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TCR transgenic mice are valuable tools for dissecting the role of autoantigen-specific T cells in the pathogenesis of type 1 diabetes but are time-consuming to generate and backcross onto congenic strains. To circumvent these limitations, we developed a new approach to rapidly generate mice expressing TCR using retroviral-mediated stem cell gene transfer and a novel picornavirus-like 2A peptide to link the TCR alpha- and beta-chains in a single retroviral vector. We refer to these as retrogenic (Rg) mice to avoid confusion with conventional transgenic mice. Our approach was validated by demonstrating that Rg nonobese diabetic (NOD)-scid mice expressing the diabetogenic TCRs, BDC2.5 and 4.1, generate clonotype-positive T cells and develop diabetes. We then expressed three TCR specific for either glutamate decarboxylase (GAD) 206-220 or GAD 524-538 or for hen egg lysozyme 11-25 as a control in NOD, NOD-scid, and B6.H2(g7) mice. Although T cells from these TCR Rg mice responded to their respective Ag in vitro, the GAD-specific T cells exhibited a naive, resting phenotype in vivo. However, T cells from Rg mice challenged with Ag in vivo became activated and developed into memory cells. Neither of the GAD-reactive TCR accelerated or protected mice from diabetes, nor did activated T cells transfer or protect against diabetes in NOD-scid recipients, suggesting that GAD may not be a primary target for diabetogenic T cells. Generation of autoantigen-specific TCR Rg mice represents a powerful approach for the analysis of a wide variety of autoantigens.

Peptides bind to MHC class II molecules with a defined periodicity such that the peptide-flanking residues (PFRs) P-1 and P11, which lie outside the core binding sequence (P1-P9), are solvent exposed and accessible to the TCR. Using a novel MHC class II:peptide binding assay, we defined the binding register for nine immunogenic epitopes to formally identify the flanking residues. Seven of the nine epitopes, restricted by H-2A(k), H-2A(g7), or H-2E(k), were found to generate T cells that were completely dependent on either P-1 or P11, with dependency on P-1 favored over P11. Such PFR dependency appears to be influenced by the type of amino acid exposed, in that residues that can form salt bridges or hydrogen bonds are favored over small or hydrophobic residues. Peptides containing alanine substitutions at P-1 or P11 in place of PFRs that mediate dependency were considerably less immunogenic and mediated a substantially reduced in vitro recall response to the native protein, inferring that PFR recognition increases immunogenicity. Our data suggest that PFR recognition is a common event characteristic of all MHC class II-restricted T cell responses. This key feature, which is not shared by MHC class I-restricted responses, may underlie the broad functional diversity displayed by MHC class II-restricted T cells.