Enhancing anti-CD3 mAb-mediated diabetes remission in autoimmune diabetes through regulation of dynamin-related protein 1(Drp1)-mediated mitochondrial dynamics in exhausted CD8(+)T-cell subpopulations.

BACKGROUND: Antigen-specific immunotherapy shows potential for inducing long-term immune tolerance in type 1 diabetes (T1D), yet its clinical application is hampered by uncertainty regarding dominant epitopes. Conversely, non-antigen-specific treatments such as anti-CD3 monoclonal antibodies (mAbs) present a more straightforward approach but struggle to maintain tolerance after treatment. Addressing these issues is critical for advancing T1D therapies. METHODS: The phenotypic and metabolic properties of two subsets of exhausted CD8(+) T cells were analyzed in both humans and NOD mice. T-cell receptor (TCR) diversity and Bulk RNA sequencing provided insights into the transcriptomic profiles and TCR reactivity of these cells. Mechanistic studies were conducted using the HEK-293 T cell line and primary cells. Single-cell RNA sequencing (scRNA-seq) was applied to evaluate the characteristics of different CD8(+) T cell subsets following two types of immunotherapies. In NY8.3 mice, the effect of mitochondrial fission inhibitors on immunotherapy results was evaluated. Final validation was carried out with peripheral blood mononuclear cells (PBMCs) from T1D patients. RESULTS: Our study reveals the diversity of two distinct exhausted CD8(+) T cell subsets in T1D through flow cytometry, highlighting unique clinical features, phenotypes, and functions. Notable differences in TCR reactivity and metabolic pathways between these subsets were identified through TCR sequencing and transcriptomic analyses in NOD mice. Both antigen-specific and non-antigen-specific stimuli produced unique exhausted CD8(+) T cell subsets. Our research identified leucine-rich repeat kinase 2 (Lrrk2) as a key regulator of mitochondrial fission, influencing the interconversion of exhausted CD8(+) T cell subsets by phosphorylating dynamin-related protein 1 (DRP1) at serine 637 (Ser637) and serine 616 (Ser616). scRNA-seq confirmed that antigen-specific immunotherapy effectively suppresses T cell signaling, induces exhaustion, and promotes the development of terminally exhausted T (TEX) cells. Mitochondrial division inhibitor 1 (Mdivi-1) enhanced the therapeutic effect of anti-CD3 mAb treatment by promoting the development of more TEX cells. CONCLUSIONS: Our results point to a new immunotherapeutic approach that targets exhausted CD8(+) T cells' energy metabolism, offering valuable insights for advancing clinical strategies in T1D therapy.