Utilization of leukapheresis and CD4 positive selection in Treg isolation and the ex-vivo expansion for a clinical application in transplantation and autoimmune disorders.
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.
Department of Surgery, Section of Transplantation, University of Chicago, Chicago, USA.
Karolina
K
Gołąb
South Australian Health and Medical Research Institute, University of Adelaide, Australia.
Randall
R
Grose
Department of Clinical Immunology and Transplantology, Medical University of Gdansk, Gdansk, Poland.
Piotr
P
Trzonkowski
Department of Medicine, Section of Hematology-Oncology, Cancer Research Center, University of Chicago, Chicago, USA.
Amittha
A
Wickrema
Department of Surgery, Section of Transplantation, University of Chicago, Chicago, USA.
Martin
M
Tibudan
Department of Family Medicine, Medical University of Gdansk, Gdansk, Poland.
Natalia
N
Marek-Trzonkowska
Department of Surgery, Section of Transplantation, University of Chicago, Chicago, USA.
Sabrina
S
Matosz
Department of Surgery, Section of Transplantation, University of Chicago, Chicago, USA.
Julia
J
Solomina
Department of Surgery, Section of Transplantation, University of Chicago, Chicago, USA.
Diane
D
Ostrega
Department of Surgery, Section of Transplantation, University of Chicago, Chicago, USA.
J
J
Michael Millis
Department of Surgery, Section of Transplantation, University of Chicago, Chicago, USA.
Piotr
P
Witkowski
D015415:Biomarkers / Q000378:metabolism; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology*; D002469:Cell Separation; D005434:Flow Cytometry; D051858:Forkhead Transcription Factors / Q000378:metabolism; D006801:Humans; D053645:Interleukin-2 Receptor alpha Subunit / Q000378:metabolism; D053699:Interleukin-7 Receptor alpha Subunit / Q000378:metabolism; D007937:Leukapheresis / Q000379:methods*; D050378:T-Lymphocytes, Regulatory / Q000166:cytology*
D015415:Biomarkers; C418974:FOXP3 protein, human; D051858:Forkhead Transcription Factors; C508017:IL2RA protein, human; D053645:Interleukin-2 Receptor alpha Subunit; D053699:Interleukin-7 Receptor alpha Subunit
CD4+ cells; clinical application; immunosupressive therapy; leukapheresis; regulatory T cells