Sepsis is the leading cause of death of hospitalized children worldwide. Despite the established link between immune dysregulation and mortality in pediatric sepsis, it remains unclear which host immune factors contribute causally to adverse sepsis outcomes. Identifying modifiable pathobiology is an essential first step to successful translation of biologic insights into precision therapeutics. We designed a prospective, longitudinal cohort study of 88 critically ill pediatric patients with multiple organ dysfunction syndrome (MODS), including patients with and without sepsis, to define subphenotypes associated with targetable mechanisms of immune dysregulation. We first assessed plasma proteomic profiles and identified shared features of immune dysregulation in MODS patients with and without sepsis. We then employed consensus clustering to define three subphenotypes based on protein expression at disease onset and identified a strong association between subphenotype and clinical outcome. We next identified differences in immune cell frequency and activation state by MODS subphenotype and determined the association between hyperinflammatory pathway activation and cellular immunophenotype. Using single cell transcriptomics, we demonstrated STAT3 hyperactivation in lymphocytes from the sickest MODS subgroup and then identified an association between STAT3 hyperactivation and T cell immunometabolic dysregulation. Finally, we compared proteomics findings between patients with MODS and patients with inborn errors of immunity that amplify cytokine signaling pathways to further assess the impact of STAT3 hyperactivation in the most severe patients with MODS. Overall, these results identify a potentially pathologic and targetable role for STAT3 hyperactivation in a subset of pediatric patients with MODS who have high severity of illness and poor prognosis.

Children's Hospital of Philadelphia and the University of Pennsylvania filed a provisional US patent application on May 22, 2024 related to the prognostic molecular subphenotypes presented in this manuscript (63/650,523; Identification of High Mortality Sub-Phenotypes of Pediatric Multiple Organ Dysfunction Syndrome (MODS) for Management and Treatment Thereof). RBL, NJM, and SEH are named as co-inventors on that patent application. All other authors declare that they have no competing interests.

This study was funded in part by K12HD047349 (RBL), K08AI135091 (SEH), and pilot grants from the Thrasher Research Fund (RBL) and the CHOP Research Institute (RBL). The parent PARADIGM study is funded by R01HD095976 (MWH). Healthy control participant recruiting was funded by a Career Award for Medical Scientists from the Burroughs Wellcome Fund (SEH). Additional funding sources include the Barbara Brodsky Foundation and institutional startup funds from the Division of Critical Care Medicine.

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The Institutional Review Board of Children's Hospital of Philadelphia gave ethical approval for this work (IRB #19-017032 and IRB #18-015920). Study participant (or legal guardian, if under 18) consent (and assent, if appropriate) were obtained prior to study enrollment in accordance with our IRB approved protocols.

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