Resilience of human tolerogenic dendritic cells to physiological oxygen supports clinical application: functional stability amidst glycolytic and differentiation shifts

Tolerogenic dendritic cells (tolDCs) hold promise for treating autoimmune diseases, potentially restoring antigen-specific immune tolerance without systemic immunosuppression. However, their behavior in vivo remains incompletely understood, particularly in response to microenvironmental factors such as oxygen (O₂) tension. While tolDCs are typically generated and functionally validated under atmospheric O₂ (21%), physiological O₂ levels (physioxia) in human tissues are considerably lower (3-9%). The primary aim of this study was to assess whether tolDCs manufactured under atmospheric O₂ conditions retain their function under physioxia at 4% O₂, mimicking tissue environments encountered upon clinical administration. To contextualize these findings, we also evaluated the effect of physioxia during in vitro generation and investigated underlying metabolic adaptations. We demonstrate that tolDCs generated under atmospheric O₂ conditions remain functionally effective in physioxic environments, preserving migratory capacity and the ability to induce T cell hyporesponsiveness. Furthermore, physioxia during tolDC generation impaired monocyte-to-tolDC differentiation efficiency, whereas hallmark tolerogenic features, including low expression of CD80, CD83, and CD86, remained intact. Metabolic profiling revealed a distinct shift under physioxia, with reduced mitochondrial reserve capacity and increased glycolytic activity. This suggests metabolic plasticity without loss of function across O₂ environments. Our findings indicate that physiological O₂ shapes tolDC differentiation and metabolism but does not compromise immunoregulatory traits. Importantly, tolDCs generated under atmospheric O₂ remained functionally competent in physioxic environments, reinforcing their suitability for therapeutic use. By modeling in vivo-relevant O₂ levels, this study provides new insights into how microenvironmental O₂ may shape tolDC behavior following clinical administration.