Functional Immune Phenotyping of Septic Patients by ELISpot
CCCF ePoster library. Mazer M. 11/11/19; 283444; EP34
Monty Mazer
Monty Mazer
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Topic: Basic or Translational Science

Mazer, Monty1; Walton, Andrew2; Remy, Kenneth1,2; Hotchkiss, Richard2

1. Division of Pediatric Critical Care Medicine, Washington University School of Medicine, St. Louis, MO, USA
2. Department of Anesthesia, Washington University School of Medicine, St. Louis MO, USA

Sepsis, a dysregulated host immune response to infection, remains a leading cause of morbidity and mortality. While patients with sepsis often exhibit an initial hyper-inflammatory phase, if sepsis persists, there is development of severe immune suppression. Recently, therapeutic approaches to sepsis have included immune adjuvant agents that act to restore immune effector cell function. A key issue in instituting immune modulatory therapy in sepsis is determining the immune phenotype. Enzyme-Linked Immunospot assay (ELISpot) quantifies the ex vivo release of cytokines from immune cells over a time period and thereby provides an excellent determination of actual cellular function. Knowledge of the exact state of cell function, as opposed to transcriptomics or measurement of cytokines at a single time point, is highly advantageous, as it will provide insight on the true state of the patient's immunity. IFN-γ is a cytokine produced by adaptive immune cells (T cells) and is critical for an effective host immune response against pathogens. TNF-α is produced by innate immune cells (monocyte/macrophages) and is a key mediator of the intensity of the host innate inflammatory response and may drive the hyper-inflammatory phase of sepsis.

The objective was to determine if the ELISpot assay for TNF-α and IFN-γ would provide an accurate assessment of the functional state of host innate and adaptive immunity, and thereby enable discrimination of patients into a hyper- versus hypo-inflammatory phase of the disorder. Patients with sepsis were compared to patients who were critically ill non-septic (CINS) and with age matched healthy volunteers.

52 septic and 31 CINS ICU patients were enrolled. 30 age matched healthy volunteers were included. Blood samples were drawn and peripheral blood mononuclear cells (PBMCs) were plated onto IFN-ɣ and TNF-α precoated ELISpot wells for overnight culture. T-cells were stimulated with anti-CD3 and anti-CD28 antibodies, and monocytes were stimulated with LPS.. Results were analyzed using t-test or ANOVA where appropriate.

Septic and CINS patients had a decrease in the number of IFN-γ producing T cells compared to healthy volunteers; 192 +/- 28 cells and 265 +/- 45 cells for septic and CINS respectively versus 711 +/- 81 cells for healthy controls (p <0.0001). There was no significant difference in the number of cells producing TNF-α in the three patient groups. There was noteworthy heterogeneity in the T cell and monocyte response in septic patients consistent with variable degrees of immune suppression or hyper-inflammation.

The ELISpot assay has excellent capability and good dynamic range in distinguishing individual patient immune responses in sepsis. Patients with sepsis have immune suppression of adaptive immunity. There is significant variability in the degree of immune suppression in septic and CINS patients consistent with a heterogeneous patient population. The ELISpot assay may be an effective means to immune phenotype patients with sepsis and identify those patients who are profoundly immune suppressed and therefore candidates for immune adjuvant therapies. The ELISpot results should be correlated with clinical outcomes including secondary infections, survival, readmission and SOFA scores in order to support its predictive potential for identifying immune suppression.

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