Expansion of Human Regulatory T-Cells From Patients With Type 1 Diabetes

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(+)-CD127(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)-gamma after reactivation. IFN-gamma production was observed front 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. Diabetes 58:652-662, 2009