University of Alberta
Cryopreserved regulatory T cells (Tregs) isolated from discarded human thymus potently suppress allogeneic and xenogeneic immune responses after expansion
Esme Dijke1,2, Romy Hoeppli3, Karen Seeberger4, Ingrid Larsen1,2, Ivan Rebeyka2,4, David Ross2,4, Gregory Korbutt2,4, Megan Levings3, Lori West1,2,4.
1Pediatrics, University of Alberta, Edmonton, AB, Canada; 2Alberta Transplant Institute, University of Alberta, Edmonton, AB, Canada; 3Surgery, University of British Columbia, Vancouver, BC, Canada; 4Surgery, University of Alberta, Edmonton, AB, Canada
Background: Regulatory T cells (Tregs) modulate immune responses directed to alloantigens and xenoantigens and are therefore a promising therapeutic tool in organ transplantation. Challenges include isolation and expansion of pure Tregs to clinically relevant numbers while maintaining stable function. Clinical feasibility could improve by efficient cryopreservation of Tregs for future infusions or expansions. We previously showed that large quantities of CD25+ thymocytes can be isolated from discarded human thymuses, routinely removed during pediatric cardiac surgery, and expanded to highly suppressive and stable Tregs. Here we studied whether thymic Tregs (tTregs) can be cryopreserved and the potential of expanded tTregs to suppressive allogeneic and xenogeneic immune responses.
Methodology: Thymocytes were isolated by mechanical dissociation from thymuses obtained during pediatric cardiac surgery. CD25+ tTregs were isolated by magnetic-bead cell separation and directly expanded with α-CD3, IL-2, rapamycin and artificial antigen-presenting cells or stored in liquid nitrogen; cryopreserved cells were thawed and expanded after >3 months of storage. Treg characteristics were assessed by analyzing FOXP3 expression, cytokine production and suppressive capacity of the proliferation of α-CD3/28-stimulated T cells. Additionally, expanded tTregs were co-cultured with T cells stimulated with irradiated allogeneic monocyte-derived dendritic cells or peripheral blood mononuclear cells (PBMC) stimulated with irradiated neonatal porcine islets to define their suppressive capacity of allogeneic and xenogeneic immune responses respectively.
Results: The expansion capacity of cryopreserved tTregs was comparable to that of fresh tTregs (range: 11–36-fold expansion). Both expanded fresh and cryopreserved tTregs maintained high FOXP3 expression, contained few IFN-γ, IL-2 or IL-17-producing cells and potently suppressed proliferation of α-CD3/28-stimulated T cells. Furthermore, expanded tTregs were strong suppressors of both allogeneic and xenogeneic immune responses: >80% inhibition of proliferation of allogeneic-stimulated T cells and >70% inhibition of proliferation of xenogeneic-stimulated PBMC was observed at a Treg:responder cell ratio of 1:2.5.
Conclusion: Expansion capacity, phenotype and function of tTregs were not affected by cryopreservation. Expanded tTregs potently suppressed allogeneic and xenogeneic-stimulated responder cells. These results indicate that discarded human thymus is an excellent source of abundant Tregs for cellular therapy in both allo- and xenotransplantation. Future studies will focus on testing the ability of expanded thymic Tregs to specifically suppress allogeneic and xenogeneic immune responses in vivo in a humanized transplant model.
07:00 - 08:00
|Orals: Tolerance, Zoonosis||Cryopreserved regulatory T cells (Tregs) isolated from discarded human thymus potently suppress allogeneic and xenogeneic immune responses after expansion||Room 109|