Research Specialist 3
University of Chicago
Towards therapeutic application of regulatory T cells: clinical grade isolation, ex vivo expansion and cryopreservation
Karolina Golab1, Omid Savari1, Zehra Tekin1, Martin Tibudan1, Sabarinathan Ramachandran1, Natalia Marek-Trzonkowska2, Piotr Trzonkowski3, Piotr Witkowski1.
1Department of Surgery, University of Chicago, Chicago, IL, United States; 2Department of Family Medicine, Medical University of Gdańsk, Gdańsk, Poland; 3Department of Clinical Immunology and Transplantology, Medical University of Gdańsk, Gdańsk, Poland
Introduction: Adoptive transfer of T regulatory cells (Tregs) is of great interest as a novel immunosuppressive therapy for autoimmune disorders and in transplantation.
The major challenge to implement such therapy in clinics is to obtain sufficient numbers of stable, functional Tregs generated accordingly to the current Good Manufacturing Practice (cGMP).
Here, we present protocol for Treg production including isolation, expansion and cryopreservation, which we established and validated in our GMP facility.
Methods: Leukapheresis product obtained from healthy volunteer was used to perform CD4 positive immunomagnetic separation on CliniMACS® system. Isolated CD4+ cells were screened for sterility and subsequently stained with clinical-grade antibodies (CD4 PerCP, CD25 APC, CD127 PE) and Tregs were sorted utilizing BD FACSAria™ III cell sorter incorporated into our GMP facility. After sorting, isolated Tregs were stimulated with anti-CD3/CD28 expansion beads at cell to bead ratio 1:1 and expanded for 13 days in X-VIVO 20 medium with 10% Human Serum and 2000 IU/ml of IL-2 in the GMP facility. At day 7, Tregs were re-stimulated with fresh beads at the same ratio. Treg markers expression: CD4, CD25, CD127, FoxP3 was analyzed by flow cytometry at day 0, 7th and 13th. At the end of expansion, Tregs were collected, beads were removed on clinical-grade ClinExVivo™ MPC® magnet in the closed bag system. Next, sample of the Final Treg Product was taken for the Release Criteria Assays: sterility, mycoplasma, endotoxin, determination of residual beads, viability, Treg identity and purity (FoxP3, CD4, CD8 expression). Additionally, Treg potency was tested in suppression of proliferation assay with CFSE-stained T effectors. Tregs remained after expansion were frozen in control-rate freezer in PlasmaLiteTM with 5% human serum albumin, 10% DMSO and then kept in liquid nitrogen. After 1 year, cells were thawed and viability/apoptosis was checked by flow cytometry. After overnight culture, Tregs were expanded again and after expansion tested to pass the release criteria as described above.
Results: 1.6x109 CD4+ cells were isolated from leukapheresis product and 5x108 of CD4+ cells were used for Treg sorting. 9.7x106 of Tregs were obtained after sorting. Next, after 13-day ex vivo expansion, we achieved 1.77x109 Tregs with 182-fold increase in cell number. During the expansion, cells presented stable Treg phenotype: percentage of CD4+CD25hiCD127- and FoxP3+ Tregs was >95% and >80%, respectively. Final Treg Clinical Product passed all set release criteria. Expanded Tregs indicated also proper suppressive function in in vitro suppression of proliferation assay.
Cryopreserved Tregs just after thawing were characterized by low recovery - 45.4% and high apoptotic rate - 41%. However, after stimulation, they expanded again well with over 100x fold cell count increase, stable FoxP3 and Treg markers expression (% of CD4+FoxP3+ > 90%, CD4+CD25hiCD127-/lo > 95%). They also indicated in vitro good suppressive abilities and passed all release criteria.
Conclusions: Here, we presented optimized Treg ex vivo expansion protocol implemented in our GMP facility allowing obtaining clinical grade cell product, which meets all quality and quantity criteria for clinical application. We also presented that excess of expanded Tregs can be cryopreserved, stored and used again, offering attractive approach for future Treg therapies in autoimmune diseases and transplantation.
15:30 - 17:00
|Islet Cell Transplantation - Miscellaneous||Towards therapeutic application of regulatory T cells: clinical grade isolation, ex vivo expansion and cryopreservation||Room 111-112|