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Presenter: Megan K., Levings, Vancouver, Canada
Authors: Megan K. Levings
The role of FOXP3 in controlling the function of effector and regulatory T cells in humans.
Alicia McMurchy1, Jana Gillies1, Derek Dai1, Sara Di Nunzio2, Rosa Bacchetta2, C. Bruce Verchere1 and Megan K. Levings1
1Department of Surgery, University of British Columbia, & Child and Family Research Centre, Vancouver, Canada
2San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Division of Regenerative Medicine, Stem Cells and Gene Therapy, San Raffaele Scientific Institute, Milan, Italy
A major factor controlling the outcome of successful organ transplantation is the balance between alloantigen-specific effector and regulatory T cells (Tregs). Animal models have shown that boosting the numbers of Tregs can replace pharmacological immunosuppression and lead to long-term graft survival. Major barriers to translating this "cellular therapy" with Tregs to humans are difficulties in obtaining sufficient cells, a lack of models to evaluate their efficacy and safety in vivo, and a poor understanding of their mechanisms of action. To address these limitations we performed a series of molecular and cellular studies to better define the role of FOXP3 in the development and function of CD4+ T cell subsets.
In humans FOXP3 is not only expressed in Tregs, but also in activated T effector (Teff) cells. We showed that in order for Tregs to function as suppressive cells, FOXP3 must be expressed continually and at high levels, but the role of transiently-expressed FOXP3 in Teff cells remained unknown. We isolated a series of FOXP3null Teff cell clones from a mother of a child affected by IPEX and who is heterozygous for a null-mutation in FOXP3. We found that FOXP3null Teff cells were hypersensitive to TCR-induced proliferation and cytokine production compared to cells expressing FOXP3. Similar results were obtained using a parallel approach to knock-down FOXP3 in Teff cells by transduction with a lenti-viral vector expressing a microRNA targeting FOXP3. These data provide the first evidence for a functional role of FOXP3 outside of Tregs and suggest that cell-intrinsic expression of this transcription factor is required to limit effector T cell responses.
We also developed a method to generate human Tregs in vitro by over-expressing FOXP3 and demonstrated that the resulting cells are suppressive and stable in vitro. To test the relative capacities of FOXP3-transduced versus Tregs expanded ex vivo to suppress allograft rejection in vivo, we established a model of human islet transplantation in immunodeficient mice. In this model, human islets are transplanted under the kidney capsule of diabetic mice and restore normoglycemia. Injection of 15 million PBMCs resulted in rejection of the allogeneic islets after 14-21 days. We also developed a protocol to expand naive human Tregs that remained FOXP3+ and did not acquire the capacity to secrete cytokines, and evaluated their functional effects in vivo. Preliminary data indicate that co-injection of expanded Tregs with PBMCs results in an increase in circulating and graft infiltrating FOXP3+ Tregs and a significant delay in allograft rejection. Experiments are on going to confirm these results and determine the relative ability of FOXP3-transduced Tregs to suppress allograft rejection in vivo.
Together these data support the notion that methods to selectively enhance the expression of FOXP3 in both Teff and Treg cells will offer new ways to limit CD4+ Teff cell-mediated rejection of allografts.
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