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...
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| Veröffentlicht in: | Oncotarget 2016-11, Vol.7 (48), p.79474-79484 |
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| creator | Gołąb, Karolina Grose, Randall Trzonkowski, Piotr Wickrema, Amittha Tibudan, Martin Marek-Trzonkowska, Natalia Matosz, Sabrina Solomina, Julia Ostrega, Diane Michael Millis, J Witkowski, Piotr |
| description | 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. |
| doi_str_mv | 10.18632/oncotarget.13101 |
| format | Article |
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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.</description><identifier>ISSN: 1949-2553</identifier><identifier>EISSN: 1949-2553</identifier><identifier>DOI: 10.18632/oncotarget.13101</identifier><identifier>PMID: 27821811</identifier><language>eng</language><publisher>United States: Impact Journals LLC</publisher><subject>Biomarkers - metabolism ; CD4-Positive T-Lymphocytes - cytology ; Cell Separation ; Flow Cytometry ; Forkhead Transcription Factors - metabolism ; Humans ; Interleukin-2 Receptor alpha Subunit - metabolism ; Interleukin-7 Receptor alpha Subunit - metabolism ; Leukapheresis - methods ; Research Paper ; T-Lymphocytes, Regulatory - cytology</subject><ispartof>Oncotarget, 2016-11, Vol.7 (48), p.79474-79484</ispartof><rights>Copyright: © 2016 Gołąb et al. 2016</rights><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c356t-a9d2c9d9133a1275619b135bbd52ced37547c2b698ceedd2eff2ff39d8a988713</citedby><cites>FETCH-LOGICAL-c356t-a9d2c9d9133a1275619b135bbd52ced37547c2b698ceedd2eff2ff39d8a988713</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5346728/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5346728/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,724,777,781,882,27905,27906,53772,53774</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/27821811$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Gołąb, Karolina</creatorcontrib><creatorcontrib>Grose, Randall</creatorcontrib><creatorcontrib>Trzonkowski, Piotr</creatorcontrib><creatorcontrib>Wickrema, Amittha</creatorcontrib><creatorcontrib>Tibudan, Martin</creatorcontrib><creatorcontrib>Marek-Trzonkowska, Natalia</creatorcontrib><creatorcontrib>Matosz, Sabrina</creatorcontrib><creatorcontrib>Solomina, Julia</creatorcontrib><creatorcontrib>Ostrega, Diane</creatorcontrib><creatorcontrib>Michael Millis, J</creatorcontrib><creatorcontrib>Witkowski, Piotr</creatorcontrib><title>Utilization of leukapheresis and CD4 positive selection in Treg isolation and the ex-vivo expansion for a clinical application in transplantation and autoimmune disorders</title><title>Oncotarget</title><addtitle>Oncotarget</addtitle><description>Adoptive transfer of T regulatory cells (Tregs) is of great interest as a novel immunosuppressive therapy in autoimmune disorders and transplantation. 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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.</description><subject>Biomarkers - metabolism</subject><subject>CD4-Positive T-Lymphocytes - cytology</subject><subject>Cell Separation</subject><subject>Flow Cytometry</subject><subject>Forkhead Transcription Factors - metabolism</subject><subject>Humans</subject><subject>Interleukin-2 Receptor alpha Subunit - metabolism</subject><subject>Interleukin-7 Receptor alpha Subunit - metabolism</subject><subject>Leukapheresis - methods</subject><subject>Research Paper</subject><subject>T-Lymphocytes, Regulatory - cytology</subject><issn>1949-2553</issn><issn>1949-2553</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2016</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpVUU1PJCEQJRuNGvUHeDEcvbTbwHQ3XEzMuF-JyV70TGionmGloQV64u5P2l-5OOPXcnmV1HuvqngInZH6kvCW0c_B65BVXEG-JIzU5BM6ImIhKto0bO9DfYhOU_pVl9csOk7FATqkBQkn5Aj9vc_W2T8q2-BxGLCD-UFNa4iQbMLKG7y8WeApJJvtBnACB3rLtR7fRVhhm4LbqZ_JeQ0YnqqN3YSCk_LpuTOEiBXWznqrlcNqmlwpXm1yLLTJKZ_ffdScgx3H2QM2ZUI0ENMJ2h-US3D6gsfo_uuXu-X36vbntx_L69tKs6bNlRKGamEEYUwR2jUtET1hTd-bhmowrCu_oGnfCq4BjKEwDHQYmDBcCc47wo7R1c53mvsRjAZfNnRyinZU8bcMysr_O96u5SpsZMMWbUd5Mbh4MYjhcYaU5WiTBldOhDAnSTjrqKC1aAuV7Kg6hpQiDG9jSC23Mcv3mOU25qI5_7jfm-I1VPYPwLiswg</recordid><startdate>20161129</startdate><enddate>20161129</enddate><creator>Gołąb, Karolina</creator><creator>Grose, Randall</creator><creator>Trzonkowski, Piotr</creator><creator>Wickrema, Amittha</creator><creator>Tibudan, Martin</creator><creator>Marek-Trzonkowska, Natalia</creator><creator>Matosz, Sabrina</creator><creator>Solomina, Julia</creator><creator>Ostrega, Diane</creator><creator>Michael Millis, J</creator><creator>Witkowski, Piotr</creator><general>Impact Journals LLC</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20161129</creationdate><title>Utilization of leukapheresis and CD4 positive selection in Treg isolation and the ex-vivo expansion for a clinical application in transplantation and autoimmune disorders</title><author>Gołąb, Karolina ; 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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. 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| subjects | Biomarkers - metabolism CD4-Positive T-Lymphocytes - cytology Cell Separation Flow Cytometry Forkhead Transcription Factors - metabolism Humans Interleukin-2 Receptor alpha Subunit - metabolism Interleukin-7 Receptor alpha Subunit - metabolism Leukapheresis - methods Research Paper T-Lymphocytes, Regulatory - cytology |
| title | 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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