Identification of gene functions in cell line models is important for gene discovery and exploration of cell-intrinsic mechanisms. However, genetic manipulations of immune cells such as T cells and macrophage cell lines using CRISPR/Cas9-mediated knock-in are difficult because of the low transfection efficiency of these cells, especially in a quiescent state. To modify genes in immune cells, drug-resistance selection and viral vectors are typically used to enrich for cells expressing the CRIPSR/Cas9 system, which inevitably results in undesirable intervention of the cells. In a previous study, we designed dual fluorescent reporters coupled to CRISPR/Cas9 that were transiently expressed after electroporation. This technical solution leads to rapid gene deletion in immune cells; however, gene knock-in in immune cells such as T cells and macrophages without the use of drug-resistance selection or viral vectors is even more challenging. In this article, we show that by using cell sorting to aid selection of cells transiently expressing CRISPR/Cas9 constructs targeting the Rosa26 locus in combination with a donor plasmid, gene knock-in can be achieved in both T cells and macrophages without drug-resistance enrichment. As an example, we show how to express human ACE2, a receptor of SARS-Cov-2, which is responsible for the current Covid-19 pandemic, in RAW264.7 macrophages by performing knock-in experiments. Such gene knock-in cells can be widely used for mechanistic studies.

This protocol uses fluorescent reporters and cell sorting to simplify knock-in experiments in macrophage and T cell lines. Two plasmids are used for these simplified knock-in experiments, namely a CRISPR/Cas9- and DsRed2-expressing plasmid and a homologous recombination donor plasmid expressing EBFP2, which is permanently integrated at the Rosa26 locus in immune cells.

CRISPR/Cas9による遺伝子ノックインとマクロファージおよびT細胞株における細胞分類

Functional genomics studies of the immune system require genetic manipulations that involve both deletion of target genes and addition of elements to ...

Isolation of Low Endotoxin Content Extracellular Vesicles Derived from Cancer Cell Lines

Extracellular vesicles (EVs) are a heterogeneous population of membrane vesicles released by cells in vitro and in vivo. Their omnipresence and significant role as carriers of biological information make them intriguing study objects, requiring reliable and repetitive protocols for their isolation. However, realizing their full potential is difficult as there are still many technical obstacles related to their research (like proper acquisition). This study presents a protocol for the isolation of small EVs (according to the MISEV 2018 nomenclature) from the culture supernatant of tumor cell lines based on differential centrifugation. The protocol includes guidelines on how to avoid contamination with endotoxins during the isolation of EVs and how to properly evaluate them. Endotoxin contamination of EVs can significantly hinder subsequent experiments or even mask their true biological effects. On the other hand, the overlooked presence of endotoxins may lead to incorrect conclusions. This is of particular importance when referring to cells of the immune system, including monocytes, because monocytes constitute a population that is especially sensitive to endotoxin residues. Therefore, it is highly recommended to screen EVs for endotoxin contamination, especially when working with endotoxin-sensitive cells such as monocytes, macrophages, myeloid-derived suppressor cells, or dendritic cells.

The proposed protocol includes guidelines on how to avoid contamination with endotoxin during the isolation of extracellular vesicles from cell culture supernatants, and how to properly evaluate them.

から胸腺リンパ腫のT細胞株の導出 ATM - / -と p53の - / -マウス

から胸腺リンパ腫のT細胞株の導出 ATM^{- / -と p53の^{- / -マウス}}