Utilization of leukapheresis and CD4 positive selection in Treg isolation and the ex-vivo expansion for a clinical application in transplantation and autoimmune disorders
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Karolina Gołąb1, Randall Grose2, Piotr Trzonkowski3, Amittha Wickrema4, Martin Tibudan1, Natalia Marek-Trzonkowska5, Sabrina Matosz1, Julia Solomina1, Diane Ostrega1, J. Michael Millis1, Piotr Witkowski1
1Department of Surgery, Section of Transplantation, University of Chicago, Chicago, USA
2South Australian Health and Medical Research Institute, University of Adelaide, Australia
3Department of Clinical Immunology and Transplantology, Medical University of Gdansk, Gdansk, Poland
4Department of Medicine, Section of Hematology-Oncology, Cancer Research Center, University of Chicago, Chicago, USA
5Department of Family Medicine, Medical University of Gdansk, Gdansk, Poland
Piotr Witkowski, email: firstname.lastname@example.org
Keywords: regulatory T cells, clinical application, leukapheresis, CD4+ cells, immunosupressive therapy
Received: January 27, 2016 Accepted: October 26, 2016 Published: November 04, 2016
Adoptive transfer of T regulatory cells (Tregs) is of great interest as a novel immunosuppressive therapy in autoimmune disorders and transplantation. Obtaining a sufficient number of stable and functional Tregs generated according to current Good Manufacturing Practice (cGMP) requirements has been a major challenge in introducing Tregs as a clinical therapy. Here, we present a protocol involving leukapheresis and CD4+ cell pre-enrichment prior to Treg sorting, which allows a sufficient number of Tregs for a clinical application to be obtained. With this method there is a decreased requirement for ex- vivo expansion. The protocol was validated in cGMP conditions. Our final Treg product passed all release criteria set for clinical applications. Moreover, during expansion Tregs presented their stable phenotype: percentage of CD4+CD25hiCD127− and CD4+FoxP3+ Tregs was > 95% and > 80%, respectively, and Tregs maintained proper immune suppressive function in vitro. Our results suggest that utilization of leukapheresis and CD4 positive selection during Treg isolation improves the likelihood of obtaining a sufficient number of high quality Treg cells during subsequent ex-vivo expansion and they can be applied clinically.
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