Journal
ONCOTARGET
Volume 7, Issue 48, Pages 79460-79470Publisher
IMPACT JOURNALS LLC
DOI: 10.18632/oncotarget.13101
Keywords
regulatory T cells; clinical application; leukapheresis; CD4(+) cells; immunosupressive therapy
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Funding
- CRC-National Center for Advancing Transitional Sciences of the NIH [UL1TR000430]
- National Centre for Research and Development, Poland [STRATEGMED1/233368/1/NCBR/2014]
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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(+)CD25(hi)CD127(-) 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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