Adverse outcomes and an immunosuppressed endotype in septic patients with reduced IFN-γ ELISpot

Abstract

BACKGROUNDSepsis remains a major clinical challenge for which successful treatment requires greater precision in identifying patients at increased risk of adverse outcomes requiring different therapeutic approaches. Predicting clinical outcomes and immunological endotyping of septic patients generally relies on using blood protein or mRNA biomarkers, or static cell phenotyping. Here, we sought to determine whether functional immune responsiveness would yield improved precision.METHODSAn ex vivo whole-blood enzyme-linked immunosorbent spot (ELISpot) assay for cellular production of interferon γ (IFN-γ) was evaluated in 107 septic and 68 nonseptic patients from 5 academic health centers using blood samples collected on days 1, 4, and 7 following ICU admission.RESULTSCompared with 46 healthy participants, unstimulated and stimulated whole-blood IFN-γ expression was either increased or unchanged, respectively, in septic and nonseptic ICU patients. However, in septic patients who did not survive 180 days, stimulated whole-blood IFN-γ expression was significantly reduced on ICU days 1, 4, and 7 (all P < 0.05), due to both significant reductions in total number of IFN-γ-producing cells and amount of IFN-γ produced per cell (all P < 0.05). Importantly, IFN-γ total expression on days 1 and 4 after admission could discriminate 180-day mortality better than absolute lymphocyte count (ALC), IL-6, and procalcitonin. Septic patients with low IFN-γ expression were older and had lower ALCs and higher soluble PD-L1 and IL-10 concentrations, consistent with an immunosuppressed endotype.CONCLUSIONSA whole-blood IFN-γ ELISpot assay can both identify septic patients at increased risk of late mortality and identify immunosuppressed septic patients.TRIAL REGISTRYN/A.FUNDINGThis prospective, observational, multicenter clinical study was directly supported by National Institute of General Medical Sciences grant R01 GM-139046, including a supplement (R01 GM-139046-03S1) from 2022 to 2024.

Keywords: Adaptive immunity; Cellular immune response; Immunology; T cells.

Figure 1. Unstimulated and stimulated IFN-γ expression as determined by ELISpot in SEPSIS and CINS patients and healthy control participants on days 1, 4, and 7 following ICU admission.

Values represent medians and individual subject responses. (A–C) IFN-γ expression in unstimulated whole blood. (D–F) IFN-γ expression in anti-CD3/anti-CD28–stimulated whole blood. Note that the scales for unstimulated expression are logarithmic, whereas they are linear for stimulated expression to appropriately reflect the magnitude and heterogeneity of the individual response. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, as determined by Kruskal-Wallis ANOVA and post hoc analyses using Dunn’s test. Values are 2 sided and represent raw P values. SFU, spot-forming units; SS, spot size; TE, total IFN-γ expression.

Figure 2. ELISpot SFU and SS from unstimulated whole blood in the 3 cohorts (healthy participants, SEPSIS, and CINS).

(A–C) IFN-γ SFU. (D–F) IFN-γ SS. In unstimulated whole blood, SEPSIS and CINS cohorts demonstrated a consistent increase in the number of cells (SFU) producing IFN-γ, when compared with healthy control participants. *P < 0.05, ***P < 0.001, ****P < 0.0001, as determined by Kruskal-Wallis ANOVA and post hoc analyses using Dunn’s test. Values are 2 sided and represent raw P values. SFU, spot-forming units; SS, spot size; TE, total IFN-γ expression.

Figure 3. Anti-CD3/anti-CD28–stimulated IFN-γ expression by ELISpot in sepsis patients measured 1, 4, and 7 days after ICU admission who survived or did not survive 180 days.

(A–C) Spot number. (D–F) Spot size. (G–I) Total IFN-γ expression. Values represent medians and individual responses. The number of participants declined over time as patients were either discharged from the ICU or died. *P < 0.05, **P < 0.01, ***P < 0.001, as determined by Kruskal-Wallis ANOVA and post hoc analyses using Dunn’s test. Values are 2 sided and represent raw P values. SFU, spot-forming units; SS, spot size; TE, total IFN-γ expression.

Figure 4. Area under the receiver operator curves (AUROC) for physiologic (SOFA, Charlson comorbidity scores) and stimulated IFN-γ ELISpot responses in differentiating in-hospital and 180-day mortality.

(A) SOFA and Charlson comorbidity index. (B) Selected ELISpot parameters discriminating 180-day mortality. (C and D) Same as for A and B but discriminating in-hospital mortality. TE, IFN-γ ELISpot total expression; SFU, IFN-γ ELISpot spot-forming units; SS, IFN-γ ELISpot spot size.

Figure 5. Flow diagram for study enrollment.

SPIES, Stratifying Patient Immune Endotypes in Sepsis study.