Multiple Sclerosis is an autoimmune disease that results from a breakdown in immune tolerance. The repeated intranasal (i.n.) administration of myelin basic protein (MBP) peptide variant Acl-9[4Y] has previously been shown to induce peripheral tolerance and protection from Experimental Autoimmune Encephalomyelitis in the T cell receptor transgenic Tg4 model. This induced tolerance is characterised by the generation of an anergic, IL-l0 secreting, regulatory C04+ T cell population (IL-l0 Treg). In order to broaden our understanding of the role for IL-l0 in induced tolerance, the dynamics of IL-l0 production, from cell to whole organism, was investigated. Analysis of the localisation of IL-l0 upon secretion from the IL-l0 Treg cell, aiming to establish if IL-l0 is released in a directional manner or instead in a multi-directional pattern to influence any cells in the surrounding environment, proved to be a major technical challenge. Assessment of the spatial and temporal dynamics of the IL-l0 Treg cell was then undertaken in the IL-l0 transcriptional reporter MBP-TCR transgenic Tg4IL-10/GFP model over the course of an escalating dose MBP Acl-9[4Y] Ln. regimen. IL-l0 Treg cells were evident after the third peptide treatment onwards and accumulated in the peripheral lymphoid organs, particularly the spleen. Upon the induction of Thl mediated EAE, MBP Ac1-9[4Y] treated Tg4IL-10GFP mice were protected from EAE with IL-l0 Treg cells, found to comprise both Foxp3+ and Foxp3- populations, primarily resident in the spleen. Th1 cells adoptively transferred into MBP Ac1-9[4Y] treated mice were retained in the spleen of MBP Ac1- 9[4Y] treated mice. Taken together, these data suggest that peptide-induced tolerance regulates antigen-specific immune responses in peripheral lymphoid organs rather than in the target tissues. Collectively, these results provide novel insights into the function of IL- 10 Treg cells in vivo, which may contribute to the optimisation of the efficacy and safety of peptide immunotherapy