CD4+ T cell responses are restricted by MHC class II-encoded glycoproteins which display antigen-derived peptides. Chimeric MalE proteins expressing foreign T cell epitopes represent a potent means to induce immune responses for recombinant vaccine design. Here, we studied the influence of the non-MHC genetic background and of the processing heterogeneity displayed by various APC types on the presentation of these chimeric proteins to T cells. For this purpose, the I-Ed-restricted poliovirus CD4+ T cell epitope was inserted into five different positions on the surface of MalE protein and the immunogenicity of the recombined T cell epitope was determined in different inbred mice. Immunization of several mouse strains expressing I-Ed with these chimeric proteins induced poliovirus-specific T cell response with four out of five constructs. In vitro presentation studies of the recombined epitope to specific T cells indicated that for a given chimeric protein the fine processing is conserved, whatever the non-H-2 genetic background of APC or the type of APC. Our results show that the insertion site in MalE modulates the immunogenicity of the recombined T cell epitope, but this phenomenon is only related to the MHC genetic background.