In-vitro generation and characterisation of murine CD4+CD25+ regulatory T cells with indirect allospecificity

Naturally arising CD4(+)CD25(+) regulatory T cells play a pivotal role in the prevention of autoimmunity and in the induction of donor-specific transplantation tolerance. Harnessing regulatory cells for potential adoptive cell therapy is hampered by their lack of antigen-specificity and their limited numbers. Here we describe the generation and expansion of murine CD4(+)CD25(+) T cells with antigen-specificity for all K-d peptide as potential reagents for adoptive cell therapy in promoting donor-specific transplantation tolerance. Using bone marrow-derived autologous dendritic cells Pulsed with the K-d peptide, we generated T cell lines front purified CD4(+)CD25(+) T cells from C56BL/6 mice. The T cell lines expressed high level of CD25 and low level of CD45RB and CD69. They maintained the expression of CD62L, GITR, CTLA-4 and more importantly FoxP3. The CD4(+)CD25(+) T cell lines were anergic after TCR stimulation and produced little cytokine such as IL-2 and IFN-gamma. Importantly, they were more potent than freshly isolated CD4(+)CD25(+) T cells in suppressing proliferation and cytokine secretion by effector CD4(+) T cells. Furthermore, the CD4(+)CD25(+) T cell lines Could be expanded to large cell numbers and maintained in culture up to I year. The K-d-specific CD4(+)CD25(+) T cell lines will be invaluable in devising a strategy for the induction of cardiac transplantation tolerance in wild-type B6 mice carrying I full mismatch BALB/c heart. (c) 2006 Elsevier B.V. All Lights reserved.