私は免疫学的アッセイやマウス操作に私を導入するためビアンカMOTHE、カルラOseroff、とマリー=フランスDelGuercioに感謝したいと思います。 GAスプリッタのラボでの作業は、NIHの助成金1 - RO1 - AI - 073558、GLRCE助成1 - U54 - AI - 057153、およびBARD US - 3829から06によって運営されている。

1。予防接種によってターゲット固有のエフェクターCTLを誘発する（0日） <ol><li>ターゲットシステム：開始する前に、特定のエフェクターを決定するとき。 in vivoで CTLアッセイにおける古典のこの例では、我々は特異的CD8 + T細胞を誘発するために精製されたペプチドの予防接種を使用します。ターゲットシステム：同ペプチドは特定のエフェクターを作成し、同系の標的細胞をパルスするために使用されます。また、あなたの特定のエフェクタと目標を得るために全体の病原体やタンパク質を使用することもできます。 </li><li>注射の無菌性を確保するためにバイオセーフティフードの作業面の内側を除染。最初の2 mL試験管に含まれているPBS /マウスに100μLの10％DMSOにペプチドを精製した50μgのを配置することにより、ペプチド免疫を準備します。次に、混合するための1ミリメートルビーズで、最大0.2 mLのマークに非常に冷たい不完全フロイントアジュバントを等量を追加し、。この点から、注入するまで氷上に免疫を保つ。パラフィルムでふたを固定します。 </li><li>速やかに混合後、氷上に戻って乳剤を配置、3分間エマルジョンを混合するために最大のRPMに設定ビーズビーターを使用してください。得られたエマルジョンは、一貫性のマヨネーズのようになります。エマルジョンは、任意の分離が明記されている場合、さらにミキシング、（最大週まで）時間が許せば℃で一晩4℃で座ってすることができます。 </li><li>どんなビーズをプルアップしないように注意して乳剤をプルアップするために1 mLの注射器と20ま​​たは20.5ゲージの針を使用してください（ビーズは針のゲージよりも大きくなるが、それでも知っておく必要があります）。 </li><li>尾の基部に皮下注射のための安全なマウス。我々は安全に優しく注射のためにマウスを固定化するように変更プラスチック50 mLコニカルチューブを使用してください。エマルジョンの0.2 mLでマウスを注入する。 </li></ol> 2。収穫標的細胞（8日目） <ol><li>あなたの標的細胞に対してナイーブ同系マウスが必要になります。 AAALACの基準にしたがってナイーブマウスを安楽死させる。 </li><li>マウスの毛や他の外部の汚染物質からの汚染の可能性を最小限に抑えるために70％エタノールを噴霧することにより、マウスを消毒する。 </li><li>解剖鋏を使用すると、マウスの左側に皮膚を通して小さなスニップを行います。腹膜嚢と内臓を明らかに皮膚のフラップを上に引き出します。 </li><li>腹膜を介して切り取り、基盤となる臓器のいずれかを妥協しないように注意している。脾臓は、表面に近く、色の肝臓（暗赤色）に似ていますが、小さいと形でより多くの腎臓のBeanです。感染マウスは、一般的に感染していないコントロールよりも顕著に大きい脾臓を示しています。 </li><li>慎重になるよう最大の細胞数を回復するために複数の断片に脾臓を壊さないように穏やかなものに注意しながら、脾臓を引き出します。すぐに氷上に準備したメディアの10 MLSを含む15 mLコニカルに脾臓を置きます。残りのマウスに進みます。 </li><li>ガラスの組織グラインダーに15 mLコニカルチューブ（メディアおよび脾臓）の内容を注ぐ。アッパーアームの強度を十分に使用して脾臓を挽く。すべての組織がその色を失ったときは、結合組織の外のすべてのセルを回復していると仮定してよい。 </li><li> 50 mLコニカルチューブに70μmのセルのフィルターを通してグラインダーの内容を注ぐ。新鮮な培地でグラインダーをすすぎ、セルストレーナーを通して注ぎます。 </li><li> 4℃遠心° C、7分1300 RPM。上清を捨て。 </li><li> 7分37℃で調製した溶解試薬とインキュベート10 MLSに再懸濁します。 </li><li>上記と同じ、新鮮な培地と遠心で50 MLSまで追加。 </li><li> （洗浄ステップ）上記と同じ、50mlの新鮮な培地と遠心分離機で懸濁します。 *注：純粋な標的細胞の集団が、レシピエントマウスへの伝達のために望まれている場合は、この時点で、特定の細胞型（すなわちマクロファージ）のための磁気細胞分離を行うことができます。また、この時点では、セル固有の殺害にその治療の影響を研究するために、文化に実験的治療を導入することもできます。 </li><li>プレート治療を96ウェル丸底の組織培養で100μLに1ウェルあたりの細胞とプレート1 × 10 7を数える。 *これは、細胞/ウェルの高濃度ですが、アッセイは迅速なものであるため、我々はこの濃度で細胞の生存率には損失を見たことがありません。 </li></ol> 3。標的細胞の治療とラベリング<ol><li>パルス適切な細胞と病原体の特定のペプチドまたは無関係なペプチドの制御と複製すると、1μg/ mlのペプチドを追加。 1.5時間4℃でインキュベートする。最後の30分間37℃のインキュベーターに移動​​します。この時間の間にCFSEの試薬を準備する。 </li><li>製造業者の仕様に従ってCFSEを準備し、0.5〜25μMの間に仕事上の濃度に希釈する。 &quot;高&quot;濃度CFSEよりも低い&quot;低&quot;濃度が10倍にします。たとえば、[低]に10 MLS PBSに0.5μLCFSEを追加し、[高]のために、10mlのPBSに5μLCFSEを追加。 37〜暖かい準備CFSE ° Cラベリングの前に。 </li><li>インキュベーターとpからの細胞のプレートを取り外します室温での高速遠心分離機、7分1300 RPMでエレト。上清を除去するフリック。 </li><li> 37℃で15分間対応するウェルでインキュベートするための適切な試薬200μLを加えることによってどちらかの[低]または[高] CFSE℃で再懸濁し、細胞</li><li> 7分間、室温、1300 RPMのインキュベーターと再びペレットからの細胞のプレートを取り外します。上清を除去するフリック。 </li><li> 37℃で30分間、200μL/ウェル暖め新鮮な培地とインキュベートで再懸濁し、細胞を℃に</li><li> 7分間、室温、1300 RPMでのインキュベーターとペレットからの細胞のプレートを取り外します。上清を除去するフリック。 </li><li> 100μL/ウェルのPBSに再懸濁し、細胞。 </li><li>養子移入のために、うまくつのマウスごとにも[高]標識された細胞への[低]というラベルの付いたセルを追加します。注入の結果は、0.2 MLSの50％具体的な目標と50％無関係のセルでなければなりません。 </li><li> （図1を参照）データの解析中に、フローサイトメーターで実行されているため、非転送コントロールとして動作するいくつかの標識された細胞を取っておくことを忘れないでください。 </li></ol> 4。養子免疫伝達<ol><li>以前にペプチド免疫したマウスを麻酔（このプロトコールの一部＃1から）AAALACの基準によると。 </li><li>レシピエントマウスに眼窩経路を介して標識した標的細胞の0.2 MLSを注入する。 </li><li>レシピエントマウスを安楽死する前に6時間または他の所定の最適な時間を待つ。 </li></ol> 5。収穫標識細胞 *注：5.11を介してステップ5.1ステップ2.11を介して2.1と同じである<ol start="12"><li> 96ウェル丸底の組織培養処理されたプレートに100μLの1 × 1ウェルあたり10 6で細胞とプレートを数える。 </li><li>すべてのウェルに1％パラホルムアルデヒドの最終濃度に100μLの2％のパラホルムアルデヒドを添加することにより回収し、非導入細胞を修正。 </li></ol> 6。フローサイトメトリーおよびデータ解析<ol><li>フローサイトメーターでサンプルを実行し、データを分析する。 </li></ol> 7。代表的な結果結果として得られるヒストグラム上で逓減エピトープの特定のピークは、細胞特異的殺（図1）の指標です。細胞特異的溶解の実際の値を決定するには、次の式を（表1に適用される）を使用します。 比=関連性のない割合：エピトープ特異的割合（[低]ピーク：[高]ピーク）、 パーセント特異的溶解= [1 - （非転送制御率/実験比）] × 100 <img src="/files/ftp_upload/2250/2250fig1.jpg" alt="図1" /> 図1。 BALB / cマウスの脾細胞を回収した。番号のCFSEのプロファイルは、ハイまたはローCFSEの表現型の細胞の割合を示している。 A）コントロールを非譲渡。 [低] CFSEのピークは無関係のペプチドをパルスしたターゲットを表す一方、すべての[高] CFSEのピークは、エピトープの特定の標的細胞を表しています。 B）CFSE標識された細胞は、PBS免疫レシピエントマウス（コントロール）から回収。 C）二つの異なるペプチドの特定のレシピエントマウスから回収したCFSE標識された細胞は、減少したエピトープの特定のピークに注意してください。 <img src="/files/ftp_upload/2250/2250fig2.jpg" alt="図2" /> 図2。分析したデータのグラフィカル表現。3つのデータ点は、マウスの各グループ、PBS、ペプチドA、およびペプチドBで示すように<table border="1"><tbody><tr><td> サンプル </td><td> CFSE HIGH％のエピトープは、パルス </td><td> 無関係なCFSE LOW％ </td><td> 高：低比 </td><td> パーセントのエピトープ特異的殺害 </td></tr><tr><td> PBS </td><td> 42.8 </td><td> 57.2 </td><td> 1.34 </td><td> 16.97 </td></tr><tr><td> PBS </td><td> 43.1 </td><td> 56.9 </td><td> 1.32 </td><td> 15.94 </td></tr><tr><td> PBS </td><td> 44.3 </td><td> 55.7 </td><td> 1.26 </td><td> 11.74 </td></tr><tr><td>ペプチド</td><td> 39.7 </td><td> 60.3 </td><td> 1.52 </td><td> 32.56 </td></tr><tr><td>ペプチド</td><td> 38.6 </td><td> 61.4 </td><td> 1.59 </td><td> 35.61 </td></tr><tr><td>ペプチド</td><td> 40.6 </td><td> 59.4 </td><td> 1.46 </td><td> 29.99 </td></tr><tr><td>ペプチドB </td><td> 38.7 </td><td> 61.3 </td><td> 1.58 </td><td> 24.68 </td></tr><tr><td>ペプチドB </td><td> 38.5 </td><td> 61.5 </td><td> 1.60 </td><td> 25.32 </td></tr><tr><td>ペプチドB </td><td> 39.3 </td><td> 60.7 </td><td> 1.54 </td><td> 22.76 </td></tr><tr><td></td><td></td><td></td><td></td><td></td></tr><tr><td> PBS NTのコントロール</td><td> 47.4 </td><td> 52.6 </td&gt; <td> 1.11 </td><td></td></tr><tr><td>ペップNTのコントロール</td><td> 49.4 </td><td> 50.6 </td><td> 1.02 </td><td></td></tr><tr><td>ペップB NTのコントロール</td><td> 45.6 </td><td> 54.4 </td><td> 1.19 </td><td></td></tr></tbody></table> 表1。代表的なデータの分析。&quot;NT&quot;は、非転送です。

<ol>
	<li>Blattman, J. N., Wherry, E. J., Ha, S. J., van der Most, R. G., Ahmed, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+epitope+escape+on+PD-1+expression+and+CD8+T-cell+exhaustion+during+chronic+infection.">Impact of epitope escape on PD-1 expression and CD8 T-cell exhaustion during chronic infection.</a> J Virol. 83 (9), 4386-4394 (2009).</li><li>Jenkins, M. R., Tsun, A., Stinchcombe, J. C., Griffiths, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+strength+of+T+cell+receptor+signal+controls+the+polarization+of+cytotoxic+machinery+to+the+immunological+synapse.">The strength of T cell receptor signal controls the polarization of cytotoxic machinery to the immunological synapse.</a> Immunity. 31 (4), 621-631 (2009).</li><li>Ingulli, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracing+tolerance+and+immunity+in+vivo+by+CFSE-labeling+of+administered+cells.">Tracing tolerance and immunity in vivo by CFSE-labeling of administered cells.</a> Methods Mol Biol. 380, 365-376 (2007).</li><li>Durward, M. A., Harms, J., Magnani, D. M., Eskra, L., Splitter, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discordant+Brucella+melitensis+antigens+yield+cognate+CD8++T+cells+in+vivo.">Discordant Brucella melitensis antigens yield cognate CD8+ T cells in vivo.</a> Infect Immun. 78 (1), 168-176 (2010).</li><li>Lyons, A. B., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+lymphocyte+division+by+flow+cytometry.">Determination of lymphocyte division by flow cytometry.</a> J Immunol Methods. 171 (1), 131-137 (1994).</li><li>Weston, S. A., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+fluorescent+dyes+for+lymphocyte+migration+studies.+Analysis+by+flow+cytometry+and+fluorescence+microscopy.">New fluorescent dyes for lymphocyte migration studies. Analysis by flow cytometry and fluorescence microscopy.</a> J Immunol Methods. 133 (1), 87-97 (1990).</li><li>Akondy, 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=The+yellow+fever+virus+vaccine+induces+a+broad+and+polyfunctional+human+memory+CD8++T+cell+response.">The yellow fever virus vaccine induces a broad and polyfunctional human memory CD8+ T cell response.</a> J Immunol. 183 (12), 7919-7930 (2009).</li><li>Wallace, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+antigen-driven+responses+by+flow+cytometry:+monitoring+proliferation+by+dye+dilution.">Tracking antigen-driven responses by flow cytometry: monitoring proliferation by dye dilution.</a> Cytometry A. 73 (11), 1019-1034 (2008).</li><li>Labadi, A., Balogh, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+preferences+in+serosal+homing+and+distribution+of+peritoneal+B-cell+subsets+revealed+by+in+situ+CFSE+labeling.">Differential preferences in serosal homing and distribution of peritoneal B-cell subsets revealed by in situ CFSE labeling.</a> Int Immunol. 21 (9), 1047-1056 (2009).</li><li>Suffner, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+support+homeostatic+expansion+of+Foxp3++regulatory+T+cells+in+Foxp3.LuciDTR+mice.">Dendritic cells support homeostatic expansion of Foxp3+ regulatory T cells in Foxp3.LuciDTR mice.</a> J Immunol. 184 (4), 1810-1820 .</li></ol>

利害の衝突は宣言されません。動物実験は、ウィスコンシン大学マディソン校の動物実験使用委員会（IACUC）によって定められたガイドラインおよび規制に準拠して行った。

CFSEは子孫10,11の間で比較的均等に分割するので、このアッセイは、クローン性増殖を含む細胞の増殖能を調査するように変更することができます。また、CFSE標識と組み合わせることができる例の四量体および生物発光13あなたの仮説に応じてより適切な方法をトレースし、他のセルが、、ありますがそれは、細胞遊走6,12を調査するためにラベル付けCFSEを使用するこ?...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>1 mm glass beads</td>
<td>&nbsp;</td>
<td>Biospec Products</td>
<td>11079110</td>
<td>&nbsp;</td>
<tr>
<td>70 &mu;m cell strainer</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>352350</td>
<td>&nbsp;</td>
<tr>
<td>96-well plate</td>
<td>&nbsp;</td>
<td>Costar</td>
<td>3799</td>
<td>&nbsp;</td>
<tr>
<td>Adjulite Incomplete Freund's Adjuvant</td>
<td>&nbsp;</td>
<td>Pacific Immunology</td>
<td>n/a</td>
<td>&nbsp;</td>
<tr>
<td>DMSO</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D-5879</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>16000-077</td>
<td>&nbsp;</td>
<tr>
<td>FC 500 Flow Cytometer</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>n/a</td>
<td>&nbsp;</td>
<tr>
<td>Kontes glass tissue grinder</td>
<td>&nbsp;</td>
<td>Kontes</td>
<td>885300-0015</td>
<td>&nbsp;</td>
<tr>
<td>Mini Beadbeater</td>
<td>&nbsp;</td>
<td>Biospec Products</td>
<td>n/a</td>
<td>&nbsp;</td>
<tr>
<td>Needle</td>
<td>&nbsp;</td>
<td>B-D</td>
<td>305175</td>
<td>&nbsp;</td>
<tr>
<td>Parafilm "M"</td>
<td>&nbsp;</td>
<td>Pechiney Plastic Packaging</td>
<td>PM992</td>
<td>&nbsp;</td>
<tr>
<td>Paraformaldehyde</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>157-8</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>10010-023</td>
<td>&nbsp;</td>
<tr>
<td>PharmLyse lysing reagent</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>555899</td>
<td>&nbsp;</td>
<tr>
<td>RPMI 1640</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>11875-093</td>
<td>&nbsp;</td>
<tr>
<td>Syringe</td>
<td>&nbsp;</td>
<td>B-D</td>
<td>309602</td>
<td>&nbsp;</td>
<tr>
<td>Vybrant CFSE Kit</td>
<td>&nbsp;</td>
<td>Invitrogen (Molecular Probes)</td>
<td>V12883</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

antigen specific in vivo killing assay using cfse labeled target cells

カルボキシフルオレセインジアセテートスクシンイミジルエステル（CFSE）を容易かつ迅速に生体内での調査のための関心の細胞集団を標識するために使用することができます。この標識は、古典的な増殖と移動を研究するために使用されています。ここで紹介した方法では、我々は、エピトープの特定のCFSE標識した標的細胞を4-6の生存と殺害を見に養子移入した後にタイムラインを短縮している。その量は、誤解を招く可能性があるとしてCD8 + T細胞クローンの特定の殺害のレベルは、応答の質を示すことができます。特定のCD8 + T細胞はサイトカイン産生と7,8を殺すの低下と時間をかけて機能的に枯渇することができます。また、特定のCD8 + T細胞クローンは、異なるTCRの特異性9と同様に他人を殺すしないことがあります。効果的な細胞性免疫（CMI）の場合、抗原はT細胞の応答の適切な数字だけでなく、機能的に堅牢な応答性T細胞だけを作り出すことを識別する必要があります。ここでは、BALB / cマウスに2つのペプチド特異的T細胞クローンのパーセントの細胞特異的殺傷を評価する。

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Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

多くの感染症は、強力なCTL応答を誘発するが、時折、応答する細胞の量は、病原体の制御に相関しない<sup&gt; 1</sup&gt;。 CTLの質の1つの尺度は具体的に死滅させる能力です。<sup&gt; 2</sup&gt;。標的細胞のCFSE標識は、このCTL応答の品質を調査するために使用することができます<em&gt; in vivoで</em<sup&gt; 3,4</sup&gt;。

抗原特異的インビボでの</emCFSE標識した標的細胞を用いた>キリングアッセイ

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation.  ...

antigen-specific-in-vivo-killing-assay-using-cfse-labeled-target-cells

Research

JoVE Journal

Immunology and Infection

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

19.5K ビュー.  University of Wisconsin-Madison. 多くの感染症は、強力なCTL応答を誘発するが、時折、応答する細胞の量は、病原体の制御に相関しない 1。 CTLの質の1つの尺度は具体的に死滅させる能力です。 2。標的細胞のCFSE標識は、このCTL応答の品質を調査するために使用することができます in vivoで

ビデオ: Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Trans-vivo Delayed Type Hypersensitivity Assay for Antigen Specific Regulation

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host immune system has experienced in the recent past. DTH reactions are often divided into a sensitization phase, referring to the initial antigen experience, and a challenge phase, which usually follows several days after sensitization. The lack of a delayed-type hypersensitivity response to a recall Ag demonstrated by skin testing is often regarded as an evidence of anergy. The traditional DTH assay has been effectively used in diagnosing many microbial infections.
Despite sharing similar immune features such as lymphocyte infiltration, edema, and tissue necrosis, the direct DTH is not a feasible diagnostic technique in transplant patients because of the possibility of direct injection resulting in sensitization to donor antigens and graft loss. To avoid this problem, the human-to-mouse "trans-vivo" DTH assay was developed 1,2. This test is essentially a transfer DTH assay, in which human peripheral blood mononuclear cells (PBMCs) and specific antigens were injected subcutaneously into the pinnae or footpad of a na&iuml;ve mouse and DTH-like swelling is measured after 18-24 hr 3. The antigen presentation by human antigen presenting cells such as macrophages or DCs to T cells in highly vascular mouse tissue triggers the inflammatory cascade and attracts mouse immune cells resulting in swelling responses. The response is antigen-specific and requires prior antigen sensitization. A positive donor-reactive DTH response in the Tv-DTH assay reflects that the transplant patient has developed a pro-inflammatory immune disposition toward graft alloantigens.
The most important feature of this assay is that it can also be used to detect regulatory T cells, which cause bystander suppression. Bystander suppression of a DTH recall response in the presence of donor antigen is characteristic of transplant recipients with accepted allografts 2,4-14. The monitoring of transplant recipients for alloreactivity and regulation by Tv-DTH may identify a subset of patients who could benefit from reduction of immunosuppression without elevated risk of rejection or deteriorating renal function.
A promising area is the application of the Tv-DTH assay in monitoring of autoimmunity15,16 and also in tumor immunology 17.

We describe a valuable diagnostic assay that could potentially be used to decide the withdrawal of immunosuppression after transplant without elevated risk of graft rejection. The assay uses the principles of Delayed Type Hypersensitivity and provides accurate assessment of both donor specific effector and regulatory immune responses mounted by recipients.

抗原特異的規制用のトランス生体内遅延型過敏アッセイ

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host ...

免疫・感染学

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

効率的なためキラー人工抗原提示細胞（KaAPC）<em&gt;体外</em&gt;ヒト抗原特異的T細胞の枯渇

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

自己免疫に対する幹細胞由来の抗原特異的制御性T細胞の開発

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

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 ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

生体内で予防接種モデルを用いた抗原特異的 T 細胞細胞傷害性関数の検出のための試金

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

まれな抗原特異的 B 細胞血液の循環から弁別に対するモノクローナル抗体の生成

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in human sera and in separate preparations of polyclonal intravenous immunoglobulin (IVIg). The protocol describes the methodological steps involved in sample preparation, printing of arrays, assay procedure, and data analysis. In addition, this protocol could be further developed to incorporate diverse clinical samples including plasma and cell culture supernatants. We show how protein microarray can be used to determine a combination of isotype (IgG, IgA, IgM), subclass (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2), and strain-specific antibodies to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B-only expressing strain (CCUG 20309), a precursor form of a B fragment of binary toxin, pCDTb, ribotype-specific whole surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). During the experiment, microarrays are probed with sera from individuals with C. difficile infection (CDI), individuals with cystic fibrosis (CF) without diarrhea, healthy controls (HC), and from individuals pre- and post-IVIg therapy for the treatment of CDI, combined immunodeficiency disorder, and chronic inflammatory demyelinating polyradiculopathy. We encounter significant differences in toxin neutralization efficacies and multi-isotype specific antibody levels between patient groups, commercial preparations of IVIg, and sera before and following IVIg administration. Also, there is a significant correlation between microarray and enzyme-linked immunosorbent assay (ELISA) for antitoxin IgG levels in serum samples. These results suggest that microarray could become a promising tool for profiling antibody responses to C.difficile antigens in vaccinated or infected humans. With further refinement of antigen panels and a reduction in production costs, we anticipate that microarray technology may help optimize and select the most clinically useful immunotherapies for C. difficile infection in a patient-specific manner.

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

This article describes a simple protein microarray method for profiling humoral immune responses to a 7-plex panel of highly purified Clostridium difficile antigens in human sera. The protocol can be extended for the determination of specific antibody responses in preparations of polyclonal intravenous immunoglobulin.

蛋白質のマイクロ アレイの試金抗原特異的抗体の応答次のクロストリジウム ・ ディフィシル感染症の血清学的定量

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

病原性細菌に対する免疫学的応答を評価するための Opsonophagocytic 殺害アッセイ

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear cells (PBMCs). The development of stable, non-toxic, fluorescent dyes such as carboxyfluorescein succinimidyl ester (CFSE) allows for rare, antigen-specific T cells to be distinguished from bystanders by diminution in fluorescent staining, as detected by flow cytometry. This method has the following advantages over alternative approaches: (i) it is very sensitive to low-frequency T cells, (ii) no knowledge of the antigen or epitope is required, (iii) the phenotype of the responding cells can be analyzed, and (iv) viable, responding cells can be sorted and used for further analysis, such as T cell cloning.

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Presented here is a protocol for measuring proliferating CD4+ T cells in response to antigenic proteins or peptides using dye dilution. This assay is particularly sensitive to rare antigen-specific T cells and can be modified to facilitate cloning of antigen-specific cells.

カルボキシフルオレスチンスクシニミジルエステルを用いたヒト抗原特異的CD4+T細胞の増殖の定量化

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear ...

High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the spontaneous non-obese diabetes (NOD) mouse model, insulin is the primary target, and genetic manipulation of these animals to remove a single key insulin epitope prevents disease. Thus, selective elimination of professional antigen presenting cells (APCs) bearing this pathogenic epitope is an approach to inhibit the unwanted insulin-specific autoimmune responses, and likely has greater translational potential.
Chimeric antigen receptors (CARs) can redirect T cells to selectively target disease-causing antigens. This technique is fundamental to recent attempts to use cellular engineering for adoptive cell therapy to treat multiple cancers. In this protocol, we describe an optimized T-cell retrovirus (RV) transduction and in vitro expansion protocol that generates high numbers of functional antigen-specific CD8 CAR-T cells starting from a low number of naive cells. Previously multiple CAR-T cell protocols have been described, but typically with relatively low transduction efficiency and cell viability following transduction. In contrast, our protocol provides up to 90% transduction efficiency, and the cells generated can survive more than two weeks in vivo and significantly delay disease onset following a single infusion. We provide a detailed description of the cell maintenance and transduction protocol, so that the critical steps can be easily followed. The whole procedure from primary cell isolation to CAR expression can be performed within 14 days. The general method may be applied to any mouse disease model in which the target is known. Similarly, the specific application (targeting a pathogenic peptide/MHC class II complex) is applicable to any other autoimmune disease model for which a key complex has been identified.

This article describes a protocol for the generation of antigen-specific CD8 T cells, and their expansion in vitro, with the aim of yielding high numbers of functional T cells for use in vitro and in vivo.

自己免疫性糖尿病モデルにおける機能試験用抗原特異的一次マウス細胞傷害性T細胞の高効率生成

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the ...

抗原特異的インビボでのキリングアッセイ