The inducible co-stimulator (ICOS, CD278) is essential to the efficient development of normal and pathological immune reactions. Since ICOS-deficient mice have enhanced susceptibility to experimental allergic encephalomyelitis (EAE), we have functionally analyzed a CD4+ICOS+ population comprising 6-15% of all CD4+ T cells in secondary lymphoid organs of unmanipulated wild-type mice and checked for their ability to suppress EAE. In C57BL/6 mice, CD4+ICOS+ cells were a major source of cytokines including IFN-gamma, IL-2, IL-4, IL-10 or IL-17A. Upon activation, these cells showed preferentially enhanced production of IL-4 or IL-10 but inhibited IFN-gamma production. In contrast, CD4+ICOS- cells mainly produced IFN-gamma. Interestingly, CD4+ICOS+ cells partially suppressed the proliferation of CD4+ICOS- or CD4+CD25- lymphocytes 'in vitro' by an IL-10-dependent mechanism. Furthermore, CD4+ICOS+ activated and expanded under appropriate conditions yielded a population enriched in cells producing IL-10 and T(h)2 cytokines that also suppressed the proliferation of CD4+CD25- lymphocytes. CD4+ICOS+ cells, before or after expansion in vitro, reduced the severity of EAE when transferred to ICOS-deficient mice. In the same EAE model, lymph node cells from ICOS-deficient mice receiving ICOS+ cells showed reduced IL-17A production and enhanced IL-10 secretion upon antigen activation in vitro. Thus, naturally occurring CD4+ICOS+ cells, expanded or not in vitro, are functionally relevant cells able of protecting ICOS-deficient mice from severe EAE.