OBJECTIVE-Regulatory T-cells (Tregs) have catalyzed the field of immune regulation. However, translating Treg-based therapies from animal models of autoimmunity to human clinical trials requires robust methods for the isolation and expansion of these cells-a need forming the basis for these studies. RESEARCH DESIGN AND METHODS-Tregs from recent-onset type 1 diabetic patients and healthy control subjects were isolated by fluorescence-activated cell sorting and compared for their capacity to expand in vitro in response to anti-CD3-anti- CD28-coated microbeads and IL-2. Expanded cells were examined for suppressive function, lineage markers and FOXP3, and cytokine production. RESULTS-Both CD4 +CD127 lo/- and CD4 +CD127 lo/-CD25 + T- cells could be expanded and used as Tregs. However, expansion of CD4 +CD127 lo/- cells required the addition of rapamycin to maintain lineage purity. In contrast, expansion of CD4 +CDi27 lo/-CD25 + T-cells, especially the CD45RA + subset, resulted in high yield, functional Tregs that maintained higher FOXP3 expression in the absence of rapamycin. Tregs from type 1 diabetic patients and control subjects expanded similarly and were equally capable of suppressing T-cell proliferation. Regulatory cytokines were produced by Tregs after culture; however, a portion of FOXP3 + cells were capable of producing interferon (IFN)-γ after reactivation. IFN-γ production was observed from both CD45RO + and CD45RA + Treg populations. CONCLUSIONS-The results support the feasibility of isolating Tregs for in vitro expansion. Based on expansion capacity, FOXP3 stability, and functional properties, the CD4 +CD127 lo/-CD25 + T-cells represent a viable cell population for cellular therapy in this autoimmune disease.