We thank Dr. Peter T. Sage for his assistance in generating some of the representative images. This work was supported by an NIH R01 grant to C.V.C. (HL104006).

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The authors have nothing to disclose.

全体的に、このプロトコルは、ⅰ）understudied生理学的なAPCとして内皮細胞を調査するための方法を説明し、ii）の「平面携帯APCモデル」の新規タイプとして。前者に関しては、ますます適応免疫応答16-18を形成する上で（造血APCに比べて、 すなわち ）を、周辺非造血（または「間質&#39;）APCは、重要な、非冗長な役割を果たしていることが理解されるようになりました。このよ...

Tリンパ球は、主要組織適合遺伝子複合体（MHC）に結合した、効率的ペプチド抗原（銀）を認識する能力を特徴と適応免疫系の分岐であるが、それらのT細胞受容体（TCR）を介して1分子。ナイーブリンパ球恒常移行し、「プロのAg提示細胞」（APCは、 例えば 、樹状細胞）をスキャンするメモリ/エフェクターT細胞が効果的に末梢組織内のAPCおよび潜在的な標的細胞の非常に広い範囲を調査する必要がある一方で、リンパ節内。 APC上の同族銀の当初認識以下分では、リンパ球は、それらの移動を阻止し、（IS）「免疫シナプス」と呼ばれる特殊な親密な細胞間のインタフェースを形成し始めます。持続（ すなわち 、30〜60分）は接点が増幅し、2-7のシグナリング維持する必要がありますされています。新興の研究では、IS内、それが連続形成し、迅速なRであることを識別強度および免疫応答2-7を得られるの品質を決定する（ すなわち 、MHC / AgをTCR、F-アクチン、接着およびシグナル伝達分子を含む）は、離散サブ細胞シグナルマイクロクラスタemodeling。しかし、このプロセスの動的な詳細及び調節機構は不完全に理解されている8,9。これは、APC表面の不規則なトポロジーに関連する技術的な課題や細胞間相互作用面のコントロール不良の向き、深く必要な時空間イメージングは8月10日 （Figure1A）に近づく制限の問題から大きく茎。 <img alt="図1" src="/files/ftp_upload/53288/53288fig1.jpg" /> イメージング免疫学的シナプスのダイナミクスについては、図1のA生理学的平面内細胞APCモデル。概略的には、T細胞とprofessio間の免疫学的シナプスの伝統的なイメージングを示しています<html>最終APC（A）とT細胞とこの新規内皮平面APCモデル（C）と比較して、従来の平面脂質二重層APCモデル（B）。プロフェッショナルAPCは、生理学的、免疫学的シナプスを提供するが、不十分指向細胞間のインタフェースを提供（最適なX-Yの撮像面に対してすなわち 、;解像度〜0.2μm）で、劇的空間（Zの撮像面〜1μmの分解能）および時間的（ すなわち 、妥協します繰り返しイメージングのすべてのzイメージングプレーン）の解像度をスキャンする必要性に起因します。二層モデルは、最適な時空間分解能イメージングを提供する平面のトポロジーを持っているだけでなく、高度に簡略化された非生理学的な剛性です。この内皮細胞モデルは、生理学的な設定で最適な空間と時間的画像の解像度を提供するために、古典的なAPCの生理学的基質と脂質二重層の平面トポロジを兼ね備えています。M /ファイル/ ftp_upload / 53288 / 53288fig1large.jpg「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。 以前の研究は、部分的に最適なXYイメージングに平行である、単一の計画に、T細胞活性化の面を固定経由で最適な時空間解像度（ すなわちを提供し 、平面基板モデル（ すなわち 、脂質二重層と抗体被覆表面）、開発することによって、これらの障害物を回避していますプレーン）11-15（ 図1B）。これらのモデルは、動的なアクチン/ TCRシグナリングマイクロクラスター7,11-14の発見を含むT細胞における抗原性のシグナル伝達を制御する分子/細胞内動態に重要な洞察を容易にしました。しかし、このようなモデルは、本質的に単純化されるだけでなく、剛性（ 図1B）（3次元位相幾何学的特徴の開発/研究を排除）。したがって、PHYに、このような調査結果を関連付ける方法を依然として不透明siologic細胞 - 細胞免疫監視。 まだunderstudiedものの、血管やリンパ管の内皮細胞は、「セミプロ」のAPC 16-18の大（ すなわち 、〜1000倍により、すべてのプロフェッショナルAPCより数の大きい）周辺の区画として浮上しています。これらの細胞は、MHC-I、MHC-II-および共刺激分子の多数を表現（例えば、CD40、LFA3、ICOSL、4-1BB、OX40L、TL1A、PD-L1;ではなく、CD80およびCD86）と戦略的です彼らは16-18特殊なセンチネル機能を果たし、血液-組織界面に位置します。以前の研究では、内皮細胞が効果的にエフェクター/メモリー、しかしナイーブではない、T細胞の19-25を再刺激することができることを実証しました。従って、内皮細胞は、T細胞の活性化、分化、メモリーと寛容16,17,26のローカル影響などの末梢組織中での適応免疫応答のエフェクター相に固有のAPCの役割を果たしている可能性があります。 CRIin vitroで増殖させた場合tically、 内皮細胞は、実質的に平面状のセル表面を形成し、（蛍光タンパク質レポーターを用いて、 例えば、）容易にトランスフェクトされています。これらの機能は、細胞間相互作用19,27の間の位相幾何学的力学の高時空間分解能イメージングに最適です。従って、内皮細胞は、抗原認識を駆動し、応答（ 図1C）19,20 を制御する細胞内分子/改造メカニズムの研究のための明らかに適した生理的な「平面携帯APC」モデルとなる恐れがあります。 接着および経内皮移動27中の白血球-内皮相互作用の詳細を研究するための（形質膜と細胞質ゾルの蛍光タンパク質メーカーと内皮細胞のトランスフェクションを含む）以前に確立された補完的なイメージング技術は、白血球が積極的にダイナミックによって内皮の表面を探査することを示しました挿入ANサブミクロンスケールのd後退は、アクチン豊富な円筒状の突起が（〜直径、深さ200〜1,000 nm）をinvadosome状突起（ すなわち 、「ILPS &#39;）27,28と呼ばれます 。これらのイメージング手法は、さらに19,20を報告し、本明細書でさらに説明するように、T細胞、内皮免疫学的シナプスの高時空間分解能イメージングのための最初の方法を開発する内皮APC機能を利用するためのプロトコルの作成 ​​に伴って拡張されました。この小説平面携帯APCモデル由来中央知見は、T細胞ILPSは、初期のAg検出を促進する上で、その後のシグナル伝達を維持するには、両方の機能することです。実際、（安定化およびカルシウム流入初期に応じて計上されていた）、TCRと活性なシグナルなどのPKC-Q、ZAP-70、ホスホチロシンとHS1の示唆に富む分子でshow濃縮を複数ILPSの配列。したがって、ILPSは、TCRシグナルマイクロへの3次元生理学的同等を表しているように見えます平面二重層モデルで見られたクラスタ。このアプローチは、このように、敏感に明らかに/レポート分子と建築（および生体力学的暗黙の）ダイナミクスそうでなければ検出できません。 本明細書に記載の方法は、適応免疫応答の基本的なメカニズムを調べるために我々の能力を向上させるために、プロのAPCと人工APC基板モデル間のギャップを埋めるに有用であるはずです。ここでの焦点は、CD4 + Th1型エフェクター/メモリー細胞の活性化にある間に、以下に説明するように、この基本的なアプローチは、容易に、T細胞型とのAgの広い範囲を研究するために改変することができます。

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			<td>Stock solution 1M in H20</td>
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		<tr height="21">
			<td height="21" style="height:21px;">phosphotyrosine (4G10 Platinum)</td>
			<td style="width:149px;">Millipore</td>
			<td>50-171-463</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">Nucleofector II</td>
			<td style="width:149px;">Amaxa Biosystems</td>
			<td style="width:152px;"></td>
			<td>Required electroporator for step 4</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:392px;">Zeiss Axiovert</td>
			<td style="width:149px;">Carl Zeiss MicroImaging</td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:392px;">Zeiss LSM510&#160;</td>
			<td style="width:149px;">Carl Zeiss MicroImaging</td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:392px;">Zeiss Axiovison Software</td>
			<td style="width:149px;">Carl Zeiss MicroImaging</td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">NU-425 (Series 60) Biological Safety Cabinet</td>
			<td style="width:149px;">NuAIRE</td>
			<td style="width:152px;">Nu-425-600</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:392px;">&#160;Forma STRCYCLE 37 &#176;C, 5% CO2 Cell culture Incubator</td>
			<td>Fisher Scientific</td>
			<td>202370</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">Centrifuge 5810</td>
			<td style="width:149px;">Eppendorf</td>
			<td style="width:152px;">EW-02570-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">Hemocytometer</td>
			<td style="width:149px;">Sigma-Aldrich</td>
			<td style="width:152px;">&#160;Z359629</td>
			<td>Bright-Line Hemocytometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">Isotemp Waterbath model 202</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td style="width:152px;">15-462-2</td>
			<td></td>
		</tr>
	</tbody>
</table>

an endothelial planar cell model for imaging immunological synapse dynamics

適応免疫は、T細胞と抗原提示細胞（「APCの &#39;）との間の動的な相互作用によって調節される「免疫学的シナプス」と呼ばれます。これらの親密な細胞間のインタフェース内の個別のサブセルラーMHC / AgをTCRのクラスター、F-アクチン、接着およびシグナル伝達分子を形成し、急速に改造。これらのダイナミクスは、病理学的免疫対開発し、したがって、保護の免疫応答の効率と品質の両方の重要な決定要因であると考えられています。生理学的なAPCとの免疫学的シナプスの現在の理解を得ることができる画像解像度の不足によって制限されます。人工基質モデル （例えば、平面脂質二重膜）は、優れた解像度を提供し、非常に貴重なツールとなっているが、彼 ​​らは本質的に非生理学的および単純化し過ぎています。血管とリンパ内皮細胞は重要な末梢組織（または間質）として出現した「半専門職のコンパートメントアルのAPC」。 （プロフェッショナルAPCの分子機構のほとんどを発現する）これらのAPCは、事実上平面状細胞表面を形成するユニークな機能を持っており、（蛍光タンパク質レポーターで、 例えば ）は、容易にトランスフェクトされています。基本的な抗原性シグナル伝達プロセスの改善されたイメージング及び質問のための新規な及び生理的な「平面セルラーAPCモデル &#39;に説明するように内皮細胞を実装するために、本明細書の基本的なアプローチ。

内皮細胞を使用し、生理的な複雑さとプロフェッショナルAPCの変形能を有する平面脂質二重層モデルの解像度の利点を組み合わせた新たなイメージング手法は、（ 図1）を開発しました。2本で観察された典型的な移行、カルシウムフラックスとトポロジカルダイナミクスの例を示します。図アプローチ。内皮上のサグ不在下でサ?...

Watch this Scientific Journal Video about An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics at JoVE.com

An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics

Adaptive immunity is controlled by dynamic 'immunological synapses' formed between T cells and antigen presenting cells. This protocol describes methods for investigating endothelial cells both as understudied physiologic APCs and as a novel type of 'planar cellular APC model'.

イメージング免疫学的シナプスダイナミクス内皮平面内細胞モデル

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological ...

an-endothelial-planar-cell-model-for-imaging-immunological-synapse

Research

JoVE Journal

Immunology and Infection

8.8K ビュー.  Harvard Medical School - BIDMC. Adaptive immunity is controlled by dynamic 'immunological synapses' formed between T cells and antigen presenting cells. This protocol describes methods for investigating endothelial cells both as understudied physiologic APCs and as a novel type of 'planar cellular APC model'.

ビデオ: An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

合成脂質二重膜上でHIV-1エンベロープ誘発性ウイルス学的シナプスとシグナリングのイメージング

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

免疫・感染学

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion (STED) Nanoscopy

Natural killer cells form tightly regulated, finely tuned immunological synapses (IS) in order to lyse virally infected or tumorigenic cells. Dynamic actin reorganization is critical to the function of NK cells and the formation of the IS. Imaging of F-actin at the synapse has traditionally utilized confocal microscopy, however the diffraction limit of light restricts resolution of fluorescence microscopy, including confocal, to approximately 200 nm. Recent advances in imaging technology have enabled the development of subdiffraction limited super-resolution imaging. In order to visualize F-actin architecture at the IS we recapitulate the NK cell cytotoxic synapse by adhering NK cells to activating receptor on glass. We then image proteins of interest using two-color stimulated emission depletion microscopy (STED). This results in &#60;80 nm resolution at the synapse. Herein we describe the steps of sample preparation and the acquisition of images using dual color STED nanoscopy to visualize F-actin at the NK IS. We also illustrate optimization of sample acquisition using Leica SP8 software and time-gated STED. Finally, we utilize Huygens software for post-processing deconvolution of images.

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion STED Nanoscopy

Here we illustrate the protocol for imaging by two-color STED nanoscopy the cytotoxic immune synapse of NK cells recapitulated on glass. Using this method we obtain sub-100 nm resolution of synapse proteins and the cytoskeleton.

デュアルカラータイム·ゲーティッド誘導放出枯渇（STED）ナノスによる免疫シナプスの可視化

Natural killer cells form tightly regulated, finely tuned immunological synapses (IS) in order to lyse virally infected or tumorigenic cells. Dynamic ...

Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known as leukocyte transendothelial migration. Two routes for leukocytes to cross the endothelial monolayer have been described: the paracellular route, i.e., through the cell-cell junctions and the transcellular route, i.e., through the endothelial cell body. However, it has been technically difficult to discriminate between the para- and transcellular route. We developed a simple in vitro assay to study the distribution of endogenous VE-cadherin and PECAM-1 during neutrophil transendothelial migration under physiological flow conditions. Prior to neutrophil perfusion, endothelial cells were briefly treated with fluorescently-labeled antibodies against VE-cadherin and PECAM-1. These antibodies did not interfere with the function of both proteins, as was determined by electrical cell-substrate impedance sensing and FRAP measurements. Using this assay, we were able to follow the distribution of endogenous VE-cadherin and PECAM-1 during transendothelial migration under flow conditions and discriminate between the para- and transcellular migration routes of the leukocytes across the endothelium.

Leukocytes cross the endothelial monolayer using the paracellular or the transcellular route. We developed a simple assay to follow the distribution of endogenous junctional VE-cadherin and PECAM-1 during leukocyte transendothelial migration under physiological flow to discriminate between the two transmigration routes.

好中球遊出の下で生理的流れの間に内皮細胞 - 細胞接合のリアルタイムイメージング

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known ...

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

アン<em&gt;エクスビボ</em急性脳スライスにおけるオリゴデンドロサイト指向T細胞攻撃の&gt;モデル

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer's attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.

We describe here a combination of the glass-supported lipid bilayer technique of forming immunological synapses with the super-resolution imaging technique of stimulated emission depletion (STED) microscopy. The goal of this protocol is to provide users with the instructions necessary to successfully carry out these two techniques.

グラスサポート平面脂質二重層上のナチュラルキラー細胞免疫シナプスの超解像イメージング

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has ...

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). This process is characterized by the transmigration of activated T cells across brain endothelial cells (ECs), the main constituents of the BBB. However, the consequences on brain EC function upon interaction with such T cells are largely unknown. Here we describe an assay that allows for the evaluation of primary mouse brain microvascular EC (MBMEC) function and barrier integrity during the interaction with T cells over time. The assay makes use of impedance cell spectroscopy, a powerful tool for studying EC monolayer integrity and permeability, by measuring changes in transendothelial electrical resistance (TEER) and cell layer capacitance (Ccl). In direct contact with ECs, stimulated but not na&#239;ve T cells are capable of inducing EC monolayer dysfunction, as visualized by a decrease in TEER and an increase in Ccl. The assay records changes in EC monolayer integrity in a continuous and automated fashion. It is sensitive enough to distinguish between different strengths of stimuli and levels of T cell activation and it enables the investigation of the consequences of a targeted modulation of T cell-EC interaction using a wide range of substances such as antibodies, pharmacological reagents and cytokines. The technique can also be used as a quality control for EC integrity in in vitro T-cell transmigration assays. These applications make it a versatile tool for studying BBB properties under physiological and pathophysiological conditions.

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Here, we describe an in vitro murine model of the blood-brain barrier that makes use of impedance cell spectroscopy, with a focus on the consequences on endothelial cell integrity and permeability upon interaction with activated T cells.

アン<em&gt;インビトロ</em&gt;インピーダンス分光法を用いた血液脳関門のモデル：T細胞 - 内皮細胞の相互作用に焦点を当て

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal ...

Studying Organelle Dynamics in B Cells During Immune Synapse Formation

Recognition of surface-tethered antigens&#160;by the B cell receptor (BCR)&#160;triggers the formation of an immune synapse (IS), where both signaling&#160;and antigen uptake are coordinated. IS formation involves dynamic actin remodeling accompanied by the polarized recruitment to the synaptic membrane of the centrosome and associated intracellular organelles such as lysosomes and the Golgi apparatus. Initial stages of actin remodeling allow B cells to increase their cell surface and maximize the quantity of antigen-BCR complexes gathered at the synapse. Under certain conditions, when B cells recognize antigens associated to rigid surfaces, this process is coupled to the local recruitment and secretion of lysosomes, which can facilitate antigen extraction. Uptaken antigens&#160;are internalized into specialized endo-lysosome compartments for processing into peptides, which are loaded onto major histocompatibility complex II (MHC-II) molecules for further presentation to T helper cells. Therefore, studying organelle dynamics associated with the formation of an IS is crucial to understanding how B cells are activated. In the present article we will discuss both imaging and a biochemical technique used to study changes in intracellular organelle positioning and cytoskeleton rearrangements that are associated with the formation of an IS in B cells.

Herein we describe two approaches to characterize cell polarization events in B lymphocytes during the formation of an IS. The first, involves quantification of organelle recruitment and cytoskeleton rearrangements at the synaptic membrane. The second is a biochemical approach, to characterize changes in composition of the centrosome, which undergoes polarization to the immune synapse.

免疫シナプス形成中のB細胞におけるオルガネラダイナミクスの研究

Recognition of surface-tethered antigens&#160;by the B cell receptor (BCR)&#160;triggers the formation of an immune synapse (IS), where both ...

Implantation and Monitoring by PET/CT of an Orthotopic Model of Human Pleural Mesothelioma in Athymic Mice

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. MPM development is mainly associated with asbestos. Treatments provide only modest survival since the median survival average is 9&#8211;18 months from the time of diagnosis. Therefore, more effective treatments must be identified. Most data describing new therapeutic targets were obtained from in vitro experiments and need to be validated in reliable in vivo preclinical models. This article describes one such reliable MPM orthotopic model obtained after injection of a human MPM cell line H2052/484 into the pleural cavity of immunodeficient athymic mice. Transplantation in the orthotopic site allows studying the progression of tumor in the natural in vivo environment. Positron emission tomography/computed tomography (PET/CT) molecular imaging using the clinical [18F]-2-fluoro-2-deoxy-D-glucose ([18F]FDG) radiotracer is the diagnosis method of choice for examining patients with MPM. Accordingly, [18F]FDG-PET/CT was used to longitudinally monitor the disease progression of the H2052/484 orthotopic model. This technique has a high 3R potential (Reduce the number of animals, Refine to lessen pain and discomfort, and Replace animal experimentation with alternatives) since the tumor development can be monitored non-invasively and the number of animals required could be significantly reduced.
This model displays a high development rate, a rapid tumor growth, is cost-efficient and allows for rapid clinical translation. By using this orthotopic xenograft MPM model, researchers can assess biological responses of a reliable MPM model following therapeutic interventions.

This article describes the generation of an orthotopic mouse model of human pleural mesothelioma by implantation of H2052/484 mesothelioma cells into the pleural cavity of immunocompromised athymic mice. The longitudinal monitoring of the development of intrapleural tumors was assessed by non-invasive multimodal [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography imaging.

アチミックマウスにおけるヒト胸膜中皮腫の正形細胞モデルのPET/CTによる移植とモニタリング

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. ...

Imaging the Human Immunological Synapse

The purpose of the method is to generate an immunological synapse (IS), an example of cell-to-cell conjugation formed by an antigen-presenting cell (APC) and an effector helper T lymphocyte (Th) cell, and to record the images corresponding to the first stages of the IS formation and the subsequent trafficking events (occurring both in the APC and in the Th cell). These events will eventually lead to polarized secretion at the IS. In this protocol, Jurkat cells challenged with Staphylococcus enterotoxin E (SEE)-pulsed Raji cells as a cell synapse model was used, because of the closeness of this experimental system to the biological reality (Th cell-APC synaptic conjugates). The approach presented here involves cell-to-cell conjugation, time-lapse acquisition, wide-field fluorescence microscopy (WFFM) followed by image processing (post-acquisition deconvolution). This improves the signal-to-noise ratio (SNR) of the images, enhances the temporal resolution, allows the synchronized acquisition of several fluorochromes in emerging synaptic conjugates and decreases fluorescence bleaching. In addition, the protocol is well matched with the end point cell fixation protocols (paraformaldehyde, acetone or methanol), which would allow further immunofluorescence staining and analyses. This protocol is also compatible with laser scanning confocal microscopy (LSCM) and other state-of-the-art microscopy techniques. As a main caveat, only those T cell-APC boundaries (called IS interfaces) that were at the right 90&#176; angle to the focus plane along the Z-axis could be properly imaged and analyzed. Other experimental models exist that simplify imaging in the Z dimension and the following image analyses, but these approaches do not emulate the complex, irregular surface of an APC, and may promote non-physiological interactions in the IS. Thus, the experimental approach used here is suitable to reproduce and to confront some biological complexities occurring at the IS.

This protocol images both the immunological synapse formation and the subsequent polarized secretory traffic towards the immunological synapse. Cellular conjugates were formed between a superantigen-pulsed Raji cell (acting as an antigen-presenting cell) and a Jurkat clone (acting as an effector helper T lymphocyte).

ヒト免疫シナプスのイメージング

The purpose of the method is to generate an immunological synapse (IS), an example of cell-to-cell conjugation formed by an antigen-presenting cell (APC) ...

An Immunological Model for Heterotopic Heart and Cardiac Muscle Cell Transplantation in Rats

Heterotopic heart transplantation in rats has been a commonly used model for diverse immunological studies for more than 50 years. Several modifications have been reported since the first description in 1964. After 30 years of performing heterotopic heart transplantation in rats, we have developed a simplified surgical approach, which can be easily taught and performed without further surgical training or background.
After dissection of the ascending aorta and the pulmonary artery and ligation of superior and inferior caval and pulmonary veins, the donor heart is harvested and subsequently perfused with ice-cold saline solution supplemented with heparin. After clamping and incising the recipient abdominal vessels, the donor ascending aorta and pulmonary artery are anastomosed to the recipient abdominal aorta and inferior vena cava, respectively, using continuous running sutures.
Depending on different donor-recipient combinations, this model allows analyses of either acute or chronic rejection of allografts. The immunological significance of this model is further enhanced by a novel approach of in-ear injection of vital cardiac muscle cells and subsequent analysis of draining cervical lymphatic tissue.

We describe a model of heterotopic abdominal heart transplantation in rats, implying modifications of current strategies, which lead to a simplified surgical approach. Additionally, we describe a novel rejection model by in-ear injection of vital cardiac muscle cells, allowing further transplant immunological analyses in rats.

ラットにおける異所性心臓と心筋細胞移植のための免疫学的モデル

Heterotopic heart transplantation in rats has been a commonly used model for diverse immunological studies for more than 50 years. Several modifications ...