この作品はアメリカの消化器協会（AGA）研究学者賞とクローン病とアメリカの大腸炎財団（SZSへ）（CCFA）キャリア開発賞、（ECS）は、NIH NIDDK F30 DK089692、胃腸生物学ノースカロライナ大学のセンターによってサポートされていましたと疾病グラントのP30のDK34987（組織学コア）。 UNCフローサイトメトリーコアファシリティは、UNCラインバーガー総合がんセンターのNCIセンターコアサポートグラント（P30CA016086）によって部分的にサポートされています。私たちは、病理組織学的分析および免疫組織化学との彼の助けを獣医のノースカロライナ州立大学からルークB.ボーストに感謝。

注：すべての動物のプロトコルはによっておよび施設内動物管理使用委員会（IACUC）規制や実験動物の管理と使用に関する米国学術研究会議·ガイドに準拠して承認されていることを確認します。ドナーマウスは、男性または女性のいずれでもよいが、レシピエントマウスは、雄であるべきである。女性の受信者が使用する場合、ドナーマウスは、雌5でなければならない。 、したがって、マウス5,6の大腸炎の表現型を、これらが腸内細菌叢に影響を与える可能性があるように、定期的に、非滅菌寝具と非酸性化した水を使用してコロニーを維持し、。 1。実験の準備<ol><li>氷のように冷たいメディアとバッファを使用。実験を通して氷上で細胞を保管してください。 </li><li>無菌技術を用いた無菌のバイオハザードフード中で実験を行います。 </li></ol>脾臓T細胞の2の分離<ol><li>子宮頸dislocatio続くCO 2チャンバー内でドナーマウス/マウスを安楽死させるN。 70％エタノールで腹部スプレー。 </li><li>腹部の横切開を行い、腹膜を露出するために皮膚をはがし。腹膜離れて鉗子で内臓からのホールドと脾臓を露出し、切除する左腹部腹膜の切開を作る。 </li><li>ペトリ皿の中で完全なメディアの10ミリリットルで脾臓を置きます。脾臓から余分な組織を除去し、廃棄する。 </li><li>つぶすと単一細胞懸濁液に脾臓を離れていじめる2滅菌ガラススライドを使用してください。 50ミリリットルに70μmのストレーナーを通してフィルタ細胞懸濁液円錐管に完全メディア5mlでストレーナーをすすぐ。 1 50ミリリットルコニカルチューブに5脾臓まで置きます。 </li><li> 7分間450×gで遠心分離した細胞。オフ注ぐことによって、または廃棄物容器に真空吸引により上清を捨てる。 </li><li>穏やかに室温で10分間、赤血球を溶解するために溶解緩衝液の脾臓当たり5mlの細胞を再懸濁。チューブへの完全なメディアの等量の（脾臓あたり5ミリリットル）を追加します。 </li><l7分間450×gでI&gt;遠心細胞。オフ注ぐことによって、または廃棄物容器に真空吸引により上清を捨てる。 </li><li>静かにラベル緩衝液10mlで細胞を懸濁します。 </li><li>トリパンブルー排除により、細胞を数える。 <ol><li>細胞懸濁液の20μlのを削除し、マイクロチューブに180μlのトリパンブルーに加え、十分に混合する。 5分後、顕微鏡下で非青色細胞を血球計数器とカウントする10μlの標識細胞を追加します。生存細胞の総数を決定します。セル/パンブルーミックスを破棄します。 </li></ol></li><li> 7分間450×gで標識化緩衝液10mlで遠心細胞。オフ注ぐことによって、または廃棄物容器に真空吸引により上清を捨てる。 </li></ol> CD4 + T細胞の3濃縮注：このセクションで使用される特定の製品については、製造元の指示に従ってください。 <ol><li>穏やかに冷たいラベル20×10 6細胞/ mLの単一細胞懸濁液に細胞を再懸濁INGバッファー。 </li><li>ビオチン化CD4 T細胞濃縮抗体の1×10 6細胞あたり5μlを添加する。 15分間氷上で細胞をインキュベート。 </li><li>ラベルバッファーの10倍のボリュームを追加します。 7分間450×gで遠心した細胞。慎重に廃棄物容器内に真空吸引を使用してすべての上清を吸引する。 </li><li>徹底的にストレプトアビジン結合磁性粒子をボルテックス。 1×10 6細胞当たりの粒子の5μlを添加する。 </li><li>徹底的になく穏やかに混合。 30分間6-12℃でミックスしてください。 </li><li> 20-80×10 6細胞/ mlの濃度に標識バッファーを加える。 12×75ミリメートルの丸底試験管当たりの標識細胞を1.0mlにまで転送する（「ポジティブフラクションチューブ」と称する）。 </li><li> 6-8分間磁石上の各ポジティブフラクションチューブを置きます。 </li><li>まだ磁石に正のフラクションチューブを、慎重に新しい無菌の50mlの円錐チューブにガラスパスツールピペットでポジティブフラクションチューブから上清を移す（referre「濃縮画分」などからd）。この濃縮画分は、CD4 + T細胞が含まれています。磁石に引きつけ標識細胞を破壊しないように注意してください。 </li><li>精力的に上下にピペッティングすることによって、ステップ3.6のようにラベル表示のバッファの同じ体積でポジティブフラクションチューブに残って細胞を再懸濁し。 6-8分間背面磁石に正のフラクションチューブを置きます。 </li><li>まだ磁石に正のフラクションチューブを、慎重に磁石に付着した細胞を破壊することなく、ステップ3.8から50ミリリットルのコニカルチューブを無菌ガラスパスツールピペットでポジティブフラクションチューブから上清（濃縮画分、CD4 +）を転送。 </li><li>繰り返して得られたCD4 + T細胞の収量を増加させるために3.9から3.10までを繰り返す。濃縮画分（CD4 +細胞）を使用してプロトコルを継続します。 </li></ol> 4.ラベル付けとソートセル7 <ol><li>遠心7分間450×gで細胞を濃縮。または真空suctをオフに傾けて上清を除去廃棄物容器にイオンを打ち込む。 </li><li>ラベル緩衝液1mlに細胞を再懸濁。カウントするとステップ2.9のようにトリパンブルー排除により、細胞生存率のために評価するために、細胞のアリコートを削除します。 </li><li> 10×10 6細胞/ mlに標識バッファーの容量を追加する。細胞が既にある場合は&lt;10×10 6、7分間450×gで遠心分離は、廃棄物容器に捨てるか、真空吸引により上清を捨て、そして10×10 6細胞/ mlに標識バッファーの容量を追加する。 <ol><li>マイクロチューブ内アイソタイプ染色し、染色されていない、および単一染色対照細胞のために約5〜10×10 5個の細胞それぞれの個別の分量を設定します。 </li></ol></li><li>細胞に5μg/ mlのCD4-FITCおよび1μg/ mlのCD45RB-PEを追加します。マイクロチューブ中でアリコートを適切な同じ濃度で、アイソタイプ対照汚れやシングルの汚れを追加します。うまくしかし穏やかに混合し、30分間、光から保護氷上でインキュベートする。 </li><li>細胞とコントロールにラベルバッファーの10倍のボリュームを追加します。S。 7分間遠心450 XG。廃棄物容器に真空吸引により上清を捨てる。 </li><li>ステップ4.5でボリュームにラベルバッファーに懸濁します。 7分間遠心450 XG。廃棄物容器に真空吸引により上清を捨てる。 </li><li> 10×10 6細胞/ mlに標識化緩衝液に再懸濁する。 FACSチューブに70μmのストレーナーを通して細胞を渡します。 FACSの準備が整うまで、光から保護氷上に保管してください。 </li><li>設定し、非染色細胞と単染色コントロールとセルソーター上の適切な補償を決定する。 </li><li>順方向及び側方散乱ゲーティング（ 図1A）と生育不能細胞を除外します。アイソタイプ染色コントロールとCD4 +およびCD45RB +細胞のためのゲーティングを設定します。 CD4 +集団上のゲート細胞。 </li><li>完全なメディアの2ミリリットルにチューブにPE染色細胞のための単純なヒストグラムを用いた高 CD45RBとCD45RB 低集団にソートCD4 +細胞。 高 CD45RB </supは&gt;集団はCD4 + CD45RB +細胞（ 高 CD45RB）の最高40％を表し、CD45RB 低集団はCD4 + CD45RB +細胞の最低20％である（ 低 CD45RB; 図1B）。 </li></ol><img alt="図1" src="/files/ftp_upload/52533/52533fig1.jpg" /> 図1：FACS分析中CD4 + CD45RB T細胞集団のプロット代表的なフローサイトメトリー（A - C）FITC CD4-、ドナーからPE CD45RB染色した脾細胞C57BL / 6マウスを、CD4 +高 CD45RBおよびCD4 +にFACSによって選別した。 CD45RB 低い T細胞集団。 （A）ダブレットイベントが前方散乱プロット上除外された。 （B）、リンパ球を前方および側方散乱プロットにゲートした。 （C）CD4 + </商標&gt; T細胞をゲートし、および（D）CD4 + CD45RB + T細胞を、PEに対するイベントヒストグラムにプロットした。 CD4 + CD45RB low細胞をCD45RB +細胞の最低20％であると考えられた。 CD4 + CD45RB high細胞をCD45RB +細胞の最大40％と定義した。 <ol start="11"><li>集団の純度を評価するためにFACSマシン上の各細胞集団のアリコートを実行する。 </li><li>遠心7分間450×gで細胞を選別した。 1mlのPBSで再懸濁します。カウントするとステップ2.9のようにトリパンブルー排除により、細胞生存率のために評価するために、細胞のアリコートを削除します。 </li></ol>受信者への細胞の5インジェクション<ol><li>再懸 ​​濁し、4×10 6細胞/ ml（CD45RB 高 ）またはPBS中の2×10 6細胞/ ml（ 低 CD45RB）に細胞を選別した。 </li><li>新しい滅菌チューブに受信者ごとCD45RB high細胞の100μlのを転送します。 PBSあたり100μlのを追加します。このチューブに受信者。したがって、動物あたりの総注射容量は200μlであり、受信者ごとの細胞の総量は、4×10 5個のCD4 + CD45RB 高ナイーブT細胞である。 </li><li>制御性T細胞を受けた実験群が必要な場合は、新しい滅菌チューブに受信者ごとCD45RB 高い細胞100μlを移す。同じチューブに受信者ごとCD45RB 低細胞100μlを追加。動物あたりの総注射容量は200μlである。 CD45RB 高の比率：CD45RB low細胞は2：1である。 </li><li> ハイ / CD45RB 低 CD4 +細胞の腹腔内各受信者の腹部（200μLの合計）の右側と左側にCD45RB 高またはCD45RB100μlのを注入。 </li></ol>レシピエント動物における疾患進行の6モニタリング<ol><li>レシピエント動物の臨床状態を評価するために、以下のparameteの集計臨床スコアを割り当てる注射の日のRS 8、その後毎週、および屠殺時： <ol><li>重量損失を測定することによって無駄に判断します。0 - なし減量を。 1 - 初期体重の0.1〜10％の損失。 2 -最初の体重（ 図2A）の10％以上の損失。 </li></ol></li></ol><img alt="図2" src="/files/ftp_upload/52533/52533fig2highres.jpg" width="650" /> 図2：炎症の臨床的および肉眼的病理学的徴候は、RAG1に野生型CD4 + CD45RB high T細胞の転写後に起こる- / -およびNRDKOレシピエントマウス11（A）NRDKO受信者5によってその初期体重の平均は10％で失わ週間後転送、RAG1のに対し、- / -レシピエントは、この時点で疾患の臨床徴候を示さなかった。各点は、平均±SEMコホートの初期体重の平均パーセンテージを表す。 **、P &lt;0.005。直腸脱の存在によって示されるように（B）いくつかのマウスは、重篤な腸炎を発症した。これはNRDKOレシピエントマウスにおける直腸脱の代表画像です。 （C）肉眼、からコロン両方RAG1 - / -とNRDKOレシピエントマウスは、RAG1からコロンに比べて厚くなったと短縮されている- / -およびT細胞の養子移入せずにNRDKOマウス。 NRDKOレシピエントマウスからのコロンが重度の炎症と増加したコロンの重み（データは示していない）を示している。 <a href="https://www.jove.com/files/ftp_upload/52533/52533fig2highres.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <ol><li><ol start="2"><li>それが経過するまで、清潔な容器内に動物を置くことによって、糞便の質を決定するスツール：0 - なし。 1 - ソフトスツール。 2 - 水っぽい及び/または血便。 </li><li>病気の以下の徴候の存在によって動物の全般的な健康状態を決定します。0 - なし猫背の姿勢、毛皮、または皮膚病変を毛。 1 - 次の本のいずれか：猫背の姿勢は、毛皮、皮膚病変を剛毛。 </li><li>直腸脱の存在の決定：0 - 不在を。 1 -現在（ 図2B）。 </li></ol></li><li>彼らは彼らの開始体重の20％を失ったか、いずれか早い方所望の時点、でてきたとき頸椎脱臼に続いてCO 2吸入により動物を生け贄に捧げる。臨床疾患は週5ポスト充満から始まる通常は明らかである。 </li><li>以前に5,7を説明するように、疾患の重症度について評価します。 <ol><li>ステップ6.1 8のように、臨床スコアを割り当てます。 </li><li>結腸の長さと重さ（ 図2C）を測定します。 </li><li>炎症の組織学的分析を実行します5。 </li><li>腸組織外培養における自発的なサイトカイン発現9、腸間膜リンパ節10、および/ ​​または血清11を決定します。 <ol><li>簡単に言えば、外植片培養9のために、縦方向に開いて、屠殺後のコロンを削除して、PBSで洗浄してください。室温で30分間完全培地中で、250rpmでオービタルシェーカーセットにコロンをインキュベートします。 </li><li>組織を小片にチョップし、24時間完全培地中で37℃でインキュベートした。上清を収集し、ELISAにより100 mgの組織あたりのサイトカインの定量のために使用します。 </li></ol></li><li> T細胞の表現型および/ ​​または機能10のex vivoでの特性評価を行います。 </li></ol></li></ol>

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L., Powrie, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+bowel+disease+in+C.B-17+scid+mice+reconstituted+with+the+CD45RBhigh+subset+of+CD4++T+cells.">Inflammatory bowel disease in C.B-17 scid mice reconstituted with the CD45RBhigh subset of CD4+ T cells.</a> Am J Pathol. 148 (5), 1503-1515 (1996).</li><li>Powrie, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+Th1+responses+prevents+inflammatory+bowel+disease+in+scid+mice+reconstituted+with+CD45RBhi+CD4.">Inhibition of Th1 responses prevents inflammatory bowel disease in scid mice reconstituted with CD45RBhi CD4.</a> T cells. Immunity. 1 (7), 553-562 (1994).</li><li>Read, S., Malmstrom, V., Powrie, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxic+T+lymphocyte-associated+antigen+4+plays+an+essential+role+in+the+function+of+CD25(+)CD4(+)+regulatory+cells+that+control+intestinal+inflammation.">Cytotoxic T lymphocyte-associated antigen 4 plays an essential role in the function of CD25(+)CD4(+) regulatory cells that control intestinal inflammation.</a> J Exp Med. 192 (2), 295-302 (2000).</li><li>Rogers, G. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+divergence+in+gastrointestinal+microbiota+in+physically-separated+genetically+identical+mice.">Functional divergence in gastrointestinal microbiota in physically-separated genetically identical mice.</a> Sci Rep. 4, 5437 (2014).</li><li>Fukata, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+myeloid+differentiation+factor+88+(MyD88)+is+required+for+CD4++T+cell+effector+function+in+a+murine+model+of+inflammatory+bowel+disease.">The myeloid differentiation factor 88 (MyD88) is required for CD4+ T cell effector function in a murine model of inflammatory bowel disease.</a> J Immunol. 180 (3), 1886-1894 (2008).</li><li>Kurtz, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+adenosine+regulates+colitis+through+effects+on+lymphoid+and+nonlymphoid+cells.">Extracellular adenosine regulates colitis through effects on lymphoid and nonlymphoid cells.</a> Am J Physiol Gastrointest Liver Physiol. 307 (3), 338-346 (2014).</li><li>Naganuma, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+edge:+Critical+role+for+A2A+adenosine+receptors+in+the+T+cell-mediated+regulation+of+colitis.">Cutting edge: Critical role for A2A adenosine receptors in the T cell-mediated regulation of colitis.</a> J Immunol. 177 (5), 2765-2769 (2006).</li><li>Ranatunga, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protective+role+for+human+IL-10-expressing+CD4++T+cells+in+colitis.">A protective role for human IL-10-expressing CD4+ T cells in colitis.</a> J Immunol. 189 (3), 1243-1252 (2012).</li><li>Srikrishna, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carboxylated+glycans+mediate+colitis+through+activation+of+NF-kappa.">Carboxylated glycans mediate colitis through activation of NF-kappa.</a> B. J Immunol. 175 (8), 5412-5422 (2005).</li><li>Wang, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IFN-gamma-induced+TNFR2+expression+is+required+for+TNF-dependent+intestinal+epithelial+barrier+dysfunction.">IFN-gamma-induced TNFR2 expression is required for TNF-dependent intestinal epithelial barrier dysfunction.</a> Gastroenterology. 131 (4), 1153-1163 (2006).</li></ol>

The authors declare that they have no competing financial interests.

ここでは、免疫不全マウスにCD4 + CD45RB + T細胞の養子移入によってマウスでの大腸の炎症を誘導するステップバイステップのプロトコルを記述します。 - / -レシピエントマウス、他の系統であるが（ 例えば 、BALB / C、129S6 / SvEv、非肥満糖尿病（NOD））及び免疫不全の遺伝的モデル（ 例えば 、SCID、 が、Rag2我々はC57BL / 6ドナー脾臓と同系RAG1を</em...

The inflammatory bowel diseases (IBD) Crohn&#8217;s disease and ulcerative colitis result from an incompletely defined and complex interaction between host immune responses, genetic susceptibility, environmental factors, and the enteric luminal contents1. Recent genome-wide association studies report associations between immune cell regulatory genes and IBD susceptibility2,3. Both innate and adaptive immune cell intrinsic genes are represented in these studies, indicating a central role for these cell populations in IBD pathogenesis.
There currently exist more than 50 animal models of human IBD. While no one model perfectly phenocopies human IBD, many are useful for studying various aspects of human disease, including disease onset and progression and the wound-healing response. In the method described here, intestinal inflammation is initiated with syngeneic splenic CD4+CD45RBhigh T cell adoptive transfer into T and B cell deficient recipient mice4. The CD4+CD45RBhigh T cell population contains mainly na&#239;ve T cells primed for activation that are capable of inducing chronic small bowel and colonic inflammation. This method allows the researcher to modify key experimental variables, including both innate and adaptive immune cell populations, to answer biologically relevant questions relating to disease pathogenesis. Additionally, this method provides precise initiation of disease onset and a well-characterized experimental time course. This permits the kinetic study of clinical features of disease progression in mice. Intestinal inflammation induced by this method shares many features with human IBD, including chronic large and small bowel transmural inflammation, pathogenesis driven by cytokines such as TNF and IL-12, and systemic symptoms such as wasting5. Thus, it is an ideal model system for studying the pathogenesis of human IBD.
The method here describes in detail the protocol for inducing experimental colitis by adoptive transfer of CD4+CD45RBhigh T cells into Rag1-/- mice. We discuss key technical steps, expected results, optimization, and trouble-shooting. We address the required elements for the successful development of this murine model of intestinal inflammation for research purposes.

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Name of Reagent/ Equipment</td>
			<td >Company</td>
			<td >Catalog Number</td>
			<td >Comments/Description</td>
		</tr>
		<tr >
			<td >10x PBS</td>
			<td >Gibco</td>
			<td >14200075</td>
			<td></td>
		</tr>
		<tr >
			<td >12x75mm round-bottom tube</td>
			<td >Falcon</td>
			<td >352052</td>
			<td></td>
		</tr>
		<tr >
			<td >15 ml conical</td>
			<td >Corning</td>
			<td >430790</td>
			<td></td>
		</tr>
		<tr >
			<td >26g x 3/8 Needle</td>
			<td >BD Biosciences</td>
			<td >305110</td>
			<td></td>
		</tr>
		<tr >
			<td >50 ml conical</td>
			<td >Corning</td>
			<td >430828</td>
			<td></td>
		</tr>
		<tr >
			<td >70 um Cell Strainer</td>
			<td >Fisherbrand</td>
			<td >22363548</td>
			<td></td>
		</tr>
		<tr >
			<td >BD IMagnet</td>
			<td >BD Biosciences</td>
			<td >552311</td>
			<td></td>
		</tr>
		<tr >
			<td >&#946;-mercaptoethanol</td>
			<td >Thermo Scientific</td>
			<td >35602</td>
			<td></td>
		</tr>
		<tr >
			<td >CD4-FITC IgG2b</td>
			<td >eBioscience</td>
			<td >11-0041</td>
			<td></td>
		</tr>
		<tr >
			<td >CD45RB-PE IgG2a</td>
			<td >BD Pharminogen</td>
			<td >553101</td>
			<td></td>
		</tr>
		<tr >
			<td >Complete Media</td>
			<td ></td>
			<td ></td>
			<td >RPMI-1640, 1% Pen/Strep, 10% FBS, 0.0004% &#946;-ME</td>
		</tr>
		<tr >
			<td >FACS tube + strainer</td>
			<td >BD Falcon</td>
			<td >352235</td>
			<td></td>
		</tr>
		<tr >
			<td >Glass Microscope Slides</td>
			<td >Fisherbrand</td>
			<td >12550A3</td>
			<td></td>
		</tr>
		<tr >
			<td >Heat-inactivated FBS</td>
			<td >Gemini</td>
			<td >100-106</td>
			<td></td>
		</tr>
		<tr >
			<td >Labeling Buffer</td>
			<td ></td>
			<td ></td>
			<td >1x PBS, 0.5% BSA, 2 mM EDTA</td>
		</tr>
		<tr >
			<td >Lysis Buffer</td>
			<td ></td>
			<td ></td>
			<td >0.08% NH4Cl, 0.1% KHCO3, 1 mM EDTA</td>
		</tr>
		<tr >
			<td >MoFlo XDP</td>
			<td >Beckman Coulter</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Mouse CD4 T lymphocyte Enrichment Set - DM</td>
			<td >BD Biosciences</td>
			<td >558131</td>
			<td></td>
		</tr>
		<tr >
			<td >Mouse IgG2a-PE</td>
			<td >BD Pharminogen</td>
			<td >553457</td>
			<td></td>
		</tr>
		<tr >
			<td >Mouse IgG2b-FITC</td>
			<td >eBioscience</td>
			<td >11-4732</td>
			<td></td>
		</tr>
		<tr >
			<td >Pasteur pipet</td>
			<td >Fisherbrand</td>
			<td >13-678-20D</td>
			<td></td>
		</tr>
		<tr >
			<td >Penicillin-Streptomycin Solution, 100X</td>
			<td >Corning Cellgro</td>
			<td >30-002-CI</td>
			<td></td>
		</tr>
		<tr >
			<td >Petri Dish</td>
			<td >Fisherbrand</td>
			<td >875713</td>
			<td></td>
		</tr>
		<tr >
			<td >Pure Ethanol 200 Proof</td>
			<td >Decon Labs</td>
			<td >2705-HC</td>
			<td></td>
		</tr>
		<tr >
			<td >RPMI-1640</td>
			<td >Gibco</td>
			<td >11-875-093</td>
			<td></td>
		</tr>
		<tr >
			<td >Syringe</td>
			<td >BD Biosciences</td>
			<td >309597</td>
			<td></td>
		</tr>
		<tr >
			<td >Trypan blue</td>
			<td >Corning Cellgro</td>
			<td >25-900-CI</td>
			<td></td>
		</tr>
		<tr >
			<td >Wash Media</td>
			<td ></td>
			<td ></td>
			<td >RPMI-1640, 1% Pen/Strep, 0.0004% &#946;-ME</td>
		</tr>
	</tbody>
</table>

induction of murine intestinal inflammation by adoptive transfer of effector cd4+cd45rbhigh t cells into immunodeficient mice

ヒト炎症性腸疾患（IBD）、それぞれに長所と短所の病因を研究するために利用可能な多くの異なる動物モデルがある。ここではTおよびB細胞欠損レシピエントマウスに同系の脾臓CD4 + CD45RB high T細胞の養子移入によって開始される実験的大腸炎モデルを記述する。主にナイーブ、エフェクター細胞からなるCD4 + CD45RB 高 T細胞集団は、密接に、ヒトIBDの重要な側面に似た、慢性的腸炎を誘導することができる。この方法は、疾患の発症および進行の側面を研究するために操作することができる。さらに、それは腸の炎症、すなわち 、先天性、適応性の機能、及び調節免疫細胞集団、および環境曝露の役割は、微生物を研究するために使用することができる。この記事では、ステップバイステップのプロトコルで大腸炎を誘発するための方法論を示している。このINCludes成功裏に研究目的のために実験的大腸炎のこのマウスモデルを開発するために必要なキー技術的な側面のデモビデオ。

約成体C57BL / 6ドナーマウスから脾臓を10から10×10 6個のCD4 + CD45RB high T細胞を確実に分離されている。この数は、年齢や歪みドナーマウスの研究者の習熟度に応じて変化するであろう。 - / - 4×10 5 C57BL / 6 CD4 + CD45RB high T細胞をC57BL / 6 RAG1に転送されたとき、レシピエントマウスマウスは、より重篤な疾患に遺伝的に感受性である場?...

Watch this Scientific Journal Video about Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice at JoVE.com

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

エフェクターCD4の養子移入によるマウス腸の炎症の誘導<sup&gt; +</sup&gt; CD45RB<sup&gt;高い</sup&gt;免疫不全マウスへのT細胞

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

induction-murine-intestinal-inflammation-adoptive-transfer-effector

Research

JoVE Journal

Immunology and Infection

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

16.3K ビュー.  University of North Carolina at Chapel Hill. Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

ビデオ: Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into mouse tail veins and colonize in the lungs, thereby, resembling the last steps of tumor cell spontaneous metastasis: survival in the circulation, extravasation and colonization in the distal organs. From a therapeutic point of view, the experimental metastasis model is the simplest and ideal model since the target of therapies is often the end point of metastasis: established metastatic tumor in the distal organ. In this model, tumor cells are injected i.v into mouse tail veins and allowed to colonize and grow in the lungs. Tumor-specific CTLs are then injected i.v into the metastases-bearing mouse. The number and size of the lung metastases can be controlled by the number of tumor cells to be injected and the time of tumor growth. Therefore, various stages of metastasis, from minimal metastasis to extensive metastasis, can be modeled. Lung metastases are analyzed by inflation with ink, thus allowing easier visual observation and quantification.

An experimental lung metastasis and CTL immunotherapy mouse model for analysis of tumor cells-T cell interaction in vivo.

実験的転移とCTLの養子移入免疫療法のマウスモデル

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into ...

免疫・感染学

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

マウス大動脈内の免疫細胞のフローサイトメトリー分析

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of human Type 1 diabetes (T1D). Cell transfer studies in irradiated recipient mice have established that T cells are pivotal in T1D pathogenesis in this model. We describe herein a simple method to rapidly induce T1D by adoptive transfer of purified, primary CD4+ T cells from pre-diabetic NOD mice transgenic for the islet-specific T cell receptor (TCR) BDC2.5 into NOD.SCID recipient mice. The major advantages of this technique are that isolation and adoptive transfer of diabetogenic T cells can be completed within the same day, irradiation of the recipients is not required, and a high incidence of T1D is elicited within 2 weeks after T cell transfer. Thus, studies of pathogenesis and therapeutic interventions in T1D can proceed at a faster rate than with methods that rely on heterogenous T cell populations or clones derived from diabetic NOD mice.

We provide a reproducible method to induce type 1 diabetes (T1D) in mice within two weeks by the adoptive transfer of islet antigen-specific, primary CD4+ T cells.

糖尿病CD4の養子移入+ T細胞によるマウスの加速された1型糖尿病の誘導

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of ...

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer&#39;s patch (PP). Common dendritic cell progenitors (CDPs, lin- c-kitlo CD115+ Flt3+) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1+ and recipients being CD45.2+. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.

This protocol describes experimental procedures to assess the differentiation of plasmacytoid dendritic cells in Peyer&#8217;s patch from common dendritic cell progenitors, using techniques involving FACS-mediated cell isolation, hydrodynamic gene transfer, and flow analysis of immune subsets in Peyer&#8217;s patch.

造血前駆細胞の養子移入を使用して、パイエル板におけるマウス形質細胞様樹状細胞の開発を評価

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...

Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo to express chimeric antigen receptors specific for glioma antigens (CAR T cells). The expansion and function of adoptively transferred CAR T cells can be potentiated by the lymphodepletive and tumoricidal effects of standard of care chemotherapy and radiotherapy. We describe a method for generating CAR T cells targeting EGFRvIII, a glioma-specific antigen, and evaluating their efficacy when combined with a murine model of glioblastoma standard of care. T cells are engineered by transduction with a retroviral vector containing the anti-EGFRvIII CAR gene. Tumor-bearing animals are subjected to host conditioning by a course of temozolomide and whole brain irradiation at dose regimens designed to model clinical standard of care. CAR T cells are then delivered intravenously to primed hosts. This method can be used to evaluate the antitumor efficacy of CAR T cells in the context of standard of care.

The lymphodepletive and immunomodulatory effects of chemotherapy and radiation standard of care can be leveraged to enhance the antitumor efficacy of T cell immunotherapy. We outline a method for generating EGFRvIII-specific chimeric antigen receptor (CAR) T cells and administering them in the context of glioblastoma standard of care.

ケアの膠芽腫の標準のコンテキストにおける養子療法のためのCAR T細胞の生成

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo ...

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

解析<em&gt;腸炎エルシニア</em&gt;エフェクター転β-ラクタマーゼエフェクター融合を用いて宿主細胞に

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

マウスの脾臓から不変ナチュラルキラーT細胞を単離および拡張するための最適化方法

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

マウスにおける実験的細菌性肺炎の誘導と表現型のための非侵襲的および技術的に非集中的な方法

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human central nervous system (CNS) autoimmune demyelinating disease, creation of an AQP4-targeted model with both clinical and histologic manifestations of CNS autoimmunity has proven challenging. Immunization of wild-type (WT) mice with AQP4 peptides elicited T cell proliferation, although those T cells could not transfer disease to na&#239;ve recipient mice. Recently, two novel AQP4 T cell epitopes, peptide (p) 135-153 and p201-220, were identified when studying immune responses to AQP4 in AQP4-deficient (AQP4-/-) mice, suggesting T cell reactivity to these epitopes is normally controlled by thymic negative selection. AQP4-/- Th17 polarized T cells primed to either p135-153 or p201-220 induced paralysis in recipient WT mice, that was associated with predominantly leptomeningeal inflammation of the spinal cord and optic nerves. Inflammation surrounding optic nerves and involvement of the inner retinal layers (IRL) were manifested by changes in serial optical coherence tomography (OCT). Here, we illustrate the approaches used to create this new in vivo model of AQP4-targeted CNS autoimmunity (ATCA), which can now be employed to study mechanisms that permit development of pathogenic AQP4-specific T cells and how they may cooperate with B cells in NMO pathogenesis.

Here, we present a protocol to induce paralysis and opticospinal inflammation by transfer of aquaporin-4 (AQP4)-specific T cells from AQP4-/- mice into WT mice. In addition, we demonstrate how to use serial optical coherence tomography to monitor visual system dysfunction.

麻痺、視神経脊髄炎自己抗原アクアポリン 4 の T 細胞特定のマウスの視覚系障害の誘導

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...

Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce efficient therapies have been proposed over the last decade. Among the promising candidates for designing novel therapeutic strategies, cellular immunotherapies have been targeted to eliminate highly invasive and chemo-radioresistant tumor cells, likely involved in a rapid and fatal relapse of this cancer. Thus, administration(s) of allogeneic GBM-reactive immune cell effectors, such as human V&#978;9V&#948;2 T lymphocytes, in the vicinity of the tumor would represents a unique opportunity to deliver efficient and highly concentrated therapeutic agents directly into the site of brain malignancies. Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a preclinical proof-of-concept for both the feasibility and the antitumor efficacy of these cellular immunotherapies that rely on stereotactic injections of allogeneic human lymphocytes within intrabrain tumor beds.

Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a proof-of-concept for both the feasibility and the antitumor efficacy of intrabrain-delivered cellular immunotherapies.

同所性同種人間神経膠腫異種移植片のマウスモデルの細胞傷害性免疫細胞の定位の養子転送

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce ...

エフェクターCD4の養子移入によるマウス腸の炎症の誘導

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