私たちは、博士に感謝します。有益なコメントをロバートBonneauとニールクリステンセン。 この作品は、医学資金のペンシルバニア州立大学によってサポートされていました。

1。 BDC2.5マウスの脾臓およびリンパ節由来のT細胞の単離<ol><li>糖尿病誘発性CD4 + T細胞のドナーとして6週齢前糖尿病女性BDC2.5マウスを使用してください。尿糖測定（下記参照）によって決定されたマウスは糖尿病フリーでなければなりません。 </li><li> CO 2窒息を使用して、各マウスを安楽死させると脾臓を除去し、無菌条件下で腋窩と上腕リンパ節。脾臓を削除するには、70％エタノールで毛皮を浸し、その後カットして肌を撤回。脾臓は、マウスの左側にある暗赤色器官として見えるようになります。小さなハサミを使って腹膜に1インチ切開を加え、そっと小さなピンセットで中央に脾臓を把握する。慎重にできるだけ結合組織および添付脂肪をトリミングおよび脾臓を除去。 </li><li>氷上で15 mlコニカルチューブに10ミリリットルダルベッコ修飾イーグル培地（DMEM）中で脾臓とリンパ節を収集します。 </li><li>第を使用して、単一細胞懸濁液を調製しプランジ​​ャが優しく同じ50ミリリットルコニカルチューブに70μmのセルストレーナー（こしにつき1脾臓、ストレーナ当たり4-6リンパ節）を通ってリンパ器官を押して滅菌を10mlの注射器の電子終わり。 </li></ol>プロセス中に、ストレーナからの細胞の回収を最大化するために1ミリリットルDMEMで数回各ストレーナーをすすいでください。 <ol start="5"><li>室温で7分間300-400 XGで15 mlコニカルチューブと遠心分離機に50ミリリットルコニカルチューブから採取した細胞を移す。 </li><li>赤血球を溶解するために、上清を捨て5mlの塩化アンモニウムカリウム（ACK）緩衝液（pH7.2）中で細胞を再懸濁、及び5分間RTでACK細胞懸濁液をインキュベートする。 </li><li> ACK-細胞懸濁液にDMEMを10mlを加え、上記のようにチューブを遠心。一回10ミリリットルDMEMで細胞ペレットを洗浄します。 </li></ol> 2。糖尿病CD4の蛍光活性化細胞選別BDC2.5 +マウスからのT細胞<ol><li> 5ミリリットルFACS s内の再懸濁細胞バッファ寧トリパンブルー色素排除により生存細胞の数（位相差顕微鏡と血球計数器を用いて）数える。 </li><li> FACS緩衝液を用いて、5×10 7細胞/ mlの細胞懸濁液の音量を調節する。 〜1染色コントロールあたり×10 6細胞（染色1いや、3つのシングル汚れ）を取り外します。非活性化されたトランスジェニックCD4 +抗CD4とT細胞（APC）、抗TCRVβ4（FITC）及び15 mlチューブ中の抗CD62L（PE）モノクローナル抗体（モノクローナル）のためのサンプルを染色。暗闇の中で4℃で20〜30分間、製造元が推奨するのmAb濃度°Cを用いた細胞染色を行う。 </li><li> &gt; 3X染色反応音量でサンプルし、FACSバッファーを持つ単一の染色コントロールを洗ってください。 （1-2×10 7細胞/サンプルをソートするためのMLと300μlの単染色コントロール用）と氷の上で店の隣まで300-400 xgでチューブを遠心、上清を除去し、FACSバッファーで細胞を再懸濁ステップ。 </li><li> 35μmのセルを介して細胞サンプルを渡す細胞塊を除去するストレーナーキャップチューブ。訓練されたオペレータとセルソーターでCD4 + TCRVβ4+ CD62L +細胞のためのサンプルをソートします。を15mlチューブに3ミリリットルDMEMに選別された細胞を収集します。 2×10 4合計イベント/秒のソート率で1.5×10 8細胞（3ドナーマウスから得られた細胞の概数）ソートするには、セットアップを含む2.5-3時間を許可します。 〜2.5×10 6非活性ジェニックCD4 +細胞選別後のマウス当たりT細胞を期待しています。 </li><li>ソートの末尾に選別された細胞の絶対数を記録し、300から400×gで7分間のためにそれらを遠心。上清を廃棄し、再懸濁細胞（2×10 6細胞/ ml）中の滅菌リン酸緩衝養子T細胞転送用生理食塩水（PBS）マグネシウム（Mg 2 + / Ca 2 +の自由）。 </li></ol> 3。糖尿病CD4の養子移入+ BDC2.5マウス由来のT細胞<ol><li>再懸濁FACS精製CD4優しく、1ミリリットルの注射器と18-1½ゲージ針を用いて+1ミリリットルのシリンジをT細胞とをロードします。注射の準備のために、27½ゲージ針で18-1½ゲージの針を交換してください。 </li><li>彼らはグルーミング行動を示すまで加熱ランプに6-8週齢の雌NOD.SCIDレシピエントマウスを公開します。 </li><li>拘束のレシピエントマウスを抑制し、注射部位を消毒するために70％（v / v）のエタノールで自分の尾を拭いてください。 </li><li>外側尾静脈のいずれかに1〜2×10 6 FACS選別された細胞/マウスを（最大で500μlのPBS）に注入します。 </li></ol>プランジ​​ャーを無理に押し込まないでください。針が静脈に適切に配​​置されている場合は、注入はほとんど抵抗なく行われる。 4。高血糖のためにレシピエントマウスの監視とT1D <ol><li> T細胞移入後5日から開始して、NOD.SCIDレシピエントマウスは、製造業者の指示に従って、試薬ストリップ（バイエルDiastix）を使用して、上昇した尿糖を毎日監視される。二つの連続urin持つマウス電子グルコース測定値は&gt; 250ミリグラム/ dlと、糖尿病と考えられている。 </li></ol>

<ol>
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すべてのマウスは、ペンシルベニア州立大学の動物実験のガイドラインに準拠し、委員会を設置し、使用中の薬の特定病原体フリー（SPF）施設のペンシルベニア州立大学で飼育した。 著者は、彼らが競合する経済的利益を持っていないことを宣言します。

T1Dは、糖尿病のNODマウスの糖尿病又はT細胞クローンに由来するTCRトランスジェニックマウスからの脾臓細胞全体またはT細胞サブセットの養子移入することにより効率を変化させることで、レシピエントマウスに誘導することができる。我々はここにNOD.SCIDマウスにFACS精製CD62L + BDC2.5ジェニックCD4 + T細胞を転送することにより、100％の発生率で2週間以内に、レシピエントマウスにT1D誘導する...

NODマウスは、自発的に自己免疫性糖尿病を発症し、広く人間T1D 1,2のための動物モデルとして使用されている。 NODマウスにおけるT1Dの病因は、T細胞とB細胞が続き、樹状細胞やマクロファージによるランゲルハンスの膵島の、生後3-4週で始まる、浸潤によって特徴付けられる。非破壊ペリ膵島炎のこの段階では、3歳の4〜6ヶ月まであからさまな糖尿病になり、インスリン産生膵β細胞の遅い、進行性の破壊につながる。 4,5脾細胞、CD4 +糖尿病NODマウスからの6,7またはCD8 + 8,9 T細胞の転送は、膵島反応性T細胞がT1D病因において中心的な役割を果たすことを示す、免疫不全NODマウスにおいて糖尿病を媒介することが示されている。実験条件に応じて、糖尿病は、これらの研究で数週間にわたって、徐々にレシピエントマウスで開発された。同様に、様々なT細胞クローンは時間とコストがかかることによって導出糖尿病誘発T細胞の培養は、数週間レシピエントマウス7,10への移行後に糖尿病を媒介することが報告されている。 CD4-CD8または拘束性糖尿病誘発T細胞クローンに由来するTCRを発現するトランスジェニックマウスの可用性と、いくつかの研究室は、その後、例えば、マウスからの脾臓T細胞は、レシピエント11-13に糖尿病を転送することができたことを示している。具体的には、BDC2.5 NODマウスは、クロモグラニンA、膵β細胞におけるタンパク質14-16に特異的なTCR BDC2.5、トランスジェニックである。あからさまに糖尿病または前糖尿病BDC2.5マウスからin vitroで活性化または非活性化の全体または画し脾臓細胞での転送効率を11,17-19を変えることで、新生児や免疫不全NODマウスに糖尿病を譲渡。 私たちは、精製されたトランスジェニックCD4を利用し簡単な方法+高効率とconsisteでレシピエントマウスにT1D誘導する前糖尿病BDC2.5マウスからT細胞を記述NCY。トランスジェニックTCRVβ4鎖を発現するナイーブ、膵島抗原特異的CD4 + T細胞はCD4ための蛍光活性化細胞選別（FACS）によって、これらのマウスから単離され+ CD62L + T細胞の多数。精製されたトランスジェニックT細胞を、次いで、官能性T細胞およびB細胞を欠き、膵島炎および糖尿病フリー20アールNOD.SCIDマウスに活性化せずに転送される。レシピエントマウスはT細胞移入後2週間以内に急速に発達T1Dを示す尿糖濃度の上昇のために監視されています。 他の方法とは対照的に不均一な特異性を有する糖尿病誘発性T細胞を移すことを、我々のプロトコルは、FACSソートしたCD4 +ほぼ独占的に糖尿病BDC2.5 TCRを発現したT細胞を使用しています。彼らの均質性に起因して、移入されたT細胞（〜1×10 6細胞/マウス）のほんの数は100％の発生率で2週間以内に急速T1D開発のために必要とされる。我々のプロトコルの別の利点は、irradiatioあるレシピエントマウスのnが、それはいくつかの他の方法のためにあるようにする必要はありません。この方法の潜在的な制限は、糖尿病の両方のCD4とCD8 T細胞サブセットまたは特にCD8 T細胞の寄与の調査を許可していないということです。 記述されたプロトコルは、ナイーブ、単一特異的CD4 +標的臓器への膵島抗原特異Th細胞のホーミングに介入するT細胞と同様に、治療戦略により仲介急速T1D開発を研究するために有用であろう。

<table><tbody><tr><td>BDC 2.5 TCR transgenic NOD mice (NOD.Cg-Tg(Tcr&amp;alpha;BDC 2.5, Tcr&amp;#946;BDC 2.5)</td><td>JAX</td><td>004460</td><td></td></tr><tr><td>NOD.SCID mice (NOD.CB17-&lt;em&gt;Prkdc&lt;sup&gt;scid&lt;/sup&gt;&lt;/em&gt;/J)</td><td>JAX</td><td>001303</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium (DMEM)</td><td>Themo Scientific</td><td>SH30022.01</td><td></td></tr><tr><td>Bayer Diastix</td><td>Fisher Scientific</td><td>AM2803</td><td></td></tr><tr><td>15 ml conical tubes</td><td>Falcon</td><td>352095</td><td></td></tr><tr><td>50 ml conical tubes</td><td>Falcon</td><td>352070</td><td></td></tr><tr><td>Sterile surgical tweezers</td><td></td><td></td><td></td></tr><tr><td>Sterile small pair scissors</td><td></td><td></td><td></td></tr><tr><td>Sterile large pair scissors</td><td></td><td></td><td></td></tr><tr><td>70 &amp;mu;m cell strainers</td><td>Fisher Scientific</td><td>22363548</td><td></td></tr><tr><td>35 &amp;mu;m cell strainer cap tubes</td><td>BD Biosciences</td><td>352235</td><td></td></tr><tr><td>Ammonium-Chloride-Potassium (ACK) buffer</td><td></td><td></td><td>0.15 M NH&lt;sub&gt;4&lt;/sub&gt;Cl, 1 mM KHCO&lt;sub&gt;3, &lt;/sub&gt;0.1 mM Na&lt;sub&gt;2&lt;/sub&gt;EDTA, pH 7.2 in dH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>BD FACSFlowTM sheath fluid</td><td>BD Biosciences</td><td>342003</td><td></td></tr><tr><td>FACS staining buffer</td><td></td><td></td><td>PBS, 0.2 mM EDTA, 0.5% BSA/FCS, filter sterilized</td></tr><tr><td>Phase contrast microscope</td><td></td><td></td><td></td></tr><tr><td>Trypan blue</td><td></td><td></td><td></td></tr><tr><td>Hemocytometer</td><td></td><td></td><td></td></tr><tr><td>Anti-CD4 (APC) mAb</td><td>Biolegend</td><td>1005616</td><td>clone RM4-5</td></tr><tr><td>Anti-TCR V&amp;#946;4 (FITC) mAb</td><td>BD Biosciences</td><td>553365</td><td>clone KT4</td></tr><tr><td>Anti-CD62L (PE) mAb</td><td>BD Biosciences</td><td>553151</td><td>clone MEL-14</td></tr><tr><td>Cell sorter</td><td>BD Biosciences</td><td></td><td>&lt;em&gt;e.g.&lt;/em&gt; BD FACSAria III</td></tr><tr><td>Heat lamp</td><td></td><td></td><td></td></tr><tr><td>Mouse restrainer</td><td></td><td></td><td></td></tr><tr><td>1 ml syringes</td><td>Becton Dickinson</td><td>309602</td><td></td></tr><tr><td>18-1&amp;frac12; gauge needles (sterile)</td><td>Becton Dickinson</td><td>305196</td><td></td></tr><tr><td>27&amp;frac12; gauge needles (sterile)</td><td>Becton Dickinson</td><td>305109</td><td></td></tr></tbody></table>

accelerated type 1 diabetes induction in mice by adoptive transfer of diabetogenic cd4+ t cells

非肥満糖尿病（NOD）マウスは自発的に生後12週間後に自己免疫性糖尿病を開発し、人間の1型糖尿病（T1D）の中で最も広範囲に研究の動物モデルである。照射レシピエントマウスでセル転送研究はT細胞が、このモデルでT1D発症に極めて重要であることを確立した。我々は、本明細書に急速に精製され、プライマリCD4 +膵島特異的T細胞受容体（TCR）NOD.SCIDレシピエントマウスへのBDC2.5トランスジェニック前糖尿病NODマウス由来のT細胞の養子免疫伝達によってT1D誘導するための簡単​​な方法を述べる。糖尿病誘発T細胞の単離および養子移入は、同じ日以内に完了することができるこの技術の主な利点は、受信者の照射が必要とされないし、T1Dの高い発生率は、T細胞の転送後2週間以内に誘発される。したがって、T1Dにおける病因および治療介入の研究では、不均一なT細胞集団またはクローンに頼る方法よりも速い速度で進むことができます糖尿病NODマウス由来。

我々の結果は、このような膵臓リンパ節などの二次リンパ器官への家へのT細胞のために重要であるCD62Lを発現するトランスジェニックBDC2.5細胞の単離を示す。今回の知見は、さらに急速に転送し、効率的にNOD.SCIDレシピエントマウスにT1Dにするには、この単一特異的T細胞集団の強力な能力を実証する。 糖尿病CD4 + BDC2.5マウス由来のT細胞の単離は、図2に示さ?...

Watch this Scientific Journal Video about Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells at JoVE.com

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

私たちは、膵島抗原特異、プライマリーCD4 + T細胞の養子移入によって2週間以内に、マウスにおける1型糖尿病（T1D）を誘導するための再現可能な方法を提供する。

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 ...

accelerated-type-1-diabetes-induction-mice-adoptive-transfer

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

16.6K ビュー.  Pennsylvania State University College of Medicine. 私たちは、膵島抗原特異、プライマリーCD4 + T細胞の養子移入によって2週間以内に、マウスにおける1型糖尿病（T1D）を誘導するための再現可能な方法を提供する。

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

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse models allow us to study how diseases unfold, often providing a good replica of the same processes occurring in humans. For some autoimmune diseases, like type 1A diabetes, there are models (the NOD mouse) that spontaneously develop a disease similar to the human counterpart. For many other autoimmune diseases, however, the model needs to be induced experimentally. A common approach in this regard is to inject the mouse with a dominant antigen derived from the organ being studied. For example, investigators interested in autoimmune thyroiditis inject mice with thyroglobulin2, and those interested in myasthenia gravis inject them with the acetylcholine receptor3. If the autoantigen for a particular autoimmune disease is not known, investigators inject a crude protein extract from the organ targeted by the autoimmune reaction. For autoimmune hypophysitis, the pathogenic autoantigen(s) remain to be identified4, and thus a crude pituitary protein preparation is used. In this video article we demonstrate how to induce experimental autoimmune hypophysitis in SJL mice.

This video shows how to induce autoimmune hypophysitis in SJL mice and how to assess its severity by histopathology.

SJLマウスにおける実験的自己免疫性下垂体炎の誘導

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse ...

免疫・感染学

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

ヒトのインビトロ</em免疫バランスの初期状態の認識のためのスクリーニングツールとして>抑制

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

Transplantation of Pancreatic Islets Into the Kidney Capsule of Diabetic Mice

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice. We found that it was easier to concentrate the islets or cells into pellets in the final delivery tubing (PE50) used to transplant the cells under the kidney capsule. This technique provides both speed and ease while reducing any undue stress to the cells or to the mouse.

Loading: Settled, hand picked, islets or pelleted cells are carefully aspirated off the bottom of a 1.5 mL microcentrifuge tube using a p200 pipetteman and a straight, thin-wall pipette tip. A length of PE50 tubing is attached to the pipette tip using a small silicone adapter tubing. Cells are allowed to settle, in the tip, and then are transferred to the PE50 tubing by slowly dialing the pipetteman. Once the cells are near the end of the PE50 tubing, a kink is made and the silicone adaptor tubing is placed over the kink. The PE50 tubing is transferred to a 15 mL conical containing a cut 5 mL pipet, and the PE50 tubing is taped over the side of the 5 mL pipet to prevent curling during centrifuging. Cells are allowed to reach 1,000 rpm and stopped.
Transplantation: Recipient mice are anesthetized, shaved, and cleaned. A small incision is made on the left flank of the mouse and the kidney is exposed. The kidney, fat, and tissue are kept moist with normal saline swab. The distal end of the PE50 is attached to a Hamilton screw drive syringe, containing a pipette tip, using the silicone adaptor tubing. A small nick is made on the right flank side of the kidney, not too large nor too deep. The beveled end of the PE50 tubing, nearest the cells, is carefully placed under the capsule, the tubing is moved around gently to make space while swabbing normal saline; a dry capsule can tear easily. A small air bubble is delivered under the capsule by slowly dialing the syringe screw drive. Islets are then slowly delivered behind the air bubble. Once the islets have been delivered kidney homeostasis is maintained and the knick is cauterized with low heat. The kidney is placed back into the cavity and the peritoneum and skin are sutured and stapled. Mice are immediately treated with Flunixin and Buprenorphine s.q. and placed in a cage on a heating pad.

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of mice. Cells are concentrated into pellets in the final tubing used for transplanting the cells under the kidney capsule. The ease of this technique reduces stress to the cells and the mouse.

糖尿病マウスの腎臓のカプセルに膵島の移植

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice.  We found that it was easier ...

生物学

Neo-Islet Formation in Liver of Diabetic Mice by Helper-dependent Adenoviral Vector-Mediated Gene Transfer

Type 1 diabetes is caused by T cell-mediated autoimmune destruction of insulin-producing cells in the pancreas. Until now insulin replacement is still the major therapy, because islet transplantation has been limited by donor availability and by the need for long-term immunosuppression. Induced islet neogenesis by gene transfer of Neuogenin3 (Ngn3), the islet lineage-defining specific transcription factor and Betacellulin (Btc), an islet growth factor has the potential to cure type 1 diabetes.
Adenoviral vectors (Ads) are highly efficient gene transfer vector; however, early generation Ads have several disadvantages for in vivo use. Helper-dependent Ads (HDAds) are the most advanced Ads that were developed to improve the safety profile of early generation of Ads and to prolong transgene expression1. They lack chronic toxicity because they lack viral coding sequences2-5 and retain only Ad cis elements necessary for vector replication and packaging. This allows cloning of up to 36 kb genes.
In this protocol, we describe the method to generate HDAd-Ngn3 and HDAd-Btc and to deliver these vectors into STZ-induced diabetic mice. Our results show that co-injection of HDAd-Ngn3 and HDAd-Btc induces 'neo islets' in the liver and reverses hyperglycemia in diabetic mice.

We describe hepatic neo-islet formation in STZ (streptozotocin)-induced diabetic mice by gene transfer of Neurogenin3 (Ngn3) and Betacellulin (Btc) using helper-dependent adenoviral vector (HDAd) and the reversal of hyperglycemia. Our method takes advantages of helper-dependent adenoviral vectors with their highly efficient in vivo transduction and the long lasting gene expression.

ヘルパー依存型アデノウイルスベクターによる遺伝子導入による糖尿病マウスの肝臓におけるネオ·膵島形成

Type 1 diabetes is caused by T cell-mediated autoimmune destruction of insulin-producing cells in the pancreas. Until now insulin replacement is still the ...

医学

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 ...

Scaffold-supported Transplantation of Islets in the Epididymal Fat Pad of Diabetic Mice

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy may incur acute whole blood reactions and result in poor islet engraftment. Here, we report a robust protocol for the transplantation of islets at the extrahepatic transplantation site-the epididymal fat pad (EFP)-in a diabetic mouse model. A protocol to isolate and purify islets at high yields from C57BL/6J mice is described, as well as a transplantation method performed by seeding islets onto a decellularized scaffold (DCS) and implanting them at the EFP site in syngeneic C57BL/6J mice rendered diabetic by streptozotocin. The DCS graft containing 500 islets reversed the hyperglycemic condition within 10 days, while the free islets without DCS required at least 30 days. The normoglycemia was maintained for up to 3 months until the graft was explanted. In conclusion, DCS enhanced the engraftment of islets into the extrahepatic site of the EFP, which could easily be retrieved and might provide a reproducible and useful platform for investigating the scaffold materials, as well as other transplantation parameters required for a successful islet engraftment.

This protocol demonstrates murine islet isolation and seeding onto a decellularized scaffold. Scaffold-supported islets were transplanted into the epididymal fat pad of streptozotocin (STZ)-induced diabetic mice. Islets survived at the transplantation site and reversed the hyperglycemic condition.

糖尿病マウスの精巣上体脂肪細胞における膵島支持骨格移植

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy ...

生物工学

High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the spontaneous non-obese diabetes (NOD) mouse model, insulin is the primary target, and genetic manipulation of these animals to remove a single key insulin epitope prevents disease. Thus, selective elimination of professional antigen presenting cells (APCs) bearing this pathogenic epitope is an approach to inhibit the unwanted insulin-specific autoimmune responses, and likely has greater translational potential.
Chimeric antigen receptors (CARs) can redirect T cells to selectively target disease-causing antigens. This technique is fundamental to recent attempts to use cellular engineering for adoptive cell therapy to treat multiple cancers. In this protocol, we describe an optimized T-cell retrovirus (RV) transduction and in vitro expansion protocol that generates high numbers of functional antigen-specific CD8 CAR-T cells starting from a low number of naive cells. Previously multiple CAR-T cell protocols have been described, but typically with relatively low transduction efficiency and cell viability following transduction. In contrast, our protocol provides up to 90% transduction efficiency, and the cells generated can survive more than two weeks in vivo and significantly delay disease onset following a single infusion. We provide a detailed description of the cell maintenance and transduction protocol, so that the critical steps can be easily followed. The whole procedure from primary cell isolation to CAR expression can be performed within 14 days. The general method may be applied to any mouse disease model in which the target is known. Similarly, the specific application (targeting a pathogenic peptide/MHC class II complex) is applicable to any other autoimmune disease model for which a key complex has been identified.

This article describes a protocol for the generation of antigen-specific CD8 T cells, and their expansion in vitro, with the aim of yielding high numbers of functional T cells for use in vitro and in vivo.

自己免疫性糖尿病モデルにおける機能試験用抗原特異的一次マウス細胞傷害性T細胞の高効率生成

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the ...

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

With the increase of incidence, inflammatory bowel diseases (IBD), which are chronic diseases affecting the gastrointestinal tract, impose a considerable health and financial burden on individuals and society. Therefore, it is critical to investigate the mechanisms underlying the pathogenesis and development of IBD. Here, a gut microbiota antigen-specific T cell transfer colitis model is described. CBir1 flagellin has been recognized as the immunodominant gut bacterial antigen in experimental colitis and patients with Crohn&#39;s disease. CBir1 TCR transgenic na&#970;ve CD4+ T cells, specific to CBir1 flagellin, can induce chronic colitis after adoptive transfer into immune-deficient Rag1-/- mice. The disease severity is assessed by histopathology. The CD4+ T cell phenotypes in colonic lamina propria are also determined. This model closely resembles the development of IBD, which provides an ideal murine model for investigating the mechanisms driving the pathogenesis of IBD and testing the potential drugs for treating IBD.

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

In this protocol, a gut microbiota antigen-specific T cell adoptive transfer colitis model is described. CD4+ T cells are isolated from CBir1 TCR transgenic mice. These are specific for an immunodominant gut microbiota antigen CBir1 flagellin, which is transferred into recipient Rag1-/- mice, leading to intestinal inflammation.

免疫不全マウスへのCBir1 TCRトランスジェニックCD4+ T細胞の養子移入による腸管炎症の誘導

With the increase of incidence, inflammatory bowel diseases (IBD), which are chronic diseases affecting the gastrointestinal tract, impose a considerable ...

Bioluminescent Monitoring of Graft Survival in an Adoptive Transfer Model of Autoimmune Diabetes in Mice

Type 1 diabetes is characterized by the autoimmune destruction of the insulin-producing beta cells of the pancreas. A promising treatment for this disease is the transplantation of stem cell-derived beta cells. Genetic modifications, however, may be necessary to protect the transplanted cells from persistent autoimmunity. Diabetic mouse models are a useful tool for the preliminary evaluation of strategies to protect transplanted cells from autoimmune attack. Described here is a minimally invasive method for transplanting and imaging cell grafts in an adoptive transfer model of diabetes in mice. In this protocol, cells from the murine pancreatic beta cell line NIT-1 expressing the firefly luciferase transgene luc2 are transplanted subcutaneously into immunodeficient non-obese diabetic (NOD)-severe combined immunodeficient (scid) mice. These mice are simultaneously injected intravenously with splenocytes from spontaneously diabetic NOD mice to transfer autoimmunity. The grafts are imaged at regular intervals via non-invasive bioluminescent imaging to monitor the cell survival. The survival of mutant cells is compared to that of control cells transplanted into the same mouse.

This protocol describes a straightforward and minimally invasive method for transplanting and imaging NIT-1 cells in non-obese diabetic (NOD)-severe combined immunodeficient mice challenged with splenocytes purified from spontaneously diabetic NOD mice.

マウスにおける自己免疫性糖尿病の養子移植モデルにおける移植片生存の生物発光モニタリング

Type 1 diabetes is characterized by the autoimmune destruction of the insulin-producing beta cells of the pancreas. A promising treatment for this disease ...

Induction of Graft Versus Host Disease in Mouse Models via Allogeneic Bone Marrow and Splenic Cell Transplantation

Source: Buchele, V., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/58871">Induction of Intestinal Graft-versus-host Disease and Its Mini-endoscopic Assessment in Live Mice.</a> J. Vis. Exp. (2019).
This video demonstrates the induction of intestinal graft-versus-host disease, GvHD, in a lethally-irradiated recipient mouse using allogeneic hematopoietic stem cells and GvHD-inducing splenic alloreactive T lymphocytes. This protocol helps establish a severe acute intestinal GvHD model to understand the mechanism of the disease and develop innovative therapeutic options.

同種骨髄移植および脾細胞移植によるマウスモデルにおける移植片対宿主病の誘導

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. GvHD ...

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

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