このプロジェクトは、健康（R01AI121180、R21AI109239とK18CA151798）、米国糖尿病協会の国立研究所からの助成金の下で、一部には、資金を供給された（1月16日 - IBS-281）、および健康のペンシルベニア州省（たばこ和解基金）へJS

<ol>
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The authors have nothing to disclose.

このプロトコルでは、重要なステップは、TCR / FoxP3の遺伝子形質iPS細胞のインビトロ分化である。 インビトロで Notchシグナル伝達は、T細胞系統に向けて開発を誘導します。 CD4 + FoxP3の+ TregのにiPS細胞を区別するために、我々は非常にMHC II（IA b）の分子を発現するOP9-DL1 / DL4 / IA B細胞を、使用していました。 iPS細胞の大部分は、CD4 +細胞に分化します。しかし、表面TCR発現の後、多くの分化プレT細胞が分化し、最終的には死ぬ能力を失います。その結果、IPSC由来の機能的Tregの細胞数は劇的インビトロ分化の4週間後に減少します。これを回避するために、IL-2の添加は、これらの時点での細胞生存率を向上させることができます。 Ag特異的プレTregの成熟および生存を駆動する別の方法は、マウスに一週間のために、in vitroで分化した前のTregを転送することです。これらの予備Tregは共存することができntinue差別と別の3週間のためにin vivoで成熟。この方法を使用して、機能的なAg特異的Treg細胞集団はnTregs状であり、マウスモデルにおいて自己免疫性関節炎を抑制する能力を有するiPS細胞から生成することができます。 Ag特異的IPSC - Tregのin vivoでの発達の効力を改善するために、別の化合物（ 例えば 、レチノイン酸、gelectin）13,14または抑制性サイトカイン（ 例えば 、TGF-β、IL-10）は、養子移入後に使用することができます前Tregの。細胞の生存を増加させることに加えて、この組み合わせアプローチは、Foxp3発現14,15を維持し、Ag特異的IPSC - Tregの品質を向上させることができます。 in vivoでのTreg開発のために生じ得る潜在的な問題は、感染またはその後の体重減少時の合併症で、その結果、全体的な免疫抑制です。 viの中で開発Ag特異的Tregの多数VOは、感染症を悪化させる可能性があります。この場合、自殺遺伝子は、誘導性カスパーゼ9 （iCasp9）15,16は 、TCR / FoxP3のベクターに組み込むことができます。このアプローチは、生体不活性小分子二量体化剤（AP1903）の注射による幹細胞由来のTregの除去はこの潜在的な問題を克服します幹細胞由来のTregの生成を「遮断」することができます。 自己Agが、個々の自己免疫疾患を患っているホストの免疫系によって認識典型的なタンパク質です。この自己Agが免疫系の標的であり、相関T細胞が削除されません。自己のAg特異的Tregを生成するには、TCR伝達の使用は良い選択です。自己のAg（ 例えば 、熱ショックタンパク質）は、特定のTCRは、末梢血単核細胞（PBMC）から成熟したCD4 + CD25 + Tregsのに導入することができ、このアプローチは、臨床試験において利用されています。また、DESなど遺伝子のFoxP3と自己のAg特異的TCRの伝達およびNotchシグナル伝達による刺激を使用して、このプロトコルでcribed、細胞由来のTregをステムは、自己のAg特異的Tregすることができます。 工学のTregを利用し、自己免疫疾患のための細胞ベースの治療は、17有用である可能性があります。 T細胞におけるTCR形質導入は、安全な実行可能、及び臨床試験においても適用可能であることが判明しているが、原因で健康な組織18との交差反応性によって引き起こされる自己免疫の主要な安全性の問題は依然として存在します。さらに、現在の方法を用いて、遺伝的に改変されたTregは通常、 生体内 19 の短期的な持続性を持っています。あるいは、モノクローナルAg特異的Tregの多数を開発するための有望な供給源は、幹細胞です。胚性幹細胞（ESC）は、最高の多分化能および自己再生を持っているが、患者からそれらを得ることは不可能です。簡単に末梢血から単離したが、造血幹細胞（HSC）は、マルチです強力な幹細胞とESCの20と同様に、細胞培養物中で増殖することができます。本IPSC技術は、異なる転写因子の形質導入によって、患者の体細胞からPSCのより効率的な生成に進んでいます。方法論的な改良の数が最大にかかる免疫原性および腫瘍原性のような潜在的な危険を減少させることによりiPS細胞を生成するために、近年開発されてきました。 ESCのに比べ、性IPSCは、同一の多能性と自己複製を持っています。その結果、IPSC技術はpatient-および/または疾患特異のPSCを開発する利点を提供することができます。 Ag特異性Tregを開発するiPS細胞の使用は、自己免疫疾患10,11のための細胞ベースの治療の分野を進めることができます。

Autoimmune arthritis is a systemic disease characterized by hyperplasia of synovial tissue and progressive destruction of articular cartilage, bone, and ligaments1. The defective generation or function of Tregs in autoimmune arthritis contributes to chronic inflammation and tissue injury because Tregs play a crucial role in preventing the development of auto-reactive immune cells. 
Manipulation of Tregs is an ideal strategy for the development of therapies to suppress inflammation in an Ag-dependent manner. For Treg-based immunotherapy, the specificity of the transferred Tregs is important for the treatment of ongoing autoimmunity2. To exhibit the suppressive activity, Tregs need to migrate and be retained at the afflicted region, which can be directed by the specificity of the TCR for the Ag at that location3. Although polyclonal Tregs may contain a small population containing this Ag specificity from their TCRs, the numbers of these Ag-specific Tregs are usually low. Consequently, cell-based therapies using polyclonal Tregs against autoimmune disorders require adoptive transfers of a large number of Tregs4,5. Because pluripotent stem cells (PSCs) have the ability to develop into any type of cell, Ag-specific PSC-Tregs may prove to be good candidates for Treg-based immunotherapy. Previous studies have shown the successful development of PSC-derived T cells, including Tregs6-8. 
Here, we describe a protocol to develop Ag-specific iPSC-Tregs. We further describe a cell-based therapy of autoimmune arthritis in a murine model using such Tregs. This method is based upon genetically modifying murine iPSCs with Ag-specific TCRs and the transcriptional factor FoxP3. The engineered iPSCs then differentiate into Ag-specific Tregs on the OP9 stromal cells expressing Notch ligands DL1, DL4, and MHC-II (I-Ab) molecules in the presence of cytokines mFlt3L and mIL-7. These Ag-specific iPSC-Tregs can produce suppressive cytokines, such as TGF-&#946; and IL-10, when stimulated with the Ag, and adoptive transfer of such Tregs has the ability to suppress AIA development in a murine model. The described protocol can be used to develop stem cell-derived Ag-specific Tregs for potential therapeutic interventions.

<table><tbody><tr><td>C57BL/6j mice</td><td>Jackson Laboratory</td><td>664</td><td></td></tr><tr><td>B6.129S7 Rag1tm1Mom/J</td><td>Jackson Laboratory</td><td>2216</td><td></td></tr><tr><td>Anti-CD3 (2C11) antibody</td><td>BD Pharmingen</td><td>553058</td><td></td></tr><tr><td>Anti-CD28 (37.51) antibody</td><td>BD Pharmingen</td><td>553295</td><td></td></tr><tr><td>Anti-CD4 (GK1.5) antibody</td><td>Biolegend</td><td>100417</td><td></td></tr><tr><td>Anti-CD8 (53&amp;ndash;6.7) antibody</td><td>Biolegend</td><td>100714</td><td></td></tr><tr><td>Anti-CD25 (3C7) antibody</td><td>Biolegend</td><td>101912</td><td></td></tr><tr><td>Anti-TCR-&amp;beta; (H57597) antibody</td><td>Biolegend</td><td>109220</td><td></td></tr><tr><td>Anti-IL10</td><td>Biolegend</td><td>505010</td><td></td></tr><tr><td>Anti-TGF&amp;beta;</td><td>Biolegend</td><td>141402</td><td></td></tr><tr><td>DMEM</td><td>Invitrogen</td><td>ABCD1234</td><td></td></tr><tr><td>&amp;alpha;-MEM</td><td>Invitrogen</td><td>A10490-01</td><td></td></tr><tr><td>FBS</td><td>Hyclone</td><td>SH3007.01</td><td></td></tr><tr><td>Brefeldin A</td><td>Sigma</td><td>B7651</td><td></td></tr><tr><td>Polybrene</td><td>Sigma</td><td>107689</td><td></td></tr><tr><td>Genejammer</td><td>Integrated science</td><td>204130</td><td></td></tr><tr><td>ACK Lysis buffer</td><td>Lonza</td><td>10-548E</td><td></td></tr><tr><td>mFlt-3L</td><td>peprotech</td><td>250-31L</td><td></td></tr><tr><td>mIL-7</td><td>peprotech</td><td>217-17</td><td></td></tr><tr><td>Gelatin</td><td>Sigma</td><td>G9391</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P6148-500G</td><td>Caution: Allergenic, Carcenogenic, Toxic</td></tr><tr><td>Permeabilization buffer</td><td>Biolegend</td><td>421002</td><td></td></tr><tr><td>mBSA</td><td>Sigma</td><td>A7906</td><td></td></tr><tr><td>Ova albumin</td><td>Avantor</td><td>0440-01</td><td></td></tr><tr><td>CFA</td><td>Difco</td><td>2017014</td><td></td></tr><tr><td>Tailveiner restrainer</td><td>Braintree scientific</td><td>RTV 150-STD</td><td></td></tr></tbody></table>

development of stem cell-derived antigen-specific regulatory t cells against autoimmunity

自己免疫疾患は、免疫学的自己寛容の損失に起因し発生します。制御性T細胞（Treg）免疫学的自己寛容の重要なメディエーターです。末梢血中を循環するものTregの2％ -約1で、マウスおよびヒトにおける成熟したCD4 + T細胞亜集団の10％ - Tregは約5を表します。人工多能性幹細胞（iPS細胞）は、自己免疫疾患の細胞ベースの治療のために使用される可能性を持っている機能のTregへ分化させることができます。ここでは、iPS細胞からの抗原（Ag）から固有のTreg（ すなわち 、IPSC-のTreg）を開発するための方法を提示します。この方法は、iPS細胞への転写因子のFoxP3及びAg特異的T細胞受容体（TCR）を組み込み、次いでノッチを発現OP9間質細胞上で分化に基づいてデルタ様（DL）1とDL4のリガンド。 in vitroでの分化に続いて、IPSC-Tregは、CD4、CD8、CD3、CD25、FoxP3の、およびAg特異的TCRを発現し、Agの刺激に応答することができます。この方法は、正常マウスモデルにおいて自己免疫性関節炎の細胞ベースの治療に適用されています。マウス-bearingのAg誘導関節炎（AIA）にこれらのAg特異的IPSC - Tregの養子移入は、関節の炎症および腫脹を軽減し、骨損失を防止する能力を有します。

28日目に、ここに示されるように、Ag特異的Tregは、実質的にCD3およびAg特異的TCR、2つのT細胞マーカーを発現しました。 CD3 +TCRVβ5+集団は、CD4を発現しました。 + CD3 +TCRVβ5+ CD4のほとんどの細胞はまた、CD25、CD127、およびCTLA-4、一般的に天然に存在するTのREGS（nTregs）および異所のFoxP3を発現するT細胞で上昇したレベルで発現される表現しました。フローサイトメトリー（図1）によって分析し、細胞内染色によって検出されるIPSC由来細胞中でFoxp3発現は、Notchリガンドでin vitro刺激後も長期間持続しました。 IPSC-Tregは潜在的な抑制活性を持っていた示し、Agをパルス脾細胞（図2） を用いてインビトロで刺激した場合また、Ag特異的IPSC-Tregは、そのようなTGF-βおよびIL-10などの抑制サイトカインを産生しました。蛍光顕微鏡は、明らかに<html>より多くのFoxP3 +細胞は、PBS処置膝よりもOVA処理した中に存在していたこと。何のFoxP3 + DsRedを+ベクター形質導入iPS細胞を受けたマウスでは膝に存在する細胞が存在しませんでした。さらに多くのCD4 + MIDR-FoxP3のよりMIDR-TCR-のFoxP3で形質導入iPS細胞を受けたマウスの膝に提示のFoxP3 +TCRVβ5+細胞（図3）。これらの観察は、Ag特異的IPSC-Tregはレシピエントマウスに養子移入した後、AIAの膝に移行することを示唆しています。転送IPSC由来細胞は、実質的にOVAが存在したときに腫れ炎症性膝を減少させたが、唯一のマウスモデル（図4）でのmBSAを注射した対照膝に影響を及ぼしませんでした。高解像度のマイクロCTシステム（図5）によって可視化セル転送膝の骨の損失を減少させました。 tp_upload / 54720 / 54720fig1.jpg &quot;/&gt; 図1：Ag 特異的IPSC-Tregの分化マウスiPS細胞を構築物で形質導入した：MIDR-TCRα-2A-TCRβ-2A-のFoxP3は、OVA特異的TCRとのFoxP3の遺伝子を含みます。遺伝子導入細胞（DsRedを+）は mFlt3LおよびMIL-7の存在下でOP9-DL1〜DL4-IAのB細胞で共培養した。0日目のT regsは細胞の分化、7、14の（A）モルフォロジーと当日のIPSC由来細胞のタンパク質発現のための22（B）フローサイトメトリー分析28. CD3 +TCRVβ5+細胞が示されているように（R1）ゲーティングおよびCD25、CD127、CTLA-で、CD4とCD8の発現について分析しました4、とのFoxP3は、CD4 + CD8としてゲート細胞に対して示さ-細胞（R2）（暗線;斜線部分はアイソタイプ対照を示している）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54720/54720fig1large.jpg" target="_blank">ラーグを表示するには、こちらをクリックしてください。</a>この図のERバージョン。 <img alt="図2" src="/files/ftp_upload/54720/54720fig2.jpg" /> 図2：I nはインビトロ 分化したAg特異的Treg の機能解析マウスIPSC-のTregは、脾臓細胞によって刺激した。抗原提示細胞（APC; Tのregsは / APCに= 1：4）およびOVA 323-339ペプチドをパルス。細胞内サイトカイン産生（TGF-βおよびIL-10）は、細胞+ライブCD4 + CD25でゲーティングした後、フローサイトメトリーによって分析した（暗線;斜線部分はアイソタイプ対照を示している）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54720/54720fig2large.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> <img alt="図3" src="/files/ftp_upload/54720/54720fig3.jpg" /> 図3：グラム固有の膝関節へのIPSC-のTreg潜入。Ag特異的IPSC-Tregが養子C57BL / 6マウスに移しました。まもなく関節炎誘導（日7から14）の後、膝を除去し、免疫組織学のために染色された（スケールバー：20μm）を。 OVA特異的IPSC-のTregを受けたマウスは、膝でTCRVβ5+細胞の数が多い。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54720/54720fig3large.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> <img alt="図4" src="/files/ftp_upload/54720/54720fig4.jpg" /> 図4：Ag 特異的IPSC-Tregの養子移入は、マウスにおけるAIAを改善するマウスiPS細胞は、レトロウイルス構築物MIDR、MIDR-のFoxP3、またはMIDR-TCR-のFoxP3としたOP9-DL1上の共培養/ DL4 /形質導入しました。 IA B細胞。 7日目に、遺伝子導入細胞（3×10 6 /マウス）となりましたdoptively 2週間細胞移入後AIAで誘導した雌のC57BL / 6マウスに移しました。関節炎の誘導後の日に、関節炎の重症度は、膝の直径を測定することによりモニターした。膝の直径（AC）％増加。膝の直径の増加0関節炎スコアは盲検様式で両膝を調べることにより評価した日に注射する前に、各マウスのためのパイロット噴射膝直径に基づいて計算しました。各膝はスコア割り当てられていた（0：目に見える腫れや変色を; 1：または変色することなく、腫れ目に見える; 2：変色して腫れ適度な; 3：重度の変色と腫れ）。各群において、5匹のマウスを使用し、データは3つの独立した実験の代表です。データは、平均±SDとして表される。（D）平均は5匹のマウスから両膝のための7日目に得点します。データは、3つの独立した実験（** はp &lt;からの平均±SDとして表されます0.01、*** P &lt;0.001、双方向ANOVA）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54720/54720fig4large.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> <img alt="図5" src="/files/ftp_upload/54720/54720fig5.jpg" /> 図5：OVA にAg特異的IPSC-Tregの移行が膝を注入し、骨損失を低減 IPSC-のTregが養子C57BL / 6マウスに移しました。 21日後、関節炎誘導の中では、マウスを麻酔し、マウスの膝の周りの骨のアーキテクチャは、マイクロCTシステムによって画像化しました。 OT-II TCR / FoxP3の遺伝子を形質導入したマウスIPSC-Tregは、ベクター形質導入iPS細胞を受けたマウスを制御するために比較して、骨損失の大幅な減少を示す受けたマウス。（A）スキャンが2.2ミリメートルの長さを用いて行った、と画像が後に捕獲されましたさらに、画像処理（ボリュームレンダリングと変換。スケールバー：。（スケールバー1ミリメートル）（B）膝関節の周りの骨量は、マイクロCT画像の3次元再構成を用いて評価し、関心ボリューム内部骨体積を計算した：1ミリメートル） <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54720/54720fig5large.jpg" target="_blank">にはこちらをクリックしてくださいこの図の拡大版を表示します。</a>

Watch this Scientific Journal Video about Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity at JoVE.com

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

自己免疫に対する幹細胞由来の抗原特異的制御性T細胞の開発

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

development-stem-cell-derived-antigen-specific-regulatory-t-cells

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

8.6K ビュー.  The Pennsylvania State University College of Medicine. We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model. 

ビデオ: Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a promising source for adoptive T cell therapy (ACT). However, classical in vitro methods for producing regenerated T cells from iPSC result in either innate-like or terminally differentiated T cells, which are phenotypically and functionally distinct from na&#239;ve T cells. Recently, a novel three-dimensional (3D) thymic culture system was developed to generate a homogenous subset of CD8&#945;&#946;+ antigen-specific T cells with a na&#239;ve T cell-like functional phenotype, including the capacity for proliferation, memory formation, and tumor suppression in vivo. This protocol avoids aberrant developmental fates, allowing for the generation of clinically relevant iPSC-derived T cells, designated as iPSC-derived thymic emigrants (iTE), while also providing a potent tool to elucidate the subsequent functions necessary for T cell maturation after thymic selection.

This article describes a novel method to generate tumor antigen-specific induced pluripotent stem cell-derived thymic emigrants (iTE) by a three-dimensional (3D) thymic culture system. iTE are a homogenous subset of T cells closely related to na&#239;ve T cells with the capacity for proliferation, memory formation, and tumor suppression.

マウス誘導多能性幹細胞由来腫瘍抗原特異的胸腺移民を生成する三次元胸腺培養システム

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a ...

がん研究

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

初代ヒトナイーブおよびメモリーT細胞から誘導した調節性T細胞の生成

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

免疫・感染学

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies 1. CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines 2-7. However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies.
TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases 8-10. However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic 11-13, HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture 14-16. Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs.
Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.

Generation of T lymphocytes from induced pluripotent stem (iPS) cells gives an alternative approach of using embryonic stem cells for T cell-based immunotherapy. The method shows that by utilizing either in vitro or in vivo induction system, iPS cells are able to differentiate into both conventional and antigen-specific T lymphocytes.

Tリンパ球に向かって誘導多能性幹細胞の分化の指示

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

Using Human Induced Pluripotent Stem Cells for the Generation of Tumor Antigen-specific T Cells

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of patients with advanced cancer. Adoptive T cell transfer (ACT) of highly enriched tumor antigen-specific T cells has been shown to cause durable regression of metastatic cancer in some patients. However, during expansion, these cells may become exhausted or senescent, limiting their effector function and persistence in vivo. Induced pluripotent stem cell (iPSC) technology may overcome these obstacles by leading to in vitro generation of large numbers of less differentiated tumor antigen-specific T cells. Human iPSC (hiPSC) have the capacity to differentiate into any type of somatic cell, including lymphocytes, which retain the original T cell receptor (TCR) genomic rearrangement when a T cell is used as a starting cell. Therefore, reprogramming of human tumor antigen-specific T cells to hiPSC followed by redifferentiation to T cell lineage has the potential to produce rejuvenated tumor antigen-specific T cells. Described here is a method for generating tumor antigen-specific CD8&#945;&#946;+ single positive (SP) T cells from hiPSC using OP9/DLL1 co-culture system. This method is a powerful tool for in vitro T cell lineage generation and will facilitate the development of in vitro derived T cells for use in regenerative medicine and cell-based therapies.

This article describes a method to generate functional tumor antigen-specific induced pluripotent stem cell-derived CD8&#945;&#946;+ single positive T cells using OP9/DLL1 co-culture system.

腫瘍抗原特異的T細胞の生成にヒト誘導多能性幹細胞を用いた

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of ...

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

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 T cells in vitro with TCR- and co-stimulation in the presence of TGF&beta; and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown.
Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44low, CD62Lhigh) and resting (CD25-, CD69-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.

Adenoviral gene transfer into naive CD4 T cells with transgenic expression of the Coxsackie adenovirus receptor enables the molecular analysis of regulatory T cell differentiation in vitro.

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 ...

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 ...

Stem Cell-Derived Viral Ag-Specific T Lymphocytes Suppress HBV Replication in Mice

Hepatitis B virus (HBV) infection is a global health issue. With over 350 million people affected worldwide, HBV infection remains the leading cause of liver cancer. This is a major concern, especially in developing countries. Failure of the immune system to mount an effective response against HBV leads to chronic infection. Although HBV vaccine is present and novel antiviral medicines are being created, eradication of virus-reservoir cells remains a major health topic. Described here is a method for the generation of viral antigen (Ag) -specific CD8+ cytotoxic T lymphocytes (CTLs) derived from induced pluripotent stem cells (iPSCs) (i.e., iPSC-CTLs), which have the ability to suppress HBV replication. HBV replication is efficiently induced in mice through hydrodynamic injection of an HBV expression plasmid, pAAV/HBV1.2, into the liver. Then, HBV surface Ag-specific mouse iPSC-CTLs are adoptively transferred, which greatly suppresses HBV replication in the liver and blood as well as prevents HBV surface Ag expression in hepatocytes. This method demonstrates HBV replication in mice after hydrodynamic injection and that stem cell-derived viral Ag-specific CTLs can suppress HBV replication. This protocol provides a useful method for HBV immunotherapy.

Presented here is a protocol for the effective suppression of hepatitis B virus (HBV) replication in mice by utilizing adoptive cell transfer (ACT) of stem cell-derived viral antigen (Ag)-specific T lymphocytes. This procedure may be adapted for potential ACT-based immunotherapy of HBV infection.

幹細胞由来ウイルスAg特異的Tリンパ球はマウスにおけるHBV複製を抑制する

Hepatitis B virus (HBV) infection is a global health issue. With over 350 million people affected worldwide, HBV infection remains the leading cause of ...

In Vivo Augmentation of Gut-Homing Regulatory T Cell Induction

Inflammatory bowel disease (IBD) is an inflammatory chronic disease in the gastrointestinal tract (GUT). In the United States, there are approximately 1.4 million IBD patients. It is generally accepted that a dysregulated immune response to gut bacteria initiates the disease and disrupts the mucosal epithelial barrier. We recently show that gut-homing regulatory T (Treg) cells are a promising therapy for IBD. Accordingly, this article presents a protocol for in vivo augmentation of gut-homing Treg cell induction. In this protocol, dendritic cells are engineered to produce locally high concentrations of two molecules de novo, active vitamin D (1,25-dihydroxyvitamin D or 1,25[OH]2D) and active vitamin A (retinoic acid or RA). We chose 1,25(OH)2D and RA based on previous findings showing that 1,25(OH)2D can induce the expression of regulatory molecules (e.g., forkhead box P3 and interleukin-10) and that RA can stimulate the expression of gut-homing receptors in T cells. To generate such engineered dendritic cells, we use a lentiviral vector to transduce dendritic cells to overexpress two genes. One gene is the cytochrome P450 family 27 subfamily B member 1 that encodes 25-hydroxyvitamin D 1&#945;-hydroxylase, which physiologically catalyzes the synthesis of 1,25(OH)2D. The other gene is the aldehyde dehydrogenase 1 family member A2 that encodes retinaldehyde dehydrogenase 2, which physiologically catalyzes the synthesis of RA. This protocol can be used for future investigation of gut-homing Treg cells in vivo.

Here we present a protocol for in vivo augmentation of gut-homing regulatory T cell induction. In this protocol, dendritic cells are engineered to locally produce high concentrations of the active vitamin D (1,25-dihydroxyvitamin D or 1,25[OH]2D) and the active vitamin A (retinoic acid or RA) de novo.

腸内調節制御T細胞誘導の生体内増強

Inflammatory bowel disease (IBD) is an inflammatory chronic disease in the gastrointestinal tract (GUT). In the United States, there are approximately 1.4 ...

Generation of Human Regulatory T Cell Clones

Human regulatory T cells (Treg) are notoriously difficult to isolate in high purity given the current methods of Treg enrichment. These methods are based on the identification of Treg through several activation-dependent cellular surface markers with varying expression levels in different physiologic and pathologic conditions. Populations isolated as &#8220;Treg&#8221; therefore often contain considerable numbers of non-Treg effector cells (i.e., Teff) which hamper the precise phenotypic and functional characterization of these cells, their genomic and proteomic characterization, their reliable enumeration in different states of health and disease, as well as their isolation and expansion for therapeutic purposes. The latter, in particular, remains a major hurdle, as the inadvertent expansion of effector cells homing in Treg-relevant cellular compartments (e.g., CD4+CD25+ T cells) may render Treg-based immunotherapy ineffective, or even harmful. This work presents a method that circumvents the problems associated with population-based isolation and expansion of Treg and shows that the generation of Treg candidate clones with the subsequent selection, culture, and expansion of only carefully vetted, monoclonal cells, enables the generation of an ultrapure Treg cell product that can be kept in culture for many months, enabling downstream investigation of these cells, including for possible therapeutic applications.

This protocol describes the cloning and expansion of human regulatory T cells for the generation of ultra-high purity viable human Treg with stable demethylation at the Treg-specific demethylated region (TSDR) and Treg-specific phenotypic features.

Human regulatory T cells (Treg) are notoriously difficult to isolate in high purity given the current methods of Treg enrichment. These methods are based ...

<meta charset="utf8"></head><body>免疫制御療法のためのヒトCAR制御細胞の工学と拡大

Generation of Human Chimeric Antigen Receptor Regulatory T Cells

Chimeric antigen receptor (CAR) T-cell therapy has reshaped the face of cancer treatment, leading to record remission rates in previously incurable hematological cancers. These successes have spurred interest in adapting the CAR platform to a small yet pivotal subset of CD4+ T cells primarily responsible for regulating and inhibiting the immune response, regulatory T cells (Tregs). The ability to redirect Tregs&#39; immunosuppressive activity to any extracellular target has enormous implications for creating cell therapies for autoimmune disease, organ transplant rejection, and graft-versus-host disease. Here, we describe in detail methodologies for bona fide Treg isolation from human peripheral blood, genetic modification of human Tregs utilizing either lentivirus or CRISPR/Cas9-aided knock-in using adeno-associated virus-mediated homologous directed repair (HDR) template delivery, and ex vivo expansion of stable human CAR Tregs. Lastly, we describe the assessment of human CAR Treg phenotypic stability and in vitro suppressive function, which provides insights into how the human CAR Tregs will behave in preclinical and clinical applications.

<meta charset="utf8"></head><body>ヒトキメラ抗原受容体制御性T細胞の作製

This protocol provides a streamlined workflow to generate and test human chimeric antigen receptor regulatory T cells (CAR Tregs).

ヒトキメラ抗原受容体制御性T細胞の作製

Chimeric antigen receptor (CAR) T-cell therapy has reshaped the face of cancer treatment, leading to record remission rates in previously incurable ...