著者は、ライブイメージングのヘルプのためのロチェスター大学の多光子顕微鏡中核施設に感謝します。 DJFにNIH AI072690とAI02851によってサポートされています。 MGOへAGおよびAI089079にAI114036。

著者らは、開示することは何もありません。

意義 ここでは、無傷のマウス耳真皮における転送、抗原特異的なエフェクターTh1細胞の4D可視化のための完全なプロトコルを提示します。この方法は、いくつかの理由のために、いくつかの現在の撮像モダリティ上の利点を提供します。腹側の耳の真皮を画像化することによって、我々は他の皮膚部位を含むイメージング・プロトコルのために必要とされる脱毛を見送ることができます。脱毛剤は、一般的に軽度であるが、それらは皮膚バリア42、免疫応答43,44を刺激することができるプロセスに混乱を引き起こすことが示されています。また、真皮や皮下組織を露出させるために侵襲的な外科的処置を回避することにより、このプロトコルは、損傷誘発炎症と真皮への好中球37および他の免疫細胞の迅速な動員を防止します。重要な分子に対するブロッキング抗体を提供するために、このシステムにおける静脈カテーテルの使用は、DYNAのリアルタイム尋問を可能にしますCD4 T細胞のマイク行動。この撮像プロトコールを使用することは、 インビトロ系8で検出されなかったCD4 T細胞間質運動30のための重要な要件を明らかにしました。 手順における重要なステップ 任意の撮影プロトコルに不可欠なステップは、イメージングのための安定した組織標本を保証することです。気泡がないことを耳とカバーガラスの間に十分なPBSがあることを確認することが重要であると耳は、テープがガラスから脱付着しなくなる場合がするように、ガラスと接触しているが、それほどではありません。同様に、プラットフォームおよび任意の真空グリースにカバースリップを保持するテープとの接触を避けることが時間の経過とともに緩みからテープを防ぐことができます。温度はまた、振動を避け、熱収縮またはプラットフォーム材料の拡大からドリフトさ一定に保たなければなりません。 制限事項および修正 、45生体タイムラプスイメージングのためのモデルとして使用されています。しかし、耳の居住免疫人口は脇腹やフットパッド46上の皮膚から区別され、他のサイト47と比較した場合、マウスの耳には、明確な血管の特性を有しています。したがって、いくつかの用途のために、他の皮膚部位に耳撮像データの比較は困難であり得ます。 このプロトコルはまた、血流から出て、真皮に移しTエフェクター細胞の効果的な移動を必要とします。これは、彼らが、間質スペースを入力することができなくなりますようホーミングまたは溢出に欠陥を有する細胞を使用する能力が制限されます。溢出これは生体内血管外漏出に受ける細胞の局在や動作を再現しないことがあり、このようにいくつかの機械的損傷および細胞の送達を引き起こしますが、直接耳真皮37,48に細胞を注入することによってバイパスすることができます。 このようなここで使用したBALB / c系統またはアルビノC57BL / 6 のTyr のc-2Jマウスなどのイメージング実験のための非着色レシピエントマウスを使用することを検討することも重要です。着色されたマウスにおけるメラニンは、高い自己蛍光であることに加えて、多光子レーザ37からの比較的低電力の励起下で加熱します。これは、結果を複雑に、皮膚およびその後の炎症36や蛍光斑点31への熱損傷を引き起こす可能性があります。これは、マルチパラメータ画像化実験に使用され得るフルオロフォアを制限することができます。しかし、いくつかの非常に明るいまたは高度に発現蛍光分子はすべて、低レーザパワーで効果的に励起することができます着色されたマウスでの効果的な可視化のために起因します。 将来の応用 これは、非侵襲的な手順であるため、容易に長期間にわたってシーケンシャルイメージングを有するマウスの縦断的研究のために適合させることができます。ここで説明したように蛍光標識が3-4日超の期間にわたってフェードだろうが、内因的に蛍光細胞または蛍光レポーター細胞の使用は、この問題を解消します。確かに、我々は以前にIFNγレポーターイエティ30,49を含むサイトカインの記者を、ベアリングvivo-生成された抗原特異的なエフェクターで追跡するために、このプロトコルを使用していたし、IL-4レポーターは50を 4get。我々はさらに、CD4-CreをROSA26・ストップ・フロックスのeYFP蛍光レポーターマウス30で内因性CD4細胞を可視化しています。 eYFPおよび他の蛍光タンパク質の蛍光特性は、CFSEなどの化学染料とは異なるように、撮像パラメータの修正が必要になることがあります効率的な可視化のため。そのようないくつかの薄暗い蛍光団の蛍光シグナルを増強することができる画素の滞留時間を増加し、観察することができる任意の光退色を軽減することができるタイムラプス画像の長さを減少させるように変化します。 900 nm励起では、我々は以前に短い撮影間隔でCFSE、CMTMR、またはのeYFPの有意な光退色を確認されていません。 この手順は、CD4エフェクターT細胞の運動性のダイナミクスを測定することに焦点を当てているが、それはこの用途に限定されるものではありません。研究は、抗原の前イメージング51,52への細胞または組織構造を標識するために真皮内に蛍光レポーターマウスおよび蛍光標識抗体の注射の使用を介して提示細胞とエフェクターT細胞の動的相互作用を測定するために現在進行中です。このプロトコルは、遮断抗体を送達するためのカテーテルの使用を示し、一方また、一方の撮像は、小分子阻害剤を含む他の化合物は、缶投与することができます。最終的に、このプロトコルは、非侵襲的な方法で、 生体内で 、経時的免疫動態を測定するための柔軟なプラットフォームを提供します。

The effector function of CD4 T cells is critically dependent on their ability to rapidly enter and traverse a wide variety of peripheral tissues to survey for damage, locate foci of infection, or cause pathology from chronic infection or autoimmunity. While the processes of homing to inflamed sites1-4 and extravasation5-7 from the vasculature into tissues have been well-characterized, the factors that drive and regulate the interstitial motility of T cells remain undefined. The migration of T cells in complex 3D environments has been studied in vitro through the use of artificial matrices8-10 or microfluidic devices11,12, but these fail to recapitulate the complex and dynamic environment of an in vivo system. It is only recently, with the advent of high-resolution multi-color intravital imaging that it has become possible to study the dynamic behavior of immune cells in situ, allowing for a better understanding of intact immune responses.
Over a decade ago, several influential studies were published that first utilized multiphoton microscopy to address immunological questions. Early studies focused on the behavior of immune cells within explanted lymphoid organs13-16, which were soon followed by techniques to image exposed lymph nodes in anesthetized mice17. Imaging allowed for new fundamental observations about the stages of lymph node priming of T cells18, the mechanisms by which T cells migrate in secondary lymphoid organs19, T cell interactions with other immune cells20,21, and dynamic T cell positioning within the lymph node22. Although many early studies focused on lymph node dynamics, intravital imaging has been since been utilized to image the immune response in many peripheral tissues, including the brain23-25, liver26, lung27, and skin28-30. 
The mouse ear dermis is particularly well poised for imaging, due to the thinness of ear skin, a relative lack of hair, and the ease with which it can be isolated from respiratory movements31. Indeed, the ear dermis has been used to image the interstitial behavior of dendritic cells32,33, T cells28,29,34,35, and neutrophils36,37, and is a well-established site for studying dermal inflammation. Increasingly, non-invasive procedures have been replacing surgical preparations of the skin, including split dermis38,39, flank39,40, or dorsal skin flap window39,41 models, that can induce changes to the local inflammatory milieu. The use of transferred, in vitro-primed, antigen-specific CD4 effector T cells allows for the study of a homogenous population of cells in the context of a dermal inflammatory response30. Here we describe a non-invasive imaging procedure that allows for the visualization of antigen-specific effector CD4 T cells in the dermal interstitium of the inflamed mouse ear, and the ability to manipulate these cells in real-time by introducing blocking antibodies through a venous catheter. We show that this model is effective for tracking the movement of CD4 T cells in the dermis and for querying the mechanisms that govern this motility.

<table border="0" cellpadding="0" cellspacing="0" width="1060">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">BALB/c mice</td>
			<td style="width:145px;">Jackson Laboratories</td>
			<td style="width:196px;">000651</td>
			<td style="width:472px;">Mice used were bred in-house</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">DO11.10 mice</td>
			<td>Jackson Laboratories</td>
			<td>003303</td>
			<td>Mice used were bred in-house</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">HBSS</td>
			<td>Fisher</td>
			<td>10-013-CV</td>
			<td>Multiple Equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Newborn Calf Serum (NCS)</td>
			<td>Thermo/HyClone</td>
			<td>SH30118.03</td>
			<td>Heat inactivated at 56 &#176;C for 30 minutes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Guinea Pig Complement</td>
			<td>Cedarlane</td>
			<td>CL-5000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">anti-CD8 antibody</td>
			<td>ATCC</td>
			<td>3.155 (ATCC TIB-211)</td>
			<td>Antibodies derived from&#160; this hybridoma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">anti-MHC Class II antibody</td>
			<td>ATCC</td>
			<td>M5/114.15.2 (ATCC TIB-120)</td>
			<td>Antibodies derived from&#160; this hybridoma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">anti-CD24 antibody</td>
			<td>ATCC</td>
			<td>J11d.2 (ATCC TIB-183)</td>
			<td>Antibodies derived from&#160; this hybridoma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">anti-Thy1.2 antibody</td>
			<td>ATCC</td>
			<td>J1j.10 (ATCC TIB-184)</td>
			<td>Antibodies derived from&#160; this hybridoma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Ficoll (Fico/Lite-LM)</td>
			<td>Atlanta Biologicals</td>
			<td>I40650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">PBS</td>
			<td>Fisher</td>
			<td>21-040-CV</td>
			<td>Multiple Equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">EDTA</td>
			<td>Fisher</td>
			<td>15323591</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">biotinylated anti-CD62L antibody (clone MEL-14)</td>
			<td>BD</td>
			<td>553149</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">streptavidin magnetic separation beads</td>
			<td>Miltenyi</td>
			<td>130-048-101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">MACS LS Separation Column</td>
			<td>Miltenyi</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">recombinant human IL-2</td>
			<td>Peprotech</td>
			<td>200-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">recombinant mouse IL-4</td>
			<td>Peprotech</td>
			<td>214-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">recombinant mouse IL-12</td>
			<td>Peprotech</td>
			<td>210-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">anti-IFNg antibody (clone XMG 1.2)</td>
			<td>eBioscience</td>
			<td>16-7311-85</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">anti-IL-4 antibody (clone 11b11)</td>
			<td>eBioscience</td>
			<td>16-7041-85</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">RPMI</td>
			<td>VWR</td>
			<td>45000-412</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Penicillin/Streptomycin</td>
			<td>Fisher</td>
			<td>15303641</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">L-glutamine</td>
			<td>Fisher</td>
			<td>15323671</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">2-mercaptoethanol</td>
			<td>Bio-Rad</td>
			<td>161-0710</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">ovalbumin peptide</td>
			<td>Biopeptide</td>
			<td></td>
			<td>ISQAVHAAHAEINEAGR-OH peptide</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Fetal Calf Serum (FCS)</td>
			<td>Thermo/HyClone</td>
			<td>SV30014.03</td>
			<td>Heat inactivated at 56 &#176;C for 30 minutes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">24-well culture plate</td>
			<td>LPS</td>
			<td>3526</td>
			<td>Multiple Equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">CFSE</td>
			<td>Life Technologies</td>
			<td>C34554</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">CMTMR</td>
			<td>Life Technologies</td>
			<td>C2927</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">28 G1/2 insulin syringes, 1ml</td>
			<td>BD</td>
			<td>329420</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">28 G1/2 insulin syringes, 300&#956;l</td>
			<td>BD</td>
			<td>309301</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">27 G1/2 TB syringes, 1ml</td>
			<td>BD</td>
			<td>309623</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">30 G1/2 needles</td>
			<td>BD</td>
			<td>305106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">PE-10 medical tubing</td>
			<td>BD</td>
			<td>427400</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">cyanoacrylate veterinary adhesive (Vetbond)</td>
			<td>3M</td>
			<td>1469SB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">heating plate</td>
			<td>WPI</td>
			<td>61830</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Heating plate controller</td>
			<td>WPI</td>
			<td>ATC-2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Water blanket controller</td>
			<td>Gaymar</td>
			<td>TP500</td>
			<td>No longer in production, newer equivalent available</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">water blanket</td>
			<td>Kent Scientific</td>
			<td>TP3E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Isoflurane vaporizer</td>
			<td>LEI Medical</td>
			<td>Isotec 4</td>
			<td>No longer in production, newer equivalent available</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">isoflurane</td>
			<td>Henry Schein</td>
			<td></td>
			<td>Ordered through Veterinary staff</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">microcentrifuge tubes</td>
			<td>VWR</td>
			<td>20170-038</td>
			<td>Multiple Equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">medical tape</td>
			<td>3M</td>
			<td>1538-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">isoflurane nosecone</td>
			<td></td>
			<td></td>
			<td>Built In-house, see Fig 2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">imaging platform</td>
			<td></td>
			<td></td>
			<td>Built In-house, see Fig 2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">curved forceps</td>
			<td>WPI</td>
			<td>15915-G</td>
			<td>Multiple Equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">scissors</td>
			<td>Roboz</td>
			<td>RS-6802</td>
			<td>Multiple Equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">glass coverslips</td>
			<td>VWR</td>
			<td></td>
			<td>Multiple Equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">high vacuum grease</td>
			<td>Fisher</td>
			<td>146355D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">cotton swabs</td>
			<td></td>
			<td></td>
			<td>Multiple Equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">delicate task wipes</td>
			<td>Fisher</td>
			<td>34155</td>
			<td>Multiple Equivalent</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:247px;">Olympus Fluoview 1000 AOM-MPM upright microscope with Spectra-Physics MaiTai HP DeepSee Ti:Sa laser</td>
			<td>Olympus</td>
			<td></td>
			<td>call for quote</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">optical table with vibration control</td>
			<td>Newport</td>
			<td></td>
			<td>call for quote</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">25x NA 1.05 water immersion objective for multiphoton imaging</td>
			<td>Olympus</td>
			<td>XLPLN25XWMP2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">objective heater</td>
			<td>Bioptechs</td>
			<td>PN 150815</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Detection filter cube</td>
			<td>Olympus</td>
			<td>FV10-MRVGR/XR</td>
			<td>Proprietary cube, can be approximated from individual filters/dichroics</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">anti-integrin &#946;1 antibody (clone hMb1-1)</td>
			<td>eBioscience</td>
			<td>16-0291-85</td>
			<td>Azide free, low endotoxin</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">anti-integrin &#946;3 antibody (clone 2C9.G3)</td>
			<td>eBioscience</td>
			<td>16-0611-82</td>
			<td>Azide free, low endotoxin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Texas Red Dextran (70,000 MW)</td>
			<td>Life Technologies</td>
			<td>D-1830</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging cd4 t cell interstitial migration in the inflamed dermis

エフェクター機能を実行するCD4 T細胞の能力は、未未定義の機構を介して炎症の末梢組織におけるこれらの細胞の迅速かつ効率的な移行に依存します。免疫系の研究に多光子顕微鏡の応用は、無傷の組織内の免疫応答の動態を測定するためのツールを提供しています。ここでは、炎症を起こしたマウス耳真皮におけるCD4 T細胞の非侵襲的な生体内多光子イメージングのためのプロトコルを提示します。カスタムイメージングプラットフォームの使用及び静脈カテーテルは、運動性に関与する重要な分子成分に対する抗体をブロッキング添加によってリアルタイムでこれらの細胞に問い合わせする能力を、皮膚間質におけるCD4 T細胞の動態の可視化を可能にします。このシステムは、in vitroモデルおよび外科的侵襲的な画像化手順の両方に勝る利点を提供します。運動のためのCD4 T細胞によって使用される経路を理解することは、最終的にバシへの洞察を提供することができますCのCD4 T細胞の機能だけでなく、慢性感染症からの両方の自己免疫疾患の病因と病理。

免疫環境を変更することなく、 その場での免疫応答を研究する能力は、炎症組織でTエフェクター細胞のリアルタイム相互作用の研究に不可欠です。 図1AおよびBで概説し、このプロトコルにより、無傷の耳の真皮のイメージングは、皮膚の間質における転送蛍光標識されたTエフェクター細胞の可視化を可能にします。これは、高解像度（ 図1C）とタイムラプス（ 図1D、 映画1）炎症を起こし、真皮内のエフェクターT細胞動態の画像の両方を可能にします。 <img alt="図1" src="/files/ftp_upload/53585/53585fig1.jpg" /> 図1.高解像度と無傷の皮膚の間質におけるTエフェクター細胞の4Dイメージング 。 （A）実験概要。耳preparの（B）写真エマルジョンの部位とイメージング（黒破線）およびイメージングのための最適な領域（赤線）のためのedが示されました。 （C）最大の3D CFSE標識Th1細胞（緑）を示す高解像度のスタックの投影、線維状コラーゲン（青）からの第二高調波信号およびテキサスレッドデキストラン標識された血管系（赤）。白い矢印は、いくつかの自家蛍光毛包のいずれかを示します。スケールバーは、CFA-炎症を起こし真皮に30分間追跡Th1細胞の50ミクロン（D）回遊パスを表す。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53585/53585fig1large.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> この撮影プロトコルは、社内で構築した特殊なイメージングプラットフォームだけでなく、撮像しながら吸入麻酔の送達のための適応ノーズコーンを使用する必要があります。イメージングプラットフォーム（ 図2A-B）は Oで構成さ隆起した中央部分でアルミベースプレートF。この隆起部分はアクリルと並ぶはめ込み部分が圧縮して、潜在的に薄い組織に損傷を与えることなく、耳へのサポートを提供するために感じています。私たちのセットアップの幾何学的形状は、撮像中に吸入麻酔を提供するために、柔軟で調整可能なノーズコーンの構築を必要としました。フレキシブルチューブ（ 図2C）の50ミリ部に接続された変更されたマイクロチューブからなるこのノーズコーンは、修正されたマイクロチューブからなるホルダー（ 図2D）で適所に固定されたフックとループファスナーを介して撮影台に固定されています。マウスのいずれかの耳を撮像することができ、最適な個々のマウスのために配置することができるように面ファスナーの使用は、ノーズコーンアセンブリの再配置を可能にします。 <img alt="図2" src="/files/ftp_upload/53585/53585fig2.jpg" /> 図2.エクイ生体内イメージングのためのpment。トップ（A）および側面（B）ビューのカスタム構築されたイメージングプラットフォーム。イソフルラン投与（C）とノーズコーンホルダー（D）のためのノーズコーン。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53585/53585fig2large.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> 尾静脈のカテーテル法は、抗体と血管系の外に拡散することができる他の小分子を投与するために、循環への継続的なアクセスを可能にする、またはそのような高分子量のデキストランのようなより大きな蛍光分子は、血管を標識します。抗β1およびカテーテルを通してブロッキング抗体抗β3インテグリンを100μgを投与した後、以前に運動性細胞は、真皮（ 動画2）内で逮捕します。これらの細胞は、抗体後に減少した平均速度を持っています<html>管理（ 図3A）、ならびに蛇行指数の有意な減少、総トラック長（ 図3B）への総変位の比率。 <img alt="図3" src="/files/ftp_upload/53585/53585fig3.jpg" /> 抗β1 および抗β3 抗体 の 図3. 投与は、CFA 炎症を起こした皮膚におけるTh1細胞の遊走を阻害する。（A）Th1細胞の平均速度を前にし、100μgの抗β1および抗β3インテグリンの投与後ブロッキング抗体を。 （B）蛇行前Th1細胞の指標と抗体遮断後。約100追跡された細胞は前と1代表的な実験における単一のマウスから抗体遮断後の画像からのものです。マン・ホイットニー別統計。エス/ ftp_upload / 53585 / 53585fig3large.jpg &quot;ターゲット=&quot; _空白 &quot;&gt;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。 毛髪は、耳の表面から除去されないので、毛髪からの撮像アーチファクトが一般的です。彼らはT細胞を曖昧にし、自動画像解析ソフトウェアを妨害することができるように、毛包（ 図4A）及びその上毛（ 図4B）からシャドーイングおよび自己蛍光からの自家蛍光は、可能な限り避けるべきです。同様に、耳の表面とカバーガラスとの間に捕捉された気泡は、撮像アーチファクト（ 図4C）をもたらすことができます。耳の適切な準備が残っている泡の数とサイズを最小限に抑える必要があります。 <img alt="図4" src="/files/ftp_upload/53585/53585fig4.jpg" /> 図4。 髪の自家蛍光と貧しい耳の前から一般的な成果物paration。（A）自己蛍光毛包。画像内の暗線を引き起こし、毛の影の上にあると（B）自己蛍光の毛と毛包（緑）、コラーゲン（白）。 （C）気泡からアーティファクト、変位、自家蛍光角化表皮（点線）のエッジを示します。スケールバーは50μmで表す。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53585/53585fig4large.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> さらに、熱の複数のソースを使用すると、熱膨張収縮に起因する組織の安定性の問題につながることができます。不適切なサーモスタットの設定は、例えば、（ 動画3）難しい結果の解釈を行うことができます撮影時の大きな振動につながることができます。一定の温度を提供し、安定性を最大化する任意のシステムに最適な設定を決定することが必須です。 Ultimatエリー、温度制御された画像化チャンバは、温度変化から変動を除去するための最良の方法です。 <img alt="映画1" src="/files/ftp_upload/53585/53585movie1.jpg" /> <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53585/gaylomovie1.mov" target="_blank">CFA-炎症真皮に移行ムービー1. Th1細胞は、（右クリックしてダウンロード）。</a> 30分のタイムラプス画像は、真皮コラーゲンネットワークに移行するTh1細胞（緑）（第二高調波発生、青）を示します。 <img alt="ムービー2" src="/files/ftp_upload/53585/53585movie2.jpg" /> <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53585/gaylomovie2.mov" target="_blank">β1の遮断と3インテグリンをβ時のムービー2. Th1細胞の停止（右クリックしてダウンロード）。</a>細胞（緑）administ前に30分間画像化しましたブロッキング抗体の比と同じ場所にさらに20分間撮像しました。 <img alt="ムービー3" src="/files/ftp_upload/53585/53585movie3.jpg" /> <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53585/gaylomovie3.mov" target="_blank">コントロール不良の加熱プレートからムービー3.振動（右クリックしてダウンロード）。</a> 30分タイムラプスTh1細胞（緑）の画像と第2高調波発生（青）。この画像を収集した後、使用される加熱プレートを画像化プラットフォームとそれに続く熱膨張と収縮の温度で振動を引き起こし、不良サーモスタットを有することが見出されました。

Watch this Scientific Journal Video about Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis at JoVE.com

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

炎症真皮における撮像CD4 T細胞間質への移行

炎症部位でのCD4エフェクターT細胞の間質運動を支配するメカニズムは、比較的知られていません。我々は、in vitroで視覚化して操作するための非侵襲的なアプローチは、その場でこれらの細胞の動的挙動の研究を可能にする、炎症を起こした耳の真皮中のCD4 T細胞を-primed提示します。

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

imaging-cd4-t-cell-interstitial-migration-in-the-inflamed-dermis

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

10.6K ビュー.  University of Rochester School of Medicine and Dentistry, Rochester, NY. 炎症部位でのCD4エフェクターT細胞の間質運動を支配するメカニズムは、比較的知られていません。我々は、in vitroで視覚化して操作するための非侵襲的なアプローチは、その場でこれらの細胞の動的挙動の研究を可能にする、炎症を起こした耳の真皮中のCD4 T細胞を-primed提示します。 

ビデオ: Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

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

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

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

マウスT細胞におけるT細胞受容体のレトロウイルス導入

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

免疫・感染学

Competitive Homing Assays to Study Gut-tropic T Cell Migration

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial cells and tissue extravasation is a multistep process controlled by different adhesion molecules (homing receptors) expressed on lymphocytes and their respective ligands (addressins) displayed on endothelial cells 1 2. Even though the function of these adhesion receptors can be partially studied ex vivo, the ultimate test for their physiological relevance is to assess their role during in vivo lymphocyte adhesion and migration. Two complementary strategies have been used for this purpose: intravital microscopy (IVM) and homing experiments. Although IVM has been essential to define the precise contribution of specific adhesion receptors during the adhesion cascade in real time and in different tissues, IVM is time consuming and labor intensive, it often requires the development of sophisticated surgical techniques, it needs prior isolation of homogeneous cell populations and it permits the analysis of only one tissue/organ at any given time. By contrast, competitive homing experiments allow the direct and simultaneous comparison in the migration of two (or even more) cell subsets in the same mouse and they also permit the analysis of many tissues and of a high number of cells in the same experiment.
Here we describe the classical competitive homing protocol used to determine the advantage/disadvantage of a given cell type to home to specific tissues as compared to a control cell population. We chose to illustrate the migratory properties of gut-tropic versus non gut-tropic T cells, because the intestinal mucosa is the largest body surface in contact with the external environment and it is also the extra-lymphoid tissue with the best-defined migratory requirements. Moreover, recent work has determined that the vitamin A metabolite all-trans retinoic acid (RA) is the main molecular mechanism responsible for inducing gut-specific adhesion receptors (integrin a4b7and chemokine receptor CCR9) on lymphocytes. Thus, we can readily generate large numbers of gut-tropic and non gut-tropic lymphocytes ex vivoby activating T cells in the presence or absence of RA, respectively, which can be finally used in the competitive homing experiments described here.

Competitive homing experiments allow to directly assessing the migratory properties of two different cell populations in a single mouse. Here we illustrate this procedure by comparing the migration of ex vivo-generated gut-tropic versus non-gut tropic T cells.

腸管指向性T細胞の移動を研究するために競争力のあるホーミングアッセイ

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial ...

A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles1,2,3. Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread4,5. Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell6,7,8. However, many biological questions about cell migration remain unanswered.
The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers.
The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip9,10. With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells11,12, fibroblasts9, neutrophils13, skeletal muscle cells14, keratinocytes15, trophoblasts16, endothelial cells17, and monocytes10,18-22. The protocol involves the creation of slides coated with gold nanoparticles (Au&deg;) that are generated by a reduction of chloroauric acid (Au3+) by sodium citrate. This method was developed by Turkevich et al. in 195123 and then improved in the 1970s by Frens et al.24,25. As a result of this chemical reduction step, gold particles (10-20 nm in diameter) precipitate from the reaction mixture and can be applied to glass coverslips, which are then ready for use in cellular migration analyses9,26,27.
In general, the phagokinetic track motility assay is a quick, quantitative and easy measure of cellular motility. In addition, it can be utilized as a simple high-throughput assay, for use with cell types that are not amenable to time-lapsed imaging, as well as other uses depending on the needs of the researcher. Together, the ability to quantitatively measure cellular motility of multiple cell types without the need for expensive microscopes and software, along with the use of common laboratory equipment and chemicals, make the phagokinetic track motility assay a solid choice for scientists with an interest in understanding cellular motility.

The phagokinetic motility track assay is a method used to assess the movement of cells. Specifically, the assay measures chemokinesis (random cell motility) over time in a quantitative manner. The assay takes advantage of the ability of cells to create a measurable track of their movement on colloidal gold-coated coverslips.

Phagokineticトラック運動アッセイによる細胞遊走の定量的評価

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular ...

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

複合体形成シグナリングT細胞のリアルタイムのライブイメージング

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

使用して樹状細胞の移行の監視<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H磁気共鳴イメージング

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

離とナイーブCD4 Tリンパ球のTh17分化

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10R&#945; and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 &#176;C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

We developed an in vitro assay to investigate the role of immunoregulatory pathways in the regulation of cytokine secretion by HIV-specific CD4 T cells.

インビトロアッセイで HIV特異的CD4 T細胞エフェクター機能に対する免疫調節経路の影響を評価する

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

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

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

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

マウスにおける同種のCD4 T細胞応答を研究する尾皮膚の移植

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

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

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

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 ...

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

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

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

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

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

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