Ex Vivo Immune Profiling Defines a Continuous Functional Immune Axis and a Sepsis-Enriched Low-Response State in Critical Illness

BackgroundImmune dysfunction in sepsis and critical illness is biologically heterogeneous, yet available stratification frameworks leave many patients unclassified. We hypothesized that ex vivo cytokine-induction responses would define a continuous axis of functional immune responsiveness and identify a low-response state enriched in sepsis. MethodsIn this prospective observational study, 39 critically ill adults enrolled within 48 hours of ICU admission and 6 healthy controls underwent standardized whole-blood stimulation with lipopolysaccharide, anti-CD3/anti-CD28 antibodies, and PMA/ionomycin, with selected wells additionally supplemented with interleukin-7 or granulocyte-macrophage colony-stimulating factor. Interleukin-6, tumor necrosis factor, and interferon-gamma responses were quantified and referenced to subject-specific unstimulated baselines. A patient-anchored primary feature matrix was used to derive a continuous immune axis by principal component analysis, and a cross-validated 5-feature MiniResponder score was developed as a portable summary measure. ResultsAmong critically ill patients, induced cytokine responses organized along a dominant continuous axis of functional immune responsiveness; the first principal component explained 53.3% of between-patient variance. MiniResponder captured this axis and showed a lower-shifted distribution in sepsis. Using a control-referenced threshold defined by the 10th percentile of the healthy-control distribution, 19 of 39 patients (48.7%) were classified as low-response, including 15 of 21 patients with sepsis (71.4%) and 4 of 18 critically ill patients without sepsis (22.2%) (odds ratio 8.75, Fisher exact P=0.004). In exploratory analyses, lower MiniResponder scores were associated with greater unadjusted improvement in Sequential Organ Failure Assessment score from day 1 to days 3-9 (rho=-0.33; P=0.046), but this association attenuated after adjustment for baseline SOFA score (beta=-0.10; 95% CI-0.36 to 0.27). ConclusionsEx vivo immune profiling identified a continuous patient-anchored axis of functional immune responsiveness in critical illness that can be summarized by a compact 5-feature score. A control-referenced low-response state was enriched in sepsis. This framework may complement existing biomarker-based stratification approaches and support future enrichment strategies in sepsis trials.