Recognition of a splice variant neo-epitope by CD4+ T cells in subjects with type 1 diabetes

Eddie James (Benaroya Research Institute)

Perrin Guyer, David Arribas-Layton, Cate Speake, Carla Greenbaum, Decio Enzirik, Sally Kent, Roberto Mallone, Eddie James

A recent discovery effort investigated tissue specific mRNA splice variants and other novel secretory granule antigens within human islets, demonstrating that unique peptide sequences from these proteins are present within HLA-class I peptidome of human β cells and documenting their recognition by CD8+ T Cells from peripheral blood and human islets. Our goal was to investigate the relevance of CD4+ T cell recognition of epitopes derived from these target antigens.

In this study, we applied a systematic epitope discovery process to identify novel CD4+ T cell epitopes derived from mRNA splice variants and novel secretory granule antigens. We first predicted potential epitopes spanning unique junctions of mRNA splice variants and within conventional secretory granule antigens contained in the data set. Peptides with DRB1*04:01 motifs were screened for in vitro binding and used to generate HLA class II tetramers. The corresponding tetramers were used to assess peptide immunogenicity, isolate T cell clones, and label and detect CD4+ T cells specific for these putative epitopes in peripheral blood. We further investigated the relevance of these epitopes by examining their characteristics in subjects with established T1D and by investigating their recognition by islet derived T cell lines.

Summary of Results
We observed detectable populations of T cells that recognize three novel epitopes in the peripheral blood of subjects with T1D at frequencies that were similar to an immunodominant proinsulin epitope. T cells that recognized these epitopes were present in peripheral blood at higher frequencies in subjects with T1D than in controls and predominantly exhibited a Th1-like surface phenotype. Among the three novel epitopes, responses to a peptide derived from the CCNI-008 splice variant tended to be the most frequent. T cells with this specificity also exhibited a more differentiated memory phenotype. Furthermore, T cells that respond to these epitopes were present among islet infiltrating T cells.

These results reveal novel epitopes that are recognized by CD4+ T cells in human T1D. This further establishes alternative splicing as a mechanism that contributes to the loss of tolerance in T1D.