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RATIONALE Sepsis is the leading cause of death in adult intensive care units. At present, sepsis diagnosis relies on non-specific clinical features. It could transform clinical care to have immune cell biomarkers that could predict sepsis diagnosis and guide treatment. For decades, neutrophil phenotypes have been studied in sepsis, but a diagnostic cell subset has yet to be identified. OBJECTIVES To identify an early specific immune signature of sepsis severity that does not overlap with other inflammatory biomarkers, and that distinguishes patients with sepsis from those with non- infectious inflammatory syndrome. METHODS Mass cytometry combined with computational high-dimensional data analysis were used to measure 42 markers on whole blood immune cells from sepsis patients and controls, and automatically and comprehensively characterize circulating immune cells, which enables identification of novel, disease-specific cellular signatures. MEASUREMENTS AND MAIN RESULTS Unsupervised analysis of high-dimensional mass cytometry data characterized previously unappreciated heterogeneity within the CD64+ immature neutrophils and revealed two new subsets distinguished by CD123 and PD-L1 expression. These immature neutrophils exhibited diminished activation and phagocytosis functions. The proportion of CD123-expressing neutrophils correlated with clinical severity. CONCLUSIONS This study showed that these two new neutrophil subsets were specific to sepsis and detectable by routine flow cytometry using seven markers. The demonstration here that a simple blood test distinguishes sepsis from other inflammatory conditions represents a key biological milestone that can be immediately translated into improvements in patient care.