A recent discovery effort identified novel splice variant and secretory granule antigens within the HLA class I peptidome of human islets and documented their recognition by CD8+ T cells from peripheral blood and human islets. In the present study, we applied a systematic discovery process to identify novel CD4+ T cell epitopes derived from these candidate antigens. We predicted 145 potential epitopes spanning unique splice junctions and within conventional secretory granule antigens and measured their in vitro binding to DRB1*04:01. We generated HLA class II tetramers for the 35 peptides with detectable binding and used these to assess immunogenicity and isolate T cell clones. Tetramers corresponding to peptides with verified immunogenicity were then used to label T cells specific for these putative epitopes in peripheral blood. T cells that recognize distinct epitopes derived from a cyclin I splice variant, neuroendocrine convertase 2, and urocortin-3 were detected at frequencies that were similar to an immunodominant proinsulin epitope. Cells specific for these novel epitopes predominantly exhibited a Th1-like surface phenotype. Among the three epitopes, responses to the cyclin I peptide exhibited a distinct memory profile. Responses to neuroendocrine convertase 2 were detected among pancreatic infiltrating T cells. These results further establish the contribution of unconventional antigens to the loss of tolerance in autoimmune diabetes.
This work was supported by JDRF grant numbers 1-SRA-2020-978-S-B and 2-SRA-2014-297-Q-R to Dr. James. Work in Dr. Kent’s laboratory was supported by National Institutes of Health grant UC4DK116284 to Dr. Kent. Work in Dr Eizirik’s laboratory was supported by Welbio and Fonds National de la Recherche Scientifique (FNRS), Belgium, grants CR-2015A-06 and CR-2019C-04 and Innovate2CureType1-Dutch Diabetes Research Fundation (DDRF). Work in Dr Mallone’s laboratory was supported by The Leona M. and Harry B. Helmsley Charitable Trust (1901-03689), Agence Nationale de la Recherche (ANR-19-CE15-0014-01), Fondation pour la Recherche Medicale (EQU20193007831) and IdEx Université Paris 2019. Drs. Eizirik and Mallone received funding from the Innovative Medicines Initiative 2 Joint Undertaking under grant agreements 115797 and 945268 (INNODIA and INNODIA HARVEST), which receive support from the EU Horizon 2020 program, the European Federation of Pharmaceutical Industries and Associations, JDRF, and The Leona M. & Harry B. Helmsley Charitable Trust. Dr. Speake’s work was supported by JDRF under grant # 3-SRA-2019-791-S-B. This research was performed with the support of the Network for Pancreatic Organ Donors with Diabetes (nPOD; RRID:SCR_014641), a collaborative type 1 diabetes research project supported by JDRF (nPOD: 5-SRA-2018-557-Q-R) and The Leona M. & Harry B. Helmsley Charitable Trust (Grant#2018PG-T1D053, G-2108-04793). These funders had no role in study design, data collection and analysis, or decisions made to prepare or publish the manuscript. The content and views expressed are the responsibility of the authors and do not necessarily reflect the official view of nPOD. Organ Procurement Organizations (OPO) partnering with nPOD to provide research resources are listed at http://www.jdrfnpod.org/for-partners/npod-partners/.