Mice immunized with neuroantigens in incomplete Freund’s adjuvant (IFA) are resistant to subsequent induction of experimental autoimmune encephalomyelitis (EAE). The mechanisms involved in this protection are complex. Studies on relevant CD4(+) or CD8(+) T cells, including effective and regulatory T cells, have been performed by others. In this work, the effects of CD4(-)-, CD8(-)- splenocytes on protection from EAE in C57BL/6 mice which were immunized with myelin oligodendrocyte glycoprotein 35-55 (MOG)35-55 in IFA were evaluated. We observed that MOG-reactive CD4(+) T cells failed to be activated and proliferate when CD4(-)-, CD8(-)- splenocytes from MOG/IFA-immunized mice were regarded as antigen-presenting cells (APC). It was shown that these APC expressed lower levels of major histocompatibility complex class II (MHC-II), CD80, and CD86 than nave cells. In addition, CD4(-)-, CD8(-)- splenocytes from MOG/IFA-immunized mice showed significantly higher levels of IL-10 mRNA expression. When the immunized-mice were induced to develop EAE, these cells secreted significantly higher levels of IL-10 and produced lower levels of IL-6, leading to decreased secretion of IL-17 and IFN- from MOG-specific CD4(+) T cells. The transfer of CD4(-)-, CD8(-)- splenocytes from MOG/IFA-immunized mice was able to ameliorate the subsequent induction of EAE in recipient mice. Thus, MOG/IFA immunization can modulate CD4(-)-, CD8(-)- splenocytes by reducing the expression of antigen-presenting molecules and altering the levels of secreted cytokines. Our study reveals an additional mechanism involved in the protective effects of MOG/IFA pre-immunization in an EAE model.