EAK 16-II / EAKIIH6自己組織化ヒドロゲルとFACS精製した胸腺上皮細胞の3-D集約を推進
Summary
This video demonstrates a protocol to enrich thymic epithelial cells (TECs) with density gradient for FACS isolation. It also shows the use of EAK16-II/EAKIIH6 peptides to promote the TEC aggregate formation. The microenvironments of EAK16-II/EAKIIH6 hydrogel provide the 3-D configuration necessary to maintain the survival and function of the TECs.
Abstract
Thymus involution, associated with aging or pathological insults, results in diminished output of mature T-cells. Restoring the function of a failing thymus is crucial to maintain effective T cell-mediated acquired immune response against invading pathogens. However, thymus regeneration and revitalization proved to be challenging, largely due to the difficulties of reproducing the unique 3D microenvironment of the thymic stroma that is critical for the survival and function of thymic epithelial cells (TECs). We developed a novel hydrogel system to promote the formation of TEC aggregates, based on the self-assembling property of the amphiphilic EAK16-II oligopeptides and its histidinylated analogue EAKIIH6. TECs were enriched from isolated thymic cells with density-gradient, sorted with fluorescence-activated cell sorting (FACS), and labeled with anti-epithelial cell adhesion molecule (EpCAM) antibodies that were anchored, together with anti-His IgGs, on the protein A/G adaptor complexes. Formation of cell aggregates was promoted by incubating TECs with EAKIIH6 and EAK16-II oligopeptides, and then by increasing the ionic concentration of the medium to initiate gelation. TEC aggregates embedded in EAK hydrogel can effectively promote the development of functional T cells in vivo when transplanted into the athymic nude mice.
Introduction
胸腺は、獲得免疫系の機能に必須である病原体反応、自己寛容T細胞の多様な集団の生成を担う主要なリンパ器官です。それは、胸腺細胞の開発、リンパ球前駆細胞として骨髄から移住動的な器官である、胸腺間質のスポンジ状の三次元（3-D）マトリックスを通って移動し、系統仕様と分化を起こし、最終的にのように移住します成熟T細胞。このよくプログラムされたプロセスの成功は、移行胸腺細胞や住宅胸腺上皮細胞（TECを）、胸腺微小環境の整合性を確立し、維持するために必須である胸腺間質の優勢な集団間のクロストークに大きく依存します。
その解剖学的位置とユニークな機能に基づいて、TECのは、2つのサブセットに分けることができます。皮質でのTEC responある（cTECs）1,2- T細胞（正の選択）を制限し、自己MHC（主要組織適合遺伝子複合体）を選択するためのsible、および自己反応性T細胞を除去するために不可欠である髄質（mTECs）中のTEC（負の選択）。多くの要因（ 例えば 、老化、感染症、放射線照射、薬物治療）は妥協適応免疫が得られ、胸腺上皮に不可逆的な損傷を引き起こす可能性があります。多くの試みにもかかわらず、胸腺機能を回復することは、胸腺微小環境を再現する難しさに挑戦してきました。 2次元環境で培養のTECが急激胸腺形成3,4のための重要な遺伝子の発現を減少させる一方、特に、胸腺三次元（3-D）の構成は、のTECの生存および機能に重要です。
EAK16-II（AEAEKAKAEAEAKAK）およびそのC末端histidinylatedアナログEAKIIH6（AEAEKAKAEAEAKAKHHHHHH）は低分子量であり、脱イオンWATEに可溶性である両親媒性オリゴペプチドRが、20mMのNaClを（ヒト体液中の正常な塩濃度は、154 mMで）より高いイオン強度に曝されたとき、β原線維を形成するためにゲル化を受けます。この環境応答性は、3D構造を形成するように多目的なビルディングブロックを作ります。複合タンパク質薬物および他の生体分子を固定するためのアダプタとして機能することができる（：EAKII-H6上のHisタグは、ドッキング機構を、これにより抗HisのIgG / Fc結合組換えタンパク質A / G（PA / GはαH6）提供します例えばヒドロゲル複合5-8に、抗体）。
我々は以前に、ヒドロゲルに固定蛍光標識されたIgG分子は、最大13日間9のための注入部位に保持することができることを実証しました。 αH6時更に、抗CD4 IgGを用いて固定PA / GアダプタがEAK16-II / EAKIIH6（EAK）ヒドロゲルに添加し、CD4 + T細胞を特異的に10を捕捉しました。同様の技術を使用して、我々は最近のTEC Cの3-Dの凝集を実証しています（：PA / G：αEpCAMαH6）TEC-特定の抗EpCAM抗体を運ぶアダプター複合体とEAKハイドロゲルに昇格するウルド。ヌードマウスの腎臓カプセル下に移植した場合、EAKハイドロゲルに埋め込 まれたTECクラスタが効果的に生体内11,12に機能的なT細胞の開発を支援することができます。
ここでは、迅速かつ効果的に蛍光活性化細胞選別（FACS）でのTECを浄化し、EAKハイドロゲルシステムと3次元TEC凝集体を生成するために、我々の方法を示します。
Protocol
Representative Results
Discussion
TECは、胸腺間質の優勢な集団であると胸腺の構造と機能のために重要な役割を果たしているが、それらは、総胸腺細胞充実の約0.1から0.5パーセントを表します。彼らはまた、細胞死の高いパーセンテージは時折すなわち 、治療は、細胞外マトリックス（ECM）からのTECを解離するために、コラゲナーゼ消化後に観察されている脆弱な細胞です。その希少性（マウス胸腺あたり200,000）お?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
この作品は、国立衛生研究所によって部分的にサポートされていたR21 AI113000（WSM）とR01 AI123392（YF）を付与します。
Materials
| 1. TEC Isolation | |||
| 70% Ethanol | Decon Laboratories | 2701(1 Gallon) | Ethanol 200 Proof, deionized water |
| Dissecting scissors, straight | Fine Science Tools, Inc. | 91460-11 | |
| Graefe forceps, straight | Fine Science Tools, Inc. | 11053-10 | |
| Graefe forceps, curved | Fine Science Tools, Inc. | 11052-10 | |
| Washing solution | 1X PBS, 0.1% BSA, 2mM EDTA | ||
| 1x PBS (Phosphate buffered saline) | Gibco | 10010-023 | |
| BSA (Bovine serum albumin) | Sigma | A1470-100G | |
| EDTA (ethylenediaminetetraacetic acid) | Invitrogen | 15575-038 | |
| Digestion solution | 9 mL RPMI-1640, 0.025 mg/mL Liberase TM Research grade, 10 mM HEPES, 0.2 mg/mL DNaseI | ||
| RPMI-1640 | Gibco | 11879-020 | |
| Liberase TM Research Grade | Roche | 05 401 127 001 | referred as "purified collagenase" |
| 1M HEPES | Lonza | 17-737E | |
| Dnase I | Roche | 10 104 159 001 | |
| 50mL Centrifuge tube | Corning | 430290 | |
| 60mm tissue culture dish | Falcon | 353002 | |
| 1/2cc U-100 Insulin syringe 28G1/2 | Becton Dickinson | 329461 | |
| 5mL Polystyrene round-bottom tube | Falcon | 352058 | |
| 5ml glass pipet | Fisher Healthcare | 13-678-27E | Use for rinsing the thymic fragments. Thymic fragments tend to stick to the wall with plastic pipets. |
| MACSmix tube rotator | Miltenyi | 130-090-753 | |
| 100um Cell strainer | Falcon | 352360 | |
| Density gradient medium: OptiPrep | Axis-Shield | ||
| Name | Company | Catalog Number | Comments |
| 2. Cell sorting | |||
| 5mL Polypropylene round-bottom tube | Falcon | 352063 | |
| Anti-mouse CD16/CD32 (Fc Block) | BD Biosciences | 553142 | Use as undiluted, 2uL per sample |
| Anti-mouse CD45-PercpCy5.5 | eBioscience | 45-0451-80 | Use at 1:150, 10uL per sample |
| Anti-mouse CD326 (EpCAM)-PE | eBioscience | 12-5791-82 | Use at 1:100, 10uL per sample |
| BD Influx | BD Biosciences | ||
| Single cell analysis software | FlowJo | ||
| Name | Company | Catalog Number | Comments |
| 2. EAK gel assembly | |||
| Anti-His-Tag | AnaSpec | 29673 | "anti-His-Tag IgG" |
| Purified anti-mouse CD326 (EpCAM) | BioLegend | 118202 | "anti-EpCAM IgG" |
| Recombinant protein A/G | Pierce Biotechnology | ||
| 1.5mL Safe-Lock Tubes, Biopur, Sterile | Fisher Healthcare | 05-402-24B | referred as "1.5mL microcentrifuge tube" |
| 96-well, Tissue culture plate, Round-bottom with low evaporation lid | BD Falcon | 353917 | |
| Rocking platform: Nutator Mixer no.1105 | BD Clay Adams | ||
| 10% sucrose | Sigma | S0389 | Prepare with sterile distilled water |
| EAK16-II (AcNH-AEAEAKAKAEAEAKAK-CONH2) | American Peptide Company | custom synthesized, 10mg/mL | |
| EAKIIH6 (AcNH-AEAEAKAKAEAEAKAKHHHHHH-CONH2) | American Peptide Company | custom synthesized, 7.5mg/mL | |
| Complete medium | RPMI-1640, 10% FBS, 1% Pen/Strep, 1% L-glutamine, 1% NEAA, 5mM HEPES, 50uM 2-Mercaptoethanol | ||
| RPMI-1640 | Gibco | 11879-020 | |
| FBS (Fetal Bovine Serum) | Atlanta Biologicals | S11150 | Heat inactivated before use |
| Pen/Strep | Gibco | 15140-122 | |
| L-glutamine 200mM (100x) | Gibco | 25030-081 | |
| NEAA (non-essential amino acid) 100x | Gibco | 11140-050 | |
| 1M HEPES | BioWhittaker | 17-737E | |
| 2-Mercaptoethanol (100X) | Millipore | ES-007-E | |
| Platform shaker: The Belly Dancer | Stovall Life Sciences Inc. | model: USBDbo |
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