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Experimental autoimmune encephalomyelitis (EAE) is commonly regarded as an animal model of the human disease multiple sclerosis (MS). Pertussis toxin (PTX) is routinely used for EAE induction in mice. Besides opening the blood–brain barrier, it acts as an adjuvant causing strong expansion of antigen-specific cells after coinjection with neuroantigens in IFA. Using an IL-17 ELISPOT assay we developed previously, we investigated the capability of PTX to induce proteolipid protein peptide 139–151(PLPp)-specific Th-17 cells in the immune periphery and in the thymus after coinjection with PLPp/IFA. PTX was found to induce peripheral PLPp-specific Th-17 cells in the draining lymph node and in the spleen, but not in the thymus. Our study indicates a new mechanism by which microbial agents can initiate or maintain autoimmune reactions and supports the growing role in particular for Th-17 cells in organ-specific autoimmune diseases like multiple sclerosis or EAE.