Immunometabolic Regulation of CD8 T Cell Dynamics in Human Critical Illness and Sepsis - PROJECT SUMMARY/ABSTRACT: Critical illness syndromes, particularly sepsis, are a leading cause of morbidity and mortality worldwide spanning all demographics. Sepsis arises from a dysregulated immune response to infection leading to systemic end-organ injury. CD8 T cells, critical for pathogen defense, are profoundly impaired during sepsis, exhibiting high rates of attrition, reduced cytokine production, and exhaustion-like phenotypes. Systemic metabolic dysfunction, a hallmark of critical illness driven by hypoxemia, tissue hypoperfusion, and inflammation, exacerbates immune cell dysfunction. However, the mechanisms linking metabolic stress to CD8 T cell failure in human critical illness with and without sepsis remain poorly understood. Our preliminary studies reveal that effector CD8 T cells from critically ill and septic patients exhibit abnormal mitochondrial phenotypes, including heightened glutamine metabolism that correlates with elevated exhaustion markers, impaired effector function, and worse clinical outcomes. Using single-cell RNA sequencing, we further identified a distinct effector CD8 T cell subset (CXCR4hi IL7Rlo) unique to critical illness, characterized by high levels of terminal exhaustion markers TIM-3 and TOX, reduced cytotoxic features, and profound hypometabolism. Among the most highly downregulated genes in this CXCR4hi IL7Rlo subset was phosphoglycerate mutase 1 (PGAM1), a glycolytic enzyme that regulates both ATP production and biosynthesis. Accordingly, we hypothesize that metabolic alterations in effector CD8 T cells drive their failure in human critical illness with and without sepsis. Aim (1) will determine how glutamine metabolism modulates effector CD8 T cell fitness and function. Using patient-derived CD8 T cells, we will employ advanced techniques, including high-dimensional spectral flow cytometry, 13C-glutamine tracing, and pharmacologic manipulation of glutaminase activity. These will assess how blocking glutaminolysis affects cytokine production, cytotoxicity, and exhaustion marker expression, and directly trace glutamine usage for energy, biosynthesis, and antioxidant generation. Aim (2) will test the role of PGAM1 as a metabolic switch controlling energy production and biosynthesis in effector CD8 T cells. We will evaluate whether PGAM1 inhibition recapitulates the functional and metabolic impairments observed in CXCR4hi IL7Rlo CD8 T effector cells, test whether rescuing glycolysis and biosynthetic pathways with pyruvate and ribose-5-phosphate, respectively, can restore effector function in CXCR4hi IL7Rlo CD8 T cells, and correlate CXCR4hi IL7Rlo CD8 T effector frequencies and phenotypes with clinical metrics in critical illness with and without sepsis. These studies hold the promise of directly linking systemic metabolic dysfunction to CD8 T cell immune failure in human critical illness and sepsis, providing key insights into immunometabolic regulation and identifying new therapeutic targets. This award will provide essential training in translational immunometabolism and human critical illness research, supported by mentorship and guidance from leading investigators in these content areas, to develop the PI into a successful, independent physician-scientist.
