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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 recentonset 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-antiCD28-coated microbeads and IL-2. Expanded cells were examined for suppressive function, lineage markers and FOXP3, and cytokine production.
RESULTS- Both CD4^sup +^CD127^sup lo/-^ and CD4^sup +^CD127^sup lo/-^CD25^sup +^ Tcells could be expanded and used as Tregs. However, expansion of CD4^sup +^CD127^sup lo/-^ cells required the addition of rapamycin to maintain lineage purity. In contrast, expansion of CD4^sup +^CD127^sup lo/-^CD25^sup +^ T-cells, especially the CD45RA^sup +^ 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^sup +^ cells were capable of producing interferon (IFN)-γ after reactivation. IFN-γ production was observed from both CD45RO^sup +^ and CD45RA^sup +^ 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^sup +^CD127^sup lo/-^CD25^sup +^ Tcells represent a viable cell population for cellular therapy in this autoimmune disease. Diabetes 58:652-662, 2009
Multiple defects within the innate and adaptive immune systems are associated with the de- velopment of type 1 diabetes (1). Collectively, these defects lead to an imbalance in immune regulation that facilitates the expansion of pathogenic autoreactive T- and B-cells, resulting in the eventual destruction of insulin-producing ß-cells (2,3). In recent years, appreciation has grown for the critical role Tregs play in maintaining immune tolerance (2). Studies in animal models and humans indicate a failure of Tregs to develop or function results in the development of systemic autoimmune disease (4,5). This is most apparent in humans with mutations in the gene encoding the transcription factor FOXP3, which is necessary for the proper development and function of Tregs (6)....