Pediatric Thymic Tissue as a Source of Regulatory T Cells for Cellular Therapy

Abstract

Purpose Infant heart transplant (HTx) recipients have better graft survival than older recipients. Nonetheless, due to need for lifelong therapy, infants carry a heavier immunosuppressive burden. Cellular therapy using regulatory T cells (Tregs) to suppress graft-directed immune responses would greatly benefit these infants. A major challenge is generating a large quantity of stable, suppressive Tregs. During infant cardiac surgery, thymectomy is usually performed to gain exposure of the retrosternal operative field. We studied the potential of thymic tissue as a source of CD25+FOXP3+ Tregs. Methods and Materials Thymuses (n=8) were obtained during pediatric cardiac surgery; thymocytes were recovered through mechanical dissociation. FOXP3+ cells were sorted by automated magnetic cell separation of CD25+ cells and expanded with αCD3, IL-2, rapamycin and CD32+ L-cells; CD25- cells were used as controls. FOXP3 and intracellular cytokine staining were done to define characteristics. Suppressive capacity was determined by co-culturing expanded cells with αCD3/CD28-stimulated peripheral blood mononuclear cells (PBMC) and analyzing proliferative responses by Cell Proliferation ELISA. Results FOXP3+ cell frequency within total thymocytes ranged from 2.2 to 3.2%. Isolated CD25+ cells were 72% positive for FOXP3 (median, range: 60-81%). After two weeks of culture, we observed a 4 to 48-fold expansion of CD25+ cells with >95% viability; 0.4 to 25-fold expansion was observed for control cells with 49–88% viability. Expanded CD25+ cells were >90% FOXP3+ and produced no IL-2 or IFN-γ, whereas control cells were 50% at a 1:10 ratio of Tregs:PBMC. Conclusions Highly suppressive FOXP3+ Tregs can be expanded from CD25+ thymocytes isolated from pediatric thymic tissue, indicating that explanted thymuses may be a source of Tregs for cellular therapy for infant HTx recipients.