We have recently discovered a new form of beta-cell autoantigen, which is recognized by CD4 T cells, and is formed through a novel post-translational modification of beta-cell peptides that involves the fusion between insulin peptide fragments and peptides of other secretory granule proteins. The hybrid insulin peptides (HIPs) are highly antigenic for autoreactive and pathogenic CD4 T cell clones derived from the nonobese diabetic (NOD) mouse. Importantly, three of these HIPs have been shown to exist in nature, as we detected them by mass spectrometry of antigenic fractions from beta cell extracts. Moreover, we have also identified two HIPs that are antigenic for T cells clones derived from the islets of T1D deceased donors. Thus, our data support the involvement of HIPs in human T1D. In this project, our goal is to extend our study of human T cell responses to HIPs by isolating T cells from spleen and pancreatic lymph nodes (PLN) of nPOD donors and then testing these T cells on a selected panel of HIPs. We will initially test the T cells through a sensitive assay (called ELISpot) for the inflammatory cytokine, interferon-γ, produced when CD4 T cells encounter antigen. Long-term goals are to identify HIPs that stimulate autoreactive T cells from human patients and to develop reagents with these HIPs that can detect the autoreactive T cells in the blood (or other tissues).