n. MBP Ac1–9[4K], [4A] or [4Y] treatment revealed an association between peptide affinity and the ability to activate CD4+ T cells in vivo. This translates into an affinity-dependant loss of responsiveness to antigenic stimulation by CD4+ T cells following repeated peptide treatment, which is most likely due to the decreased

ability of these cells to secrete IL-2. Indeed, the non-responsive state of CD4+ T cells from i.n. MBP Ac1–9[4Y]-treated mice could be reversed by the addition of exogenous IL-2 5. Exogenous IL-2 also reverses the anergy of CD4+ T cells from i.n. MBP Ac1–9[4K]- and [4A]-treated mice (Supporting Information Fig. 1). Lack of secreted IFN-γ in CD4+ T-cell cultures from i.n. Ac1–9[4Y]-treated mice is in turn likely to be the result of their anergy. This is supported by the observation that CD4+ T cells remain able to produce IFN-γ upon PMA and ionomycin stimulation. Interestingly, although anergy abrogates Inhibitor high throughput screening the production of IL-2 and IFN-γ in these cells, it allows the production of BIBW2992 ic50 IL-10. By studying the effect of repeated i.n. administration of either MBP Ac1–9[4K], [4A] or [4Y], we reveal a correlation between the affinity of peptide binding to H-2 Au and acquisition of a regulatory phenotype by CD4+ T cells, as demonstrated by IL-10 secretion and naïve T-cell suppression, both in vitro and

in vivo. Of note, the mechanism of in vitro suppression by CD4+ T cells from i.n. MBP Ac19[4Y]-treated mice has been shown to be cell contact-dependent, as determined by loss of suppression when using a transwell cell culture system, and cytokine independent, since neither anti-IL-10R or anti-TGF-β (or both) reversed suppression 6. Moreover, Vieira et al. showed reduced IL-2 expression

in co-cultures, indicating that CD4+ T cells from i.n. Ac19[4Y]-treated mice actively suppress naïve T cells in vitro7. Interestingly, there is no direct correlation between anergy Mirabegron and in vitro suppression 13; cells from Ac1–9[4K]-treated mice were anergic but failed to suppress in vitro. Conversely, the observed EAE protection 4 and inhibition of T-cell proliferation in vivo afforded by i.n. MBP Ac1–9[4Y] treatment 6 has previously been attributed to IL-10. Our results show that i.n. treatment with high affinity peptides, which drive the production of IL-10 amongst CD4+ T cells, correlates with their ability to mediate suppression, both in vitro and in vivo, and to protect against EAE development. However, administration of i.n. MBP Ac1–9[4K], which does not lead to IL-10 secretion, can also limit disease, albeit to a lesser degree. Thus, other facets of tolerance apart from IL-10, such as anergy and/or reduction in the ability to secrete IL-2 and IFN-γ, are likely to play a role. Taken together, our data point to a model in which repeated treatment with peptide antigen induces anergy in T cells, which is sufficient for debilitating their own effector function.