Objective: To determine the antigenic determinants and specific molecular requirements for the generation of autoregulatory neuroantigen-specific CD81 T cells in models of multiple sclerosis (MS). Methods: We have previously shown that MOG35-55-specific CD81 T cells suppress experimental autoimmune encephalomyelitis (EAE) in the C57BL/6 model. In this study, we utilized multiple models of EAE to assess the ability to generate autoregulatory CD81 T cells. Results: We demonstrate that alternative myelin peptides (PLP178-191) and other susceptible mouse strains (SJL) generated myelin-specific CD81 T cells, which were fully capable of suppressing disease. The disease-ameliorating function of these cells was dependent on the specific cognate myelin antigen. Generation of these autoregulatory CD81 T cells was not affected by thymic selection, but was dependent on the presence of both CD41 and CD81 T-cell epitopes in the immunizing encephalitogenic antigen. Conclusions: These studies show that the generation of autoregulatory CD81 T cells is a more generalized, antigen-specific phenomenon across multiple neuroantigens and mouse strains, with significant implications in understanding disease regulation.