Dysregulated T Cell Cytokine Responses Characterize a High-Risk Pediatric Sepsis Subphenotype

Background Sepsis is the leading cause of pediatric in-hospital mortality worldwide, driven in part by immune dysregulation. As sepsis biology is marked by immune heterogeneity, precision medicine approaches to identify actionable phenotypes are crucial to improving patient outcomes. Among three molecular subphenotypes we previously identified in children with sepsis, Group C patients have poor clinical outcomes and a unique immune profile involving dysregulated STAT3 signaling. In this analysis, we sought to evaluate cytokine production in CD8+ T cells from pediatric sepsis patients across molecular subphenotypes. Methods We measured T cell cytokine production by flow cytometry in pediatric sepsis samples (n = 17) after 24-hour stimulation with αCD3/αCD28. We calculated absolute and relative change in geometric mean fluorescent intensity from baseline for both conditions for six cytokines: IL-2, IL-13, IL-17, IL-21, TNF-α, and IFN-γ. Samples were acquired via a Cytek Aurora spectral flow cytometer and analyzed in FlowJo and Rstudio. We compared cytokine expression across sepsis subphenotypes by ANOVA. Results Baseline, unstimulated cytokine production was minimal in all groups. In response to αCD3/αCD28 stimulation, CD8+ T cells from Group C patients demonstrated significantly increased IL-2 production (p = 0.012) compared with Groups A and B, and a trend toward sustained TNF-α production (p = 0.11). Conversely, Group C showed an exaggerated reduction in IL-17 production in response to stimulation (p = 0.03). CD4+ T cells from Group C patients similarly demonstrated significantly increased IL-2 production (p = 0.003) after stimulation compared with Groups A and B, and a similar trend toward sustained TNF-α production (p = 0.11). IFN-γ, IL-13, and IL-21 expression increased with stimulation in all groups but did not vary by subphenotype. Conclusions Group C patients demonstrate increased pro-inflammatory cytokine production in CD8+ T cells following αCD3/αCD28 stimulation compared with Groups A and B. This dysregulated response to T cell stimulation in the most severe molecular sepsis subphenotype is a potentially reversible cause of organ dysfunction in pediatric patients with sepsis.