Name: transplantation of tail skin to study allogeneic cd4 t cell responses in mice
Uploaded: 2014-07-25T13:00:00
Description: Watch this Scientific Journal Video about Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice at JoVE.com

This work was supported by SNF grants PPOOA-_119204 and PPOOP3_144918 to S.W.R. We thank E. Palmer and B.T.H. Hausmann for mice and technical expertise.

このビデオ出版物およびプロトコルでは、すべての動物の手順はカントン当局バーゼルシュタット、スイスで承認された動物プロトコルに従って行った。可能な限り無菌状態ですべての手順を実行します。 外科1。準備<ol><li>使用前にすべての手術器具やガーゼをオートクレーブ。 </li><li>暖かいパッドを加熱して、テーブル（ 材料/方法の表 ）に手術器具を...

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N., Palmer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+contribution+of+mutant+amino+acids+to+alloantigenicity.">The contribution of mutant amino acids to alloantigenicity.</a> The Journal of experimental medicine. 170 (3), (1989).</li><li>Monaco, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunosuppression+and+tolerance+for+clinical+organ+allografts.">Immunosuppression and tolerance for clinical organ allografts.</a> Current Opinion in Immunology. 1 (6), 1174-1177 (1989).</li><li>Rosenberg, A. 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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+acceptance+of+skin+and+cardiac+allografts+after+blocking+CD40+and+CD28+pathways.">Long-term acceptance of skin and cardiac allografts after blocking CD40 and CD28 pathways.</a> Nature. 381 (6581), 434-438 (1996).</li><li>Tomura, M., Nakatani, I., Murachi, M., Tai, X. G., Toyo-oka, K., Fujiwara, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+allograft+responses+induced+by+interleukin-6,+which+selectively+modulates+interferon-gamma+but+not+interleukin-2+production.">Suppression of allograft responses induced by interleukin-6, which selectively modulates interferon-gamma but not interleukin-2 production.</a> Transplantation. 64 (5), 757-763 (1997).</li><li>Ring, G. 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皮膚移植拒絶反応およびT細胞依存耐性を研究するために広く用いられる方法である。皮膚移植モデルが確立されて以来、いくつかの適応および変更が適用されている。記載された手順では、IA BM12尾の皮膚移植が麻酔ガス（イソフルラン）を用いて行われる。ガス麻酔を使用すると、移植されたマウスへのストレスを減少させる実行し、マウスの回復の時間が短縮されます。手順は、...

このような皮膚、心臓、腎臓などの固形臓器の移植は現在、世界の医療行為1の標準的な手順である。正常に移植された臓器は、ドナーの主要組織適合性抗原を認識するレシピエントの免疫系の活性化によって拒絶することができる。このため、移植患者は免疫抑制剤2を用いた治療を必要としています。マウスでの同種皮膚移植は1955年にメダウォアらによって設立され、後に主要組織適合遺伝子複合体（MHC）クラスIおよびIIとして記述対象となる分子を同定するために有用だった。それ以来、皮膚移植モデルを連続的に変更され、T細胞サブセットの役割および移植片拒絶2-4を抑制するのに化学的および生物学的介入の関連性を研究するために適合されている。耳とトランクから肌を準備するのがより困難であると尾の皮膚5よりも低酸素症や壊死を受けやすい。しかし、移植手順は同様である。また尾の皮膚移植のモニタリングは、皮膚の特徴的な髪の質感が容易です。 この記事では、CD4 + T細胞媒介同種移植片拒絶およびマウスにおける寛容の様々な側面の研究を可能にし、MHCクラスIIミスマッチ尾皮膚移植のための詳細な手順を提供します。 （IA BM12と呼ばれる）MHCクラスII分子IA b は6-9の天然の三点突然変異は、C57BL / 6マウス8における皮膚同種移植片の拒絶反応を誘導するのに十分である。 IAのBM12分子は、種々のαβ-T細胞受容体Vα2Vβ8-TCR特異的T細胞は、TCR-トランスジェニックマウス10を生成するために、同定され、そのうちC57BL / 6マウスからの（TCR）の鎖を用いてCD4 + T細胞を活性化する。 Vα2Vβ8-TCR特異的T細胞の養子移入は、免疫不全C57Bに拒絶モデルを確立するために使用されているL / 6 RAG2 - / - IA BM12皮膚を移植したマウス。 ドナーとレシピエント間の遺伝的差異は、移植受容と拒絶の結果に影響を与える。移植の異なる種類があります。自家移植は、レシピエント個体自体からの移植である。 syngraftsおよび同種移植片は、それぞれ遺伝的に同一と遺伝的に無関係な個人からの移植である。別の同種臓器移植の受け入れは、患者とマウスモデル11,3,4における化学的および生物学的介入によって実証されている。基本的なアプローチでは、抗CD3抗体で処理したC57BL / 6マウスIA BM12尾皮膚（未発表データ）の生存期間の延長を示した。レシピエントマウスにおける移植の前にCD4 +およびCD8 + T細胞の枯渇は、MHCクラスIおよびIIミスマッチ移植片（12改訂）の受入れをもたらした。興味深いことに、皮膚移植片の拒絶反応は、CD4 +の存在に依存する</sup&gt; T細胞（12牧師）。このモデルでは、調節性T細胞と抗体または抑制との共刺激分子を遮断することによって、異なる免疫細胞間の特異的相互作用を標的とする（未発表データ）寛容を誘導し得る。実際、CD40およびCD28の両方を遮断することは、長期的な皮膚同種移植片寛容13,14につながった。 尾の皮膚移植は他の臓器の移植に比べて実行​​が容易であり、監視が容易です。また、尾の皮膚移植は、調製が容易であり、他の皮膚組織よりも虚血を受けにくい。注入された麻酔薬とは対照的に、移植時の麻酔ガス（イソフルラン）の使用は、プロシージャおよび受信者の回復時間の両方を短縮します。不完全な創傷治癒および移植片拒絶につながる可能性尾皮膚移植のカールは、組織接着剤の適用によって防止される。さらに、IA BM12尾皮膚移植モデルでは、排他的に、CD4を活性化させる<sup&gt; +（同じ遺伝的背景の）免疫応答性および免疫不全マウスの両方においてT細胞は、結果の解釈を容易にする。 このプロトコルは、化学的、生物学的な介入を可能にし、信頼性の高い再現性があり、容易に監視マウスモデルを説明しています。モデルは、尾の皮膚移植の拒絶と寛容の誘導を調査対象としています。

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			<td height="21" style="height:21px;">Cotton swab</td>
			<td style="width:183px;">Carl Roth GmbH</td>
			<td style="width:119px;">31025060</td>
			<td></td>
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			<td height="21" style="height:21px;width:299px;">Dafalgan , UPSA</td>
			<td>Bristol Myers Squibb SA</td>
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			<td height="21" style="height:21px;">Hansaplast Finger Strips</td>
			<td>Beierdorf AG</td>
			<td style="width:119px;">REF.76861</td>
			<td></td>
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			<td height="21" style="height:21px;">Histoacryl Tissue adhesive</td>
			<td style="width:183px;">Braun</td>
			<td style="width:119px;">REF.1050052</td>
			<td></td>
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			<td height="21" style="height:21px;">Leukotape classic , 2cm x 10 m</td>
			<td>BSN Medical SAS</td>
			<td>REF.02204-00</td>
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			<td height="21" style="height:21px;">PBS, Phosphate-Buffered Saline, pH 7.4</td>
			<td style="width:183px;">Invitrogen</td>
			<td style="width:119px;">10010015</td>
			<td>GIBCO</td>
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			<td height="21" style="height:21px;">Sterile gauze, 5 x 5cm, 8ply</td>
			<td>MaiMed GmbH</td>
			<td>21010</td>
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			<td height="21" style="height:21px;">Surgical instruments&#160;</td>
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			<td style="width:119px;">14094-11</td>
			<td>Fine iris scissor&#160;</td>
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			<td style="width:183px;"></td>
			<td style="width:119px;">14010-15</td>
			<td>Mayo scissors,</td>
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			<td style="width:119px;">14080-11</td>
			<td>Artery scissors ball tip 11.5 cm&#160;</td>
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			<td style="width:119px;">11021-14</td>
			<td>Tissue forceps</td>
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			<td height="21" style="height:21px;width:299px;">Surgical Blade No.20</td>
			<td style="width:183px;">Swann-Morton LTD</td>
			<td style="width:119px;">3006</td>
			<td>Carbon Steel</td>
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			<td height="21" style="height:21px;width:299px;">Surgical blade Handles</td>
			<td style="width:183px;">Swann-Morton LTD</td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="21" style="height:21px;">Syringe, 1ml</td>
			<td style="width:183px;">ARTSANA</td>
			<td style="width:119px;"></td>
			<td>disposable</td>
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			<td height="21" style="height:21px;">Temgesic , Buprenorphine</td>
			<td style="width:183px;">ESSEX Chemie AG</td>
			<td style="width:119px;"></td>
			<td>0.3mg/ml</td>
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			<td height="21" style="height:21px;width:299px;">Tissue Culture dishes 10 cm, 60.1 cm2</td>
			<td style="width:183px;">TPP</td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:299px;">Vaseline</td>
			<td style="width:183px;">Vifor SA</td>
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			<td style="width:183px;">Solis</td>
			<td style="width:119px;"></td>
			<td>Type 223</td>
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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.

最初のアプローチで、C57BL / 6マウスのIA BM12同種移植片およびIA B型syngraftsを移植した。包帯除去後の、C57BL / 6マウス（ 図1A）における接触ゾーンの閉鎖せずに移植片、創傷治癒の徴候をマニフェスト。 syngraftsながら包帯を除去した後、CD4 + T細胞媒介炎症は、移植後13日以内に、C57BL / 6マウス（キャラクターいっぱい図1B-C）で壊死領域（赤い?...

Watch this Scientific Journal Video about Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice at JoVE.com

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

Research

JoVE Journal

Immunology and Infection

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

同種造血幹細胞移植後のウイルス感染を予防/治療するMultivirus -特異的T細胞の生成

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

免疫・感染学

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

糖尿病CD4の養子移入+ T細胞によるマウスの加速された1型糖尿病の誘導

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

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

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 ...

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

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

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 ...

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

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

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

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

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 ...

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

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 ...

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

体外と体内の T, B, 骨髄細胞の抑制活性と移植から体液性応答の評価

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms ...

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

勉強する人間の T 細胞のリコール反応体内吸引水疱プロトコル

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model ...

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

ジカ ウイルス特異 Immunoprivileged 臓器感染 Ifnar1-/-マウスの T 細胞応答の評価

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...