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他のすべてのモデルと同様に、上記の抗原駆動型大腸炎モデルは、技術を実行研究者が知っておく必要がありますいくつかの問題を提示することができます。 OT-IIホストで/レッド+ CD4 + T CD62L +細胞を注入すると、研究者は腹腔内に注射針を挿入することは非常に穏やかで注意しなければなりません。これを怠ると、死亡につながる可能性がマウスの腸、または任意の?...

腸は、外部環境にさらされる体の最大の表面です。常駐微生物の広大な配列は、腸内細菌叢（または微生物叢）を形成するために、ヒトの腸にコロニーを形成します。これは、最大100000000000000微生物細胞からなると推定され、生物学1-3で知られている最も人口密度の高い細菌の生息地の1を構成しています。 GITでは細菌は、彼らが生き残ると4を掛け腸のニッチを植民地化。リターンでは、微生物は、そのゲノム1でエンコードされていない追加の機能的特徴を持つホストを付与します。例えば、微生物は、上皮細胞の増殖を刺激し、ホストは自分で作り出すことができないビタミンを生成し、代謝を調節し、病原体4-6から保護します。この有益な関係を考えると、いくつかの著者は、人間が「超生物」または細菌とヒト遺伝子7,8のミックスである「holobionts」であることを示唆しています。 （人間の）ホスト上の微生物叢の有益な影響を考えると、腸管免疫系はルーメンでその存在を可能にするだけでなく、腔側9-11から侵入する病原体を殺すために共生微生物を容認する必要があります。腸管免疫系は無害と潜在的に有害な管腔の微生物を区別するためのメカニズムを開発しました。しかし、これらのメカニズムはまだよく12を理解されていません。腸の整合性を維持することは寛容と免疫の13の間のバランスを保つために厳密に調節免疫恒常性を必要とします。免疫恒常性のアンバランスは、炎症性腸疾患（IBD）3,14などの腸疾患の誘導に寄与する。 クローン病（CD）および潰瘍性大腸炎（UC）：IBDの2つの主要なタイプがあります。これらの疾患を有する患者は、通常、直腸出血、重度の下痢や腹痛15,16苦しみます。 IBDの一つの原因はまだです不明ですが、遺伝的要因の組み合わせ、環境の影響および調節不全免疫応答が疾患の発症15のための重要な事象であるかもしれません。 IBDの動物モデルは、50年以上にわたって使用されてきました。過去数十年間、新しいIBDモデル系は、IBD 17,18の病因に関するさまざまな仮説をテストするために開発されてきました。慢性大腸炎の最もよく特徴付けられたモデルは、T細胞ホメオスタシス19,20の破壊を誘導し、T細胞移入モデルです。 16,21このモデルは、（ - / -およびSCIDマウスのようなRAGなど）TおよびB細胞を欠いている宿主に免疫したマウスからのナイーブT細胞を転送することを含みます。このモデルにおける疾患の発症は、下痢の存在を評価することによって、3-10週間モニター身体活動の減少、及び体重減少です。これには、消耗症候群16と呼ばれています。健康なマウスと比較して移植したホストの大腸組織は、シックですR、16短いと重いです。 T細胞移入モデルを使用して、T細胞集団は、IBD 22の病因に寄与することができる方法の異なる理解することが可能です。 T細胞移入モデルでは、抗原特異的様式で疾患プロセスにおけるAPCおよびT細胞の間の相互作用を分析しません。骨髄細胞およびリンパ系細胞間の相互作用は、腸の炎症23の発症の原因であることが示されています。 IBDの多くの側面が明らかにされてきたが、病気の開発につながる初期のイベントは、まだ明確に理解する必要があります。 微生物転写大腸炎の非存在下で24を確立できないことが示されています。最近、いくつかの説は、IBDは、共生細菌25に対する免疫応答の結果であることを示唆しています。著者らは、共生細菌は、遠位腸内の炎症を誘導するのに不可欠であることも提案しています26。生殖無料（GF）動物では腸管免疫系は、一般的に27,28を低下しているが、完全に有能な腸管免疫系29の開発における特定の病原体を含まない細菌の結果の混合物とこれらのマウスの植民地化。したがって、微生物叢は、いずれかの素因または腸の炎症30,31の開発から保護するメカニズムとして、IBDの病因における重要な要素であると思われます。現在の理論は、遺伝的素因の患者32に、IBDは、腸内毒素症と呼ばれる、微生物の不均衡の結果であることを示唆しているが、それは明らかではないが、まだ腸内毒素症は、原因や病気12の結果である場合。 IBDの発症における微生物の役割を考慮すると、 インビトロ実験において CD4 + T細胞は、腸内細菌33,34でパルスしたAPCにより活性化することができることを示しました。 また、より抗原ことが示されています例えば、Eなど、さまざまな共生細菌種、 大腸菌、バクテロイデス、ユーバクテリウムおよびプロテウスは 、CD4 + T細胞35を活性化することができます。これは、T細胞への細菌抗原の提示は、IBDの開発のために重要であることを示しています。疾患過程における微生物叢によって誘導される複数の抗原の複雑さを軽減するために、 大腸菌株は、OVA抗原を産生するように作成されています。転写性大腸炎は、RAGにOVA特異的T細胞を注射することによって誘導した- / - OVAを発現する大腸菌を保菌動物大腸菌 。 このモデルは、CX 3 CR1 +議員、結腸粘膜固有層（CLP）36の主要な細胞サブセットは、転送37を大腸炎の間にCD4 + T細胞と相互作用していることを示唆している最近の証拠に基づいています。国会議員は、その樹状突起36、38,39を使用して、細菌のような、粒子状抗原のための腸管腔をサンプリング。以前の研究国会議員はまた、腸管腔40,41に導入され、このようなOVAなどの可溶性抗原を、取ることができることを実証しました。 CLPにおけるCX 3 CR1 + MPの存在量を考えると、これらの細胞が管腔細菌をサンプリングし、CD4 T細胞と相互作用することが可能です。 E.が定着OVA特異的CD4 + T細胞を移植したマウスの共焦点イメージングコリ CFP-OVAは、CX 3 CR1 + MPSは抗原駆動型大腸炎の開発中OT-II CD4 + T細胞と接触していることを示しています。このモデルは、腸のAPCおよび唯一の腸管内腔における特定の抗原を発現する細菌に特異的なT細胞間の抗原提示プロセスの研究を可能にします。

<table border="0" cellpadding="0" cellspacing="0" width="801">
	<tbody>
		<tr height="23">
			<td height="23" style="height:23px;width:216px;">LB Broth, Miller (Luria-Bertani)</td>
			<td style="width:200px;">Difco</td>
			<td style="width:219px;">244620</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rotary Shake</td>
			<td style="width:200px;">Reiss Laborbedarf e. K.</td>
			<td style="width:219px;">Model 3020 GFL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2 mm gap couvettes&#160;</td>
			<td style="width:200px;">Peqlab Biotechnologie GmbH</td>
			<td style="width:219px;">71-2020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycerol</td>
			<td style="width:200px;">Sigma-Aldrich</td>
			<td style="width:219px;">G5516-100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gene Pulser Xcell system&#160;</td>
			<td style="width:200px;">BioRad Laboratories GmbH</td>
			<td style="width:219px;">1652660</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LB Agar, Miller (Luria-Bertani)</td>
			<td style="width:200px;">Difco</td>
			<td style="width:219px;">244510</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ampicillin</td>
			<td style="width:200px;">Sigma-Aldrich</td>
			<td style="width:219px;">A9393-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SOC Medium</td>
			<td style="width:200px;">Sigma-Aldrich</td>
			<td>S1797-100ML</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;width:216px;">High Pure Plasmid Isolation Kit</td>
			<td style="width:200px;">Roche</td>
			<td style="width:219px;">11754777001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Agarose</td>
			<td style="width:200px;">Carl Roth GmbH &#38; Co</td>
			<td style="width:219px;">3810.1</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:216px;">EDTA</td>
			<td style="width:200px;">Sigma-Aldrich</td>
			<td style="width:219px;">E9884-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris-HCl</td>
			<td style="width:200px;">Sigma-Aldrich</td>
			<td style="width:219px;">T5941</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glacial acetic acid</td>
			<td style="width:200px;">Sigma-Aldrich</td>
			<td style="width:219px;">537020&#160;</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Gel chamber&#160;</td>
			<td style="width:200px;">PEQLAB Biotechnology GmbH</td>
			<td style="width:219px;">40-0708</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Loading Dye</td>
			<td style="width:200px;">Thermo Fisher</td>
			<td style="width:219px;">R0611</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GeneRuler 1 kb DNA Ladder&#160;</td>
			<td style="width:200px;">Thermo Fisher</td>
			<td style="width:219px;">SM0312</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethidium bromide solution</td>
			<td style="width:200px;">Carl Roth GmbH &#38; Co. KG</td>
			<td style="width:219px;">2218.3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Photo-documentation system&#160;</td>
			<td style="width:200px;">Decon Science Tech GmbH</td>
			<td style="width:219px;">DeVision G&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DNA sequencing&#160;</td>
			<td style="width:200px;">MWG-Biotech GmbH</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Phosphate buffered saline (PBS)</td>
			<td style="width:200px;">Biochrom</td>
			<td>L182-50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fluorescent microscope&#160;</td>
			<td style="width:200px;">Zeiss</td>
			<td style="width:219px;">HBO 100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mini-PROTEAN Tetra System</td>
			<td style="width:200px;">Bio-Rad Laboratories GmbH</td>
			<td style="width:219px;">1658005</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PageRuler Prestained Protein Ladder&#160;</td>
			<td style="width:200px;">Fermentas, St. Leon-Rot, Germany</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">IstanBlue Solution</td>
			<td style="width:200px;">Expedeon, Cambridgeshire, United Kingdom</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nitrocellulose membrane&#160;</td>
			<td style="width:200px;">Macherey-Nagel GmbH &#38; Co. KG</td>
			<td>741280</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Electro blotter&#160;</td>
			<td style="width:200px;">Biometra GmbH</td>
			<td style="width:219px;">846-015-600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bovine Serum Albumins (BSA)</td>
			<td style="width:200px;">Sigma-Aldrich</td>
			<td style="width:219px;">A6003-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-Ovalbumin antibody&#160;</td>
			<td style="width:200px;">Abcam</td>
			<td style="width:219px;">ab181688</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-rabbit IgG&#160; HRP</td>
			<td style="width:200px;">Sigma-Aldrich</td>
			<td style="width:219px;">A0545&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pierce ECL Plus Western Blotting Substrate</td>
			<td style="width:200px;">Pierce Biotechnology, Thermo Fischer Scientific Inc</td>
			<td style="width:219px;">32132</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Forene</td>
			<td>Abbott</td>
			<td style="width:219px;">2594.00.00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FBS</td>
			<td style="width:200px;">Invitrogen</td>
			<td style="width:219px;">10500-064</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Falcon Cell Strainers</td>
			<td style="width:200px;">Fischer Scientific&#160;</td>
			<td style="width:219px;">08-771-19</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ammonium chloride</td>
			<td style="width:200px;">Sigma-Aldrich</td>
			<td style="width:219px;">254134-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris Base</td>
			<td style="width:200px;">Sigma-Aldrich</td>
			<td style="width:219px;">10708976001</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:216px;">CD4+ CD62 L+ T isolation kit&#160;</td>
			<td style="width:200px;">Miltenyi Biotec</td>
			<td style="width:219px;">130-093-227&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MACS LS Columns&#160;</td>
			<td style="width:200px;">Miltenyi Biotec</td>
			<td style="width:219px;">130-042-401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MACS MS Columns&#160;</td>
			<td style="width:200px;">Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MidiMACS Separator</td>
			<td style="width:200px;">Miltenyi Biotec</td>
			<td style="width:219px;">130-042-302</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MiniMACS Separator</td>
			<td style="width:200px;">Miltenyi Biotec</td>
			<td style="width:219px;">130-042-102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MACS MultiStand</td>
			<td style="width:200px;">Miltenyi Biotec</td>
			<td style="width:219px;">130-042-303</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Feeding Needle 20G</td>
			<td style="width:200px;">SouthPointe Surgical Supply, Inc</td>
			<td style="width:219px;">FN-7903</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Formalin solution, neutral buffered, 10%</td>
			<td style="width:200px;">Sigma-Aldrich</td>
			<td style="width:219px;">HT501128</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Paraffin</td>
			<td style="width:200px;">Sigma-Aldrich</td>
			<td style="width:219px;">1496904</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hematoxylin</td>
			<td style="width:200px;">Sigma-Aldrich</td>
			<td style="width:219px;">H9627</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Eosin Y</td>
			<td style="width:200px;">Sigma-Aldrich</td>
			<td style="width:219px;">230251&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dithiothreitol</td>
			<td style="width:200px;">Sigma-Aldrich</td>
			<td style="width:219px;">D9779&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Collagenase type VIII</td>
			<td style="width:200px;">Sigma-Aldrich</td>
			<td style="width:219px;">C-2139</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Roswell Park Memorial Institute (RPMI) medium</td>
			<td style="width:200px;">AppliChem</td>
			<td style="width:219px;">A2044, 9050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Percoll (density 1.124 g/ml)</td>
			<td style="width:200px;">Biochrome</td>
			<td style="width:219px;">L-6145</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium azide</td>
			<td style="width:200px;">Sigma-Aldrich</td>
			<td style="width:219px;">438456</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mouse BD Fc Block</td>
			<td style="width:200px;">BD Pharmingen</td>
			<td style="width:219px;">553141</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">FITC-conjugated mAb binding V&#223; 5.1, 5.2&#160;</td>
			<td style="width:200px;">BD Pharmingen</td>
			<td style="width:219px;">553189</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">APC-conjugated mAb binding CD4 GK1.5&#160;</td>
			<td style="width:200px;">eBioscience</td>
			<td style="width:219px;">17-0041-83</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FACS Calibur&#160;</td>
			<td style="width:200px;">BD Biosciences</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FCS Express V3 software</td>
			<td style="width:200px;">DeNovo</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Meta scanning confocal microscope&#160;</td>
			<td style="width:200px;">Zeiss</td>
			<td style="width:219px;">LSM 710&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Zeiss Workstation</td>
			<td style="width:200px;">Zeiss</td>
			<td style="width:219px;">LSM 7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Zeiss ZEM software&#160;</td>
			<td style="width:200px;">Zeiss</td>
			<td style="width:219px;">v4.2.0.121</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Maxisorp immuno plates&#160;</td>
			<td style="width:200px;">NUNC, Roskilde</td>
			<td style="width:219px;">442404</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Streptavidin conjugated alkaline phosphatase</td>
			<td style="width:200px;">Jackson Immuno Research</td>
			<td style="width:219px;">016-050-084</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Alkaline phosphatase substrate 4-Nitrophenyl phosphate disodium salt hexahydrate</td>
			<td style="width:200px;">Sigma-Aldrich</td>
			<td style="width:219px;">71768-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">mAb R4-6A2</td>
			<td style="width:200px;">BD Biosciences</td>
			<td style="width:219px;">551216</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">mAb XMG1.2&#160;</td>
			<td style="width:200px;">BD Biosciences</td>
			<td>554410</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TECAN microplate-ELISA reader</td>
			<td style="width:200px;">Tecan</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EasyWin software</td>
			<td style="width:200px;">Tecan</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

development of an antigen-driven colitis model to study presentation of antigens by antigen presenting cells to t cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/- recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts.
This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

抗原駆動型大腸炎モデルEを確立するために、 大腸菌株 CFPの遺伝子は、ニワトリオボアルブミンタンパク質のコード配列に融合され、融合構築物は、強力な構成的プロモーターP ハイパー （ 図1A）の制御下で発現されるプラスミドを含むように構成されています。蛍光顕微鏡は、組換えE.ことを示しています?...

Watch this Scientific Journal Video about Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells at JoVE.com

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

T細胞への抗原提示細胞による抗原の提示を研究するための抗原駆動性大腸炎モデルの開発

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...

development-an-antigen-driven-colitis-model-to-study-presentation

Research

JoVE Journal

Immunology and Infection

8.9K ビュー.  University College Cork. In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

ビデオ: Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals may exhibit suppressive immune microenvironment and, in contrast to activating CTL, their autologous antigen presenting cells may tend to tolerize or anergize antigen specific CTL. As a result, although still in the experimental phase, CTL-based adoptive immunotherapy has evolved to become a promising treatment for various diseases such as cancer and virus infections. In initial experiments ex vivo expanded CMV (cytomegalovirus) specific CTL have been used for treatment of CMV infection in immunocompromised allogeneic bone marrow transplant patients. While it is common to have life-threatening CMV viremia in these patients, none of the patients receiving expanded CTL develop CMV related illness, implying the anti-CMV immunity is established by the adoptively transferred CTL1. Promising results have also been observed for melanoma and may be extended to other types of cancer2. 
While there are many ways to ex vivo stimulate and expand human CTL, current approaches are restricted by the cost and technical limitations. For example, the current gold standard is based on the use of autologous DC. This requires each patient to donate a significant number of leukocytes and is also very expensive and laborious. Moreover, detailed in vitro characterization of DC expanded CTL has revealed that these have only suboptimal effector function 3. 
Here we present a highly efficient aAPC based system for ex vivo expansion of human CMV specific CTL for adoptive immunotherapy (Figure 1). The aAPC were made by coupling cell sized magnetic beads with human HLA-A2-Ig dimer and anti-CD28mAb4. Once aAPC are made, they can be loaded with various peptides of interest, and remain functional for months. In this report, aAPC were loaded with a dominant peptide from CMV, pp65 (NLVPMVATV). After culturing purified human CD8+ CTL from a healthy donor with aAPC for one week, CMV specific CTL can be increased dramatically in specificity up to 98% (Figure 2) and amplified more than 10,000 fold. If more CMV-specific CTL are required, further expansion can be easily achieved by repetitive stimulation with aAPC. Phenotypic and functional characterization shows these expanded cells have an effector-memory phenotype and make significant amounts of both TNF&alpha; and IFN&gamma; (Figure 3).

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

A new DC independent method for induction and expansion of antigen-specific T cells is described. HLA A2-Ig based artificial Antigen Presenting Cells (aAPC) are loaded with HLA-A2 restricted peptides to efficiently expand CTL of diverse antigen specificity. This technology holds great potential for CTL-based adoptive immunotherapy.

HLA - Igは効率のためのベースの人工抗原提示細胞生体外で</emヒトCTLの>拡大

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals ...

免疫・感染学

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

細胞運命を以下に<em&gt; E大腸菌の</emラムダファージによる感染後&gt;

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules2. NKT cells express an invariant TCR&alpha; chain rearrangement: V&alpha;14J&alpha;18 in mice and V&alpha;24J&alpha;18 in humans, which is associated with V&beta; chains of limited diversity3-6, and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d 7. The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with &alpha;-Galactosylceramide (&alpha;-GalCer) and a variety of cytokines8. Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines9,10. In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients11. However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells12, 13. Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is a fundamental requirement of NKT cell activation, antigen: CD1d-Ig complexes provide a reliable method to isolate, activate, and expand effector NKT cell populations.

Artificial Antigen Presenting Cell aAPC Mediated Activation and Expansion of Natural Killer T Cells

Here we describe a method for activating and expanding human NKT cells from bulk T cell populations using artificial antigen presenting cells (aAPC). The use of CD1d-based aAPC provides a standardized method for generating high numbers of functional NKT cells.

ナチュラルキラーT細胞の細胞（AAPC）媒介活性化と増殖を提示する人工抗原

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike ...

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

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

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

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

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

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

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGF&beta; and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown.
Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44low, CD62Lhigh) and resting (CD25-, CD69-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.

Adenoviral gene transfer into naive CD4 T cells with transgenic expression of the Coxsackie adenovirus receptor enables the molecular analysis of regulatory T cell differentiation in vitro.

ナイーブCD4のアデノウイルス形質導入Tregの分化を研究するためにT細胞

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

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 ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

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

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

T細胞は、樹状細胞からTransinfectionによって細菌をキャプチャ

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

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 ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

超高感度単一分子の開発と検証配列人間のインターフェロン-α のデジタルの酵素免疫測定法

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...