T cell autoreactivity is one of the major factors in the pathogenesis of type 1 diabetes (T1D). Antigen-specific immunotherapy (ASI) aims to regulate these T cell populations without inducing broad immunosuppression. Previously, we showed that administration of liposomes encapsulating a T1D-relevant CD4 epitope (2.5mi) together with the immunomodulator, calcitriol delayed T1D progression and cross-regulated CD8 T cells in a mouse model. Here, we characterised the response of multiple islet-specific CD4+ and CD8+ T cells to 2.5mi/calcitriol ASI. We used high-dimensional flow cytometry and clustering algorithms to examine inhibitory and exhaustion phenotypes of antigen-specific CD4+ and CD8+ T cells in the pancreatic draining lymph node and spleens of treated mice. Endogenous islet-specific T cell responses were evaluated using MHC class I and II tetramers recognizing IGRP and 2.5mi-specific cells, respectively. We found that IGRP-specific CD8 T cells upregulated markers of exhaustion, including high intracellular EOMES, and reduced DNAM-1 surface expression after ASI. These IGRP-specific CD8+ T cells were functionally impaired, as intracellular granzyme B expression was reduced in response to PMA/ionomycin stimulation in vitro. Although CD4+ Foxp3+ Treg proportions did not change, activation markers, including T-bet and CD49b increased, and Foxp3- IFN-γ+ IL-10+ Tr1-like T cells expanded after ASI. Consistent with their antigen experience in response to ASI, the target population of conventional 2.5mi-specific CD4+ Foxp3- T cells expanded and expressed high levels of DNAM-1 and PD-1. These findings suggest that 2.5mi/calcitriol liposome ASI regulates both 2.5mi-specific CD4+ T cells and bystander IGRP-specific CD8+ T cells. The ASI-induced exhaustion-like phenotype of islet-specific CD8+ T cells resembles a signature identified after anti-CD3 immunotherapy in T1D patients, suggesting common regulatory mechanisms.