Biological sex defines regulatory T-cell reprogramming trajectories in immune-related adverse events and autoimmunity

Abstract

Immune checkpoint inhibitors (ICIs) targeting the PD-1/PD-L1 and CTLA-4 pathways have revolutionized cancer therapy but are frequently associated with immune-related adverse events (irAEs), i.e. inflammatory toxicities that resemble classical autoimmune diseases and reflect disruption of peripheral tolerance. Regulatory T cells (Tregs), key mediators of immune homeostasis, are directly affected by checkpoint blockade and have previously been shown to undergo extensive inflammatory and metabolic reprogramming during irAEs. Given the established role of biological sex in shaping immune regulation, inflammatory signaling and Treg biology, we investigated in this study whether sex-dependent mechanisms contribute to Treg dysfunction during irAE development and whether these responses differ from those observed in spontaneous autoimmune disease. We performed a sex-based integrative transcriptomic analysis of bulk RNA sequencing datasets derived from peripheral blood Tregs isolated from patients with advanced melanoma and additional solid tumors treated with anti-PD-1 immunotherapy, as well as from individuals with chronic autoimmune diseases and healthy controls. Differential gene expression, pathway enrichment and network-level analyses were conducted separately in male and female cohorts to characterize inflammatory, metabolic and tolerance-associated transcriptional programs linked to irAEs and autoimmune disease. Sex-based analyses revealed both shared and distinct transcriptional programs associated with irAEs and autoimmunity. Female-derived Tregs exhibited broader transcriptional remodeling during autoimmune disease, characterized by extensive enrichment of immune regulatory, inflammatory, metabolic and oxidative stress-associated biological processes, whereas male-derived Tregs displayed comparatively more restricted remodeling involving both immune-related and metabolic pathways. In contrast, irAE-associated Tregs exhibited a distinct pattern of sex-dependent transcriptional organization, indicating that biological sex shapes Treg adaptation differently between the two settings of peripheral tolerance breakdown. Comparative analyses further demonstrated that although autoimmune disease and irAEs shared common biological processes, the degree, directionality and organization of these transcriptional responses differed substantially between the sexes, supporting disease-specific rather than uniform Treg adaptation. These findings demonstrate that biological sex significantly influences Treg-associated transcriptional remodeling during peripheral tolerance breakdown. Rather than imposing a common regulatory program, biological sex shapes disease-specific transcriptional trajectories according to the inflammatory context in which immune tolerance is disrupted. These results provide new insight into sex-dependent mechanisms of immune regulation and highlight the importance of incorporating biological sex as a biological variable in studies investigating immune-mediated disease, biomarker development and personalized immunology.