Kynurenine-Driven Metabolic Adaptations Rewire CD4 T Cells in a Subset-Specific Manner in Human Critical Illness and Severe Sepsis

Allison Sewell – Department of Pathology, Microbiology, and Immunology – Vanderbilt University Medical Center; Erin Mwizerwa, BSN – Department of Medicine – Vanderbilt University Medical Center; Chooi Ying Sim, BSN – Department of Medicine – Vanderbilt University Medical Center; Samantha Tanner, BSN – Department of Medicine – Vanderbilt University Medical Center; Casey Nichols – Department of Pathology, Microbiology, and Immunology – Vanderbilt University Medical Center; Heather Durai – Department of Pathology, Microbiology, and Immunology – Vanderbilt University Medical Center; Erin Jennings, PhD – Department of Pathology, Microbiology, and Immunology – Vanderbilt University Medical Center; Paul Lindau, MD, PhD – Department of Medicine – Vanderbilt University Medical Center; Erin Wilfong, MD, PhD – Department of Medicine – Vanderbilt University Medical Center; Dawn Newcomb, PhD – Department of Medicine – Vanderbilt University Medical Center; Julie Bastarache, MD – Department of Medicine – Vanderbilt University Medical Center; Lorraine Ware, MD – Department of Medicine – Vanderbilt University Medical Center; Jeffrey Rathmell, PhD – Cornelius Vanderbilt Professor of Immunobiology and Director of the Vanderbilt Center for Immunobiology, Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center

Abstract Text: Host immunity in sepsis is characterized by the progressive development of immunosuppression, particularly among CD4 T cells, leading to serious secondary infections, impaired organ recovery, and significant mortality. Bioenergetic dysfunction is archetypal in sepsis, but its mechanistic link to human CD4 T cell dysfunction remains unclear. To investigate, we generated a 644,147-cell scRNA-seq map of critical illness from PBMCs collected from ICU patients at Vanderbilt. In ICU patients, particularly with sepsis, regulatory T cells (Tregs) were preserved in frequency and function while conventional CD4 subsets (Tconv) underwent substantial attrition, reflecting progressive immunosuppression. Reaction-level metabolic modeling by in silico flux balance analysis identified elevated kynurenine metabolic signatures in septic Tregs compared to Tconv. Kynurenine is a biomarker of disease severity in sepsis, but the mechanisms underlying this are unclear. Using an ex vivo single cell energetic assay (SCENITH), we demonstrated that ICU patient Tregs exhibited higher basal metabolic rates, increased metabolic plasticity with bolstered glycolytic capacity, and elevated kynurenine uptake. Acquired glycolytic capacity directly correlated with Treg survival and supported suppressive phenotypes, marked by stabilized TIGIT and FOXP3 expression amid mitochondrial stress. Moreover, Treg glycolytic capacity in critical illness associated with key clinical metrics including higher SOFA scores, worse shock, longer hospitalizations, and mortality. Supplementation of healthy Tregs with kynurenine increased their glycolytic capacity, whereas pharmacologic kynurenine 3-monooxygenase inhibition partially reversed the suppressive adaptations of septic Tregs. Collectively, these findings establish immunometabolic dysfunction as a driver of CD4 T cell remodeling in sepsis and highlight therapeutic opportunities to restore immune balance.