INTRODUCTION: 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 highly suppressive and stable Tregs can be expanded from discarded pediatric thymuses using a protocol that included rapamycin (RAPA). Thymus contains mainly naive T cells, thus contamination risk with CD25+ effector T cells after isolation is low. We therefore questioned whether RAPA is required for expansion of thymic (t)Tregs.

METHODS: Thymuses (n=4) were obtained during pediatric cardiac surgery. After mechanical dissociation, CD25+ tTregs were isolated by magnetic-bead based cell separation and expanded with α-CD3, IL-2 and artificial APC without and with RAPA (-RAPA and +RAPA, respectively). After 7 days, RAPA was removed from cultures; cells were further expanded for 4-5 days. Tregs were assessed by analyzing phenotype and suppressive capacity.

RESULTS: tTregs +RAPA had significantly lower expansion capacity than tTregs -RAPA (6 – 18-fold vs. 145 – 270-fold respectively; p=0.005). Both expanded cultures were FOXP3+, Helios+, CTLA-4+. Yet, tTregs +RAPA had fewer CD45RO+ and more LAP+ cells than Tregs -RAPA (p=0.005 and p=0.06 respectively). Moreover, both 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; Fig 1).

CONCLUSION: Although tTregs +RAPA had lower expansion capacity than tTregs -RAPA, these cells demonstrated stronger suppression of in vitro proliferation and IFN-γ production of activated T cells, indicating that RAPA enhances the suppressive function of ex vivo expanded tTregs. RAPA is thus a favourable additive to ex vivo expansion protocols of therapeutic Tregs isolated from pediatric thymuses.

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