(383) - Rapamycin Enhances the Suppressive Capacity of Ex Vivo Expanded Regulatory T Cells (Tregs) Isolated From Pediatric Thymus

Abstract

Treg-based cellular therapy to suppress graft-directed immunity could reduce the need for life-long immunosuppressive drugs. Challenges include isolation and expansion of pure Tregs to clinically relevant numbers while maintaining stable function. We showed that large quantities of stable, highly suppressive Tregs can be expanded from discarded pediatric thymus using an expansion protocol that included rapamycin (RAPA). The thymus contains naïve T cells, thus contamination of isolated thymic Tregs (tTregs) with CD25+ effector T cells is low. We therefore questioned whether RAPA is needed for the expansion of therapeutic tTregs. Thymuses (n=4) were obtained during pediatric cardiac surgery. After mechanical dissociation, CD25+ tTregs were isolated by magnetic-bead based cell isolation and expanded with α-CD3, IL-2 and artificial APC without (-) or with (+) RAPA. After 7 days, RAPA was removed from the +RAPA culture and all cells were further expanded for 4-5 days. Treg characteristics were assessed by analyzing phenotype and suppressive capacity. Purity of isolated CD25+ tTregs was 82-95%; >70% of these cells were FOXP3+. tTregs +RAPA expanded significantly less than tTregs -RAPA (range: 6 to 18-fold vs. 145 to 270-fold, respectively; p=0.005). Both cultures were FOXP3+, Helios+ and CTLA-4+. Yet, tTregs +RAPA had less CD45RO+ cells and more LAP+ cells than Tregs -RAPA (p=0.005 and p=0.06, respectively). Moreover, both the in vitro proliferation and IFN-γ production of α-CD3/CD28-stimulated allogeneic T cells were more highly suppressed by tTregs +RAPA than tTregs -RAPA (p<0.001; Figure 1). Although tTregs +RAPA had a lower expansion capacity than tTregs -RAPA, these cells showed a stronger suppression of activated T-cell function, indicating that RAPA enhances the suppressive capacity of expanded tTregs. RAPA is therefore a favorable additive to the expansion protocol of therapeutic Tregs isolated from pediatric thymuses.