Summary

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

Published: November 08, 2016
doi:

Summary

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.

Abstract

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.

Introduction

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-β 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.

Protocol

Alle Tierversuche werden von der Pennsylvania State University College of Medicine Animal Care Committee (IACUC Protokoll # 45470) zugelassen und werden in Übereinstimmung mit den Richtlinien der Vereinigung für die Bewertung und Zulassung von Labortierpflege durchgeführt. 1. Stammzellkultur Inkubieren einer 10-cm-Schale mit 10 ml 0,1% Gelatine für mindestens 30 Minuten bei 37 ° C (Inkubator), um die Platte zu beschichten. Entfernen Gelatine aus der Schale und der P…

Representative Results

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 i…

Discussion

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, verl…

Disclosures

The authors have nothing to disclose.

Acknowledgements

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

Materials

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

References

  1. Firestein, G. S. Evolving concepts of rheumatoid arthritis. Nature. 423, 356-361 (2003).
  2. Ferraro, A., et al. Interindividual variation in human T regulatory cells. Proc Natl Acad Sci U S A. 111, E1111-E1120 (2014).
  3. Tang, Q., et al. In vitro-expanded antigen-specific regulatory T cells suppress autoimmune diabetes. J Exp Med. 199, 1455-1465 (2004).
  4. van Herwijnen, M. J., et al. Regulatory T cells that recognize a ubiquitous stress-inducible self-antigen are long-lived suppressors of autoimmune arthritis. Proc Natl Acad Sci U S A. 109, 14134-14139 (2012).
  5. Wright, G. P., et al. Adoptive therapy with redirected primary regulatory T cells results in antigen-specific suppression of arthritis. Proc Natl Acad Sci U S A. 106, 19078-19083 (2009).
  6. Schmitt, T. M., et al. Induction of T cell development and establishment of T cell competence from embryonic stem cells differentiated in vitro. Nat Immunol. 5, 410-417 (2004).
  7. La Motte-Mohs, R. N., Herer, E., Zuniga-Pflucker, J. C. Induction of T-cell development from human cord blood hematopoietic stem cells by Delta-like 1 in vitro. Blood. 105, 1431-1439 (2005).
  8. Lei, F., Haque, R., Weiler, L., Vrana, K. E., Song, J. T lineage differentiation from induced pluripotent stem cells. Cell Immunol. 260, 1-5 (2009).
  9. Lei, F., Haque, R., Xiong, X., Song, J. Directed differentiation of induced pluripotent stem cells towards T lymphocytes. J Vis Exp. , e3986 (2012).
  10. Lei, F., et al. In vivo programming of tumor antigen-specific T lymphocytes from pluripotent stem cells to promote cancer immunosurveillance. Cancer Res. 71, 4742-4747 (2011).
  11. Haque, R., et al. Programming of regulatory T cells from pluripotent stem cells and prevention of autoimmunity. J Immunol. 189, 1228-1236 (2012).
  12. Chi, V., Chandy, K. G. Immunohistochemistry: paraffin sections using the Vectastain ABC kit from vector labs. J Vis Exp. , (2007).
  13. Lu, L., et al. Critical role of all-trans retinoic acid in stabilizing human natural regulatory T cells under inflammatory conditions. Proc Natl Acad Sci U S A. 111, E3432-E3440 (2014).
  14. Wu, C., et al. Galectin-9-CD44 interaction enhances stability and function of adaptive regulatory T cells. Immunity. 41, 270-282 (2014).
  15. Di Stasi, A., et al. Inducible apoptosis as a safety switch for adoptive cell therapy. N Engl J Med. 365, 1673-1683 (2011).
  16. Ramos, C. A., et al. An inducible caspase 9 suicide gene to improve the safety of mesenchymal stromal cell therapies. Stem Cells. 28, 1107-1115 (2010).
  17. Haque, R., Lei, F., Xiong, X., Wu, Y., Song, J. FoxP3 and Bcl-xL cooperatively promote regulatory T cell persistence and prevention of arthritis development. Arthritis Res Ther. 12, R66 (2010).
  18. van Loenen, M. M., et al. Mixed T cell receptor dimers harbor potentially harmful neoreactivity. Proc Natl Acad Sci U S A. 107, 10972-10977 (2010).
  19. Kim, Y. C., et al. Engineered antigen-specific human regulatory T cells: immunosuppression of FVIII-specific T- and B-cell responses. Blood. 125, 1107-1115 (2015).
  20. Himburg, H. A., et al. Pleiotrophin regulates the expansion and regeneration of hematopoietic stem cells. Nat Med. 16, 475-482 (2010).

Play Video

Cite This Article
Haque, M., Fino, K., Sandhu, P., Song, J. Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity. J. Vis. Exp. (117), e54720, doi:10.3791/54720 (2016).

View Video