Lewis rats can be rendered unresponsive to experimental autoimmune encephalomyelitis by immunization with myelin basic protein (MBP), or MBP68-86, the dominant encephalitogenic MBP epitope for this strain, administered in IFA. However, protected rats harbor potentially encephalitogenic T cells, which are maintained in an inactive state. We investigated whether these quiescent effector cells could be activated in vitro. Although these T cells respond poorly to MBP68-86, they proliferate vigorously whether cocultured with MBP68-86 and either IL-2 or IL-12, suggesting that the T cells are in a state of anergy. Moreover, we could activate these anergic T cells with peptide and cytosine-guanine dinucleotide (CpG) oligonucleotide, but not control oligonucleotide, suggesting that products of the innate immune response are capable of activating anergic autoreactive T cells. The activated T cells produced the proinflammatory cytokine, IFN-gamma in response to IL-12, and IL-6 was secreted in response to CpG oligonucleotide. IL-6 has been reported to play a role in T cell activation by blocking T regulatory/suppressor (Treg) cell-mediated suppression through a Toll-like receptor-dependent pathway. However, anti-IL-6 mAb did not block CpG activation of the anergized cells. In contrast, anti-TGF-beta(1) Ab released the unresponsive T cells from the anergic state in the presence of MBP68-86, whereas TGF-beta(1) inhibited proliferation of MBP68-86- plus CpG-activated T cells. Because TGF-beta(1) has previously been implicated in Treg activity, this finding is consistent with a role for Treg cells in maintaining autoreactive T cells in the anergic state.