Objective Systemic lupus erythematosus (SLE) is characterized by persistent stimulation of the adaptive immune response and oxidative stress from Reactive Oxygen Species (ROS) generation. Mitochondria, central to energy metabolism through Oxidative Phosphorylation (OXPHOS), are implicated in the pathogenesis of SLE. This study aimed to assess abnormal mitochondrial function and associated ROS production in the adaptive immune response of SLE patients.

Methods Initially peripheral blood mononuclear cells (PBMCs) were isolated from SLE patients (n=37) and age/sex-matched healthy controls (HC, n=20). Flow cytometry (FC) was employed to quantify mitochondrial mass and mitochondria-derived ROS generation in CD4⁺, CD8⁺, and CD19⁺ lymphocytes. Next, we sought to evaluate the influence of soluble serum mediators on cellular function. Healthy PBMCs were isolated and cultured with 10% serum from either SLE patients (n=17) or healthy donors (n=12), before quantify ROS with FC. To evaluate CD4⁺ T cell derived cytokines on cellular metabolism, following magnetic bead isolation, CD4+ T cells were stimulated with anti-CD3/CD28 for 24 hours (HC=13, SLE=13). Following this, healthy PBMCs were culture in this cellular supernatant and ROS was again quantified by FC. Finally real-time CD4+ T cell mitochondrial metabolic function was evaluated using Seahorse Respirometry MitoStress Test.

Results When adjusted for mitochondrial mass, individual mitochondria-derived ROS production was markedly increased in CD4⁺, CD8⁺, and CD19⁺ cells in SLE when compared with HC (figure 1A). Following co-culture with donor serum, healthy PBMCs cultured with SLE serum showed significantly higher ROS generation in CD4⁺, CD8⁺ and CD19⁺ lymphocytes when compared with HC (figure 1B). Following co-culture with supernatant from stimulated CD4⁺ T cells; CD8⁺ and CD19⁺ cells cultured with SLE CD4⁺ T cell supernatant showed higher ROS formation than those cultured with HC CD4⁺ supernatant (figure 1C). Seahorse Respirometry indicated higher basal CD4+ respiration (figure 1D), increased proton leak (figure 1E), and enhanced mitochondrial ATP production (figure 1F) in SLE, suggesting closer proximity to maximal function with limited upregulation potential.

Conclusion These findings underscore the significance of abnormal immune cell metabolic pathways in SLE, highlighting potential therapeutic targets. Targeting CD4⁺ T cell mitochondrial dysfunction may offer a novel approach for future therapeutic intervention.

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Abstract P120 Figure 1