Dieses Projekt wurde finanziert, zum Teil unter Zuschüsse aus den National Institutes of Health (R01AI121180, R21AI109239 und K18CA151798), der American Diabetes Association (1-16-IBS-281) und der Pennsylvania Department of Health (Tobacco Settlement Funds) zu JS

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The authors have nothing to disclose.

In diesem Protokoll ist ein kritischer Schritt der in vitro Differenzierung von TCR / FoxP3 - Gen-Transduktion iPSCs. In vitro Notch - Signalweg induziert Entwicklung in Richtung der T - Zell - Abstammungslinie. Um iPSCs in CD4 + FoxP3 + Tregs unterscheiden, haben wir die OP9-DL1 / DL4 / IA b - Zellen, die hoch express MHC II (IA b) Moleküle. Die meisten der iPSCs differenzieren sich in CD4 + -Zellen. Doch nach der Oberfläche TCR-Expression, verlieren viele differenzierte pre-T-Zellen die Fähigkeit zu unterscheiden, und schließlich sterben. Als Ergebnis verringert sich die Zellzahl der iPSC abgeleiteten funktionellen Treg dramatisch nach vier Wochen in vitro - Differenzierung. Um dies zu vermeiden, Zugabe von IL-2 kann das Überleben der Zelle zu diesen Zeitpunkten zu verbessern. Eine alternative Methode , die Reifung und das Überleben von Ag-spezifische Pre-Tregs zu fahren, um die Pre-Tregs zu übertragen , die für eine Woche in Mäuse in vitro differenziert haben. Diese Pre-Tregs können continue Differenzierung und Reifung in vivo für weitere drei Wochen. Mit dieser Methode kann eine funktionelle Ag-spezifischen Treg Population von iPSCs erzeugt werden, die nTregs artig und haben die Fähigkeit zur Unterdrückung von Autoimmun arthritis in dem murinen Modell sind. Um die Wirksamkeit der in vivo Entwicklung von Ag-spezifischen iPSC-Treg, verschiedene Verbindungen (beispielsweise Retinsäure, gelectin) 13,14 oder suppressive Zytokine (zB TGF-β, IL-10) verbessern kann nach dem adoptiven Transfer verwendet werden , des vorge Treg. Zusätzlich das Überleben der Zelle zu erhöhen, können diese kombinierte Ansatz FoxP3 Expression 14,15 erhalten und die Qualität der Ag-spezifischen iPSC-Treg verbessern. Ein mögliches Problem , das für eine in vivo Treg Entwicklung entstehen könnte , ist insgesamt Immunsuppression, was zu Komplikationen bei Infektionen oder nach Gewichtsverlust. Eine große Anzahl von Ag-spezifische Tregs in vi Entwicklungvo kann Infektionen verschlimmern. In diesem Fall ist ein Suizid-Gen, die induzierbare Caspase 9 (ICasp9) 15,16 kann in den TCR / FoxP3 Vektor eingebaut werden. Dieser Ansatz ermöglicht die Entfernung der Stammzellen abgeleiteten Tregs durch die Injektion eines bioinerter kleinen Molekül-Dimerisierung Mittel (AP1903) auf &quot;abgeschaltet&quot; die Erzeugung von Stammzellen abgeleiteten Tregs, die dieses Potential Problem überwinden. Ein selbst Ag ist ein typisches Protein, das durch das Immunsystem des Wirten erkannt von einer einzelnen Autoimmunerkrankung leidet. Diese Selbst Ag ist das Ziel des Immunsystems und die korrelierten T-Zellen werden nicht gelöscht. Selbst Ag spezifische Tregs, die Verwendung von TCR Übertragung ist eine gute Wahl zu erzeugen. Ein selbst Ag (zB Hitzeschock - Protein) spezifische TCR kann in reifen CD4 + CD25 + Tregs aus peripheren mononukleären Blutzellen (PBMCs) transduziert werden, und dieser Ansatz wurde in klinischen Studien verwendet worden. Alternativ kann, wie described in diesem Protokoll können selbst Ag spezifische Tregs, die Gentransduktion der Selbst Ag spezifischen TCR mit FoxP3 und Stimulation mit Notch-Signalweg, Stammzell-abgeleiteten Tregs verwenden. Zellbasierten Therapien für Autoimmunerkrankungen entwickelt Treg Verwendung kann 17 nützlich sein. Obwohl TCR Transduktion in T - Zellen , die in klinischen Studien sicher, machbar und anwendbar zu sein, gibt es noch immer große Sicherheitsbedenken wegen der Autoimmunität verursacht durch Kreuzreaktivität mit gesundem Gewebe 18 bewährt hat. Darüber hinaus aktuelle Methoden, genetisch veränderten Tregs haben in der Regel eine kurzfristige Persistenz in vivo 19. Alternativ kann eine vielversprechende Quelle einer großen Anzahl von monoklonalen Ag-spezifischen Treg für die Entwicklung von Stammzellen ist. Embryonale Stammzellen (ES-Zellen) haben die beste Pluripotenz und Selbsterneuerung, aber es ist nicht möglich, sie von Patienten zu erhalten. Obwohl leicht aus dem peripheren Blut isoliert, sind hämatopoetischen Stammzellen (HSCs) multi-potenten Stammzellen und kann ähnlich wie WSR 20 in Zellkultur expandiert werden. IPSC vorliegenden Technologie zu einer effizienteren Erzeugung von PSC von Patienten somatischen Zellen durch Transduktion von verschiedenen Transkriptionsfaktoren vorgerückt. Eine Reihe von methodischen Verbesserungen wurden in den letzten Jahren entwickelt iPSCs zu erzeugen, indem maximal mögliche Gefahren wie Immunogenität und tumorigenicity reduzieren. Im Vergleich zu WSR haben iPSCs identisch Pluripotenz und Selbsterneuerung. Als Ergebnis kann iPSC Technologie einen Vorteil bei der Entwicklung von Patienten- und / oder krankheitsspezifische PSCs bereitzustellen. Die Verwendung von iPS - Zellen Ag-spezifische Tregs kann vorrücken auf dem Gebiet der zellbasierten Therapien für Autoimmunerkrankungen 10,11 zu entwickeln.

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

Autoimmunerkrankungen entstehen aufgrund der Verlust der immunologischen Selbsttoleranz. Regulatorische T-Zellen (Tregs) sind wichtige Vermittler von immunologischer Selbsttoleranz. Tregs repräsentieren etwa 5 - 10% der reifen CD4 + T - Zell - Subpopulation bei Mäusen und Menschen, mit etwa 1 bis 2% der Tregs im peripheren Blut zirkulieren. Induzierte pluripotenter Stammzellen (iPS) in funktionelle Treg unterschieden werden, die ein Potential für zellbasierte Therapie von Autoimmunerkrankungen eingesetzt werden. Hier präsentieren wir eine Methode Antigen (Ag) -spezifische Tregs von iPS - Zellen zu entwickeln (dh iPS-Tregs). Das Verfahren basiert auf den Transkriptionsfaktor FoxP3 enthält und eine Ag-spezifischen T-Zell-Rezeptor (TCR) in iPSCs und dann Notch Differenzierung auf Zellen OP9 Stromazellen exprimieren Liganden delta-like (DL) 1 und DL4. Folgende in vitro Differenzierung exprimieren die iPSC-Treg CD4, CD8, CD3, CD25, FoxP3 und Ag-spezifischen TCR und können Ag - Stimulation reagiert.Diese Methode wurde zur zellbasierte Therapie von Autoimmun Arthritis in einem Mausmodell erfolgreich angewendet. Adoptive Übertragung dieser Ag-spezifischen iPSC-Treg in Ag-induzierten Arthritis (AIA) -haltigen Mäusen hat die Fähigkeit, Gelenkentzündung und Schwellungen zu reduzieren und Knochenverlust zu verhindern.

Wie hier gezeigt, am Tag 28, Ag-spezifischen Treg ausgedrückt wesentlichen CD3 und Ag-spezifischen TCR, zwei T-Zell-Marker. Die CD3 + TCRVβ5 + Bevölkerung ausgedrückt CD4. Die meisten der CD3 + TCRVβ5 + CD4 + Zellen auch CD25, CD127 exprimiert und CTLA-4, die in erhöhten Mengen exprimiert werden , typischerweise in natürlich T regs (nTregs) und in T - Zellen exprimieren FoxP3 ektopisch auftritt. Foxp3 - Expression in iPSC abgeleiteten Zellen blieb sogar nach einer Langzeit - in vitro - Stimulation mit dem Notch - Liganden, wie durch Durchflusszytometrie (Figur 1) analysiert , indem die intrazelluläre Färbung nachgewiesen. Darüber hinaus erzeugt die Ag-spezifische iPSC-Treg suppressive Zytokine, wie TGF-β und IL-10, wenn in vitro mit Ag-gepulste Splenozyten (Abbildung 2) stimuliert, was anzeigt , das iPSC-Treg Potential suppressive Aktivitäten hatten. Die Fluoreszenzmikroskopie ergab,<html> dass mehr FoxP3 + Zellen in der OVA-behandelten vorhanden waren , als die PBS-behandelten Knie; es gab keine FoxP3 + Zellen in den Knien in Mäusen bestehenden die DsRed + Vektor-transduzierten iPS - Zellen zu erhalten. Viele mehr CD4 + FoxP3 + TCRVβ5 + in den Knien von Mäusen dargestellt Zellen iPSCs mit dem Midr-TCR-FoxP3 transduziert Empfangen als die Midr-FoxP3 (Abbildung 3). Diese Beobachtungen legen nahe, dass Ag-spezifischen iPS-Tregs wandern an die AIA Knie nach dem adoptiven Transfer in Empfängermäuse. Die übertragenen iPSC abgeleiteten Zellen wesentlich verringert die entzündliche Schwellung Knie wenn OVA vorhanden war, hatte aber keine Wirkung auf die Steuer Knie , die nur mit mBSA in dem murinen Modell (Figur 4) eingespritzt wurde; reduzierte Knochenverlust in der Übertragungskniezelle durch die hochauflösende Mikro-CT - System (Figur 5) sichtbar gemacht . tp_upload / 54720 / 54720fig1.jpg &quot;/&gt; Abb . 1: Die Differenzierung der Ag-spezifischen iPS-Tregs Murine iPSCs wurden mit einem Konstrukt transduziert: Midr-TCR &amp; agr;-2A-TCR &amp; bgr;-2A-FoxP3 enthält Gene von OVA-spezifischen TCR und FoxP3. Die Gen-transduzierten Zellen (DsRed +) wurden cokultiviert auf OP9-DL1-DL4-IA - B - Zellen in Gegenwart von mFlt3L und mIL-7. (A) Morphologie der T regs Zelldifferenzierung am Tag 0, 7, 14 und 22 (B) durchflusszytometrische Analyse für die Proteinexpression von iPSC abgeleiteten Zellen am Tag 28 CD3 + TCRVβ5 + Zellen , wie angegeben (R1) gated wurden und für die Expression von CD4 und CD8 analysiert, mit CD25, CD127, CTLA 4 und FoxP3 für Zellen als CD4 + CD8 gated gezeigt - Zellen (R2). (dunkle Linien, schraffierten Flächen zeigen Isotypkontrollen) <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54720/54720fig1large.jpg" target="_blank">Bitte klicken Sie hier</a> , <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54720/54720fig1large.jpg" target="_blank">um eine larg</a>er Version dieser Figur. <img alt="Figur 2" src="/files/ftp_upload/54720/54720fig2.jpg" /> Abbildung 2: Funktionelle Analyse von I n Vitro Differentiated Ag-spezifischen Treg Murine iPSC-Treg von Splenozyten stimuliert wurden. (APCs; T regs / APCs = 1: 4) und gepulst mit OVA 323-339 - Peptid. Die intrazellulären Zytokin - Produktion (TGF-β und IL-10) durch die Strömung analysiert Zytometrie nach + Zellen , die auf Live - CD4 + CD25 Gating. (Dunkle Linien, schraffierten Flächen Isotypkontrollen angeben) <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54720/54720fig2large.jpg" target="_blank">Bitte hier klicken</a> , um <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54720/54720fig2large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a> <img alt="Figur 3" src="/files/ftp_upload/54720/54720fig3.jpg" /> Abbildung 3: Ag spezifischen iPS-Tregs Infiltrieren in die Kniegelenke. Ag-spezifischen iPS-Tregs adoptiv in C57BL / 6 - Mäuse übertragen wurden. Kurz nach Arthritis Induktion (Tage 7-14) wurden die Knie entfernt und gefärbt für Immunhistologie (Maßstabsbalken: 20 &amp; mgr; m). Mäuse OVA-spezifischen iPS-Tregs haben eine große Anzahl von TCRVβ5 + Zellen in den Knien zu erhalten. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54720/54720fig3large.jpg" target="_blank">Bitte klicken Sie hier</a> , um <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54720/54720fig3large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a> <img alt="Abbildung 4" src="/files/ftp_upload/54720/54720fig4.jpg" /> Abbildung 4: adoptiven Transfer von Ag-spezifischen iPS-Tregs Verbessert AIA bei Mäusen Murine iPSCs wurden mit dem retroviralen Konstrukt transduziert Midr, Midr-FoxP3 oder Midr-TCR-FoxP3 und waren co-kultiviert auf der OP9-DL1 / DL4 /. IA - B - Zellen. Am Tag 7 werden die Gen-transduzierten Zellen (3 × 10 6 / Maus) wurden adoptively übertragen in weibliche C57BL / 6-Mäuse, die mit AIA zwei Wochen nach der Zellübertragung induziert wurden. Am Tag nach der Arthritis Induktion wurde die Schwere der Arthritis durch Messung des Knies Durchmesser. (AC) Prozent Anstieg der Knie Durchmesser überwacht. Erhöhung der Knie Durchmesser wurde durch die Untersuchung beide Knie in verblindet ausgewertet für jede Maus vor der Injektion am Tag 0 Arthritis-Score basiert auf Voreinspritzung Knie Durchmesser berechnet; jedes Knie wurde eine Punktzahl zugeordnet (0: keine sichtbare Schwellung oder Verfärbung, 1: sichtbare Schwellung mit oder ohne Verfärbung, 2: moderat mit Verfärbung Schwellung, 3: schwere mit Verfärbung Schwellungen). In jeder Gruppe wurden fünf Mäuse verwendet, und die Daten sind repräsentativ für drei unabhängige Experimente. Die Daten werden als Mittelwert ± SD dargestellt. (D) Der Mittelwert an Tag scoring 7 für beide Knie von fünf Mäusen. Die Daten werden als Mittelwert ± SD dargestellt aus drei unabhängigen Experimenten (** p &lt;0,01, *** p &lt;0,001, Zwei-Wege - ANOVA). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54720/54720fig4large.jpg" target="_blank">Bitte klicken Sie hier</a> , um <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54720/54720fig4large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a> <img alt="Abbildung 5" src="/files/ftp_upload/54720/54720fig5.jpg" /> Abbildung 5:. Ag-spezifischen iPS-Tregs Migrate auf die OVA injizierte Knie und reduzieren den Knochenverlust iPS-Tregs wurden adoptiv in C57BL / 6 - Mäuse übertragen. Innerhalb von 21 Tagen post-Arthritis Induktion wurden die Mäuse anästhesiert und der Knochenarchitektur um die Maus Knie wurde durch die Mikro-CT-System abgebildet wird. Mäuse , die OT-II TCR / FoxP3 Gen-transduzierten murinen iPS-Tregs signifikante Reduktion der Knochenschwund aufweisen , im Vergleich Mäuse , die Vektor-transduzierten iPS - Zellen zu steuern. (A) Die Scans mit einem 2,2 mm Länge durchgeführt wurden, und die Bilder wurden gefangen genommen , nachdem weitere Bildverarbeitung (Volumen-Rendering und Transformation;Maßstabsbalken:. 1 mm) (B) Knochenvolumen um Kniegelenk bewertet dreidimensionale Rekonstruktion von Mikro-CT - Bilder verwenden und im Inneren des Volumens von Interesse Knochenvolumen wurde (Maßstabsbalken berechnet. 1 mm) <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54720/54720fig5large.jpg" target="_blank">Bitte klicken Sie hier</a> , <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54720/54720fig5large.jpg" target="_blank">um</a> die <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54720/54720fig5large.jpg" target="_blank">sehen eine größere Version dieser Figur.</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.

Entwicklung von Stammzellen abgeleiteten Antigen-spezifische regulatorische T-Zellen gegen Autoimmunität

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

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

8.6K Aufrufe.  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. 

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

Regulatory T cells: Therapeutic Potential for Treating Transplant Rejection and Type I Diabetes

My name's Jeff Bluestone. I'm the director of the UCSF Diabetes Center. We're at a laboratory here and oversee the immune tolerance network.My work is involved in basic understanding of how the immune system distinguishes self from non-self and how we can use therapies to try to induce what we call immune tolerance, which is the ability of the immune system being modulated and protect itself from recognizing itself as foreign and attacking things like its own pancreas or brain or joints. That happens in many autoimmune diseases. We're particularly interested in therapies, both drug and cellular therapies that might be used to educate or reeducate the immune system so that we don't block or destroy our own ability To protect our own tissue.So whenever A a T cell comes through the thymus or B cell comes out of the bone marrow, it has to learn to distinguish self from non-self, and this is what we call immune tolerance, which is that ability to tolerate one's own tissue and not destroy it or attack it. What often happens in many disease settings is that the immune system because of environmental and genetic reasons, starts attacking one's own tissue and tolerance is broken and it's that system that we're trying to reestablish with some of our therapies. And the ultimate goal of immune tolerance therapies as opposed to generalized immunosuppression is to try to develop therapies or drugs that will in fact reestablish tolerance without requirement for ongoing therapy.So that today where somebody with a kidney transplant or a liver transplant might require 20 or 30 pills a day to keep the immune system from attacking the, the kidney or pancreas or, or or liver in the future. And we might be able to just provide some kind of therapy that would change the immune system so that it becomes tolerant to the kidney or liver And doesn't reject it. So one of the more Interesting elements of immune tolerance has been to discovery that there are specialized cells in the body whose job it is to protect the organs of the body and and to regulate the immune system so that it doesn't attack those tissues.These so-called regulatory T cells have become a very important and central part of understanding immune tolerance over the last decade or so. In fact, regulatory T cells are often known as suppressor. T cells have been around for 30 years since early discovery and and descriptions by people like Dick Han.Unfortunately, in the eighties, these cells came in some disrepute because of the absence of unique biological or biochemical markers that could distinguish them from other cells and data that was difficult to interpret given our current understanding of how T cells recognize proteins and how the immune system works. But then starting in the mid nineties through seminal work by investigators like Shimon Succi and others, we started to understand both the cellular as well as biochemical basis for regulatory T cells, such that when a transcription factor called Fox P three was discovered in early two thousands, we're able to finally put a specialized tag on these cells and really understand them. And ever since we've really been able to exploit those cells by expanding em, isolating em, and reintroducing em into animals to demonstrate that they are capable of inducing immune tolerance, which is what we are All about.One of the Most important questions that is challenge the field is trying to understand what the mechanism of tregs are, both in immune responses as well as during tolerance induction. And there have been a number of questions related to do the cells act on other T cells that are pathogenic? Do they act on antigen presenting cells or do they make cytokines that ultimately regulate the immune response?Our own studies have suggested based on in vi, in vivo imaging that in fact these cells act directly on the major antigen presenting cell in in the pancreas, and that is a dendritic cell. And we demonstrated that in fact we could label regulatory T cells one color green while the dendritic cells were labeled another color, and show that in fact these regulatory T cells interacted directly with the dendritic cells and by doing so, fundamentally altered the ability of those dendritic cells to activate the pathogenic T cell response. So we think that either directly or through some intermediate cytokine like IL 10 or TGF beta that the tregs educate or change the dendritic cells to make them less able to activate a pathogenic T cell response.Yeah, So one of the more interesting aspects of regulatory T cells has been the notion that in addition to this very specialized subset that exists in the thymus and develops during thymic development, the so-called natural T-Rex that during the course of an immune response, other T cells may in fact turn into regulatory T cells either through direct upregulation of Fox P three due to cytokines like TGF beta, which are known to turn on FOX P three or even other cells which are not Fox P three positive, but take on regulatory T-cell characteristics. Those so-called adaptive tregs are generally very important in in regulating an inflammatory response because they're interacting with the antigens that are present locally in that inflamed tissue. And those adaptive cells are probably critical in maintaining tolerance locally in a targeted tissue, whereas the natural tregs are probably more useful or important in maintaining a, a, a immune homeostasis or generalized suppression of auto reactivity in the whole animal Or patient.So now that we know That these regulatory cells are essential in in maintaining immune homeostasis, we can imagine what would happen if they're disrupted. And in fact, if you destroy Fox P three and either mouse models using knockout technology, or in fact there's a human disease called I pex, the individual animals die in a short period of time of a massive autoimmune disease where each targeted tissue, liver, kidney, heart are in infiltrated with T cells and the tissues are destroyed. So that's the fundamental immuno immune homeostasis that's required that these cells Really protect the individual.One of the important Discoveries with regards to regulatory T cells is the possibility that we might be able to both isolate them and expand them in a specific way to reintroduce into individuals with diseases to bring the immune system back into a homeostatic equilibrium. And in fact, work in our laboratory and others have been able to take these cells out and expand them up to a thousand fold and show that they retain their suppressor activity and can be reintroduced into individual animals to induce an a regulatory response. Where that's been most successful at, at this time has been in treating autoimmune disease and animal models of diabetes, for instance, we can prevent or even reverse diabetes by putting in small numbers of these antigen specific eyelid antigen specific regulatory T cells that we've expanded ex vivo and we hope that we can get into people soon with similar populations that have been expanded in in a similar manner.Now the future hopefully will go beyond autoimmune diseases and into more broad based immunological settings and perhaps the most daunting of which is transplantation. Today we transplant over a half a dozen organs into people, but still are required to give them lifelong immunosuppression to prevent those transplants from being rejected. The hope is that we can take regulatory cells that see the donor tissue from these kidney, liver, pancreas, eyelet transplants and be able to target those regulatory cells to those tissues and prevent the immune system from recognizing those tissues as far and now take this alloimmune response and convert it into a safe self response and prevent rejection without ongoing immunosuppressive Drug therapy.So What we hope is, is that this particular therapy is going to be very appropriate, appropriate for treating people with type one diabetes. Once all of the insulin producing beta cells have been destroyed, the only way we're ultimately gonna be able to treat the disease is by both reconstituting the individual with an eyelet or some insulin producing cell, and at the same time prevent the autoimmune and alloimmune responses from destroying it. So our approach is to take people with long-term diabetes and transplant eyelets at present from cric donors, but in the future, perhaps from stem cells or other sources to actually replace the insulin producing capability, we hope to introduce regulatory T cells into that setting to prevent those eyelets that are coming from foreign sources, from being rejected.As I mentioned, this notion that we can induce tolerance in these individuals will require not just the source of an insulin producing cell, but some combination of drugs and cellular therapies. We hope like regulatory cells to put the individual into a state of tolerance in homeostasis so that these foreign tissues are not recognized and destroyed once introduced into the diabetic patient. Specifically, our approach to actually reconstituting insulin producing beta cells in patients starts out with the kind of experiments we're doing in animal models, which is to take eyelets that have been prepared from a pancreas, remembering that only about 0.5%of all of the cells in the pancreas actually are the eyelets to make insulin to isolate those cells and purify them in a way that we can reintroduce them into a recipient.We reintroduce them either by putting them under the kidney capsule, which is routinely the way we do it in animal models are directly into the liver, which is the way we do it in humans. And once those cells go into that site, either kidney or liver, they then seed that site and start getting the blood and other nutrients that they need in order to survive and make the insulin that then gets circulated to other tissues and organs as needed. And the goal is to obviously, once those cells are in that tissue, to then treat with our various immunotherapeutics to prevent the immune system from attacking the cells Within those tissues.So of course there are a Number of challenges for all novel immunotherapies and tregs are no exception. We need to worry about a number of things as we try to this for a therapeutic. We need to worry about cell numbers at present, that we can only identify small numbers of cells that have the antigen specificity that we need to block both auto and allo immunity.So we'll need to figure out ways to both isolate those cells and expand them to sufficient numbers to treat an individual. In addition, we need to be careful that these cells are truly pure regulatory T cells. We don't wanna make the mistake of putting in cells that have the ability to be pathogenic and attack the foreign tissue as opposed to the regulatory cells which protect.And so understanding the stability and the robustness of these cells and how how they survive in various inflammatory settings will be critical. The third thing we need to worry about is non-specific immunosuppression that could be caused. These regulatory cells are designed to shut down broadly in immune response.And so we don't want to, at the same time we're blocking graft rejection, block the ability of an individual to recognize a bacteria or a virus or cancer cells that might come up, but instead stay targeted and focused on the tissue that we've transplanted into the individual. And then finally, I think the, the other challenge with any cellular therapy is being able to do it robustly and routinely many, many, many times over because each patient is gonna require their own set of cells. This is not a drug you can just take off the shelf.And so we're gonna have to work technologies out that are, use good manufacturing practices that are approved by the food and drug administration that allow us to grow these up the same way every time and be sure that we're working with a, a truly homogeneous and and well-defined population of Cells. When one Thinks about funding for projects like this, it's always a mix between the federal funding that we get through the NIH to support the basic science that's important in moving this forward. And then for us, fortunately, other organizations such as the Juvenile Diabetes Foundation who have been very supportive having raised an enormous amount of money from the community to help in these highly risky projects.And so we've been fortunate in being able to partner with both the NIH and JDRF as well as with pharmaceutical companies on occasion to be able to move these kinds of projects along. So at present, I think the funding is actually adequate and we really hope that we will be able to drive this project forward Effectively. We've been lucky in the diabetes community having such a strong, both federal and non-federal interest in the disease.Of course, that's not inappropriate given the fact that disease is really growing and reaching epidemic proportions, but we hope that the funding that exists will be sufficient for us to move This project. So Like all scientists, we always feel as if we are underfunded and underappreciated when you look at how much money is being spent to, to deal with problems around the world, to deal with war in Iraq, to deal with our, our military, and know that just a couple of days of that funding could support the NIH for a year. It's hard to imagine how we've placed our priorities because we think as scientists that there are a lot of things that we can do to change human health, human, the human state.So we only continue to hope that the advances in the science look in in 50 years. We're not gonna be talking about health insurance, we're not gonna be talking about HMOs and management. We're gonna talk about the amazing discoveries that have been made that are gonna change health in in our country that's gonna change health in the world.And just like the industrial revolution of turn of the last century, that in this century the, the major impact is gonna be on how we've changed diseases, both acute and chronic diseases. And so we hope that the public appreciates that and appreciates our efforts to try to make that kind of Difference.

Regulatorische T-Zellen: Therapeutische Potenzial zur Behandlung von Transplantatabstoßung und Typ-I-Diabetes

Forschung

Biologie

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.

Ein dreidimensionales thymisches Kultursystem zur Generierung von murine induzierten pluripotenten Stammzell-abgeleiteten Tumor-Antigen-spezifischen thymischen Auswanderern

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

Krebsforschung

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.

Generation der induzierte regulatorische T-Zellen aus primären humanen naiven und Gedächtnis-T-Zellen

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

Immunologie und Infektion

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.

Gerichtete Differenzierung von induzierten pluripotenten Stammzellen in Richtung T-Lymphozyten

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.

Verwendung von humaninduzierten pluripotenten Stammzellen zur Erzeugung von Tumorantigen-spezifischen T-Zellen

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.

Adenovirale Transduktion von Naive CD4 T-Zellen zu Treg Differenzierung Studieren

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.

Hocheffiziente Generierung von Antigen-spezifischen primären Maus-Zytotoxischen T-Zellen für Funktionstests in einem Autoimmun-Diabetes-Modell

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.

Stammzell-abgeleitete virale Ag-spezifische T-Lymphozyten unterdrücken HBV-Replikation bei Mäusen

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

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>Entwicklung und Ausbau von humanen CAR-Tregs für Immunregulationstherapien

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>Erzeugung humaner chimärer Antigenrezeptor-regulatorischer T-Zellen

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

Erzeugung humaner chimärer Antigenrezeptor-regulatorischer T-Zellen

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