2010 - TTS International Congress


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T Cell Diversity and Functional Biology

87.6 - Allogeneic Treg generated in vitro with retinoic acid and TGF-β control cytokine production and allograft rejection.

Presenter: Carolina, Moore, Santiago, Chile
Authors: Moore C., Fuentes C., Ramirez V., Bono M., Fierro J., Wood K., Bushell A., Rosemblatt M.

ALLOGENEIC TREG GENERATED IN VITRO WITH RETINOIC ACID AND TGF-? CONTROL CYTOKINE PRODUCTION AND ALLOGRAFT REJECTION.

T CELL DIVERSITY AND FUNCTIONAL BIOLOGY

C. Moore1, C. Fuentes2, V. Ramirez2, M.R. Bono2, J.A. Fierro3, K.J. Wood4, A. Bushell4, M. Rosemblatt5
1, UNAB - MIFAB, Santiago/CHILE, 2, Universidad de Chile, Santiago/CHILE, 3, Clinica Las Condes, Santiago/CHILE, 4Transplantation Research Immunology Group, Nuffield Department of Surgery, University of Oxford, Oxford/UNITED KINGDOM, 5, UNAB and MIFAB, Santiago/CHILE

Body: Introduction: CD4+CD25+ FoxP3+ regulatory T cells (Treg) mediate immunological self-tolerance and suppress immune responses. Under specific conditions naiveCD4+CD25-Foxp3- T cells can be converted into Foxp3+ adaptive regulatory T cells. In the gut, a subset of dendritic cells (DCs) is specialized to induceTreg in a TGF-β and retinoic acid (RA)-dependent manner. We have exploited this effect to generate alloantigen reactive Treg in vitro. The resultant population has profound effects oncytokine production by effector T cells in vitro and when co-transferred into syngeneic recipients can prevent skin graft rejection. Methods: Splenic naive T cells from WT C57BL/6 or CBAwere co-cultured with BALB/c or C57BL/6 splenic DCs in the presence of TGF-β, RA and/or IL-2. On day 6, cells were analyzed for expression of FoxP3 and characteristic Tregs markers orflow-sorted. Sorted CD4+CD25high cells were co-cultured at different ratios with CD4+CD25- CFSE-labeled responder T cells and CD11c+ dendritic cells in asuppression assay. On day 3, T cell proliferation was analyzed by CFSE-dilution by flow cytometry. Cytokine secretion during Treg generation and in the suppression assay was determined by CytokineBead Assay. For examination of Treg function in vivo, non-sorted CBA RA-Treg were adoptively transferred into immunodeficients CBA.Rag mice together with syngeneicCD4+CD25- effector cells. One day later, reconstituted recipients were transplanted with C57BL/6 or third party skin grafts. Results: In vitro, the combination ofTGF-β and RA act results in a striking enrichment of Foxp3+ cells (80% cf. 1-3% in the starting population) compared to 2% in the absence of RA and TGF-β. These RA-Tregs showhigh expression of CD101, CD103, CD39, FR4, CD49d and P-selectin. Furthermore, these RA-Tregs do not secrete Th1, Th2 and Th17 cytokines compared with the respective controls, which may contribute toa further Treg enrichment or generation by infectious tolerance. In vitro, RA-Tregs suppress T cell proliferation and inhibit the production of IL-6, IL-17, IFN-γ and IL-4 and showincreased expression of IL-10. Most importantly, adoptive transfer of RA-Tregs 1 day before skin transplantation had a striking effect on graft outcome. Mice reconstituted with effector cells onlyrejected B6 skin grafts acutely (MST 11 days, n=3). However, reconstitution with effector cells plus 2x105 B6-driven RA-Treg resulted in 60% B6 skin graft survival with normal hair growthand normal macroscopic appearance (MST >50 days, n=5). The regulation of rejection was alloantigen specific since mice reconstituted with the same effector and Treg populations rejectedthird-party BALB/c grafts at control rates (MST 11 days, n=4). Conclusion: The results show that co-culturing naive T cells with donor presenting cells in the presence of TGF-β and RA generatesde novo regulatory T cells. RA-Tregs modify the cytokine secretion of effector T cells are able to extend the survival of allogeneic skin grafts in an antigen specific manner. Generation ofRA-Tregs represents a new powerful approach to generate allogeneic-specific Treg cells with in vivo suppressive capacity.

Disclosure: All authors have declared no conflicts of interest.


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