Abstract The genetic predisposition to multiple sclerosis (MS) is most strongly conveyed by MHC class II haplotypes, possibly by shaping the autoimmune CD4 T cell repertoire. Whether Ag-processing enzymes contribute to MS susceptibility by editing the peptide repertoire presented by these MHC haplotypes is unclear. Thymus-specific serine protease (TSSP) is expressed by thymic epithelial cells and thymic dendritic cells (DCs) and, in these two stromal compartments, TSSP edits the peptide repertoire presented by class II molecules. We show in this article that TSSP increases experimental autoimmune encephalomyelitis severity by limiting central tolerance to myelin oligodendrocyte glycoprotein. The effect on experimental autoimmune encephalomyelitis severity was MHC class II allele dependent, because the lack of TSSP expression conferred protection in NOD mice but not in C57BL/6 mice. Importantly, although human thymic DCs express TSSP, individuals segregate into two groups having a high or 10-fold lower level of expression. Therefore, the level of TSSP expression by thymic DCs may modify the risk factors for MS conferred by some MHC class II haplotypes.