Expansion of Highly Suppressive FOXP3+ Regulatory T Cells from Pediatric Thymic Tissue

Abstract

Introduction: Infant heart transplant (HTx) recipients have better graft survival than patients transplanted at older ages. Nonetheless, due to lifelong need for therapy, infant HTx recipients can expect to carry an immunosuppressive heavier burden, resulting in substantial morbidities from adverse drug effects. Development of a cellular therapy using regulatory T cells (Tregs) to suppress graft-directed immune responses would greatly benefit these infants. A major challenge for such a therapy, however, is generating a large quantity of stable, highly suppressive Tregs. Infants undergoing HTx usually have thymectomy in order to gain adequate exposure of the retrosternal operative field. We investigated the potential of explanted thymic tissue as a source for isolation and expansion of highly suppressive CD4+CD25+CD127lowFOXP3+ Tregs. Methodology: Thymic tissue (n=3) was obtained from thymectomy during pediatric cardiac surgery and thymocytes were recovered through mechanical dissociation. FOXP3+ cells were isolated by automated magnetic cell separation of CD4+CD127lowCD25+ thymocytes. CD4+CD127low-depleted cells were used as controls. Cells were expanded for two weeks by stimulation with anti-CD3, IL-2, rapamycin and CD32+ L cells. FOXP3 and intracellular cytokine staining was performed to define characteristics of expanded cells. The suppressive capacity after expansion was determined by co-culturing the expanded cells with PKH-labeled anti-CD3/CD28-stimulated peripheral blood mononuclear cells (PBMC) and analyzing proliferative responses by flow cytometry. Results: CD4+CD25+CD127lowFOXP3+ cell frequency within the total thymocyte population ranged from 2.8 to 7.9%. Isolated CD4+CD25+CD127low cell populations were 65-84% positive for FOXP3. After two weeks of culture, we observed a 4 to 40-fold expansion of CD4+CD25+CD127low cells with >95% viability; 0.4 to 25-fold expansion was observed for control cells with 49-88% viability. The expanded CD4+CD25+CD127low cells were >95% FOXP3+ and produced no IL-2 or IFN-γ, whereas control cells were < 14% FOXP3+ and 58-65% produced IFN-γ. Moreover, in contrast to control cells, expanded CD4+CD25+CD127lowFOXP3+ cells were highly potent suppressors, efficiently suppressing the frequency of proliferating PBMC >80% even at a 1:20 ratio of Tregs:PBMC. Conclusion: In this preliminary study, we showed that highly suppressive FOXP3+ Tregs can be expanded from CD4+CD25+CD127low T cells isolated from pediatric thymic tissue, indicating that explanted thymuses may be a potential source for isolation and expansion of Tregs for cellular therapy. Future work includes defining the stability of expanded Tregs and their suppressive capacity of alloantigen immune responses.