ABSTRACT:The presentation of antigenic peptides by major histocompatibility complex (MHC) class II to CD4 ϩ T cells is crucial to initiate immune responses. We developed a new system for delivery of an antigenic peptide to the MHC class II pathway, using the invariant chain (Ii). We designed a mutated human p33-form Ii, CLIP-substituted Ii, in which streptococcal M12p55-68 (RDLEQAYNELSGEA) was substituted for CLIP (class II associated invariant chain peptide) . We examined the peptide presenting function of this construct, in comparison with the previously reported C-terminal fused Ii, in which a cathepsin cleavage site and M12p54-68 was ligated to the C-terminus of Ii. Mouse L cell transfectants expressing either of these two mutated Ii along with HLA-DR4 could process and present M12p55-68 to the peptide specific and DR4-restricted CD4 ϩ T cell clone. CLIP-substituted Ii was much more efficient in antigen presentation than was the C-terminal fused Ii. Similar to the wild-type Ii, the CLIP-substituted Ii was associated intracellularly with DR4 molecules. These results indicate that the peptide substituted for CLIP of Ii p33 bound to the groove of DR molecules in the same manner as CLIP and it was preferentially presented to the CD4 ϩ T cell clone in the absence of HLA-DM molecules. This system may prove useful for immunotherapy with DNA vaccines or for construction of an antigen presenting cell library with diverse peptides.

Accumulating evidence indicates that recognition by TCRs is far more degenerate than formerly presumed. Cross-recognition of microbial Ags by autoreactive T cells is implicated in the development of autoimmunity, and elucidating the recognition nature of TCRs has great significance for revelation of the disease process. A major drawback of currently used means, including positional scanning synthetic combinatorial peptide libraries, to analyze diversity of epitopes recognized by certain TCRs is that the systematic detection of cross-recognized epitopes considering the combinatorial effect of amino acids within the epitope is difficult. We devised a novel method to resolve this issue and used it to analyze cross-recognition profiles of two glutamic acid decarboxylase 65-autoreactive CD4+ T cell clones, established from type I diabetes patients. We generated a DNA-based randomized epitope library based on the original glutamic acid decarboxylase epitope using class II-associated invariant chain peptide-substituted invariant chains. The epitope library was composed of seven sublibraries, in which three successive residues within the epitope were randomized simultaneously. Analysis of agonistic epitopes indicates that recognition by both TCRs was significantly affected by combinations of amino acids in the antigenic peptide, although the degree of combinatorial effect differed between the two TCRs. Protein database searching based on the TCR recognition profile proved successful in identifying several microbial and self-protein-derived mimicry epitopes. Some of the identified mimicry epitopes were actually produced from recombinant microbial proteins by APCs to stimulate T cell clones. Our data demonstrate the importance of the combinatorial nature of amino acid residues of epitopes in molecular mimicry.

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