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B. Matta, P. Jha, P. S. Bora, N. S. Bora; CD4+CD25+t Regulatory Cells Play a Critical Role in Induction of Tolerance Against EAAU. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2513.
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To investigate the role of CD4+CD25+ regulatory T-cells and IL-2 in tolerance against EAAU
EAAU was induced in Lewis rats by melanin associated antigen (MAA). Tolerance was induced by i.v. administration of 400 µg of MAA before immunization with MAA (emulsified with complete Freund’s adjuvant) for induction of EAAU. Animals were sacrificed at the onset of EAAU and mixed lymphocytes harvested from the popliteal lymph nodes were cultured in the presence or absence of MAA. Mixed lymphocytes were also stained with anti-CD4, anti-CD25 and anti-IL2 (intracellular) for flow cytometry. CFSE stained, sorted CD4+CD25- T cells from LN of tolerized and non-tolerized animals were cocultured separately with CD4+CD25+ T regulatory cells from tolerized or non-tolerized animals. After six days of culture the proliferation of CD4+CD25- T cells was analyzed by flow cytometry. For adoptive transfer of tolerance, T cells obtained from the popliteal lymph nodes of tolerized Lewis rats, with or without CD4+CD25+ T regulatory cells were injected into naïve syngenic animals 24 h before immunization.
The number of CD4+CD25+ Tregs increased as early as 48 hours after first i.v. administration of MAA and increased in the popliteal lymph nodes of tolerized animals. The number of CD4+CD25+ Tregs increased only after the induction of the disease (day 6) in non-tolerized animals. Flow cytometric analysis further demonstrated that the numbers of IL-2 expressing CD4+ T cells were drastically decreased in popliteal lymph nodes of tolerized animals compared to non-tolerized animals at the onset of EAAU. Interestingly rIL-2 treatment (day 8, 10 and 12) completely reversed the tolerance. Our results from coculture experiments further established that the CD4+CD25+ T regs isolated from tolerized animals are suppressive. Furthermore tolerance could not be transferred adoptively by mixed lymphocytes depleted of CD4+CD25+ T cells.
Our data demonstrated CD4+CD25+ T regulatory cells play a critical role in tolerance induction in EAAU. Our results further suggested that IL-2 expressing cells are reduced and that IL-2 replenishment reverses the tolerance.
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